U.S. patent application number 14/796509 was filed with the patent office on 2016-01-07 for genetic products differentially expressed in tumors and use thereof.
The applicant listed for this patent is BioNTech AG. Invention is credited to Michael Koslowski, Ugur Sahin, Ozlem Tureci.
Application Number | 20160002738 14/796509 |
Document ID | / |
Family ID | 27771250 |
Filed Date | 2016-01-07 |
United States Patent
Application |
20160002738 |
Kind Code |
A1 |
Sahin; Ugur ; et
al. |
January 7, 2016 |
GENETIC PRODUCTS DIFFERENTIALLY EXPRESSED IN TUMORS AND USE
THEREOF
Abstract
The invention relates to the identification of genetic products
that are expressed in association with a tumor and the nucleic acid
coding therefor. The invention relates to the therapy and diagnosis
of diseases in which the genetic products that are expressed in
association with a tumor are expressed in an aberrant manner. The
invention also relates to proteins, polypeptides, and peptides
which are expressed in association with a tumor and the nucleic
acids coding therefor.
Inventors: |
Sahin; Ugur; (Mainz, DE)
; Tureci; Ozlem; (Mainz, DE) ; Koslowski;
Michael; (Mainz, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioNTech AG |
Mainz |
|
DE |
|
|
Family ID: |
27771250 |
Appl. No.: |
14/796509 |
Filed: |
July 10, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13796094 |
Mar 12, 2013 |
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14796509 |
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13184719 |
Jul 18, 2011 |
8551490 |
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13796094 |
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12197956 |
Aug 25, 2008 |
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13184719 |
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10506443 |
Sep 2, 2004 |
7429461 |
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PCT/EP2003/002556 |
Mar 12, 2003 |
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12197956 |
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Current U.S.
Class: |
435/6.11 ;
435/6.12 |
Current CPC
Class: |
A61P 35/04 20180101;
A61K 39/00 20130101; C07K 16/30 20130101; A61P 43/00 20180101; C07K
2317/34 20130101; C07K 16/3053 20130101; A61P 37/04 20180101; C12Q
1/6886 20130101; A61K 39/39558 20130101; C12Q 2600/158 20130101;
A61P 35/00 20180101; C07K 16/3023 20130101; C07K 14/4748 20130101;
A61K 48/00 20130101; C07K 2317/76 20130101; A61P 31/00 20180101;
C07K 16/3069 20130101; G01N 33/574 20130101; A61K 38/00 20130101;
C12Q 2600/112 20130101; C07K 16/3015 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 13, 2002 |
DE |
102-11-088.3 |
Claims
1.-32. (canceled)
33. A method of diagnosing a disease characterized by expression or
abnormal expression of a tumor-associated antigen, which method
comprises detection of a nucleic acid which codes for the
tumor-associated in a sample isolated from a patient, with said
tumor-associated antigen having a sequence encoded by a nucleic
acid which comprises a nucleic acid sequence of SEQ ID NO: 39, or a
part thereof, wherein the part thereof is a sequence selected from
the group consisting of ESTs AW137203, BF327792, BF327797,
BE069044, and BF330665.
34. The method as claimed in claim 33, in which the detection
comprises (i) contacting the sample with an agent which binds
specifically to the nucleic acid coding for the tumor-associated
antigen, and (ii) detecting the formation of a complex between the
agent and the nucleic acid.
35. The method as claimed in claim 34, in which the detection is
compared to detection in comparable normal sample.
36. The method as claimed in any of claim 33, in which the disease
is characterized by expression or abnormal expression of the
tumor-associated antigens and in which detection comprises
detection of the nucleic acid coding for said tumor-associated
antigen.
37. The method as claimed in claim 33, in which the nucleic acid is
detected using a polynucleotide probe prepared by amplification
with a primer set comprising SEQ ID NO: 44 and SEQ ID NO: 45 or SEQ
ID NO: 46 and SEQ ID NO: 47 which hybridizes specifically to said
nucleic acid.
38. The method as claimed in claim 37, in which the polynucleotide
probe comprises a sequence of 6-50 contiguous nucleotides of the
nucleic acid coding for the tumor-associated antigen.
39. The method as claimed in claim 33, in which the nucleic acid is
detected by selectively amplifying said nucleic acid with a primer
set comprising SEQ ID NO: 44 and SEQ ID NO: 45 or SEQ ID NO: 46 and
SEQ ID NO: 47.
40. (canceled)
41. (canceled)
42. (canceled)
43. (canceled)
44. A method for determining regression, course or onset of a
disease characterized by expression abnormal expression of a
tumor-associated antigen, 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 the amount of
nucleic acid which codes for the tumor-associated antigen, said
tumor-associated antigen having a sequence encoded by a nucleic
acid which comprises a nucleic acid sequence of SEQ ID NO: 39, or a
part thereof, wherein the part thereof is a sequence selected from
the group consisting of ESTs AW137203, BF327792, BF327797,
BE069044, and BF330665.
45. The method as claimed in claim 44, wherein the monitoring is
determined 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.
46. The method as claimed in claim 44, in which the disease is
characterized by expression or abnormal expression of the
tumor-associated antigens and in which monitoring comprises
monitoring the amount of the nucleic acids which code for said
tumor-associated antigen.
47. The method as claimed in claim 44, in which the amount of the
nucleic acid is monitored using a polynucleotide probe prepared by
amplification with a primer set comprising SEQ ID NO: 44 and SEQ ID
NO: 45 or SEQ ID NO: 46 and SEQ ID NO: 47 which hybridizes
specifically to said nucleic acid.
48. The method as claimed in claim 47, in which the polynucleotide
probe comprises a sequence of 6-50 contiguous nucleotides of the
nucleic acid coding for the tumor-associated antigen.
49. The method as claimed in claim 44, in which the amount of the
nucleic acid is monitored by selectively amplifying said nucleic
acid with a primer set comprising SEQ ID NO: 44 and SEQ ID NO: 45
or SEQ ID NO: 46 and SEQ ID NO: 47.
50. (canceled)
51. (canceled)
52. (canceled)
53. (canceled)
54. (canceled)
55. The method as claimed in claim 33, in which the sample
comprises body fluid and/or body tissue and the disease is breast
cancer.
56.-117. (canceled)
118. The method as claimed in claim 44, in which the sample
comprises body fluid and/or body tissue and the disease is breast
cancer.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/796,094 filed on Mar. 12, 2013, which is a divisional
of U.S. patent application Ser. No. 13/184,719, filed on Jul. 18,
2011, which is a continuation of U.S. patent application Ser. No.
12/197,956, which was filed on Aug. 25, 2008, as a divisional
application of U.S. patent application Ser. No. 10/506,443, now
U.S. Pat. No. 7,429,461, which was filed Sep. 2, 2004, as a
National Stage Entry of PCT/EP03/02556, which was filed on Mar. 12,
2003, and claimed priority to German Patent Application Number
102-11-088.3, which was filed on Mar. 13, 2002. The contents of
each of the preceding applications 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 molecule/1y 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
at 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. The class of
cancer/testis antigens (CTA) is of great interest here. CTA and
genes encoding them (cancer/testis genes or CTG) are defined by
their characteristic expression pattern [Tureci et al, Mol Med
Today. 3:342-9, 1997]. They are not found in normal tissues, except
testis and germ cells, but are expressed in a number of human
malignomas, not tumor type-specifically but with different
frequency in tumor entities of very different origins (Chen &
Old, Cancer J. Sci. Am. 5:16-7, 1999). Serum reactivities against
CTA are also not found in healthy controls but only in tumor
patients. This class of antigens, in particular owing to its tissue
distribution, is particularly valuable for immunotherapeutic
projects and is tested in current clinical patient studies
(Marchand at al., Int. J. Cancer 80:219-30, 1999; Knuth et al.,
Cancer Chemother. Pharmacol. 46:p 46-51, 2000).
[0004] 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
[0005] It was the object of the present invention to provide target
structures for a diagnosis and therapy of cancers.
[0006] According to the invention, this object is achieved by the
subject matter of the claims.
BRIEF DESCRIPTIONS OF THE FIGURES
[0007] FIG. 1: Diagrammatic representation of the cloning of eCT.
The strategy comprises identifying candidate genes (GOI="Genes of
interest") in databases and testing said genes by means of
RT-PCR.
[0008] FIG. 2: Splicing of LDH C. Alternative splicing events
result in the absence of exon 3 (SEQ ID NO:2), of the two exons 3
and 4 (SEQ ID NO:3), of the exons 3, 6 and 7 (SEQ ID NO:4) and of
exon 7 (SEQ ID NO:5). ORF=open reading frame, as=amino acid.
[0009] FIG. 3: Alignment of possible LDH-C proteins. SEQ ID NO:8
and SEQ ID NO:10 are truncated portions of the prototype protein
(SEQ ID NO:6). The protein sequences of SEQ ID NO:7, SEQ ID NO:9,
SEQ ID NO:11, SEC ID NO:12 and SEQ ID NO:13 are additionally
altered and contain only tumor-specific epitopes (printed in bold
type). The catalytic centre is framed.
[0010] FIG. 4: Quantification of LDH C in various tissues by means
of real time PCR. No transcripts were detected in normal tissues
other than testis, but significant levels of expression were
detected in tumors.
[0011] FIG. 5: Exon composition of TPTE variants. According to the
invention, splice variants were identified (SEQ ID NO:20, SEQ ID
NO:21, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57)
which are expressed in testicular tissues and in tumors and which
have frame shifts and thus altered sequence regions.
[0012] FIG. 6: Alignment of the possible TPTE proteins. Alternative
splicing events result in alterations of the encoded proteins, with
the reading frame being retained in principle. The putative
transmembrane domains are printed in bold type, the catalytic
domain is framed.
[0013] FIG. 7: Alignment of TSBP variants at the nucleotide level.
The differences in the nucleotide sequences of the TSBP variants
found according to the invention (SEQ) ID NO:31, SEQ ID NO:32, SEQ
ID NO:33) to the known sequence (NM.sub.--006781, SEQ ID NO: 29)
are printed in bold type.
[0014] FIG. 8: Alignment of TSBP variants at the protein level. In
the proteins encoded by the TSBP variants found according to the
invention (SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36), frame shifts
cause substantial differences to the previously described protein
(SEQ ID NO:30, NM.sub.--006781) and are indicated by bold type.
[0015] FIG. 9: RT-PCR for MS4Al2. Expression was detected in the
tissues tested only in testis, colon and colorectal carcinomas
(colon ca's). In one of the 6 liver tissue samples shown, a
positive detection was carried out for MS4Al2, since this sample
has been infiltrated by a colon carcinoma metastasis. Later studies
also demonstrated distinct expression in colon carcinoma
metastases.
[0016] FIG. 10: RT-PCR for BRC01. BRCO1 is distinctly overexpressed
in breast tumors in comparison with expression in normal mammary
gland tissue.
[0017] FIG. 11: RT-PCR for MORC, TPX1, LDHC, SGY-1. A study of
various normal tissues reveals expression only in testis (1 skin, 2
small intestine, 3 colon, 4 liver, 5 lung, 6 stomach, 7 breast, 8
kidney, 9 ovary, 10 prostate, 11 thyroid, 12 leukocytes, 13 thymus,
14 negative control, 15 testis). The examination of tumors (1-17
lung tumors, 18-29 melanomas, 30 negative control, 31 testis)
reveals ectopic expression in said tumors with different
frequencies for the individual eCT.
[0018] FIG. 12: Mitochondrial localization of LDHC in the MCF-7
breast cancer cell line. MCF-7 cells were transiently transfected
with an LDHC expression plasmid. The antigen was detected with
LDHC-specific antibodies and showed distinct colocalization with
the mitochondrial respiratory chain enzyme cytochrome
C-oxidase.
[0019] FIG. 13: Predicted topology of TPTE and subcellular
localization on the cell surface of MCF-7 cells. The diagram on the
left-hand side depicts the 4 putative TPTE transmembrane domains
(arrows). MCF-7 cells were transiently transfected with a TPTE
expression plasmid. The antigen was detected using TPTE-specific
antibodies and showed distinct colocalization with MHC I molecules
located on the cell surface.
[0020] FIG. 14: MS4Al2 localization on the cell membrane. Tumor
cells were transiently transfected with a GFP-tagged MS4Al2
construct and showed complete colocalization with plasma membrane
markers in confocal immunofluorescence microscopy.
DETAILED DESCRIPTION OF THE INVENTION
[0021] According to the invention, 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 actually testis- and thus germ cell-specific genes
which are usually silent in adult tissues are reactivated in tumor
cells in an ectopic and forbidden manner First, data mining
produces a list as complete as possible of all known
testis-specific genes which are then evaluated for their aberrant
activation in 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).
[0022] The concept of the invention, which has proved much more
successful, however, is based on utilizing data mining for
electronically extracting all testis-specific genes and then
evaluating said genes for ectopic expression in tumors.
[0023] The invention thus relates in one aspect to a strategy for
identifying genes differentially expressed in tumors. Said strategy
combines data mining of public sequence libraries ("in silico")
with subsequent evaluating laboratory-experimental ("wet bench")
studies.
[0024] According to the invention, a combined strategy based on two
different bioinformatic scripts enabled new members of the
cancer/testis (CT) gene class to be identified. These have
previously been classified as being purely testis-, germ cell- or
sperm-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
invention, these tumor-associated genes and the genetic products
encoded thereby were identified and provided independently of an
immunogenic action.
[0025] The tumor-associated antigens identified according to the
invention have an amino acid sequence encoded by a nucleic; acid
which is selected from the group consisting of (a) a nucleic acid
which comprises a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1-5, 19-21, 29, 31-33, 37, 39, 40, 54-57,
62, 63, 70, 74, 85-88, a part or derivative thereof, b) a nucleic
acid which hybridizes with the nucleic acid of (a) under stringent
conditions, c) a nucleic acid which is degenerate with respect to
the nucleic acid of (a) or (b), and (d) a nucleic acid which is
complementary to the nucleic acid of (a), (b) or (c). In a
preferred embodiment, a tumor-associated antigen identified
according to the invention has an amino acid sequence encoded by a
nucleic acid which is selected from the group consisting of SEQ ID
NOs: 1-5, 19-21, 29, 31-33, 37, 39, 40, 54-57, 62, 63, 70, 74,
85-88. In a further preferred embodiment, a tumor-associated
antigen identified according to the invention comprises an amino
acid sequence selected from the group consisting of SEQ ID NOs:
6-13, 14-18, 22-24, 30, 34-36, 38, 41, 58-61, 64, 65, 71, 75,
80-84, 89-100, a part or derivative thereof.
[0026] The present invention generally relates to the use of
tumor-associated antigens identified according to the invention or
of parts 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 invention or parts 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.
[0027] Preferred diseases for a therapy and/or diagnosis are those
in which one or more of the tumor-associated antigens identified
according to the invention are selectively expressed or abnormally
expressed.
[0028] The invention also relates to nucleic acids and genetic
products which are expressed in association with a tumor cell and
which are produced by altered splicing (splice variants) of known
genes or by altered translation with utilization of alternative
open reading frames. Said nucleic acids comprise the sequences
according to (SEQ ID NO: 2-5, 20, 21, 31-33, 54-57, 85-88) of the
sequence listing. Furthermore, the genetic products comprise
sequences according to (SEQ ID NO: 7-13, 23, 24, 34-36, 58-61,
89-100) of the sequence listing. The splice variants of the
invention can be used according to the invention as targets for
diagnosis and therapy of neoplastic diseases.
[0029] Very different mechanisms may cause splice variants to be
produced, for example [0030] utilization of variable transcription
initiation sites [0031] utilization of additional exons [0032]
complete or incomplete splicing out of single or two or more exons,
[0033] splice regulator sequences altered via mutation (deletion or
generation of new donor/acceptor sequences), [0034] incomplete
elimination of intron sequences.
[0035] 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 invention, for example, after
extraction of nucleic acids by PCR amplification with splice
variant-specific oligonucleotides. According to the invention, all
sequence-dependent detection systems are suitable for detection.
These are, apart from PCR, for example gene chip/microarray
systems, Northern blot, RNAse protection assays (RDA) and others.
All detection systems have in common that detection is based on a
specific hybridization with at least one splice variant-specific
nucleic acid sequence. However, tumor cells may also be detected
according to the invention by antibodies which recognize a specific
epitope encoded by the splice variant. Said antibodies may be
prepared by using for immunization peptides which are specific for
said splice variant. Suitable for immunization are particularly the
amino 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 invention 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 for utilization as
pharmaceutically active substances in active immunotherapy
(vaccination, vaccine therapy).
[0036] In one aspect, the invention relates to a pharmaceutical
composition comprising an agent which recognizes the
tumor-associated antigen identified according to the invention and
which is preferably selective for cells which have expression or
abnormal expression of a tumor-associated antigen identified
according to the invention. 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
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 invention. Recognition needs not be
accompanied directly with inhibition of activity or expression of
the antigen. In this aspect of the invention, 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 cell
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.
[0037] In one aspect, the invention relates to a pharmaceutical
composition comprising an agent which inhibits expression or
activity of a tumor-associated antigen identified according to the
invention, 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 invention.
[0038] The invention 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 invention, 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 invention.
[0039] The invention furthermore relates to a pharmaceutical
composition which comprises one or more components selected from
the group consisting of (i) a tumor-associated antigen identified
according to the invention or a part thereof, (ii) a nucleic acid
which codes for a tumor-associated antigen identified according to
the invention or for a part thereof, (iii) an antibody which binds
to a tumor-associated antigen identified according to the invention
or to a part thereof, (iv) an antisense nucleic acid which
hybridizes specifically with a nucleic acid coding for a
tumor-associated antigen identified according to the invention, (v)
a host cell which expresses a tumor-associated antigen identified
according to the invention or a part thereof, and (vi) isolated
complexes between a tumor-associated antigen identified according
to the invention or a part thereof and an HLA molecule.
[0040] A nucleic acid coding for a tumor-associated antigen
identified according to the invention or for a part thereof may be
present in the pharmaceutical composition in an expression vector
and functionally linked to a promoter.
[0041] A host cell present in a pharmaceutical composition of the
invention 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,
monocyte or a macrophage.
[0042] An antibody present in a pharmaceutical composition of the
invention 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.
[0043] An antisense nucleic acid present in a pharmaceutical
composition of the invention may comprise a sequence of 6-50, in
particular 10-30, 15-30 and 20-30, contiguous nucleotides of the
nucleic acid coding for the tumor-associated antigen identified
according to the invention.
[0044] In further embodiments, a tumor-associated antigen, provided
by a pharmaceutical composition of the invention 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.
[0045] A pharmaceutical composition of the invention 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
invention 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.
[0046] The invention 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 invention.
[0047] In one aspect, the invention relates to a method of
diagnosing a disease characterized by expression or abnormal
expression of a tumor-associated antigen identified according to
the invention. 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.
[0048] In a further aspect, the invention 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 invention, 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.
[0049] According to the invention, 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.
[0050] 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 invention, 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.
[0051] 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.
[0052] According to the invention, detection of an antibody or
monitoring the amount of antibodies may be carried cut using a
protein or peptide binding specifically to said antibody.
[0053] According to the invention, 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.
[0054] 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.
[0055] In a further aspect, the invention 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 invention, which method comprises
administering an antibody which binds to said tumor-associated
antigen or to a part thereof and which is coupled to a therapeutic
or diagnostic agent. The antibody may be a monoclonal antibody. In
further embodiments, the antibody is a chimeric or humanized
antibody or a fragment of a natural antibody.
[0056] The invention 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
invention, 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 invention 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.
[0057] 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.
[0058] In a further aspect, the invention relates to a method of
treating a patient having a disease characterized by expression or
abnormal expression of a tumor-associated antigen, which method
comprises (i) identifying a nucleic acid which codes for a
tumor-associated antigen identified according to the invention 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.
[0059] The invention also relates to a method of treating a disease
characterized by expression or abnormal expression of a
tumor-associated antigen identified according to the invention,
which method comprises (i) identifying cells from the patient which
express abnormal amounts of the tumor-associated antigen, (ii)
isolating a sample of said cells, (iii) culturing said cells, and
(iv) introducing said cells into the patient in an amount suitable
for triggering an immune response to the cells.
[0060] Preferably, the host cells used according to the invention
are nonproliferative or are rendered nonproliferative. A disease
characterized by expression or abnormal expression of a
tumor-associated antigen is in particular cancer.
[0061] The present invention furthermore relates to a nucleic acid
selected from the group consisting of (a) a nucleic acid which
comprises a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 2-5, 20-21, 31-33, 39, 54-57, 62, 63,
85-88, a part or derivative thereof, (b) a nucleic acid which
hybridizes with the nucleic acid of (a) under stringent conditions,
(c) a nucleic acid which is degenerate with respect to the nucleic
acid of (a) or (b), and (d) a nucleic acid which is complementary
to the nucleic acid of (a), (b) or (c). The invention furthermore
relates to a nucleic acid, which codes for a protein or polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 7-13, 14-18, 23-24, 34-36, 58-61, 64, 65,
89-100, a part or derivative thereof.
[0062] In a further aspect, the invention relates to promoter
sequences of nucleic acids of the invention. 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.
[0063] In a further aspect, the invention relates to a recombinant
nucleic acid molecule, in particular DNA or RNA molecule, which
comprises a nucleic acid of the invention.
[0064] The invention also relates to host cells which contain a
nucleic acid of the invention or a recombinant nucleic acid
molecule comprising a nucleic acid of the invention.
[0065] 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 invention or a part thereof. Preferably, the host cell is
nonproliferative. In a preferred embodiment, the host cell is an
antigen-presenting cell, in particular a dendritic cell, a monocyte
or a macrophage.
[0066] In a further embodiment, the invention relates to
oligonucleotides which hybridize with a nucleic acid identified
according to the invention and which may be used as genetic probes
or as "antisense" molecules. Nucleic acid molecules in the form of
oligonucleotide primers or competent samples, which hybridize with
a nucleic acid identified according to the invention or parts
thereof, may be used for finding nucleic acids which are homologous
to said nucleic acid identified according to the invention. 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 invention refers to
conditions which allow specific hybridization between
polynucleotides.
[0067] In a further aspect, the invention relates to a protein or
polypeptide 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:
2-5, 20-21, 31-33, 39, 54-57, 62, 63, 85-88, a part or derivative
thereof, (b) a nucleic acid which hybridizes with the nucleic acid
of (a) under stringent conditions, (c) a nucleic acid which is
degenerate with respect to the nucleic acid of (a) or (b), and (d)
a nucleic acid which is complementary to the nucleic acid of (a),
(b) or (c). In a preferred embodiment, the invention relates to a
protein or polypeptide which comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 7-13, 14-18,
23-24, 34-36, 58-61, 64, 65, 89-100, a part or derivative
thereof
[0068] In a further aspect, the invention relates to an immunogenic
fragment of a tumor-associated antigen identified according to the
invention. Said fragment preferably binds to a human HLA receptor
or to a human antibody. A fragment of the invention preferably
comprises a sequence of at least 6, in particular at least 8, at
least 10, at least 12, at least 15, at least 20, at least 30 or at
least 50, amino acids.
[0069] In a further aspect, the invention relates to an agent which
binds to a tumor-associated antigen identified according to the
invention or to a part thereof. In a preferred embodiment, the
agent is 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 invention relates to an antibody which binds
selectively to a complex of (i) a tumor-associated antigen
identified according to the invention or a part thereof and (ii) an
MHC molecule to which said tumor-associated antigen identified
according to the invention or said part thereof binds, with said
antibody not binding to (i) or (ii) alone. An antibody of the
invention may be a monoclonal antibody. In further embodiments, the
antibody is a chimeric or humanized antibody or a fragment of a
natural antibody.
[0070] The invention furthermore relates to a conjugate between an
agent of the invention which binds to a tumor-associated antigen
identified according to the invention or to a part thereof or an
antibody of the invention and a therapeutic or diagnostic agent. In
one embodiment, the therapeutic or diagnostic agent is a toxin.
[0071] In a further aspect, the invention relates to a kit for
detecting expression or abnormal expression of a tumor-associated
antigen identified according to the invention, which kit comprises
agents for detection (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.
[0072] According to the invention, genes are described which are
expressed in tumor cells selectively or aberrantly and which are
tumor-associated antigens.
[0073] According to the invention, these genes or their derivatives
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 pathogenicity 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.
[0074] "Derivative" of a nucleic acid means according to the
invention 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.
[0075] According to the invention, a nucleic acid is preferably
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic
acids comprise according to the invention genomic DNA, cDNA, mRNA,
recombinantly produced and chemically synthesized molecules.
According to the invention, a nucleic acid may be present as a
single-stranded or double-stranded and linear or covalently
circularly closed molecule.
[0076] The nucleic acids described according to the invention have
preferably been isolated. The term "isolated nucleic acid" means
according to the invention that the nucleic acid was (i) amplified
in vitro, for example by polymerase chain reaction (PCR), (ii)
recombinantly produced by cloning, (iii) purified, for example by
cleavage and gel-electrophoretic fractionation, or (iv)
synthesized, for example by chemical synthesis. An isolated nucleic
acid is a nucleic acid which is available for manipulation by
recombinant DNA techniques.
[0077] A nucleic acid is "complementary" to another nucleic acid if
the two sequences are capable of hybridizing and forming a stable
duplex with one another, with hybridization preferably being
carried out under conditions which allow specific hybridization
between polynucleotides (stringent conditions). Stringent
conditions are described, for example, in Molecular Cloning: A
Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold
Spring Harbor Laboratory press, Cold Spring Harbor, N. Y., 1989 or
Current Protocols in Molecular Biology, F. M. Ausubel et al.,
Editors, John Wiley & Sons, Inc., New York and refer, for
example, to hybridization at 65.degree. C. in hybridization buffer
(3.5.times.SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02%
bovine serum albumin, 2.5 mM NaH.sub.2PO.sub.4 (pH 7), 0.5% SDS, 2
mM EDTA). SSC is 0.15 M sodium chloride/0.15 M sodium citrate, pH
7. After hybridization, the membrane to which the DNA has been
transferred is washed, for example, in 2.times.SSC at room
temperature and then in 0.1-0.5.times.SSC/0.1.times.SDS at
temperatures of up to 68.degree. C.
[0078] According to the invention, complementary nucleic acids have
at least 40%, in particular at least 50%, at least 60%, at least
70%, at least 80%, at least 90% and preferably at least 95%, at
least 98 or at least 99%, identical nucleotides.
[0079] Nucleic acids coding for tumor-associated antigens may,
according to the invention, be present alone or in combination with
other nucleic acids, in particular heterologous nucleic acids. In
preferred embodiments, a nucleic acid is functionally linked to
expression control sequences or regulatory sequences which may be
homologous or heterologous with respect to said nucleic acid. A
coding sequence and a regulatory sequence are "functionally" linked
to one another, if they are covalently linked to one another in
such a way that expression or transcription of said coding sequence
is under the control or under the influence of said regulatory
sequence. If the coding sequence is to be translated into a
functional protein, then, with a regulatory sequence functionally
linked to said coding sequence, induction of said regulatory
sequence results in transcription of said coding sequence, without
causing a frame shift in the coding sequence or said coding
sequence not being capable of being translated into the desired
protein or peptide.
[0080] The term "expression control sequence" or "regulatory
sequence" comprises according to the invention promoters, enhancers
and other control elements which regulate expression of a gene. In
particular embodiments of the invention, the expression control
sequences can be regulated. The exact structure of regulatory
sequences may vary as a function of the species or cell type, but
generally comprises 5' untranscribed and 5' untranslated sequences
which are involved in initiation of transcription and translation,
respectively, such as TATA box, capping sequence, CAAT sequence,
and the like. More specifically, 5' untranscribed regulatory
sequences comprise a promoter region which includes a promoter
sequence for transcriptional control of the functionally linked
gene. Regulatory sequences may also comprise enhancer sequences or
upstream activator sequences.
[0081] 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 invention, be combined with any other genes. This
allows the selective activity of these promoters to be
utilized.
[0082] According to the invention, 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
invention, 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.
[0083] In a preferred embodiment, a recombinant DNA molecule is
according to the invention a vector, where appropriate with a
promoter, which controls expression of a nucleic acid, for example
a nucleic acid coding for a tumor-associated antigen of the
invention. The term "vector" is used here in its most general
meaning and comprises any intermediary vehicle for a nucleic acid
which enables said nucleic acid, for example, to be introduced into
prokaryotic and/or eukaryotic cells and, where appropriate, to be
integrated into a genome. Vectors of this kind are preferably
replicated and/or expressed in the cells. An intermediary vehicle
may be adapted, for example, to the use in electroporation, in
bombardment with microprojectiles, in liposomal administration, in
the transfer with the aid of agrobacteria or in insertion via DNA
or RNA viruses. Vectors comprise plasmids, phagemids or viral
genomes.
[0084] The nucleic acids coding for a tumor-associated antigen
identified according to the invention may be used for transfection
of host cells. Nucleic acids here mean both recombinant DNA and
RNA. Recombinant RNA may be prepared by in-vitro transcription of a
DNA template. Furthermore, it may be modified by stabilizing
sequences, capping and polyadenylation prior to application.
According to the invention, the term "host cell" relates to any
cell which can be transformed or transfected with an exogenous
nucleic acid. The term "host cells" comprises according to the
invention prokaryotic (e.g. E. coli) or eukaryotic cells (e.g.
dendritic cells, B cells, CHO cells, COS cells, K562 cells, yeast
cells and insect cells). Particular preference is given to
mammalian cells such as cells from humans, mice, hamsters, pigs,
goats, primates. The cells may be derived from a multiplicity of
tissue types and comprise primary cells and cell lines. Specific
examples comprise keratinocytes, peripheral blood leukocytes, stem
cells of the bone marrow and embryonic stem cells. In further
embodiments, the host cell is an antigen-presenting cell, in
particular a dendritic cell, monocyte or a macrophage. A nucleic
acid may be present in the host cell in the form of a single cop or
of two or more copies and, in one embodiment is expressed in the
host cell.
[0085] According to the invention, the term "expression" is used in
its most general meaning and comprises the production of RNA or of
RNA and protein. It also comprises partial expression of nucleic
acids. Furthermore, expression may be carried out transiently or
stably. Preferred expression systems in mammalian cells comprise
pcDNA3.1 and pRc/CMV (Invitrogen, Carlsbad, Calif.), which contain
a selective 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).
[0086] In those cases of the invention 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 he 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.
[0087] The invention also comprises kits for amplification of a
nucleic acid coding for a tumor-associated antigen. Such kits
comprise, for example, a pair of amplification primers which
hybridize to the nucleic acid coding for the tumor-associated
antigen. The primers preferably comprise a sequence of 6-50, in
particular 10-30, 15-30 and 20-30 contiguous nucleotides of the
nucleic acid and are nonoverlapping, in order to avoid the
formation of primer dimers. One of the primers will hybridize to
one strand of the nucleic acid coding for the tumor-associated
antigen, and the other primer will hybridize to the complementary
strand in an arrangement which allows amplification of the nucleic
acid coding for the tumor-associated antigen.
[0088] "Antisense" molecules or "antisense" nucleic acids may be
used for regulating, in particular reducing, expression of a
nucleic acid. The term "antisense molecule" or "antisense nucleic
acid" refers according to the invention to an oligonucleotide which
is an oligoribonucleotide, oligodeoxyribonucleotide, modified
oligoribonucleotide or modified 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 invention, the "antisense molecule" also comprises
a construct which contains a nucleic acid or a part thereof in
reverse orientation with respect to its natural promoter. An
antisense transcript of a nucleic acid or of a part thereof may
form a duplex with the naturally occurring mRNA specifying the
enzyme and thus prevent accumulation of or translation of the mRNA
into the active enzyme, Another possibility is the use of ribozymes
for inactivating a nucleic acid. Antisense oligonucleotides
preferred according to the invention have a sequence of 6-50, in
particular 10-30, 15-30 and 20-30, contiguous nucleotides of the
target nucleic acid and preferably are fully complementary to the
target nucleic acid or to a part thereof.
[0089] 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.
[0090] In one embodiment, an oligonucleotide of the invention
consists of ribonucleotides, deoxyribonucleotides or a combination
thereof, with the 5' end of one nucleotide and the 3' end of
another nucleotide being linked to one another by a phosphodiester
bond. These oligonucleotides may be synthesized in the conventional
manner or produced recombinantly.
[0091] In preferred embodiments, an oligonucleotide of the
invention is a "modified" oligonucleotide. Here, the
oligonucleotide may be modified in very different ways, without
impairing its ability to bind its target, in order to increase, for
example, its stability or therapeutic efficacy. According to the
invention, the term "modified oligonucleotide" means an
oligonucleotide in which (i) at least two of its nucleotides are
linked to one another by a synthetic internucleoside bond (i.e. an
internucleoside bond which is not a phosphodiester bond) and/or
(ii) a chemical group which is usually not found in nucleic acids
is covalently linked to the oligonucleotide. Preferred synthetic
internucleoside bonds are phosphorothioates, alkyl phosphonates,
phosphorodithioates, phosphate esters, alkyl phosphonothioates,
phosphoramidates, carbamates, carbonates, phosphate triesters,
acetamidates, arboxymethyl esters and peptides.
[0092] 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.
[0093] Preferably, the proteins and polypeptides described
according to the invention 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.
[0094] 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 invention may be isolated from biological samples such as
tissue or cell homogenates and may also be expressed recombinantly
in a multiplicity of pro- or eukaryotic expression systems.
[0095] For the purposes of the present invention, "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.
[0096] 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:
[0097] 1. small aliphatic, nonpolar or slightly polar residues:
Ala, Ser, Thr (Pro, Gly)
[0098] 2. negatively charged residues and their amides: Asn, Asp,
Glu, Gln
[0099] 3. positively charged residues: His, Arg, Lys
[0100] 4. large aliphatic, nonpolar residues: Met, Leu, Ile, Val,
(Cys)
[0101] 5. large aromatic residues: Phe, Tyr, Trp.
[0102] 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.
[0103] 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.
[0104] According to the invention, "derivatives" of proteins or
polypeptides also comprise single or multiple substitutions,
deletions and/or additions of any molecules associated with the
enzyme, such as carbohydrates, lipids and/or proteins or
polypeptides. The term "derivative" also extends to all functional
chemical equivalents of said proteins or polypeptides.
[0105] According to the invention, 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 invention preferably
comprises a sequence of at least 6, in particular at least 8, at
least 10, at least 12, at least 15, at least 20, at least 30 or at
least 50, consecutive amino acids of the tumor-associated
antigen.
[0106] A part or a fragment of a nucleic acid coding for a
tumor-associated antigen relates according to the invention to the
part of the nucleic acid, which codes at least for the
tumor-associated antigen and/or for a part or a fragment of said
tumor-associated antigen, as defined above.
[0107] 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.
[0108] The present invention also enables proteins binding to
tumor-associated antigens described herein to be isolated,
including antibodies and cellular binding partners of said
tumor-associated antigens.
[0109] According to the invention, 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.
[0110] 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.
[0111] The invention 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 a 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 antigens, for example by binding
to such antigens.
[0112] The invention 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.
[0113] 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, 7.sup.th 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 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.
[0114] 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.
[0115] 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.
[0116] WO 92/04381 for example, describes 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 invention 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 invention also
comprises "single-chain" antibodies.
[0118] The invention 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 invention. 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
invention 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 invention, 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, cyclpsporine,
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 invention a human
being, a nonhuman primate or another animal, in particular a mammal
such as a cow, horse, pig, sheep, goat, dog, cat or a rodent such
as a mouse and rat. In a particularly preferred embodiment, the
patient is a human being.
[0122] According to the invention, the term "disease" refers to any
pathological state in which tumor-associated antigens are expressed
or abnormally expressed. "Abnormal expression" means according to
the invention that expression is altered, preferably increased,
compared to the state in a healthy individual. An increase in
expression refers to an increase by at least 10%, in particular at
least 20%, at least 50% or at least 100%. In one embodiment, the
tumor-associated antigen is expressed only in tissue of a diseased
individual, while expression in a healthy individual is repressed.
One example of such a disease is cancer, in particular seminomas,
melanomas, teratomas, gliomas, colorectal cancer, breast cancer,
prostate cancer, cancer of the uterus, ovarian cancer and lung
cancer.
[0123] According to the invention, 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, urine, feces or
other body fluids, for use in the various methods described
herein.
[0124] According to the invention, the term "immunoreactive cell"
means a cell which can mature into an immune cell (such as B cell,
T helper cell, or cytolytic T cell) with suitable stimulation
Immunoreactive cells comprise OD34.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. This results
in propagation of specific cytotoxic T lymphocytes with high
affinity if the donor had no previous contact with the specific
complex. The high affinity T cell receptor of these propagated
specific T lymphocytes is cloned and can be transduced via gene
transfer, for example using retroviral vectors, into T cells of
other 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 invention. Cytotoxic T lymphocytes may
also be generated in vivo in a manner known per se. One method uses
nonproliferative cells expressing the complex. The cells used here
will be those which usually express the complex, such as irradiated
tumor cells or cells transfected with one or both genes necessary
for presentation of the complex (i.e. the antigenic peptide and the
presenting 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 at 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 at al., Proc Natl Aced Sci
USA 98:3299-303, 2001). It may also be carried out in combination
with reagents which facilitate uptake into dendritic cells. In vivo
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 at al., J. Immunol. 167:787-96, 2001; Appella at 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
invention may also be used as vaccines for immunization. According
to the invention, 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 stimulating particular lymphocytes. Adjuvants are
known and comprise in a nonlimiting way monophosphoryl lipid A
(MPL, SmithKline Beecham), saponin 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 at 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 no effective antigen-presenting cells (APCs) for T cells.
Induction of B7 expression would enable rumor 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 Thl-cytokine profile in a T
cell population, resulting in further enhanced T cell activity
(Gajewski et al., J. Inmunol. 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). CD4O-CD40L interaction thus
complements the interaction of signal I (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 or in situations in which T helper
epitopes have not been defined in known TRA precursors.
[0139] The invention 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 invention 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 invention 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 invention may be
administered in pharmaceutically compatible preparations. Such
preparations may usually contain pharmaceutically compatible
concentrations of salts. buffer substances, preservatives,
carriers, supplementing immunity-enhancing substances such as
adjuvants, CpG and cytokines and, where appropriate, other
therapeutically active compounds.
[0143] The therapeutically active compounds of the invention may be
administered via any conventional route, including by injection or
infusion. The administration may be carried out, for example,
orally, intravenously, intraperitonealy, intramuscularly,
subcutaneously or transdermally. Preferably, antibodies are
therapeutically administered by way of a lunch aerosol. Antisense
nucleic acids are preferably administered by slow intravenous
administration.
[0144] The compositions of the invention are administered in
effective amounts. An "effective amount" refers to the amount which
achieves a desired reaction or a desired effect alone or together
with further doses. In the case of treatment of a particular
disease or of 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 invention will
depend on the condition to be treated, the severeness of the
disease, the individual parameters of the patient, including age,
physiological condition, size and weight, the duration of
treatment, the type of an accompanying therapy (if present), the
specific route of administration and similar factors.
[0146] The pharmaceutical compositions of the invention are
preferably sterile and contain an effective amount of the
therapeutically active substance to generate the desired reaction
or the desired effect,
[0147] The doses administered of the compositions of the invention
may depend on various parameters such as the type of
administration, the condition of the patient, the desired period of
administration, etc. In the case that a reaction in a patient is
insufficient with an initial dose, higher doses (or effectively
higher doses achieved by a different, more localized route of
administration) may be used.
[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 invention are
generally administered in pharmaceutically compatible amounts and
in pharmaceutically compatible compositions. The term
"pharmaceutically compatible" refers to a nontoxic material which
does not interact with the action of the active component of the
pharmaceutical composition. Preparations of this kind may usually
contain salts, buffer substances, preservatives, carriers and,
where appropriate, other therapeutically active compounds. When
used in medicine, the salts should be pharmaceutically compatible.
However, salts which are not pharmaceutically compatible may be
used for preparing pharmaceutically compatible salts and are
included in the invention. Pharmacologically and pharmaceutically
compatible salts of this kind comprise in a nonlimiting way those
prepared from the following acids: hydrochloric, hydrobromic,
sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric,
formic, malonic, succinic acids, and the like. Pharmaceutically
compatible salts may also be prepared as alkali metal salts or
alkaline earth metal salts, such as sodium salts, potassium salts
or calcium salts.
[0150] A pharmaceutical composition of the invention may comprise a
pharmaceutically compatible carrier. According to the invention,
the term "pharmaceutically compatible carrier" refers to one or
more compatible solid or liquid fillers, diluents or encapsulating
substances, which are suitable for administration to humans. The
term "carrier" refers to an organic or inorganic component, of a
natural or synthetic nature, in which the active component is
combined in order to facilitate application. The components of the
pharmaceutical composition of the invention are usually such that
no interaction occurs which substantially impairs the desired
pharmaceutical efficacy.
[0151] The pharmaceutical compositions of the invention may contain
suitable buffer substances such as acetic acid in a salt, citric
acid in a salt, boric acid in a salt and phosphoric acid in a
salt.
[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 invention 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 invention is described in detail by the figures
and examples herein, which are used only for illustration purposes
and are not meant to be limiting, Owing to the description and the
examples, further embodiments which are likewise included in the
invention are accessible to the skilled worker.
EXAMPLES
Material and methods
[0156] The terms "in silica", "electronic" and "virtual cloning"
refer solely to the utilization of methods based on databases,
which may also be used to simulate laboratory experimental
processes.
[0157] 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.
[0158] Datamining-Based Strategy for Determining eCT
(Electronically Cloned Cancer/Testis Genes)
[0159] Two in silico strategies, namely GenBank keyword search and
the cDNAxProfiler, were combined (FIG. 1). Utilizing the NCBI
ENTREZ Search and Retrieval System (http://www.ncbi.nlm nih
gov/Entrez), a GenBank search was carried out for candidate genes
annotated as being specifically expressed in testicular tissue
(Wheeler at al., Nucleic Acids Research 28:10-14, 2000).
[0160] Carrying out queries with the keywords "testis-specific
gene", "sperm-specific gene", "spermatogonia-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.
[0161] The list of the GOI found was curated by determining
different names for the same sequence and eliminating such
redundancies.
[0162] All candidate genes obtained by the keyword search were in
turn studied with respect to their tissue distribution by the
"electronic Northern" (eNorthern) 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)
(http://www.ncbi.nlm nih.gov/BLAST). The tissue origin of each EST
which is found to be homologous to the 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
nontesticular normal tissues with the exception of placenta and
fetal tissue. 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).
[0163] 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 from testis were
assigned, excluding mixed libraries. All cDNA libraries prepared
from normal tissues other than testis, ovary or fetal tissue 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.
[0164] This combined datamining includes all of the about 13000
full-length genes in the public domain and predicts out of these
genes a total of 140 genes having potential testis-specific
expression, Among the latter were 25 previously known genes of the
CT gene class, underlining the efficiency of our strategy.
[0165] All other genes were first evaluated in normal tissues by
means of specific RT-PCR. All GOI which had proved to be expressed
in nontesticular 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
ectopically activated in tumor cells.
[0166] RNA Extraction, Preparation of Poly-d(T) Primed cDNA and
RT-PCR Analysis
[0167] 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.
[0168] 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, antisense CCTAACCAGCTGCCCAACTGTAG,
hybridization temperature 67.degree. C.).
[0169] 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 (Oiagen). 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)
[0170] 20 .mu.l of this reaction were fractionated and analyzed on
an ethidium bromide-stained agarose gel.
[0171] The following primers were used for expression analysis of
the corresponding antigens at the hybridization temperature
indicated.
[0172] LDH-C (67.degree. C.)
TABLE-US-00001 sense TGCCGTAGGCATGGCTTGTGC, antisense
CAACATCTGAGACACCATTCC TPTE (64.degree. C.) sense
TGGATGTCACTCTCATCCTTG, antisense CCATAGTTCCTGTTCTATCTG TSBP
(63.degree. C.) sense TCTAGCACTGTCTCGATCAAG, antisense
TGTCCTCTTGGTACATCTGAC MS4A12 (66.degree.) sense
CTGTGTCAGCATCCAAGGAGC, antisense TTCACCTTTGCCAGCATGTAG BRCO1
(60.degree. C.) sense CTTGCTCTGAGTCATCAGATG, antisense
CACAGAATATGAGCCATACAG TPX1 (65.degree. C.) sense
TTTTGTCTATGGTGTAGGACC, antisense GGAATGGCAATGATGTTACAG
[0173] Preparation of Random Hexamer-Primed cDNA and Quantitative
Real Time PCR
[0174] LDHC expression was quantified by means of real time
PCR.
[0175] The principle of quantitative real time PCR using the ABI
PRISM Sequence Detection System (PE Biosystems, USA) utilizes the
5'-3' exonuclease activity of Taq DNA polymerase for direct and
specific detection of PCR products via release of fluorescence
reporter dyes. In addition to sense and antisense primers, the PCR
employs a doubly fluorescently labeled probe (TaqMan probe) which
hybridizes to a sequence of the PCR product. The probe is labeled
5' with a reporter dye (e.g. FAM) and 3' with a quencher dye (e.g.
TAMRA). If the probe is intact, the spatial proximity of reporter
to quencher suppresses the emission of reporter fluorescence. If
the probe hybridizes to the PCR product during the PCR, said probe
is cleaved by the 5'-3' exonuclease activity of Taq DNA polymerase
and suppression of the reporter fluorescence is removed. The
increase in reporter fluorescence as a consequence of the
amplification of the target, is measured after each PCR cycle and
utilized for quantification. Expression of the target gene is
quantified absolutely or relative to expression of a control gene
with constant expression in the tissues to be studied. LDHC
expression was calculated by means of the .DELTA..DELTA.-C.sub.t
method (PE Biosystems, USA), after normalizing the samples to 18s
RNA as "housekeeping" gene. The reactions were carried out in
duplex mixtures and determined in duplicate. cDNA was synthesized
using the High Capacity cDNA Archive Kit (PE Biosystems, USA) and
hexamer primers according to the manufacturer's information. In
each case 5 .mu.l of the diluted cDNA were used for the PCR in a
total volume of 25 .mu.l: sense primer (GGTGTCACTTCTGTGCCTTCCT) 300
nM; antisense primer (CGGCACCAGTTCCAACAATAG) 300 nM; TaqMan probe
(CAAAGGTTCTCCAAATGT) 250 nM; sense primer 18s RNA 50 nM; antisense
primer 18s RNA 50 nM; 18s RNA sample 250 nM; 12.5 .mu.l TaqMan
Universal PCR Master Mix; initial denaturation 95.degree. C. (10
min); 95.degree. C. (15 sec); 60.degree. C. (1 min); 40 cycles. Due
to amplification of a 128 bp product beyond the border of exon 1
and exon 2, all LDHC splice variants described were included in the
quantification.
[0176] Cloning and Sequence Analysis
[0177] Full length genes and gene fragments were cloned by common
methods. The sequence was determined by amplifying corresponding
antigens by means of the pfu proofreading polymerase (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 into the TOPO-TA vector according to the
manufacturer's information. A commercial service carried out the
sequencing. The sequences were analyzed by means of common
prediction programs and algorithms.
Example 1
Identification of LDH C as a New Tumor Antigen
[0178] LDH C (SEQ ID) NO:1) and its translation product (SEQ ID
NO:6) have been described as testis-specific isoenzyme of the
lactate dehydrogenase family. The sequence has been published in
GenBank under accession number NM.sub.--017448. The enzyme forms a
homotetramer having a molecular weight of 140 kDa (Goldberg, E. et
al., Contraception 64(2):93-8, 2001; Cooker et al, Biol. Reprod.
48(6):1309-19, 1993; Gupta, G. S., Crit, Rev. Biochem, Mol. Biol.
34(6):361-85, 1999).
[0179] RT-PCR studies for expression analysis using a primer pair
(5'-TGCCGTAGGCATGGCTTGTGC-3',5'-CAACATCTGAGACACCATTCC-3') which
does not cross-amplify the related and ubiquitously expressed
isoenzymes LDH A and LDH B and which is based on the LDH C
prototype sequence NM.sub.--017448 which has previously been
described as being testis-specific, confirmed according to the
invention the lack of expression in all normal tissues tested, but
demonstrated that the stringent transcriptional repression of this
antigen in somatic cells has been removed in the case of tumors;
cf. Table 1. As has been described classically for CT genes, LDH C
is expressed in a number of tumor entities.
TABLE-US-00002 TABLE 1 Expression of LDHC in tumors Tested Tissue
in total Positive % Melanoma 16 7 44 Mammary carcinomas 20 7 35
Colorectal tumors 20 3 15 Prostate carcinomas 8 3 38 Bronchial
carcinomas 17 8 47 Kidney cell carcinomas 7 4 57 Ovarian carcinomas
7 3 43 Thyroid carcinomas 4 1 25 Cervical carcinomas 6 5 83
Melanoma cell lines 8 5 63 Bronchial carcinoma cell lines 6 2
33
[0180] The expected size of the amplification product is 824 bp,
using the PCR primers mentioned above. According to the invention,
however, amplification of multiple additional bands was observed in
tumors, but not in testis. Since this is indicative for the
presence of alternative splice variants, the complete open reading
frame was amplified using LDH-C-specific primers
(5'-TAGCGCCTCAACTGTCGTTGG-3',5'-CAACATCTGAGACACCATTCC-3') and
independent full-length clones were sequenced. Alignments with the
prototype ORF of the LDH C sequence described (SEQ ID NO:1) and the
genomic sequence on chromosome 11 confirm additional splice
variants (SEQ ID NO:2-5). The alternative splicing events result in
the absence of exon 3 (SEQ ID NO:2), of the two exons 3 and 4 (SEQ
ID NO:3), of the exons 3, 6 and 7 (SEQ ID NO:4) or of exon 7 (SEQ
ID NO:5) (cf. FIG. 2).
[0181] These new splice variants are generated exclusively in
tumors, but not in testis. Alternative splicing causes alterations
in the reading frame and results in new possible ORFS encoding the
amino acid sequences depicted in SEQ ID NO:7-13 (ORF for SEQ ID
NO:7: nucleotide position 59-214 of SEQ ID NO:2 and, respectively,
SEQ ID NO:4 ORF for SEQ ID NO:8: nucleotide position 289-939 of SEQ
ID NO:2; ORF for SEQ ID NO:9: nucleotide position 59-196 of SEQ ID
NO:3; ORF for SEQ ID NO:10: nucleotide position 535-765 of SEQ ID
NO:3; ORF for SEQ ID NO:11: nucleotide position 289-618 of SEQ ID
NO:4; ORF for SEQ ID NO:12: nucleotide position 497-697 of SEQ ID
NO:4; ORF for SEQ ID NO:13: nucleotide position 59-784 of SEQ ID
NO:5) (FIG. 2, 3). Apart from premature termination, utilization of
alternative start codons is also possible so that the encoded
proteins may be truncated both N-terminally and C-terminally.
[0182] While SEQ ID NO:8 and SEQ ID NO:10 represent truncated
portions of the prototype protein, the protein sequence of SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 are
additionally altered and contain only tumor-specific epitopes
(printed in bold type in FIG. 3). Peptide regions which could
result in tumor specific epitopes are as follows (the strictly
tumor-specific portion produced by frame shifts is underlined):
TABLE-US-00003 SEQ ID NO: 14: GAVGMACAISILLKITVYLQTPE (of SEQ ID
NO: 7) SEQ ID NO: 15: GAVGMACAISILLKWIF (of SEQ ID NO: 9) SEQ ID
NO: 16: GWIIGEHGDSSGIIWNKRRTLSQYPLCLGAEWCIRCCEN (of SEQ ID NO: 11)
SEQ ID NO: 17: MVGLLENMVILVGLYGIKEELFL (of SEQ ID NO: 12) SEQ ID
NO: 18: EHWKNIHKQVIQRDYME (of SEQ ID NO: 13)
[0183] These regions may potentially contain epitopes which can be
recognized on MHC I or MHC II molecules by T lymphocytes and which
result in a strictly tumor-specific response.
[0184] Not all of the predicted proteins have the catalytic lactate
dehydrogenase domain for NADH-dependent metabolization of pyruvate
to lactate, which represents the last step of anaerobic glycolysis.
This domain would be required for the enzymatic function as lactate
dehydrogenase (framed in FIG. 3). Further analyses, for example
using algorithms such as TMpred and pSORT (Nakai & Kanehisa,
1992), predict different subcellular localizations for the putative
proteins.
[0185] According to the invention, the level of expression was
quantified by real time PCR using a specific primer-sample set. The
amplicon is present in the junction between exon 1 and exon 2 and
thus detects all variants (SEQ ID NO:1-5). These studies too, do
not detect any transcripts in normal tissues except testis. They
confirm significant levels of expression in tumors (FIG. 4).
[0186] LDHC-specific polyclonal antibodies were produced according
to the invention by selecting a peptide from the extreme N-terminal
region MSTVKEQLIEKLIEDDENSQ (SEQ ID NO:80). LDHC-specific
antibodies were produced in rabbits with the aid of this peptide.
Subsequent studies on protein expression confirmed selective LDHC
expression in testis and in various tumors. In addition,
immunohistological studies in accordance with the invention
revealed a distinct colocalization of LDHC with cytochrome C
oxidase in mitochondria. This indicates that LDHC plays an
important part in the respiratory chain of tumors.
Example 2
Identification of TPTE as a New Tumor Antigen
[0187] The sequences of the TPTE transcript (SEQ ID NO:19) and of
its translation product (SEQ ID NO:22) have been published in
GenBank under accession number NM.sub.--013315 (Walker, S. M. at
al., Biochem. J. 360(Pt 2):277-83, 2001; Guipponi M. et al., Hum.
Genet. 107(2):127-31, 2000; Chen H. et al., Hum. Genet.
105(5):399-409, 1999) TPTE has been described as a gene coding for
a possible transmembrane tyrosinephosphatase, with testis-specific
expression located in the pericentromeric region of chromosomes 21,
13, 15, 22 and Y (Chen, H. et al., Hum. Genet. 105:399-409, 1999).
Alignment studies in accordance with the invention additionally
reveal homologous genomic sequences on chromosomes 3 and 7.
[0188] According to the invention, PCR primers
(5'-TGGATGTCACTCTCATCCTTG-3' and 5'-CCATAGTTCCTGTTCTATCTG-3') were
generated based on the sequence of TPTE (SEQ ID NO:19) and used for
RT-PCR analyses (95.degree. 15 min; 94.degree. 1 min; 63.degree. 1
min; 72.degree. 1 min; 35 cycles) in a number of human tissues.
Expression in normal tissues was shown to be limited to testis. As
described for the other eCT, TPTE variants were shown according to
the invention to be ectopically activated in a number of tumor
tissues; cf. Table 2. According to the invention, further TPTE
splice variants were identified (SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57) which are
expressed in testicular tissue and in tumors and which have frame
shifts and thus altered sequence regions (FIG. 5).
TABLE-US-00004 TABLE 2 Expression of TPTE in tumors Tested Tissue
in total Positive % Melanoma 18 9 50 Mammary carcinomas 20 4 20
Colorectal tumors 20 0 0 Prostate carcinomas 8 3 38 Bronchial
carcinomas 23 9 39 Kidney cell carcinomas 7 0 0 Ovarian carcinomas
7 2 29 Thyroid carcinomas 4 0 0 Cervical carcinomas 6 1 17 Melanoma
cell lines 8 4 50 Bronchial carcinoma cell lines 6 2 33 Mammalian
carcinoma cell lines 5 4 80
[0189] The TPTE genomic sequence consists of 24 axons (accession
number NT.sub.--029430). The transcript depicted in SEQ ID NO:19
contains all of these exons. The splice variant depicted in SEQ ID
NO:20 is produced by splicing out exon 7. The splice variant
depicted in SEQ ID NO:21 shows partial incorporation of an intron
downstream of exon 15. As the variants SEQ ID NO:54, SEQ ID NO:55,
SEQ ID NO:56, SEQ ID NO:57 indicate, it is alternatively also
possible to splice out exons 18, 19, 20 and 21.
[0190] These alternative splicing events result in alterations of
the encoded protein, with the reading frame being retained in
principle (FIG. 6). For example, the translation product encoded by
the sequence depicted in SEQ ID NO:20 (SEQ ID NO:23) has a deletion
of 13 amino acids in comparison to the sequence depicted in SEQ ID
NO:22. The translation product encoded by the sequence depicted in
SEQ ID NO:21 (SEQ ID NO:24) carries an additional insertion in the
central region of the molecule and thereby differs from the other
variants by 14 amino acids.
[0191] The translation products of the variants SEQ ID NO:54, SEQ
ID NO:55, SEQ ID N0:56, SEQ ID NO:57, namely the proteins SEQ ID
NO:58, SEQ) ID NO:59, SEQ ID NO:60, SEQ ID NO:61, are likewise
altered.
[0192] Analyses for predicting the functional domains reveal the
presence of a tyrosinephosphatase domain for SEQ ID NO:22, SEQ ID
NO:23, SEQ ID NO:24, SEQ ID NO:58, SEQ ID NO:60 but not for SEQ ID
NO:59, SEQ ID NO:61. For all variants, 3-4 transmembrane domains
are predicted (FIG. 6).
[0193] Analysis of TPTE antigen expression, using specific
antibodies, confirmed selective expression in testis and in a
number of different tumors. Colocalization studies moreover
revealed that according to the invention TPTE is located together
with class I immunoglobulins on the cell surface of tumor cells.
Previously, TPTE had been described only as a Golgi-associated
protein. Owing to TPTE expression on the cell surface of tumor
cells, this tumor antigen is suitable according to the invention as
an outstanding target for developing diagnostic and therapeutic
monoclonal antibodies. Owing to the predicted membrane topology of
TPTE, the extracellular exposed regions are particularly suitable
for this purpose according to the invention. According to the
invention, this comprises the peptides FTDSKLYIPLEYRS (SEQ ID
NO:81) and FDIKLLRNIPRWT (SEQ ID NO: 82). In addition, TPTE was
shown to promote the migration of tumor cells. To this end, tumor
cells which had been transfected with TPTE under the control of a
eukaryotic promoter and control cells were studied in "Boyden
chamber" migration experiments, as to whether they exhibit directed
migration. TPTE-transfected cells here had according to the
invention markedly (3-fold) increased migration in 4 independent
experiments. These functional data indicate that TPTE plays an
important part in the metastasizing of tumors. Thus, processes
which inhibit according to the invention endogenous TPTE activity
in tumor cells, for example by using antisense RNA, different
methods of RNA interference (RNAi) by means of expression vectors
or retroviruses, and by using small molecules, could result in
reduced metastasizing and thus be very important therapeutically. A
causal connection between the activity of a phosphatase in tumors
and increased migration and increased formation of metastases was
established recently for the PTEN tyrosinephosphastase (Iijima and
Devreotes Cell 109:599-610, 2002).
Example 3
Identification of TSBP as a New Tumor Antigen
[0194] The electronic cloning method employed according to the
invention produced TSBP (SEQ ID NO:29) and the protein derived
therefrom (SEQ ID NO:30). The gene has been described previously as
being testis-specifically regulated (accession number
NM.sub.--006781). The gene was predicted to encode a basic protein
and to be located on chromosome 6 close to a sequence coding for an
MHC complex (C6orf10) (Stammers M. et al., Immunogenetics
51(4-5):373-82, 2000). According to the invention, the previously
described sequence was shown to be incorrect. The sequence of the
invention is substantially different from the known sequence.
According to the invention, 3 different splicing variants were
cloned. The differences in the nucleotide sequences of the TSBP
variants found according to the invention (SEQ ID NO:31, SEQ ID NO:
32, SEQ ID NO:33) to the known sequence (NM.sub.--006781, SEQ ID
NO:29) are depicted in FIG. 7 (differences depicted in bold type).
They result in frame shifts so that the proteins encoded by the
TSBP variants found according to the invention (SEQ ID NO:34, SEQ
ID NO:35, SEQ ID NO:36) differ substantially from the previously
described protein (SEQ ID NO:30) (FIG. 8).
[0195] It was confirmed according to the invention that this
antigen is strictly transcriptionally repressed in normal tissues
(PCR primers 5'-TCTAGCACTGTCTCGATCAAG-3' and
5'-TGTCCTCTTGGTACATCTGAC-3'). However, in 25 normal tissues
studied, TSBP was expressed, apart from in testis, also in normal
lymph node tissue. According to the invention, ectopic activation
of TSBP in tumors was also detected, and it therefore qualifies as
a tumor marker or tumor-associated antigen (Table 3).
[0196] Although TSBP, expression is found in primary tumor tissue,
it is not found in permanent cell lines of corresponding tumor
entities. Moreover, the gene is in the direct neighborhood of Notch
4 which is specifically expressed in arteries and involved in
vascular morphogenesis. These are significant indications of this
being a marker for specific endothelial cells. TSBP may therefore
serve as a potential marker for tumor endothelia and for
neovascular targeting.
[0197] Consequently, the TSBP promoter may be cloned to another
genetic product whose selective expression in lymph nodes is
desired.
[0198] Analysis of TSBP antigen expression, using specific
antibodies, confirmed the selective localization of the protein in
testis and lymph nodes and also in melanomas and bronchial
carcinomas. In addition, immunohistological studies using
GFP-tagged TSBP revealed a distinct perinucleic accumulation.
TABLE-US-00005 TABLE 3 Expression of TSBP in tumors Tested Tissue
in total Positive % Melanoma 12 2 16 Mammary carcinomas 15 0 --
Colorectal tumors 15 0 -- Prostate carcinomas 8 0 -- Bronchial
carcinomas 7 17 41 Kidney cell carcinomas 7 0 -- Ovarian carcinomas
7 0 -- Thyroid carcinomas 4 0 -- Cervical carcinomas 6 0 --
Melanoma cell lines 8 0 -- Bronchial carcinoma cell lines 6 0
--
Example 4
Identification of MS4Al2 as a New Tumor Antigen
[0199] MS4Al2 (SEQ ID NO:37, accession number NM.sub.--017716) and
its translation product (SEQ ID NO:38) have been described
previously as members of a multigene family related to the B
cell-specific antigen CD20, the hematopoietic cell-specific protein
HTm4 and the .beta. chain of the high affinity IgE receptor. All
family members are characterized by at least four potential
transmembrane domains and both the C and the N-terminus are
cytoplasmic (Liang Y. et al., Immunogenetics 53(5):357-68, 2001;
Liang Y. & Tedder, Genomics 72(2):119-27, 2001). According to
the invention, RT-PCR studies on MS4Al2 were carried out. The
primers were selected based on the published MS4Al2 sequence
(NM.sub.--017716) (sense: CTGTGTCAGCATCCAAGGAGC, antisense:
TTCACCTTTGCCAGCATGTAG). In the tissues tested, expression was
detected only in testis, colon (6/8) and colorectal carcinomas
(colon-Ca's) (16/20) and in colonic metastases (12/15) (FIG.
9).
[0200] The high incidence in colonic metastases makes TSBP an
attractive diagnostic and therapeutic target. According to the
invention, the predicted extracellular region comprising the
protein sequence GVAGQDYWAVLSGKG (SEQ ID NO:83) is particularly
suitable for producing monoclonal antibodies and small chemical
inhibitors. According to the invention, the intracellular
localization of the MS4Al2 protein on the cell membrane was also
confirmed by fluorescence superposition using plasma membrane
markers in confocal immunofluorescence.
TABLE-US-00006 TABLE 4 Expression of MS4Al2 in normal tissues and
colorectal carcinomas and metastasis Ileum + Colon + Liver - Lung -
Lymph nodes - Stomach - Spleen - Adrenal gland - Kidney - Esophagus
- Ovary - Rectum + Testis + Thymus - Skin - Mamma - Pancreas - PBMC
- PBMC act. - Prostate - Thyroid - Tube - Uterus - Cerebrum -
Cerebellum - Colorectal tumors 16/20 Colorectal tumors metastases
12/15
[0201] Thus, MS4Al2 is a cell membrane-located differentiation
antigen for normal colon epithelia, which is also expressed in
colorectal tumors and metastases.
Example 5
Identification of BRCO1 as a New Tumor Antigen
[0202] BRCO1 and its translation product have not been described
previously. The datamining method of the invention produced the EST
(expressed sequence tag) AI668620. RT-PCR studies using specific
primers (sense: CTTGCTCTGAGTCATCAGATG, antisense:
CACAGAATATGAGCCATACAG) were carried for expression analysis.
According to the invention, specific expression was found in
testicular tissue and additionally in normal mammary gland (Table
5). In all other tissues, this antigen is transcriptionally
repressed. It is likewise detected in mammary gland tumors (20 out
of 20). BRCO1 is distinctly overexpressed in breast tumors in
comparison with expression in normal mammary gland tissue (FIG.
10). Utilizing EST contigs (the following ESTs were incorporated:
AW137203, BF327792, BF327797, BE069044, BF330665), more than 1500
bp of this transcript were cloned according to the invention by
electronic full-length cloning (SEQ ID NO:39). The sequence maps to
chromosome 10p11-12. In the same region, in immediate proximity,
the gene for a mammary differentiation antigen, NY-BR-1, has been
described previously (NM 052997; Jager, D. et al., Cancer Res.
61(5):2055-61, 2001).
TABLE-US-00007 TABLE 5 Expression of BRCO1 in normal tissues and
breast tumors Ileum - Colon - liver - Lung - Lymph nodes - Stomach
- Spleen - Adrenal gland - Kidney - Esophagus - Ovary - Rectum -
Testis + Thymus - Skin - Mamma + Pancreas - PBMC - PBMC act. -
Prostate - Thyroid - Tube - Uterus - Cerebrum - Cerebellum -
Mammary carcinomas ++ (20/20)
[0203] Matched pair (mammary carcinoma and adjacent normal tissue)
studies revealed BRCO1 overexpression in 70% of the mammary
carcinomas in comparison with the normal tissue.
[0204] Thus, BRCO1 is a new differentiation antigen for normal
mammary gland epithelia, which is overexpressed in breast
tumors.
Example 6
Identification of TPX1 as a New Tumor Antigen
[0205] The sequence of TPX1 (Acc. No. NM.sub.--003296; SEQ ID NO:
40) and of its translation product (SEQ ID NO:41, are known. The
antigen has been described previously only as being
testis-specific, that is as an element of the outer fibers and of
the acrosome of sperms. Previously, an involvement as adhesion
molecule in the attachment of sperms to Sertoli cells has been
attributed to said antigen (O'Bryan, M. K. et al., Mol. Reprod.
Dev. 58(1):116-25, 2001; Maeda, T, et al., Dev. Growth Differ.
41(6):715-22, 1999). The invention reveals, for the first time,
aberrant expression of TPX1 in solid tumors (Table 6). Owing to the
marked amino acid homology between TPX1 and the
neutrophile-specific matrix glycoprotein SGP 28 (Kjeldsen et al.,
FEBS Lett 380:246-259, 1996), TPX1-specific protein sequences
comprising the peptide SREVTTNAQR (SEQ ID NO:84) are suitable
according to the invention for preparing diagnostic and therapeutic
molecules.
TABLE-US-00008 TABLE 6 Expression of TPX1 in tumors Tested Tissue
in total Positive % Melanoma 16 1 6 Mammary carcinomas 20 3 15
Colorectal tumors 20 0 0 Prostate carcinomas 8 3 37 Bronchial
carcinomas 17 2 11 Kidney cell carcinomas 7 1 14 Ovarian carcinomas
7 1 14 Thyroid carcinomas 4 0 0 Cervical carcinomas 6 1 16 Melanoma
cell lines 8 2 25 Bronchial carcinoma cell lines 6 1 16
Example 7
Identification of BRCO2 as a New Tumor Genetic Product
[0206] BROC2 and its translation product have not been described
previously. The method of the invention produced the ESTs
(expressed sequence tag) BE069341, BF330573 and AA601511. RT-PCR
studies using specific primers (sense: AGACATGGCTCAGATGTGCAG,
antisense: GGAAATTAGCAAGGCTCTCGC) were carried out for expression
analysis. According to the invention, specific expression was found
in testicular tissue and additionally in normal mammary gland
(Table 7). In all other tissues, this genetic product is
transcriptionally repressed. It is likewise detected in mammary
gland tumors. Utilizing EST contigs (the following ESTs were
incorporated: BF330573, AL044891 and AA601511), 1300 bp of this
transcript were cloned according to the invention by electronic
full-length cloning (SEQ ID 62), The sequence maps to chromosome
10p11-12. In the same region, in immediate proximity, the gene for
a mammary differentiation genetic product, NY-BR-1, has been
described previously (NM.sub.--052997; Jager, D. et al., Cancer
Res. 61(5):2055-61, 2001), and here the BRCO1 described above under
Example 6 is located. Further genetic analyses revealed according
to the invention that the sequence listed under SEQ ID NO:62
represents the 3' untranslated region of the NY-BR-1 gene, which
has not been described previously.
TABLE-US-00009 TABLE 7 Expression of BRCO2 in normal tissues and
breast tumors Tissue Expression Testis + Mamma + Skin - liver -
Prostate - Thymus - Brain - Lung - Lymph nodes - Spleen - Adrenal
gland - Ovary - Leukocytes - Colon - Esophagus - Uterus - Skeleton
muscle - Epididymis - Bladder - Kidney - Mammary carcinoma +
[0207] BRCO2 is a new differentiation genetic product for normal
mammary gland epithelia, which is also expressed in breast
tumors.
Example 8
Identification of PCSC as a New Tumor Genetic Product
[0208] PCSC (SEQ ID NO:63) and its translation product have not
been described previously. The datamining method of the invention
produced the EST (expressed sequence tag) BF064073. RT-PCR studies
using specific primers (sense: TCAGGTATTCCCTGCTCTTAC, antisense:
TGGGCAATTCTCTCAGGCTTG) were carried out for expression analysis.
According to the invention, specific expression was found in normal
colon, and additionally in colon carcinomas (Table 5). In all other
tissues, this genetic product is transcriptionally repressed. PCSC
codes for two putative ORFs (SEQ ID 64 and SEQ ID 65) Sequence
analysis of SEQ ID 64 revealed a structural homology to CXC
cytokines. In addition, 4 alternative PCSC cDNA fragments were
cloned (SEQ ID NO:85-88). In each case, according to the invention,
each cDNA contains 3 putative ORFs which code for the polypeptides
depicted in SEQ ID NO:89-100.
TABLE-US-00010 TABLE 8 Expression of PCSC in normal tissues and
colorectal carcinomas Ileum + Colon + liver - Lung - Lymph nodes -
Stomach - Spleen - Adrenal gland - Kidney - Esophagus - Ovary -
Rectum + Testis - Thymus - Skin - Mamma - Pancreas - PBMC - PBMC
act. - Prostate - Thyroid - Tube - Uterus - Cerebrum - Cerebellum -
Colorectal tumors 19/20 Colorectal tumors metastases 15/15
[0209] Thus, PCSC is a differentiation antigen for normal colon
epithelia which is also expressed in colorectal tumors and in all
colon metastases studied. PCSC expression detected in all
colorectal metastases according to the invention renders this tumor
antigen a very interesting target for prophylaxis and treatment of
metastasizing colon tumors.
Example 9
Identification of SGY-1 as a New Tumor Antigen
[0210] The sequences of the SGY-1 transcript (SEQ ID N0:70) and of
its translation product (SEQ ID NO:71) have been published in
GenBank under accession number AF177398 (Krupnik et al., Gene 238,
301-313, 1999). Soggy-1 has previously been described as a member
of the Dickkopf protein family which act as inhibitors and
antagonists of the Wnt family of proteins. The Wnt proteins in turn
have important functions in embryonic development. Based on the
sequence of SGY-1 (SEQ ID NO:70), PCR primers
(5'-CTCCTATCCATGATGCTGACG-3' and 5'-CCTGAGGATGTACAGTAAGTG-3') were
generated according to the invention and used for RT-PCR analyses
(95.degree. 15 min; 94.degree. 1 min; 63.degree. 1 min; 72.degree.
1 min; 35 cycles) in a number of human tissues. Expression in
normal tissues was shown to be limited to testis. As described for
the other eCT, SGY-1 was shown according to the invention to be
ectopically activated in a number of tumor tissues; cf. Table
9.
TABLE-US-00011 TABLE 9 Expression of SGY-1 in tumors Tested Tissue
in total Positive % Melanoma 16 4 25 Mammary carcinomas 20 4 20
Colorectal tumors 20 0 0 Prostate carcinomas 8 1 13 Bronchial
carcinomas 32 3 18 Kidney cell carcinomas 7 0 0 Ovarian carcinomas
7 4 57 Thyroid carcinomas 4 0 0 Cervical carcinomas 6 2 33 Melanoma
cell lines 8 2 25 Bronchial carcinoma cell lines 6 2 33 Mammalian
carcinoma cell lines
Example 10
Identification of MORC as a New Tumor Antigen
[0211] The sequences of the MORC transcript (SEQ ID NO:74) and of
its translation product (SEQ ID NO:75) have been published in
GenBank under the accession number XM.sub.--037008 (Inoue et al.,
Hum Mol Genet. July: 8(7):1201-7, 1999).
[0212] MORC has originally been described as being involved in
spermatogenesis. Mutation of this protein in the mouse system
results in underdevelopment of the gonads. Based on the sequence of
MORC (SEQ ID NO:74), PCR primers (5'-CTGAGTATCAGCTACCATCAG-3' and
5'-TCTGTAGTCCTTCACATATCG-3') were generated according to the
invention and used for RT-PCR analyses (95.degree. 15 min;
94.degree. 1 min; 63.degree. 1 min; 72.degree. 1 min; 35 cycles) in
a number of human tissues. Expression in normal tissues was shown
to be limited to testis. As described for the other eCT, MORC was
shown according to the invention to be ectopically activated in a
number of tumor tissues cf. Table 10.
TABLE-US-00012 TABLE 10 Expression of MORC in tumors Tested Tissue
in total Positive % Melanoma 16 3 18 Mammary carcinomas 20 0 0
Colorectal tumors 20 0 0 Prostate carcinomas 8 0 0 Bronchial
carcinomas 17 3 18 Kidney cell carcinomas 7 0 0 Ovarian carcinomas
7 1 14 Thyroid carcinomas 4 0 0 Cervical carcinomas 6 0 0 Melanoma
cell lines 8 1 12 Bronchial carcinoma cell lines 6 1 17
Sequence CWU 1
1
10011171DNAHomo sapiens 1ctgtcgttgg tgtatttttc tggtgtcact
tctgtgcctt ccttcaaagg ttctccaaat 60gtcaactgtc aaggagcagc taattgagaa
gctaattgag gatgatgaaa actcccagtg 120taaaattact attgttggaa
ctggtgccgt aggcatggct tgtgctatta gtatcttact 180gaaggatttg
gctgatgaac ttgcccttgt tgatgttgca ttggacaaac tgaagggaga
240aatgatggat cttcagcatg gcagtctttt ctttagtact tcaaagatta
cttctggaaa 300agattacagt gtatctgcaa actccagaat agttattgtc
acagcaggtg caaggcagca 360ggagggagaa actcgccttg ccctggtcca
acgtaatgtg gctataatga aatcaatcat 420tcctgccata gtccattata
gtcctgattg taaaattctt gttgtttcaa atccagtgga 480tattttgaca
tatatagtct ggaagataag tggcttacct gtaactcgtg taattggaag
540tggttgtaat ctagactctg cccgtttccg ttacctaatt ggagaaaagt
tgggtgtcca 600ccccacaagc tgccatggtt ggattattgg agaacatggt
gattctagtg tgcccttatg 660gagtggggtg aatgttgctg gtgttgctct
gaagactctg gaccctaaat taggaacgga 720ttcagataag gaacactgga
aaaatatcca taaacaagtt attcaaagtg cctatgaaat 780tatcaagctg
aaggggtata cctcttgggc tattggactg tctgtgatgg atctggtagg
840atccattttg aaaaatctta ggagagtgca cccagtttcc accatggtta
agggattata 900tggaataaaa gaagaactct ttctcagtat cccttgtgtc
ttggggcgga atggtgtctc 960agatgttgtg aaaattaact tgaattctga
ggaggaggcc cttttcaaga agagtgcaga 1020aacactttgg aatattcaaa
aggatctaat attttaaatt aaagccttct aatgttccac 1080tgtttggaga
acagaagata gcaggctgtg tattttaaat tttgaaagta ttttcattga
1140tcttaaaaaa taaaaacaaa ttggagacct g 117121053DNAHomo sapiens
2ctgtcgttgg tgtatttttc tggtgtcact tctgtgcctt ccttcaaagg ttctccaaat
60gtcaactgtc aaggagcagc taattgagaa gctaattgag gatgatgaaa actcccagtg
120taaaattact attgttggaa ctggtgccgt aggcatggct tgtgctatta
gtatcttact 180gaagattaca gtgtatctgc aaactccaga atagttattg
tcacagcagg tgcaaggcag 240caggagggag aaactcgcct tgccctggtc
caacgtaatg tggctataat gaaatcaatc 300attcctgcca tagtccatta
tagtcctgat tgtaaaattc ttgttgtttc aaatccagtg 360gatattttga
catatatagt ctggaagata agtggcttac ctgtaactcg tgtaattgga
420agtggttgta atctagactc tgcccgtttc cgttacctaa ttggagaaaa
gttgggtgtc 480caccccacaa gctgccatgg ttggattatt ggagaacatg
gtgattctag tgtgccctta 540tggagtgggg tgaatgttgc tggtgttgct
ctgaagactc tggaccctaa attaggaacg 600gattcagata aggaacactg
gaaaaatatc cataaacaag ttattcaaag tgcctatgaa 660attatcaagc
tgaaggggta tacctcttgg gctattggac tgtctgtgat ggatctggta
720ggatccattt tgaaaaatct taggagagtg cacccagttt ccaccatggt
taagggatta 780tatggaataa aagaagaact ctttctcagt atcccttgtg
tcttggggcg gaatggtgtc 840tcagatgttg tgaaaattaa cttgaattct
gaggaggagg cccttttcaa gaagagtgca 900gaaacacttt ggaatattca
aaaggatcta atattttaaa ttaaagcctt ctaatgttcc 960actgtttgga
gaacagaaga tagcaggctg tgtattttaa attttgaaag tattttcatt
1020gatcttaaaa aataaaaaca aattggagac ctg 10533879DNAHomo sapiens
3ctgtcgttgg tgtatttttc tggtgtcact tctgtgcctt ccttcaaagg ttctccaaat
60gtcaactgtc aaggagcagc taattgagaa gctaattgag gatgatgaaa actcccagtg
120taaaattact attgttggaa ctggtgccgt aggcatggct tgtgctatta
gtatcttact 180gaagtggata ttttgacata tatagtctgg aagataagtg
gcttacctgt aactcgtgta 240attggaagtg gttgtaatct agactctgcc
cgtttccgtt acctaattgg agaaaagttg 300ggtgtccacc ccacaagctg
ccatggttgg attattggag aacatggtga ttctagtgtg 360cccttatgga
gtggggtgaa tgttgctggt gttgctctga agactctgga ccctaaatta
420ggaacggatt cagataagga acactggaaa aatatccata aacaagttat
tcaaagtgcc 480tatgaaatta tcaagctgaa ggggtatacc tcttgggcta
ttggactgtc tgtgatggat 540ctggtaggat ccattttgaa aaatcttagg
agagtgcacc cagtttccac catggttaag 600ggattatatg gaataaaaga
agaactcttt ctcagtatcc cttgtgtctt ggggcggaat 660ggtgtctcag
atgttgtgaa aattaacttg aattctgagg aggaggccct tttcaagaag
720agtgcagaaa cactttggaa tattcaaaag gatctaatat tttaaattaa
agccttctaa 780tgttccactg tttggagaac agaagatagc aggctgtgta
ttttaaattt tgaaagtatt 840ttcattgatc ttaaaaaata aaaacaaatt ggagacctg
8794811DNAHomo sapiens 4ctgtcgttgg tgtatttttc tggtgtcact tctgtgcctt
ccttcaaagg ttctccaaat 60gtcaactgtc aaggagcagc taattgagaa gctaattgag
gatgatgaaa actcccagtg 120taaaattact attgttggaa ctggtgccgt
aggcatggct tgtgctatta gtatcttact 180gaagattaca gtgtatctgc
aaactccaga atagttattg tcacagcagg tgcaaggcag 240caggagggag
aaactcgcct tgccctggtc caacgtaatg tggctataat gaaatcaatc
300attcctgcca tagtccatta tagtcctgat tgtaaaattc ttgttgtttc
aaatccagtg 360gatattttga catatatagt ctggaagata agtggcttac
ctgtaactcg tgtaattgga 420agtggttgta atctagactc tgcccgtttc
cgttacctaa ttggagaaaa gttgggtgtc 480caccccacaa gctgccatgg
ttggattatt ggagaacatg gtgattctag tgggattata 540tggaataaaa
gaagaactct ttctcagtat cccttgtgtc ttggggcgga atggtgtctc
600agatgttgtg aaaattaact tgaattctga ggaggaggcc cttttcaaga
agagtgcaga 660aacactttgg aatattcaaa aggatctaat attttaaatt
aaagccttct aatgttccac 720tgtttggaga acagaagata gcaggctgtg
tattttaaat tttgaaagta ttttcattga 780tcttaaaaaa taaaaacaaa
ttggagacct g 81151047DNAHomo sapiens 5ctgtcgttgg tgtatttttc
tggtgtcact tctgtgcctt ccttcaaagg ttctccaaat 60gtcaactgtc aaggagcagc
taattgagaa gctaattgag gatgatgaaa actcccagtg 120taaaattact
attgttggaa ctggtgccgt aggcatggct tgtgctatta gtatcttact
180gaaggatttg gctgatgaac ttgcccttgt tgatgttgca ttggacaaac
tgaagggaga 240aatgatggat cttcagcatg gcagtctttt ctttagtact
tcaaagatta cttctggaaa 300agattacagt gtatctgcaa actccagaat
agttattgtc acagcaggtg caaggcagca 360ggagggagaa actcgccttg
ccctggtcca acgtaatgtg gctataatga aatcaatcat 420tcctgccata
gtccattata gtcctgattg taaaattctt gttgtttcaa atccagtgga
480tattttgaca tatatagtct ggaagataag tggcttacct gtaactcgtg
taattggaag 540tggttgtaat ctagactctg cccgtttccg ttacctaatt
ggagaaaagt tgggtgtcca 600ccccacaagc tgccatggtt ggattattgg
agaacatggt gattctagtg tgcccttatg 660gagtggggtg aatgttgctg
gtgttgctct gaagactctg gaccctaaat taggaacgga 720ttcagataag
gaacactgga aaaatatcca taaacaagtt attcaaaggg attatatgga
780ataaaagaag aactctttct cagtatccct tgtgtcttgg ggcggaatgg
tgtctcagat 840gttgtgaaaa ttaacttgaa ttctgaggag gaggcccttt
tcaagaagag tgcagaaaca 900ctttggaata ttcaaaagga tctaatattt
taaattaaag ccttctaatg ttccactgtt 960tggagaacag aagatagcag
gctgtgtatt ttaaattttg aaagtatttt cattgatctt 1020aaaaaataaa
aacaaattgg agacctg 10476332PRTHomo sapiens 6Met Ser Thr Val Lys Glu
Gln Leu Ile Glu Lys Leu Ile Glu Asp Asp 1 5 10 15 Glu Asn Ser Gln
Cys Lys Ile Thr Ile Val Gly Thr Gly Ala Val Gly 20 25 30 Met Ala
Cys Ala Ile Ser Ile Leu Leu Lys Asp Leu Ala Asp Glu Leu 35 40 45
Ala Leu Val Asp Val Ala Leu Asp Lys Leu Lys Gly Glu Met Met Asp 50
55 60 Leu Gln His Gly Ser Leu Phe Phe Ser Thr Ser Lys Ile Thr Ser
Gly 65 70 75 80 Lys Asp Tyr Ser Val Ser Ala Asn Ser Arg Ile Val Ile
Val Thr Ala 85 90 95 Gly Ala Arg Gln Gln Glu Gly Glu Thr Arg Leu
Ala Leu Val Gln Arg 100 105 110 Asn Val Ala Ile Met Lys Ser Ile Ile
Pro Ala Ile Val His Tyr Ser 115 120 125 Pro Asp Cys Lys Ile Leu Val
Val Ser Asn Pro Val Asp Ile Leu Thr 130 135 140 Tyr Ile Val Trp Lys
Ile Ser Gly Leu Pro Val Thr Arg Val Ile Gly 145 150 155 160 Ser Gly
Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Ile Gly Glu 165 170 175
Lys Leu Gly Val His Pro Thr Ser Cys His Gly Trp Ile Ile Gly Glu 180
185 190 His Gly Asp Ser Ser Val Pro Leu Trp Ser Gly Val Asn Val Ala
Gly 195 200 205 Val Ala Leu Lys Thr Leu Asp Pro Lys Leu Gly Thr Asp
Ser Asp Lys 210 215 220 Glu His Trp Lys Asn Ile His Lys Gln Val Ile
Gln Ser Ala Tyr Glu 225 230 235 240 Ile Ile Lys Leu Lys Gly Tyr Thr
Ser Trp Ala Ile Gly Leu Ser Val 245 250 255 Met Asp Leu Val Gly Ser
Ile Leu Lys Asn Leu Arg Arg Val His Pro 260 265 270 Val Ser Thr Met
Val Lys Gly Leu Tyr Gly Ile Lys Glu Glu Leu Phe 275 280 285 Leu Ser
Ile Pro Cys Val Leu Gly Arg Asn Gly Val Ser Asp Val Val 290 295 300
Lys Ile Asn Leu Asn Ser Glu Glu Glu Ala Leu Phe Lys Lys Ser Ala 305
310 315 320 Glu Thr Leu Trp Asn Ile Gln Lys Asp Leu Ile Phe 325 330
751PRTHomo sapiens 7Met Ser Thr Val Lys Glu Gln Leu Ile Glu Lys Leu
Ile Glu Asp Asp 1 5 10 15 Glu Asn Ser Gln Cys Lys Ile Thr Ile Val
Gly Thr Gly Ala Val Gly 20 25 30 Met Ala Cys Ala Ile Ser Ile Leu
Leu Lys Ile Thr Val Tyr Leu Gln 35 40 45 Thr Pro Glu 50 8216PRTHomo
sapiens 8Met Lys Ser Ile Ile Pro Ala Ile Val His Tyr Ser Pro Asp
Cys Lys 1 5 10 15 Ile Leu Val Val Ser Asn Pro Val Asp Ile Leu Thr
Tyr Ile Val Trp 20 25 30 Lys Ile Ser Gly Leu Pro Val Thr Arg Val
Ile Gly Ser Gly Cys Asn 35 40 45 Leu Asp Ser Ala Arg Phe Arg Tyr
Leu Ile Gly Glu Lys Leu Gly Val 50 55 60 His Pro Thr Ser Cys His
Gly Trp Ile Ile Gly Glu His Gly Asp Ser 65 70 75 80 Ser Val Pro Leu
Trp Ser Gly Val Asn Val Ala Gly Val Ala Leu Lys 85 90 95 Thr Leu
Asp Pro Lys Leu Gly Thr Asp Ser Asp Lys Glu His Trp Lys 100 105 110
Asn Ile His Lys Gln Val Ile Gln Ser Ala Tyr Glu Ile Ile Lys Leu 115
120 125 Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser Val Met Asp Leu
Val 130 135 140 Gly Ser Ile Leu Lys Asn Leu Arg Arg Val His Pro Val
Ser Thr Met 145 150 155 160 Val Lys Gly Leu Tyr Gly Ile Lys Glu Glu
Leu Phe Leu Ser Ile Pro 165 170 175 Cys Val Leu Gly Arg Asn Gly Val
Ser Asp Val Val Lys Ile Asn Leu 180 185 190 Asn Ser Glu Glu Glu Ala
Leu Phe Lys Lys Ser Ala Glu Thr Leu Trp 195 200 205 Asn Ile Gln Lys
Asp Leu Ile Phe 210 215 945PRTHomo sapiens 9Met Ser Thr Val Lys Glu
Gln Leu Ile Glu Lys Leu Ile Glu Asp Asp 1 5 10 15 Glu Asn Ser Gln
Cys Lys Ile Thr Ile Val Gly Thr Gly Ala Val Gly 20 25 30 Met Ala
Cys Ala Ile Ser Ile Leu Leu Lys Trp Ile Phe 35 40 45 1076PRTHomo
sapiens 10Met Asp Leu Val Gly Ser Ile Leu Lys Asn Leu Arg Arg Val
His Pro 1 5 10 15 Val Ser Thr Met Val Lys Gly Leu Tyr Gly Ile Lys
Glu Glu Leu Phe 20 25 30 Leu Ser Ile Pro Cys Val Leu Gly Arg Asn
Gly Val Ser Asp Val Val 35 40 45 Lys Ile Asn Leu Asn Ser Glu Glu
Glu Ala Leu Phe Lys Lys Ser Ala 50 55 60 Glu Thr Leu Trp Asn Ile
Gln Lys Asp Leu Ile Phe 65 70 75 11109PRTHomo sapiens 11Met Lys Ser
Ile Ile Pro Ala Ile Val His Tyr Ser Pro Asp Cys Lys 1 5 10 15 Ile
Leu Val Val Ser Asn Pro Val Asp Ile Leu Thr Tyr Ile Val Trp 20 25
30 Lys Ile Ser Gly Leu Pro Val Thr Arg Val Ile Gly Ser Gly Cys Asn
35 40 45 Leu Asp Ser Ala Arg Phe Arg Tyr Leu Ile Gly Glu Lys Leu
Gly Val 50 55 60 His Pro Thr Ser Cys His Gly Trp Ile Ile Gly Glu
His Gly Asp Ser 65 70 75 80 Ser Gly Ile Ile Trp Asn Lys Arg Arg Thr
Leu Ser Gln Tyr Pro Leu 85 90 95 Cys Leu Gly Ala Glu Trp Cys Leu
Arg Cys Cys Glu Asn 100 105 1266PRTHomo sapiens 12Met Val Gly Leu
Leu Glu Asn Met Val Ile Leu Val Gly Leu Tyr Gly 1 5 10 15 Ile Lys
Glu Glu Leu Phe Leu Ser Ile Pro Cys Val Leu Gly Arg Asn 20 25 30
Gly Val Ser Asp Val Val Lys Ile Asn Leu Asn Ser Glu Glu Glu Ala 35
40 45 Leu Phe Lys Lys Ser Ala Glu Thr Leu Trp Asn Ile Gln Lys Asp
Leu 50 55 60 Ile Phe 65 13241PRTHomo sapiens 13Met Ser Thr Val Lys
Glu Gln Leu Ile Glu Lys Leu Ile Glu Asp Asp 1 5 10 15 Glu Asn Ser
Gln Cys Lys Ile Thr Ile Val Gly Thr Gly Ala Val Gly 20 25 30 Met
Ala Cys Ala Ile Ser Ile Leu Leu Lys Asp Leu Ala Asp Glu Leu 35 40
45 Ala Leu Val Asp Val Ala Leu Asp Lys Leu Lys Gly Glu Met Met Asp
50 55 60 Leu Gln His Gly Ser Leu Phe Phe Ser Thr Ser Lys Ile Thr
Ser Gly 65 70 75 80 Lys Asp Tyr Ser Val Ser Ala Asn Ser Arg Ile Val
Ile Val Thr Ala 85 90 95 Gly Ala Arg Gln Gln Glu Gly Glu Thr Arg
Leu Ala Leu Val Gln Arg 100 105 110 Asn Val Ala Ile Met Lys Ser Ile
Ile Pro Ala Ile Val His Tyr Ser 115 120 125 Pro Asp Cys Lys Ile Leu
Val Val Ser Asn Pro Val Asp Ile Leu Thr 130 135 140 Tyr Ile Val Trp
Lys Ile Ser Gly Leu Pro Val Thr Arg Val Ile Gly 145 150 155 160 Ser
Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Ile Gly Glu 165 170
175 Lys Leu Gly Val His Pro Thr Ser Cys His Gly Trp Ile Ile Gly Glu
180 185 190 His Gly Asp Ser Ser Val Pro Leu Trp Ser Gly Val Asn Val
Ala Gly 195 200 205 Val Ala Leu Lys Thr Leu Asp Pro Lys Leu Gly Thr
Asp Ser Asp Lys 210 215 220 Glu His Trp Lys Asn Ile His Lys Gln Val
Ile Gln Arg Asp Tyr Met 225 230 235 240 Glu 1423PRTHomo sapiens
14Gly Ala Val Gly Met Ala Cys Ala Ile Ser Ile Leu Leu Lys Ile Thr 1
5 10 15 Val Tyr Leu Gln Thr Pro Glu 20 1517PRTHomo sapiens 15Gly
Ala Val Gly Met Ala Cys Ala Ile Ser Ile Leu Leu Lys Trp Ile 1 5 10
15 Phe 1639PRTHomo sapiens 16Gly Trp Ile Ile Gly Glu His Gly Asp
Ser Ser Gly Ile Ile Trp Asn 1 5 10 15 Lys Arg Arg Thr Leu Ser Gln
Tyr Pro Leu Cys Leu Gly Ala Glu Trp 20 25 30 Cys Leu Arg Cys Cys
Glu Asn 35 1723PRTHomo sapiens 17Met Val Gly Leu Leu Glu Asn Met
Val Ile Leu Val Gly Leu Tyr Gly 1 5 10 15 Ile Lys Glu Glu Leu Phe
Leu 20 1817PRTHomo sapiens 18Glu His Trp Lys Asn Ile His Lys Gln
Val Ile Gln Arg Asp Tyr Met 1 5 10 15 Glu 192168DNAHomo sapiens
19gaatccgcgg ggagggcaca acagctgcta cctgaacagt ttctgaccca acagttaccc
60agcgccggac tcgctgcgcc ccggcggctc tagggacccc cggcgcctac acttagctcc
120gcgcccgaga gaatgttgga ccgacgacac aagacctcag acttgtgtta
ttctagcagc 180tgaacacacc ccaggctctt ctgaccggca gtggctctgg
aagcagtctg gtgtatagag 240ttatggattc actaccagat tctactgtat
gctcttgaca actatgacca caatggtcca 300cccacaaatg aattatcagg
agtgaaccca gaggcacgta tgaatgaaag tcctgatccg 360actgacctgg
cgggagtcat cattgagctc ggccccaatg acagtccaca gacaagtgaa
420tttaaaggag caaccgagga ggcacctgcg aaagaaagcc cacacacaag
tgaatttaaa 480ggagcagccc gggtgtcacc tatcagtgaa agtgtgttag
cacgactttc caagtttgaa 540gttgaagatg ctgaaaatgt tgcttcatat
gacagcaaga ttaagaaaat tgtgcattca 600attgtatcat cctttgcatt
tggactattt ggagttttcc tggtcttact ggatgtcact 660ctcatccttg
ccgacctaat tttcactgac agcaaacttt atattccttt ggagtatcgt
720tctatttctc tagctattgc cttatttttt ctcatggatg ttcttcttcg
agtatttgta 780gaaaggagac agcagtattt ttctgactta tttaacattt
tagatactgc cattattgtg 840attcttctgc tggttgatgt cgtttacatt
ttttttgaca ttaagttgct taggaatatt 900cccagatgga cacatttact
tcgacttcta cgacttatta ttctgttaag aatttttcat 960ctgtttcatc
aaaaaagaca acttgaaaag ctgataagaa ggcgggtttc agaaaacaaa
1020aggcgataca caagggatgg atttgaccta gacctcactt acgttacaga
acgtattatt 1080gctatgtcat ttccatcttc tggaaggcag tctttctata
gaaatccaat caaggaagtt 1140gtgcggtttc tagataagaa acaccgaaac
cactatcgag tctacaatct atgcagtgaa 1200agagcttacg atcctaagca
cttccataat agggtcgtta gaatcatgat tgatgatcat 1260aatgtcccca
ctctacatca gatggtggtt ttcaccaagg aagtaaatga gtggatggct
1320caagatcttg aaaacatcgt agcgattcac tgtaaaggag gcacagatag
aacaggaact 1380atggtttgtg
ccttccttat tgcctctgaa atatgttcaa ctgcaaagga aagcctgtat
1440tattttggag aaaggcgaac agataaaacc cacagcgaaa aatttcaggg
agtagaaact 1500ccttctcaga agagatatgt tgcatatttt gcacaagtga
aacatctcta caactggaat 1560ctccctccaa gacggatact ctttataaaa
cacttcatta tttattcgat tcctcgttat 1620gtacgtgatc taaaaatcca
aatagaaatg gagaaaaagg ttgtcttttc cactatttca 1680ttaggaaaat
gttcggtact tgataacatt acaacagaca aaatattaat tgatgtattc
1740gacggtccac ctctgtatga tgatgtgaaa gtgcagtttt tctattcgaa
tcttcctaca 1800tactatgaca attgctcatt ttacttctgg ttgcacacat
cttttattga aaataacagg 1860ctttatctac caaaaaatga attggataat
ctacataaac aaaaagcacg gagaatttat 1920ccatcagatt ttgccgtgga
gatacttttt ggcgagaaaa tgacttccag tgatgttgta 1980gctggatccg
attaagtata gctccccctt ccccttctgg gaaagaatta tgttctttcc
2040aaccctgcca catgttcata tatcctaaat ctatcctaaa tgttcccttg
aagtatttat 2100ttatgtttat atatgtttat acatgttctt caataaatct
attacatata tataaaaaaa 2160aaaaaaaa 2168202114DNAHomo sapiens
20gaatccgcgg ggagggcaca acagctgcta cctgaacagt ttctgaccca acagttaccc
60agcgccggac tcgctgcgcc ccggcggctc tagggacccc cggcgcctac acttagctcc
120gcgcccgaga gaatgttgga ccgacgacac aagacctcag acttgtgtta
ttctagcagc 180tgaacacacc ccaggctctt ctgaccggca gtggctctgg
aagcagtctg gtgtatagag 240ttatggattc actaccagat tctactgtat
gctcttgaca actatgacca caatggtcca 300cccacaaatg aattatcagg
agtgaaccca gaggcacgta tgaatgaaag tcctgatccg 360actgacctgg
cgggagtcat cattgagctc ggccccaatg acagtccaca gacaagtgaa
420tttaaaggag caaccgagga ggcacctgcg aaagaaagtg tgttagcacg
actttccaag 480tttgaagttg aagatgctga aaatgttgct tcatatgaca
gcaagattaa gaaaattgtg 540cattcaattg tatcatcctt tgcatttgga
ctatttggag ttttcctggt cttactggat 600gtcactctca tccttgccga
cctaattttc actgacagca aactttatat tcctttggag 660tatcgttcta
tttctctagc tattgcctta ttttttctca tggatgttct tcttcgagta
720tttgtagaaa ggagacagca gtatttttct gacttattta acattttaga
tactgccatt 780attgtgattc ttctgctggt tgatgtcgtt tacatttttt
ttgacattaa gttgcttagg 840aatattccca gatggacaca tttacttcga
cttctacgac ttattattct gttaagaatt 900tttcatctgt ttcatcaaaa
aagacaactt gaaaagctga taagaaggcg ggtttcagaa 960aacaaaaggc
gatacacaag ggatggattt gacctagacc tcacttacgt tacagaacgt
1020attattgcta tgtcatttcc atcttctgga aggcagtctt tctatagaaa
tccaatcaag 1080gaagttgtgc ggtttctaga taagaaacac cgaaaccact
atcgagtcta caatctatgc 1140agtgaaagag cttacgatcc taagcacttc
cataataggg tcgttagaat catgattgat 1200gatcataatg tccccactct
acatcagatg gtggttttca ccaaggaagt aaatgagtgg 1260atggctcaag
atcttgaaaa catcgtagcg attcactgta aaggaggcac agatagaaca
1320ggaactatgg tttgtgcctt ccttattgcc tctgaaatat gttcaactgc
aaaggaaagc 1380ctgtattatt ttggagaaag gcgaacagat aaaacccaca
gcgaaaaatt tcagggagta 1440gaaactcctt ctcagaagag atatgttgca
tattttgcac aagtgaaaca tctctacaac 1500tggaatctcc ctccaagacg
gatactcttt ataaaacact tcattattta ttcgattcct 1560cgttatgtac
gtgatctaaa aatccaaata gaaatggaga aaaaggttgt cttttccact
1620atttcattag gaaaatgttc ggtacttgat aacattacaa cagacaaaat
attaattgat 1680gtattcgacg gtccacctct gtatgatgat gtgaaagtgc
agtttttcta ttcgaatctt 1740cctacatact atgacaattg ctcattttac
ttctggttgc acacatcttt tattgaaaat 1800aacaggcttt atctaccaaa
aaatgaattg gataatctac ataaacaaaa agcacggaga 1860atttatccat
cagattttgc cgtggagata ctttttggcg agaaaatgac ttccagtgat
1920gttgtagctg gatccgatta agtatagctc ccccttcccc ttctgggaaa
gaattatgtt 1980ctttccaacc ctgccacatg ttcatatatc ctaaatctat
cctaaatgtt cccttgaagt 2040atttatttat gtttatatat gtttatacat
gttcttcaat aaatctatta catatatata 2100aaaaaaaaaa aaaa
2114212222DNAHomo sapiens 21gaatccgcgg ggagggcaca acagctgcta
cctgaacagt ttctgaccca acagttaccc 60agcgccggac tcgctgcgcc ccggcggctc
tagggacccc cggcgcctac acttagctcc 120gcgcccgaga gaatgttgga
ccgacgacac aagacctcag acttgtgtta ttctagcagc 180tgaacacacc
ccaggctctt ctgaccggca gtggctctgg aagcagtctg gtgtatagag
240ttatggattc actaccagat tctactgtat gctcttgaca actatgacca
caatggtcca 300cccacaaatg aattatcagg agtgaaccca gaggcacgta
tgaatgaaag tcctgatccg 360actgacctgg cgggagtcat cattgagctc
ggccccaatg acagtccaca gacaagtgaa 420tttaaaggag caaccgagga
ggcacctgcg aaagaaagcc cacacacaag tgaatttaaa 480ggagcagccc
gggtgtcacc tatcagtgaa agtgtgttag cacgactttc caagtttgaa
540gttgaagatg ctgaaaatgt tgcttcatat gacagcaaga ttaagaaaat
tgtgcattca 600attgtatcat cctttgcatt tggactattt ggagttttcc
tggtcttact ggatgtcact 660ctcatccttg ccgacctaat tttcactgac
agcaaacttt atattccttt ggagtatcgt 720tctatttctc tagctattgc
cttatttttt ctcatggatg ttcttcttcg agtatttgta 780gaaaggagac
agcagtattt ttctgactta tttaacattt tagatactgc cattattgtg
840attcttctgc tggttgatgt cgtttacatt ttttttgaca ttaagttgct
taggaatatt 900cccagatgga cacatttact tcgacttcta cgacttatta
ttctgttaag aatttttcat 960ctgtttcatc aaaaaagaca acttgaaaag
ctgataagaa ggcgggtttc agaaaacaaa 1020aggcgataca caagggatgg
atttgaccta gacctcactt acgttacaga acgtattatt 1080gctatgtcat
ttccatcttc tggaaggcag tctttctata gaaatccaat caaggaagtt
1140gtgcggtttc tagataagaa acaccgaaac cactatcgag tctacaatct
atgcagtatg 1200tacattactc tatattgtgc tactgtagat agaaaacaga
ttactgcacg tgaaagagct 1260tacgatccta agcacttcca taatagggtc
gttagaatca tgattgatga tcataatgtc 1320cccactctac atcagatggt
ggttttcacc aaggaagtaa atgagtggat ggctcaagat 1380cttgaaaaca
tcgtagcgat tcactgtaaa ggaggcacag atagaacagg aactatggtt
1440tgtgccttcc ttattgcctc tgaaatatgt tcaactgcaa aggaaagcct
gtattatttt 1500ggagaaaggc gaacagataa aacccacagc gaaaaatttc
agggagtaga aactccttct 1560cagaagagat atgttgcata ttttgcacaa
gtgaaacatc tctacaactg gaatctccct 1620ccaagacgga tactctttat
aaaacacttc attatttatt cgattcctcg ttatgtacgt 1680gatctaaaaa
tccaaataga aatggagaaa aaggttgtct tttccactat ttcattagga
1740aaatgttcgg tacttgataa cattacaaca gacaaaatat taattgatgt
attcgacggt 1800ccacctctgt atgatgatgt gaaagtgcag tttttctatt
cgaatcttcc tacatactat 1860gacaattgct cattttactt ctggttgcac
acatctttta ttgaaaataa caggctttat 1920ctaccaaaaa atgaattgga
taatctacat aaacaaaaag cacggagaat ttatccatca 1980gattttgccg
tggagatact ttttggcgag aaaatgactt ccagtgatgt tgtagctgga
2040tccgattaag tatagctccc ccttcccctt ctgggaaaga attatgttct
ttccaaccct 2100gccacatgtt catatatcct aaatctatcc taaatgttcc
cttgaagtat ttatttatgt 2160ttatatatgt ttatacatgt tcttcaataa
atctattaca tatatataaa aaaaaaaaaa 2220aa 222222551PRTHomo sapiens
22Met Asn Glu Ser Pro Asp Pro Thr Asp Leu Ala Gly Val Ile Ile Glu 1
5 10 15 Leu Gly Pro Asn Asp Ser Pro Gln Thr Ser Glu Phe Lys Gly Ala
Thr 20 25 30 Glu Glu Ala Pro Ala Lys Glu Ser Pro His Thr Ser Glu
Phe Lys Gly 35 40 45 Ala Ala Arg Val Ser Pro Ile Ser Glu Ser Val
Leu Ala Arg Leu Ser 50 55 60 Lys Phe Glu Val Glu Asp Ala Glu Asn
Val Ala Ser Tyr Asp Ser Lys 65 70 75 80 Ile Lys Lys Ile Val His Ser
Ile Val Ser Ser Phe Ala Phe Gly Leu 85 90 95 Phe Gly Val Phe Leu
Val Leu Leu Asp Val Thr Leu Ile Leu Ala Asp 100 105 110 Leu Ile Phe
Thr Asp Ser Lys Leu Tyr Ile Pro Leu Glu Tyr Arg Ser 115 120 125 Ile
Ser Leu Ala Ile Ala Leu Phe Phe Leu Met Asp Val Leu Leu Arg 130 135
140 Val Phe Val Glu Arg Arg Gln Gln Tyr Phe Ser Asp Leu Phe Asn Ile
145 150 155 160 Leu Asp Thr Ala Ile Ile Val Ile Leu Leu Leu Val Asp
Val Val Tyr 165 170 175 Ile Phe Phe Asp Ile Lys Leu Leu Arg Asn Ile
Pro Arg Trp Thr His 180 185 190 Leu Leu Arg Leu Leu Arg Leu Ile Ile
Leu Leu Arg Ile Phe His Leu 195 200 205 Phe His Gln Lys Arg Gln Leu
Glu Lys Leu Ile Arg Arg Arg Val Ser 210 215 220 Glu Asn Lys Arg Arg
Tyr Thr Arg Asp Gly Phe Asp Leu Asp Leu Thr 225 230 235 240 Tyr Val
Thr Glu Arg Ile Ile Ala Met Ser Phe Pro Ser Ser Gly Arg 245 250 255
Gln Ser Phe Tyr Arg Asn Pro Ile Lys Glu Val Val Arg Phe Leu Asp 260
265 270 Lys Lys His Arg Asn His Tyr Arg Val Tyr Asn Leu Cys Ser Glu
Arg 275 280 285 Ala Tyr Asp Pro Lys His Phe His Asn Arg Val Val Arg
Ile Met Ile 290 295 300 Asp Asp His Asn Val Pro Thr Leu His Gln Met
Val Val Phe Thr Lys 305 310 315 320 Glu Val Asn Glu Trp Met Ala Gln
Asp Leu Glu Asn Ile Val Ala Ile 325 330 335 His Cys Lys Gly Gly Thr
Asp Arg Thr Gly Thr Met Val Cys Ala Phe 340 345 350 Leu Ile Ala Ser
Glu Ile Cys Ser Thr Ala Lys Glu Ser Leu Tyr Tyr 355 360 365 Phe Gly
Glu Arg Arg Thr Asp Lys Thr His Ser Glu Lys Phe Gln Gly 370 375 380
Val Glu Thr Pro Ser Gln Lys Arg Tyr Val Ala Tyr Phe Ala Gln Val 385
390 395 400 Lys His Leu Tyr Asn Trp Asn Leu Pro Pro Arg Arg Ile Leu
Phe Ile 405 410 415 Lys His Phe Ile Ile Tyr Ser Ile Pro Arg Tyr Val
Arg Asp Leu Lys 420 425 430 Ile Gln Ile Glu Met Glu Lys Lys Val Val
Phe Ser Thr Ile Ser Leu 435 440 445 Gly Lys Cys Ser Val Leu Asp Asn
Ile Thr Thr Asp Lys Ile Leu Ile 450 455 460 Asp Val Phe Asp Gly Pro
Pro Leu Tyr Asp Asp Val Lys Val Gln Phe 465 470 475 480 Phe Tyr Ser
Asn Leu Pro Thr Tyr Tyr Asp Asn Cys Ser Phe Tyr Phe 485 490 495 Trp
Leu His Thr Ser Phe Ile Glu Asn Asn Arg Leu Tyr Leu Pro Lys 500 505
510 Asn Glu Leu Asp Asn Leu His Lys Gln Lys Ala Arg Arg Ile Tyr Pro
515 520 525 Ser Asp Phe Ala Val Glu Ile Leu Phe Gly Glu Lys Met Thr
Ser Ser 530 535 540 Asp Val Val Ala Gly Ser Asp 545 550
23533PRTHomo sapiens 23Met Asn Glu Ser Pro Asp Pro Thr Asp Leu Ala
Gly Val Ile Ile Glu 1 5 10 15 Leu Gly Pro Asn Asp Ser Pro Gln Thr
Ser Glu Phe Lys Gly Ala Thr 20 25 30 Glu Glu Ala Pro Ala Lys Glu
Ser Val Leu Ala Arg Leu Ser Lys Phe 35 40 45 Glu Val Glu Asp Ala
Glu Asn Val Ala Ser Tyr Asp Ser Lys Ile Lys 50 55 60 Lys Ile Val
His Ser Ile Val Ser Ser Phe Ala Phe Gly Leu Phe Gly 65 70 75 80 Val
Phe Leu Val Leu Leu Asp Val Thr Leu Ile Leu Ala Asp Leu Ile 85 90
95 Phe Thr Asp Ser Lys Leu Tyr Ile Pro Leu Glu Tyr Arg Ser Ile Ser
100 105 110 Leu Ala Ile Ala Leu Phe Phe Leu Met Asp Val Leu Leu Arg
Val Phe 115 120 125 Val Glu Arg Arg Gln Gln Tyr Phe Ser Asp Leu Phe
Asn Ile Leu Asp 130 135 140 Thr Ala Ile Ile Val Ile Leu Leu Leu Val
Asp Val Val Tyr Ile Phe 145 150 155 160 Phe Asp Ile Lys Leu Leu Arg
Asn Ile Pro Arg Trp Thr His Leu Leu 165 170 175 Arg Leu Leu Arg Leu
Ile Ile Leu Leu Arg Ile Phe His Leu Phe His 180 185 190 Gln Lys Arg
Gln Leu Glu Lys Leu Ile Arg Arg Arg Val Ser Glu Asn 195 200 205 Lys
Arg Arg Tyr Thr Arg Asp Gly Phe Asp Leu Asp Leu Thr Tyr Val 210 215
220 Thr Glu Arg Ile Ile Ala Met Ser Phe Pro Ser Ser Gly Arg Gln Ser
225 230 235 240 Phe Tyr Arg Asn Pro Ile Lys Glu Val Val Arg Phe Leu
Asp Lys Lys 245 250 255 His Arg Asn His Tyr Arg Val Tyr Asn Leu Cys
Ser Glu Arg Ala Tyr 260 265 270 Asp Pro Lys His Phe His Asn Arg Val
Val Arg Ile Met Ile Asp Asp 275 280 285 His Asn Val Pro Thr Leu His
Gln Met Val Val Phe Thr Lys Glu Val 290 295 300 Asn Glu Trp Met Ala
Gln Asp Leu Glu Asn Ile Val Ala Ile His Cys 305 310 315 320 Lys Gly
Gly Thr Asp Arg Thr Gly Thr Met Val Cys Ala Phe Leu Ile 325 330 335
Ala Ser Glu Ile Cys Ser Thr Ala Lys Glu Ser Leu Tyr Tyr Phe Gly 340
345 350 Glu Arg Arg Thr Asp Lys Thr His Ser Glu Lys Phe Gln Gly Val
Glu 355 360 365 Thr Pro Ser Gln Lys Arg Tyr Val Ala Tyr Phe Ala Gln
Val Lys His 370 375 380 Leu Tyr Asn Trp Asn Leu Pro Pro Arg Arg Ile
Leu Phe Ile Lys His 385 390 395 400 Phe Ile Ile Tyr Ser Ile Pro Arg
Tyr Val Arg Asp Leu Lys Ile Gln 405 410 415 Ile Glu Met Glu Lys Lys
Val Val Phe Ser Thr Ile Ser Leu Gly Lys 420 425 430 Cys Ser Val Leu
Asp Asn Ile Thr Thr Asp Lys Ile Leu Ile Asp Val 435 440 445 Phe Asp
Gly Pro Pro Leu Tyr Asp Asp Val Lys Val Gln Phe Phe Tyr 450 455 460
Ser Asn Leu Pro Thr Tyr Tyr Asp Asn Cys Ser Phe Tyr Phe Trp Leu 465
470 475 480 His Thr Ser Phe Ile Glu Asn Asn Arg Leu Tyr Leu Pro Lys
Asn Glu 485 490 495 Leu Asp Asn Leu His Lys Gln Lys Ala Arg Arg Ile
Tyr Pro Ser Asp 500 505 510 Phe Ala Val Glu Ile Leu Phe Gly Glu Lys
Met Thr Ser Ser Asp Val 515 520 525 Val Ala Gly Ser Asp 530
24569PRTHomo sapiens 24Met Asn Glu Ser Pro Asp Pro Thr Asp Leu Ala
Gly Val Ile Ile Glu 1 5 10 15 Leu Gly Pro Asn Asp Ser Pro Gln Thr
Ser Glu Phe Lys Gly Ala Thr 20 25 30 Glu Glu Ala Pro Ala Lys Glu
Ser Pro His Thr Ser Glu Phe Lys Gly 35 40 45 Ala Ala Arg Val Ser
Pro Ile Ser Glu Ser Val Leu Ala Arg Leu Ser 50 55 60 Lys Phe Glu
Val Glu Asp Ala Glu Asn Val Ala Ser Tyr Asp Ser Lys 65 70 75 80 Ile
Lys Lys Ile Val His Ser Ile Val Ser Ser Phe Ala Phe Gly Leu 85 90
95 Phe Gly Val Phe Leu Val Leu Leu Asp Val Thr Leu Ile Leu Ala Asp
100 105 110 Leu Ile Phe Thr Asp Ser Lys Leu Tyr Ile Pro Leu Glu Tyr
Arg Ser 115 120 125 Ile Ser Leu Ala Ile Ala Leu Phe Phe Leu Met Asp
Val Leu Leu Arg 130 135 140 Val Phe Val Glu Arg Arg Gln Gln Tyr Phe
Ser Asp Leu Phe Asn Ile 145 150 155 160 Leu Asp Thr Ala Ile Ile Val
Ile Leu Leu Leu Val Asp Val Val Tyr 165 170 175 Ile Phe Phe Asp Ile
Lys Leu Leu Arg Asn Ile Pro Arg Trp Thr His 180 185 190 Leu Leu Arg
Leu Leu Arg Leu Ile Ile Leu Leu Arg Ile Phe His Leu 195 200 205 Phe
His Gln Lys Arg Gln Leu Glu Lys Leu Ile Arg Arg Arg Val Ser 210 215
220 Glu Asn Lys Arg Arg Tyr Thr Arg Asp Gly Phe Asp Leu Asp Leu Thr
225 230 235 240 Tyr Val Thr Glu Arg Ile Ile Ala Met Ser Phe Pro Ser
Ser Gly Arg 245 250 255 Gln Ser Phe Tyr Arg Asn Pro Ile Lys Glu Val
Val Arg Phe Leu Asp 260 265 270 Lys Lys His Arg Asn His Tyr Arg Val
Tyr Asn Leu Cys Ser Met Tyr 275 280 285 Ile Thr Leu Tyr Cys Ala Thr
Val Asp Arg Lys Gln Ile Thr Ala Arg 290 295 300 Glu Arg Ala Tyr Asp
Pro Lys His Phe His Asn Arg Val Val Arg Ile 305 310 315 320 Met Ile
Asp Asp His Asn Val Pro Thr Leu His Gln Met Val Val Phe 325 330 335
Thr Lys Glu Val Asn Glu Trp Met Ala Gln Asp Leu Glu Asn Ile Val 340
345 350 Ala Ile His Cys Lys Gly Gly Thr Asp Arg Thr Gly Thr Met Val
Cys 355 360 365 Ala Phe Leu Ile Ala Ser Glu Ile Cys Ser Thr Ala Lys
Glu Ser Leu 370 375 380 Tyr Tyr Phe Gly Glu Arg Arg Thr Asp Lys Thr
His Ser Glu Lys Phe 385 390 395
400 Gln Gly Val Glu Thr Pro Ser Gln Lys Arg Tyr Val Ala Tyr Phe Ala
405 410 415 Gln Val Lys His Leu Tyr Asn Trp Asn Leu Pro Pro Arg Arg
Ile Leu 420 425 430 Phe Ile Lys His Phe Ile Ile Tyr Ser Ile Pro Arg
Tyr Val Arg Asp 435 440 445 Leu Lys Ile Gln Ile Glu Met Glu Lys Lys
Val Val Phe Ser Thr Ile 450 455 460 Ser Leu Gly Lys Cys Ser Val Leu
Asp Asn Ile Thr Thr Asp Lys Ile 465 470 475 480 Leu Ile Asp Val Phe
Asp Gly Pro Pro Leu Tyr Asp Asp Val Lys Val 485 490 495 Gln Phe Phe
Tyr Ser Asn Leu Pro Thr Tyr Tyr Asp Asn Cys Ser Phe 500 505 510 Tyr
Phe Trp Leu His Thr Ser Phe Ile Glu Asn Asn Arg Leu Tyr Leu 515 520
525 Pro Lys Asn Glu Leu Asp Asn Leu His Lys Gln Lys Ala Arg Arg Ile
530 535 540 Tyr Pro Ser Asp Phe Ala Val Glu Ile Leu Phe Gly Glu Lys
Met Thr 545 550 555 560 Ser Ser Asp Val Val Ala Gly Ser Asp 565
2521DNAArtificial SequenceDescription of artificial sequence
Oligonucleotide 25tgccgtaggc atggcttgtg c 212621DNAArtificial
SequenceDescription of artificial sequence Oligonucleotide
26caacatctga gacaccattc c 212721DNAArtificial SequenceDescription
of artificial sequence Oligonucleotide 27tggatgtcac tctcatcctt g
212821DNAArtificial SequenceDescription of artificial sequence
Oligonucleotide 28ccatagttcc tgttctatct g 21292192DNAHomo sapiens
29agctcagctg ggagcgcaga ggctcacgcc tgtaatccca tcatttgctt aggtctgatc
60aatctgctcc acacaatttc tcagtgatcc tctgcatctc tgcctacaag ggcctccctg
120acacccaagt tcatattgct cagaaacagt gaacttgagt ttttcgtttt
accttgatct 180ctctctgaca aagaaatcca gatgatgcaa cacctgatga
agacaataca tggaaaatga 240cagtcttgga aataactttg gctgtcatcc
tgactctact gggacttgcc atcctggcta 300ttttgttaac aagatgggca
cgacgtaagc aaagtgaaat gtatatctcc agatacagtt 360cagaacaaag
tgctagactt ctggactatg aggatggtag aggatcccga catgcatatc
420aacacaaagt gacacttcat atgataaccg agagagatcc aaaaagagat
tacacaccat 480caaccaactc tctagcactg tctcgatcaa gtattgcttt
acctcaagga tccatgagta 540gtataaaatg tttacaaaca actgaagaac
ctccttccag aactgcagga gccatgatgc 600aattcacagc cctattcccg
gagctacagg acctatcaag ctctctcaaa aaaccattgt 660gcaaactcca
ggacctattg tacaatatct ggatccaatg tcagatcgca tctcacacaa
720tcactggtca ccttcagcac ccgcggtcac ccatggcacc cataataatt
tcacagagaa 780ccgcaagtca gctggcagca cctataagaa tacctcaagt
tcacactatg gacagttctg 840gaaaaatcac actgactcct gtggttatat
taacaggtta catggacgaa gaacttcgaa 900aaaaatcttg ttccaaaatc
cagattctaa aatgtggagg cactgcaagg tctcagatag 960ccgagaagaa
aacaaggaag caactaaaga atgacatcat atttacgaat tctgtagaat
1020ccttgaaatc agcacacata aaggagccag aaagagaagg aaaaggcact
gatttagaga 1080aagacaaaat aggaatggag gtcaaggtag acagtgacgc
tggaatacca aaaagacagg 1140aaacccaact aaaaatcagt gaagatgagt
ataccacaag gacagggagc ccaaataaag 1200aaaagtgtgt cagatgtacc
aagaggacag gagtccaagt aaagaagagt gagtcaggtg 1260tcccaaaagg
acaagaagcc caagtaacga agagtgggtt ggttgtactg aaaggacagg
1320aagcccaggt agagaagagt gagatgggtg tgccaagaag acaggaatcc
caagtaaaga 1380agagtcagtc tggtgtctca aagggacagg aagcccaggt
aaagaagagg gagtcagttg 1440tactgaaagg acaggaagcc caggtagaga
agagtgagtt gaaggtacca aaaggacaag 1500aaggccaagt agagaagact
gaggcagatg tgccaaagga acaagaggtc caagaaaaga 1560agagtgaggc
aggtgtactg aaaggaccag aatcccaagt aaagaacact gaggtgagtg
1620taccagaaac actggaatcc caagtaaaga agagtgagtc aggtgtacta
aaaggacagg 1680aagcccaaga aaagaaggag agttttgagg ataaaggaaa
taatgataaa gaaaaggaga 1740gagatgcaga gaaagatcca aataaaaaag
aaaaaggtga caaaaacaca aaaggtgaca 1800aaggaaagga caaagttaaa
ggaaagagag aatcagaaat caatggtgaa aaatcaaaag 1860gctcgaaaag
gcgaaggcaa atacaggaag gaagtacaac aaaaaagtgg aagagtaagg
1920ataaattttt taaaggccca taagacaagt gattattatg attcccatac
tccagataca 1980aaccatatcc cagccattgc ctaaacagat tacaattata
aaatcccttt catcttcata 2040tcacagtttc tgctcttcag aagtttcacc
ctttttaatc tctcagccac aaacctcagt 2100tccaatattg ttataagtta
agacgtatat gattccgtca agaaagactg gatactttct 2160gaagtaaaac
attttaatta aagaaaaaaa aa 219230568PRTHomo sapiens 30Met Thr Val Leu
Glu Ile Thr Leu Ala Val Ile Leu Thr Leu Leu Gly 1 5 10 15 Leu Ala
Ile Leu Ala Ile Leu Leu Thr Arg Trp Ala Arg Arg Lys Gln 20 25 30
Ser Glu Met Tyr Ile Ser Arg Tyr Ser Ser Glu Gln Ser Ala Arg Leu 35
40 45 Leu Asp Tyr Glu Asp Gly Arg Gly Ser Arg His Ala Tyr Gln His
Lys 50 55 60 Val Thr Leu His Met Ile Thr Glu Arg Asp Pro Lys Arg
Asp Tyr Thr 65 70 75 80 Pro Ser Thr Asn Ser Leu Ala Leu Ser Arg Ser
Ser Ile Ala Leu Pro 85 90 95 Gln Gly Ser Met Ser Ser Ile Lys Cys
Leu Gln Thr Thr Glu Glu Pro 100 105 110 Pro Ser Arg Thr Ala Gly Ala
Met Met Gln Phe Thr Ala Leu Phe Pro 115 120 125 Glu Leu Gln Asp Leu
Ser Ser Ser Leu Lys Lys Pro Leu Cys Lys Leu 130 135 140 Gln Asp Leu
Leu Tyr Asn Ile Trp Ile Gln Cys Gln Ile Ala Ser His 145 150 155 160
Thr Ile Thr Gly His Leu Gln His Pro Arg Ser Pro Met Ala Pro Ile 165
170 175 Ile Ile Ser Gln Arg Thr Ala Ser Gln Leu Ala Ala Pro Ile Arg
Ile 180 185 190 Pro Gln Val His Thr Met Asp Ser Ser Gly Lys Ile Thr
Leu Thr Pro 195 200 205 Val Val Ile Leu Thr Gly Tyr Met Asp Glu Glu
Leu Arg Lys Lys Ser 210 215 220 Cys Ser Lys Ile Gln Ile Leu Lys Cys
Gly Gly Thr Ala Arg Ser Gln 225 230 235 240 Ile Ala Glu Lys Lys Thr
Arg Lys Gln Leu Lys Asn Asp Ile Ile Phe 245 250 255 Thr Asn Ser Val
Glu Ser Leu Lys Ser Ala His Ile Lys Glu Pro Glu 260 265 270 Arg Glu
Gly Lys Gly Thr Asp Leu Glu Lys Asp Lys Ile Gly Met Glu 275 280 285
Val Lys Val Asp Ser Asp Ala Gly Ile Pro Lys Arg Gln Glu Thr Gln 290
295 300 Leu Lys Ile Ser Glu Asp Glu Tyr Thr Thr Arg Thr Gly Ser Pro
Asn 305 310 315 320 Lys Glu Lys Cys Val Arg Cys Thr Lys Arg Thr Gly
Val Gln Val Lys 325 330 335 Lys Ser Glu Ser Gly Val Pro Lys Gly Gln
Glu Ala Gln Val Thr Lys 340 345 350 Ser Gly Leu Val Val Leu Lys Gly
Gln Glu Ala Gln Val Glu Lys Ser 355 360 365 Glu Met Gly Val Pro Arg
Arg Gln Glu Ser Gln Val Lys Lys Ser Gln 370 375 380 Ser Gly Val Ser
Lys Gly Gln Glu Ala Gln Val Lys Lys Arg Glu Ser 385 390 395 400 Val
Val Leu Lys Gly Gln Glu Ala Gln Val Glu Lys Ser Glu Leu Lys 405 410
415 Val Pro Lys Gly Gln Glu Gly Gln Val Glu Lys Thr Glu Ala Asp Val
420 425 430 Pro Lys Glu Gln Glu Val Gln Glu Lys Lys Ser Glu Ala Gly
Val Leu 435 440 445 Lys Gly Pro Glu Ser Gln Val Lys Asn Thr Glu Val
Ser Val Pro Glu 450 455 460 Thr Leu Glu Ser Gln Val Lys Lys Ser Glu
Ser Gly Val Leu Lys Gly 465 470 475 480 Gln Glu Ala Gln Glu Lys Lys
Glu Ser Phe Glu Asp Lys Gly Asn Asn 485 490 495 Asp Lys Glu Lys Glu
Arg Asp Ala Glu Lys Asp Pro Asn Lys Lys Glu 500 505 510 Lys Gly Asp
Lys Asn Thr Lys Gly Asp Lys Gly Lys Asp Lys Val Lys 515 520 525 Gly
Lys Arg Glu Ser Glu Ile Asn Gly Glu Lys Ser Lys Gly Ser Lys 530 535
540 Arg Arg Arg Gln Ile Gln Glu Gly Ser Thr Thr Lys Lys Trp Lys Ser
545 550 555 560 Lys Asp Lys Phe Phe Lys Gly Pro 565 311686DNAHomo
sapiens 31atgacagtct tggaaataac tttggctgtc atcctgactc tactgggact
tgccatcctg 60gctattttgt taacaagatg ggcacgatgt aagcaaagtg aaatgtatat
ctccagatac 120agttcagaac aaagtgctag acttctggac tatgaggatg
gtagaggatc ccgacatgca 180tattcaacac aaagtgacac ttcatatgat
aaccgagaga gatccaaaag agattacaca 240ccatcaacca actctctagc
actgtctcga tcaagtattg ctttacctca aggatccatg 300agtagtataa
aatgtttaca aacaactgaa gaacctcctt ccagaactgc aggagccatg
360atgcaattca cagcccctat tcccggagct acaggaccta tcaagctctc
tcaaaaaacc 420attgtgcaaa ctccaggacc tattgtacaa tatcctggat
ccaatgctgg tccaccttca 480gcaccccgcg gtccacccat ggcacccata
ataatttcac agagaaccgc aagtcagctg 540gcagcaccta taataatttc
gcagagaact gcaagaatac ctcaagttca cactatggac 600agttctggaa
aaatcacact gactcctgtg gttatattaa caggttacat ggatgaagaa
660cttgcaaaaa aatcttgttc caaaatccag attctaaaat gtggaggcac
tgcaaggtct 720cagaatagcc gagaagaaaa caaggaagca ctaaagaatg
acatcatatt tacgaattct 780gtagaatcct tgaaatcagc acacataaag
gagccagaaa gagaaggaaa aggcactgat 840ttagagaaag acaaaatagg
aatggaggtc aaggtagaca gtgacgctgg aataccaaaa 900agacaggaaa
cccaactaaa aatcagtgag atgagtatac cacaaggaca gggagcccaa
960ataaagaaaa gtgtgtcaga tgtaccaaga ggacaggagt cccaagtaaa
gaagagtgag 1020tcaggtgtcc caaaaggaca agaagcccaa gtaacgaaga
gtgggttggt tgtactgaaa 1080ggacaggaag cccaggtaga gaagagtgag
atgggtgtgc caagaagaca ggaatcccaa 1140gtaaagaaga gtcagtctgg
tgtctcaaag ggacaggaag cccaggtaaa gaagagggag 1200tcagttgtac
tgaaaggaca ggaagcccag gtagagaaga gtgagttgaa ggtaccaaaa
1260ggacaagaag gccaagtaga gaagactgag gcagatgtgc caaaggaaca
agaggtccaa 1320gaaaagaaga gtgaggcagg tgtactgaaa ggaccagaat
cccaagtaaa gaacactgag 1380gtgagtgtac cagaaacact ggaatcccaa
gtaaagaaga gtgagtcagg tgtactaaaa 1440ggacaggaag cccaagaaaa
gaaggagagt tttgaggata aaggaaataa tgataaagaa 1500aaggagagag
atgcagagaa agatccaaat aaaaaagaaa aaggtgacaa aaacacaaaa
1560ggtgacaaag gaaaggacaa agttaaagga aagagagaat cagaaatcaa
tggtgaaaaa 1620tcaaaaggct cgaaaagggc gaaggcaaat acaggaagga
agtacaacaa aaaagtggaa 1680gagtaa 1686321710DNAHomo sapiens
32atgacagtct tggaaataac tttggctgtc atcctgactc tactgggact tgccatcctg
60gctattttgt taacaagatg ggcacgacgt aagcaaagtg aaatgcatat ctccagatac
120agttcagaac aaagtgctag acttctggac tatgaggatg gtagaggatc
ccgacatgca 180tattcaacac aaagtgacac ttcatgtgat aaccgagaga
gatccaaaag agattacaca 240ccatcaacca actctctagc actgtctcga
tcaagtattg ctttacctca aggatccatg 300agtagtataa aatgtttaca
aacaactgaa gaacttcctt ccagaactgc aggagccatg 360atgcaattca
cagcccctat tcccggagct acaggaccta tcaagctctc tcaaaaaacc
420attgtgcaaa ctccaggacc tattgtacaa tatcctggac ccaatgtcag
atcgcatcct 480cacacaatca ctggtccacc ttcagcaccc cgcggtccac
ccatggcacc cataataatt 540tcacagagaa ccgcaagtca gctggcagca
cctataataa tttcgcagag aactgcaaga 600atacctcaag ttcacactat
ggacagttct ggaaaaacca cactgactcc tgtggttata 660ttaacaggtt
acatggatga agaacttgca aaaaaatctt gttccaaaat ccagattcta
720aaatgtggag gcactgcaag gtctcagaat agccgagaag aaaacaagga
agcactaaag 780aatgacatca tatttacgaa ttctgtagaa tccttgaaat
cagcacacat aaaggagcca 840gaaagagaag gaaaaggcac tgatttagag
aaagacaaaa taggaatgga ggtcaaggta 900gacagtgacg ctggaatacc
aaaaagacag gaaacccaac taaaaatcag tgagatgagt 960ataccacaag
gacagggagc ccaaataaag aaaagtgtgt cagatgtacc aagaggacag
1020gagtcccaag taaagaagag tgagtcaggt gtcccaaaag gacaagaagc
ccaagtaacg 1080aagagtgggt tggttgtact gaaaggacag gaagcccagg
tagagaagag tgagatgggt 1140gtgccaagaa gacaggaatc ccaagtaaag
aagagtcagt ctggtgtctc aaagggacag 1200gaagcccagg taaagaagag
ggagtcagtt gtactgaaag gacaggaagc ccaggtagag 1260aagagtgagt
tgaaggtacc aaaaggacaa gaaggccaag tagagaagac tgaggcagat
1320gtgccaaagg aacaagaggt ccaagaaaag aagagtgagg caggtgtact
gaaaggacca 1380gaatcccaag taaagaacac tgaggtgagt gtaccagaaa
cactggaatc ccaagtaaag 1440aagagtgagt caggtgtact aaaaggacag
gaagcccaag aaaagaagga gagttttgag 1500gataaaggaa ataatgataa
agaaaaggag agagatgcag agaaagatcc aaataaaaaa 1560gaaaaaggtg
acaaaaacac aaaaggtgac aaaggaaagg acaaagttaa aggaaagaga
1620gaatcagaaa tcaatggtga aaaatcaaaa ggctcgaaaa gggcgaaggc
aaatacagga 1680aggaagtaca acaaaaaagt ggaagagtaa 1710331665DNAHomo
sapiens 33atgacagtct tggaaataac tttggctgtc atcctgactc tactgggact
tgccatcctg 60gctattttgt taacaagatg ggcacgatgt aagcaaagtg aaatgtatat
ctccagatac 120agttcagaac aaagtgctag acttctggac tatgaggatg
gtagaggatc ccgacatgca 180tattcaacac aaagtgagag atccaaaaga
gattacacac catcaaccaa ctctctagca 240ctgtctcgat caagtattgc
tttacctcaa ggatccatga gtagtataaa atgtttacaa 300acaactgaag
aacctccttc cagaactgca ggagccatga tgcaattcac agcccctatt
360cccggagcta caggacctat caagctctct caaaaaacca ttgtgcaaac
tccaggacct 420attgtacaat atcctggatc caatgctggt ccaccttcag
caccccgcgg tccacccatg 480gcacccataa taatttcaca gagaaccgca
agtcagctgg cagcacctat aataatttcg 540cagagaactg caagaatacc
tcaagttcac actatggaca gttctggaaa aatcacactg 600actcctgtgg
ttatattaac aggttacatg gatgaagaac ttgcaaaaaa atcttgttcc
660aaaatccaga ttctaaaatg tggaggcact gcaaggtctc agaatagccg
agaagaaaac 720aaggaagcac taaagaatga catcatattt acgaattctg
tagaatcctt gaaatcagca 780cacataaagg agccagaaag agaaggaaaa
ggcactgatt tagagaaaga caaaatagga 840atggaggtca aggtagacag
tgacgctgga ataccaaaaa gacaggaaac ccaactaaaa 900atcagtgaga
tgagtatacc acaaggacag ggagcccaaa taaagaaaag tgtgtcagat
960gtaccaagag gacaggagtc ccaagtaaag aagagtgagt caggtgtccc
aaaaggacaa 1020gaagcccaag taacgaagag tgggttggtt gtactgaaag
gacaggaagc ccaggtagag 1080aagagtgaga tgggtgtgcc aagaagacag
gaatcccaag taaagaagag tcagtctggt 1140gtctcaaagg gacaggaagc
ccaggtaaag aagagggagt cagttgtact gaaaggacag 1200gaagcccagg
tagagaagag tgagttgaag gtaccaaaag gacaagaagg ccaagtagag
1260aagactgagg cagatgtgcc aaaggaacaa gaggtccaag aaaagaagag
tgaggcaggt 1320gtactgaaag gaccagaatc ccaagtaaag aacactgagg
tgagtgtacc agaaacactg 1380gaatcccaag taaagaagag tgagtcaggt
gtactaaaag gacaggaagc ccaagaaaag 1440aaggagagtt ttgaggataa
aggaaataat gataaagaaa aggagagaga tgcagagaaa 1500gatccaaata
aaaaagaaaa aggtgacaaa aacacaaaag gtgacaaagg aaaggacaaa
1560gttaaaggaa agagagaatc agaaatcaat ggtgaaaaat caaaaggctc
gaaaagggcg 1620aaggcaaata caggaaggaa gtacaacaaa aaagtggaag agtaa
166534561PRTHomo sapiens 34Met Thr Val Leu Glu Ile Thr Leu Ala Val
Ile Leu Thr Leu Leu Gly 1 5 10 15 Leu Ala Ile Leu Ala Ile Leu Leu
Thr Arg Trp Ala Arg Cys Lys Gln 20 25 30 Ser Glu Met Tyr Ile Ser
Arg Tyr Ser Ser Glu Gln Ser Ala Arg Leu 35 40 45 Leu Asp Tyr Glu
Asp Gly Arg Gly Ser Arg His Ala Tyr Ser Thr Gln 50 55 60 Ser Asp
Thr Ser Tyr Asp Asn Arg Glu Arg Ser Lys Arg Asp Tyr Thr 65 70 75 80
Pro Ser Thr Asn Ser Leu Ala Leu Ser Arg Ser Ser Ile Ala Leu Pro 85
90 95 Gln Gly Ser Met Ser Ser Ile Lys Cys Leu Gln Thr Thr Glu Glu
Pro 100 105 110 Pro Ser Arg Thr Ala Gly Ala Met Met Gln Phe Thr Ala
Pro Ile Pro 115 120 125 Gly Ala Thr Gly Pro Ile Lys Leu Ser Gln Lys
Thr Ile Val Gln Thr 130 135 140 Pro Gly Pro Ile Val Gln Tyr Pro Gly
Ser Asn Ala Gly Pro Pro Ser 145 150 155 160 Ala Pro Arg Gly Pro Pro
Met Ala Pro Ile Ile Ile Ser Gln Arg Thr 165 170 175 Ala Ser Gln Leu
Ala Ala Pro Ile Ile Ile Ser Gln Arg Thr Ala Arg 180 185 190 Ile Pro
Gln Val His Thr Met Asp Ser Ser Gly Lys Ile Thr Leu Thr 195 200 205
Pro Val Val Ile Leu Thr Gly Tyr Met Asp Glu Glu Leu Ala Lys Lys 210
215 220 Ser Cys Ser Lys Ile Gln Ile Leu Lys Cys Gly Gly Thr Ala Arg
Ser 225 230 235 240 Gln Asn Ser Arg Glu Glu Asn Lys Glu Ala Leu Lys
Asn Asp Ile Ile 245 250 255 Phe Thr Asn Ser Val Glu Ser Leu Lys Ser
Ala His Ile Lys Glu Pro 260 265 270 Glu Arg Glu Gly Lys Gly Thr Asp
Leu Glu Lys Asp Lys Ile Gly Met 275 280 285 Glu Val Lys Val Asp Ser
Asp Ala Gly Ile Pro Lys Arg Gln Glu Thr 290 295 300 Gln Leu Lys Ile
Ser Glu Met Ser Ile Pro Gln Gly Gln Gly Ala Gln 305 310 315 320 Ile
Lys Lys Ser Val Ser Asp Val Pro Arg Gly Gln Glu Ser Gln Val 325 330
335 Lys Lys
Ser Glu Ser Gly Val Pro Lys Gly Gln Glu Ala Gln Val Thr 340 345 350
Lys Ser Gly Leu Val Val Leu Lys Gly Gln Glu Ala Gln Val Glu Lys 355
360 365 Ser Glu Met Gly Val Pro Arg Arg Gln Glu Ser Gln Val Lys Lys
Ser 370 375 380 Gln Ser Gly Val Ser Lys Gly Gln Glu Ala Gln Val Lys
Lys Arg Glu 385 390 395 400 Ser Val Val Leu Lys Gly Gln Glu Ala Gln
Val Glu Lys Ser Glu Leu 405 410 415 Lys Val Pro Lys Gly Gln Glu Gly
Gln Val Glu Lys Thr Glu Ala Asp 420 425 430 Val Pro Lys Glu Gln Glu
Val Gln Glu Lys Lys Ser Glu Ala Gly Val 435 440 445 Leu Lys Gly Pro
Glu Ser Gln Val Lys Asn Thr Glu Val Ser Val Pro 450 455 460 Glu Thr
Leu Glu Ser Gln Val Lys Lys Ser Glu Ser Gly Val Leu Lys 465 470 475
480 Gly Gln Glu Ala Gln Glu Lys Lys Glu Ser Phe Glu Asp Lys Gly Asn
485 490 495 Asn Asp Lys Glu Lys Glu Arg Asp Ala Glu Lys Asp Pro Asn
Lys Lys 500 505 510 Glu Lys Gly Asp Lys Asn Thr Lys Gly Asp Lys Gly
Lys Asp Lys Val 515 520 525 Lys Gly Lys Arg Glu Ser Glu Ile Asn Gly
Glu Lys Ser Lys Gly Ser 530 535 540 Lys Arg Ala Lys Ala Asn Thr Gly
Arg Lys Tyr Asn Lys Lys Val Glu 545 550 555 560 Glu 35569PRTHomo
sapiens 35Met Thr Val Leu Glu Ile Thr Leu Ala Val Ile Leu Thr Leu
Leu Gly 1 5 10 15 Leu Ala Ile Leu Ala Ile Leu Leu Thr Arg Trp Ala
Arg Arg Lys Gln 20 25 30 Ser Glu Met His Ile Ser Arg Tyr Ser Ser
Glu Gln Ser Ala Arg Leu 35 40 45 Leu Asp Tyr Glu Asp Gly Arg Gly
Ser Arg His Ala Tyr Ser Thr Gln 50 55 60 Ser Asp Thr Ser Cys Asp
Asn Arg Glu Arg Ser Lys Arg Asp Tyr Thr 65 70 75 80 Pro Ser Thr Asn
Ser Leu Ala Leu Ser Arg Ser Ser Ile Ala Leu Pro 85 90 95 Gln Gly
Ser Met Ser Ser Ile Lys Cys Leu Gln Thr Thr Glu Glu Leu 100 105 110
Pro Ser Arg Thr Ala Gly Ala Met Met Gln Phe Thr Ala Pro Ile Pro 115
120 125 Gly Ala Thr Gly Pro Ile Lys Leu Ser Gln Lys Thr Ile Val Gln
Thr 130 135 140 Pro Gly Pro Ile Val Gln Tyr Pro Gly Pro Asn Val Arg
Ser His Pro 145 150 155 160 His Thr Ile Thr Gly Pro Pro Ser Ala Pro
Arg Gly Pro Pro Met Ala 165 170 175 Pro Ile Ile Ile Ser Gln Arg Thr
Ala Ser Gln Leu Ala Ala Pro Ile 180 185 190 Ile Ile Ser Gln Arg Thr
Ala Arg Ile Pro Gln Val His Thr Met Asp 195 200 205 Ser Ser Gly Lys
Thr Thr Leu Thr Pro Val Val Ile Leu Thr Gly Tyr 210 215 220 Met Asp
Glu Glu Leu Ala Lys Lys Ser Cys Ser Lys Ile Gln Ile Leu 225 230 235
240 Lys Cys Gly Gly Thr Ala Arg Ser Gln Asn Ser Arg Glu Glu Asn Lys
245 250 255 Glu Ala Leu Lys Asn Asp Ile Ile Phe Thr Asn Ser Val Glu
Ser Leu 260 265 270 Lys Ser Ala His Ile Lys Glu Pro Glu Arg Glu Gly
Lys Gly Thr Asp 275 280 285 Leu Glu Lys Asp Lys Ile Gly Met Glu Val
Lys Val Asp Ser Asp Ala 290 295 300 Gly Ile Pro Lys Arg Gln Glu Thr
Gln Leu Lys Ile Ser Glu Met Ser 305 310 315 320 Ile Pro Gln Gly Gln
Gly Ala Gln Ile Lys Lys Ser Val Ser Asp Val 325 330 335 Pro Arg Gly
Gln Glu Ser Gln Val Lys Lys Ser Glu Ser Gly Val Pro 340 345 350 Lys
Gly Gln Glu Ala Gln Val Thr Lys Ser Gly Leu Val Val Leu Lys 355 360
365 Gly Gln Glu Ala Gln Val Glu Lys Ser Glu Met Gly Val Pro Arg Arg
370 375 380 Gln Glu Ser Gln Val Lys Lys Ser Gln Ser Gly Val Ser Lys
Gly Gln 385 390 395 400 Glu Ala Gln Val Lys Lys Arg Glu Ser Val Val
Leu Lys Gly Gln Glu 405 410 415 Ala Gln Val Glu Lys Ser Glu Leu Lys
Val Pro Lys Gly Gln Glu Gly 420 425 430 Gln Val Glu Lys Thr Glu Ala
Asp Val Pro Lys Glu Gln Glu Val Gln 435 440 445 Glu Lys Lys Ser Glu
Ala Gly Val Leu Lys Gly Pro Glu Ser Gln Val 450 455 460 Lys Asn Thr
Glu Val Ser Val Pro Glu Thr Leu Glu Ser Gln Val Lys 465 470 475 480
Lys Ser Glu Ser Gly Val Leu Lys Gly Gln Glu Ala Gln Glu Lys Lys 485
490 495 Glu Ser Phe Glu Asp Lys Gly Asn Asn Asp Lys Glu Lys Glu Arg
Asp 500 505 510 Ala Glu Lys Asp Pro Asn Lys Lys Glu Lys Gly Asp Lys
Asn Thr Lys 515 520 525 Gly Asp Lys Gly Lys Asp Lys Val Lys Gly Lys
Arg Glu Ser Glu Ile 530 535 540 Asn Gly Glu Lys Ser Lys Gly Ser Lys
Arg Ala Lys Ala Asn Thr Gly 545 550 555 560 Arg Lys Tyr Asn Lys Lys
Val Glu Glu 565 36554PRTHomo sapiens 36Met Thr Val Leu Glu Ile Thr
Leu Ala Val Ile Leu Thr Leu Leu Gly 1 5 10 15 Leu Ala Ile Leu Ala
Ile Leu Leu Thr Arg Trp Ala Arg Cys Lys Gln 20 25 30 Ser Glu Met
Tyr Ile Ser Arg Tyr Ser Ser Glu Gln Ser Ala Arg Leu 35 40 45 Leu
Asp Tyr Glu Asp Gly Arg Gly Ser Arg His Ala Tyr Ser Thr Gln 50 55
60 Ser Glu Arg Ser Lys Arg Asp Tyr Thr Pro Ser Thr Asn Ser Leu Ala
65 70 75 80 Leu Ser Arg Ser Ser Ile Ala Leu Pro Gln Gly Ser Met Ser
Ser Ile 85 90 95 Lys Cys Leu Gln Thr Thr Glu Glu Pro Pro Ser Arg
Thr Ala Gly Ala 100 105 110 Met Met Gln Phe Thr Ala Pro Ile Pro Gly
Ala Thr Gly Pro Ile Lys 115 120 125 Leu Ser Gln Lys Thr Ile Val Gln
Thr Pro Gly Pro Ile Val Gln Tyr 130 135 140 Pro Gly Ser Asn Ala Gly
Pro Pro Ser Ala Pro Arg Gly Pro Pro Met 145 150 155 160 Ala Pro Ile
Ile Ile Ser Gln Arg Thr Ala Ser Gln Leu Ala Ala Pro 165 170 175 Ile
Ile Ile Ser Gln Arg Thr Ala Arg Ile Pro Gln Val His Thr Met 180 185
190 Asp Ser Ser Gly Lys Ile Thr Leu Thr Pro Val Val Ile Leu Thr Gly
195 200 205 Tyr Met Asp Glu Glu Leu Ala Lys Lys Ser Cys Ser Lys Ile
Gln Ile 210 215 220 Leu Lys Cys Gly Gly Thr Ala Arg Ser Gln Asn Ser
Arg Glu Glu Asn 225 230 235 240 Lys Glu Ala Leu Lys Asn Asp Ile Ile
Phe Thr Asn Ser Val Glu Ser 245 250 255 Leu Lys Ser Ala His Ile Lys
Glu Pro Glu Arg Glu Gly Lys Gly Thr 260 265 270 Asp Leu Glu Lys Asp
Lys Ile Gly Met Glu Val Lys Val Asp Ser Asp 275 280 285 Ala Gly Ile
Pro Lys Arg Gln Glu Thr Gln Leu Lys Ile Ser Glu Met 290 295 300 Ser
Ile Pro Gln Gly Gln Gly Ala Gln Ile Lys Lys Ser Val Ser Asp 305 310
315 320 Val Pro Arg Gly Gln Glu Ser Gln Val Lys Lys Ser Glu Ser Gly
Val 325 330 335 Pro Lys Gly Gln Glu Ala Gln Val Thr Lys Ser Gly Leu
Val Val Leu 340 345 350 Lys Gly Gln Glu Ala Gln Val Glu Lys Ser Glu
Met Gly Val Pro Arg 355 360 365 Arg Gln Glu Ser Gln Val Lys Lys Ser
Gln Ser Gly Val Ser Lys Gly 370 375 380 Gln Glu Ala Gln Val Lys Lys
Arg Glu Ser Val Val Leu Lys Gly Gln 385 390 395 400 Glu Ala Gln Val
Glu Lys Ser Glu Leu Lys Val Pro Lys Gly Gln Glu 405 410 415 Gly Gln
Val Glu Lys Thr Glu Ala Asp Val Pro Lys Glu Gln Glu Val 420 425 430
Gln Glu Lys Lys Ser Glu Ala Gly Val Leu Lys Gly Pro Glu Ser Gln 435
440 445 Val Lys Asn Thr Glu Val Ser Val Pro Glu Thr Leu Glu Ser Gln
Val 450 455 460 Lys Lys Ser Glu Ser Gly Val Leu Lys Gly Gln Glu Ala
Gln Glu Lys 465 470 475 480 Lys Glu Ser Phe Glu Asp Lys Gly Asn Asn
Asp Lys Glu Lys Glu Arg 485 490 495 Asp Ala Glu Lys Asp Pro Asn Lys
Lys Glu Lys Gly Asp Lys Asn Thr 500 505 510 Lys Gly Asp Lys Gly Lys
Asp Lys Val Lys Gly Lys Arg Glu Ser Glu 515 520 525 Ile Asn Gly Glu
Lys Ser Lys Gly Ser Lys Arg Ala Lys Ala Asn Thr 530 535 540 Gly Arg
Lys Tyr Asn Lys Lys Val Glu Glu 545 550 371182DNAHomo sapiens
37acacaggttg gagcagagaa agaggaaaca tagaggtgcc aaaggaacaa agacataatg
60atgtcatcca agccaacaag ccatgctgaa gtaaatgaaa ccatacccaa cccttaccca
120ccaggcagct ttatggctcc tggatttcaa cagcctctgg gttcaatcaa
cttagaaaac 180caagctcagg gtgctcagcg tgctcagccc tacggcatca
catctccggg aatctttgct 240agcagtcaac cgggtcaagg aaatatacaa
atgataaatc caagtgtggg aacagcagta 300atgaacttta aagaagaagc
aaaggcacta ggggtgatcc agatcatggt tggattgatg 360cacattggtt
ttggaattgt tttgtgttta atatccttct cttttagaga agtattaggt
420tttgcctcta ctgctgttat tggtggatac ccattctggg gtggcctttc
ttttattatc 480tctggctctc tctctgtgtc agcatccaag gagctttccc
gttgtctggt gaaaggcagc 540ctgggaatga acattgttag ttctatcttg
gccttcattg gagtgattct gctgctggtg 600gatatgtgca tcaatggggt
agctggccaa gactactggg ccgtgctttc tggaaaaggc 660atttcagcca
cgctgatgat cttctccctc ttggagttct tcgtagcttg tgccacagcc
720cattttgcca accaagcaaa caccacaacc aatatgtctg tcctggttat
tccaaatatg 780tatgaaagca accctgtgac accagcgtct tcttcagctc
ctcccagatg caacaactac 840tcagctaatg cccctaaata gtaaaagaaa
aaggggtatc agtctaatct catggagaaa 900aactacttgc aaaaacttct
taagaagatg tcttttattg tctacaatga tttctagtct 960ttaaaaactg
tgtttgagat ttgtttttag gttggtcgct aatgatggct gtatctccct
1020tcactgtctc ttcctacatt accactacta catgctggca aaggtgaagg
atcagaggac 1080tgaaaaatga ttctgcaact ctcttaaagt tagaaatgtt
tctgttcata ttactttttc 1140cttaataaaa tgtcattaga aacaaaaaaa
aaaaaaaaaa aa 118238267PRTHomo sapiens 38Met Met Ser Ser Lys Pro
Thr Ser His Ala Glu Val Asn Glu Thr Ile 1 5 10 15 Pro Asn Pro Tyr
Pro Pro Gly Ser Phe Met Ala Pro Gly Phe Gln Gln 20 25 30 Pro Leu
Gly Ser Ile Asn Leu Glu Asn Gln Ala Gln Gly Ala Gln Arg 35 40 45
Ala Gln Pro Tyr Gly Ile Thr Ser Pro Gly Ile Phe Ala Ser Ser Gln 50
55 60 Pro Gly Gln Gly Asn Ile Gln Met Ile Asn Pro Ser Val Gly Thr
Ala 65 70 75 80 Val Met Asn Phe Lys Glu Glu Ala Lys Ala Leu Gly Val
Ile Gln Ile 85 90 95 Met Val Gly Leu Met His Ile Gly Phe Gly Ile
Val Leu Cys Leu Ile 100 105 110 Ser Phe Ser Phe Arg Glu Val Leu Gly
Phe Ala Ser Thr Ala Val Ile 115 120 125 Gly Gly Tyr Pro Phe Trp Gly
Gly Leu Ser Phe Ile Ile Ser Gly Ser 130 135 140 Leu Ser Val Ser Ala
Ser Lys Glu Leu Ser Arg Cys Leu Val Lys Gly 145 150 155 160 Ser Leu
Gly Met Asn Ile Val Ser Ser Ile Leu Ala Phe Ile Gly Val 165 170 175
Ile Leu Leu Leu Val Asp Met Cys Ile Asn Gly Val Ala Gly Gln Asp 180
185 190 Tyr Trp Ala Val Leu Ser Gly Lys Gly Ile Ser Ala Thr Leu Met
Ile 195 200 205 Phe Ser Leu Leu Glu Phe Phe Val Ala Cys Ala Thr Ala
His Phe Ala 210 215 220 Asn Gln Ala Asn Thr Thr Thr Asn Met Ser Val
Leu Val Ile Pro Asn 225 230 235 240 Met Tyr Glu Ser Asn Pro Val Thr
Pro Ala Ser Ser Ser Ala Pro Pro 245 250 255 Arg Cys Asn Asn Tyr Ser
Ala Asn Ala Pro Lys 260 265 391948DNAHomo sapiens 39gcacgaggtt
ttgaggacca gcaacacagc aatacttcca gatctccata taacctctgt 60tcatttggga
ggggctttgt attttcaaca ggagagttca aagttcattt ttttttcagc
120aactacagtt ctaagtgaaa tctattttta ttgatacatg gtattttaca
tgtttatggg 180atacatatga gtcataatct attttaaata ataccttagt
gttgtaaaat caacagtgct 240ttttaaaaga aatatacctt gttaattatc
ccacatgtgt ctccagaagt acagcttgaa 300caaatccacc ttctgtggac
caagcaccac cctgggcatt tctagcatga gcaaaatcca 360aggtcctggc
tggactccag agatgctatt tacctcagaa gcatgacaat aggaggcaga
420aggagcaggc aaatccaagt cctttcttgt agtttccttg tttggggagg
aaaagttgag 480ttttactatt atggaaaaga aacaggaaat agagacagac
aaagagatat gacaatacag 540tcctgccacc cagatactca tttccaccta
ccattccatg catttgtttt gaatatataa 600gtatgtacat aaaggtaggt
actctcaagt ccatcagggc ttggctgtcc actgtttttg 660aagttccaga
atgtttttgc taagttgagg aaataccaaa tcaggactat gaaaattatg
720gtatatattg atgtgtcaca gaacacagat gtgacataat aaagatgtgt
aagattatat 780atataacttg tgtgtacacc tacctcatct ggggataaca
cctcaagttt aattttgagg 840cttgggtcaa tcgtgcttcc cttccctttc
ataggtcctc tatgagatat tgtcatagat 900tccatgttat gcaatagcca
tagaatatga catctctcta tgataattct atattacttt 960aattgctgca
cagaagttca ttgtatgtaa gtgccacagt atattataga tcttcttgtg
1020ggacatctat ttctagttta tgtgatagta tagcactttc atgaatgttc
ttgtacttga 1080tctttacaca ttttcttttt tccttaggat gaattctgag
agatgtaatt gatggggcaa 1140aatgtactca ctgtttgagg tttgaaattt
ttccatcaaa agctggtact cttggttttt 1200taagacaaag agcaaatcct
cccctgccag gattgacttt tggctctttt ttttcaaacc 1260tcactgcttt
ttggtttagt tgtcataaaa tgccaagcac catgaacagg gctccatgaa
1320ggggctcaga ggtaggaggg ctgtgattag gagaaggctt ggactgatgg
gcaatttgag 1380tgctcagaat tagagtgagg gggtgggggt gctgcaggga
cagatgctgg ggaaagacac 1440cctgaagggc aaagggagca acaatggctg
cagtacatgt ggcctttcag ctagcgcaga 1500ggatggaaac cagagtgggc
tgatgattgg atgccaggcc tgagccagca actgtgatcc 1560tgagctgtgc
acacttctgg ttgggattat ttctggtttc tacttcctgt ttgaagatgt
1620ggcatggaga gtgctctgct ttgacctgaa gtattttatc tatcctcagt
ctcaggacac 1680tgttgatgga attaaggcca agcacatctg caaaaaagac
attgctggag gaggtgcaaa 1740gagctggaaa ccaagtctcc agtcctggga
aaagcagtgg tatggaaaag caatggaaag 1800agcattttga aaatgccatt
ccactgtttt ctggccttta tgatttctgc tgagaaatcc 1860actgttagtc
tgatggggtc tccttcatag caccaatgac ctgaagagcc ttgttgaagg
1920aagactccat ctgatgactc agagcaag 1948401406DNAHomo sapiens
40cggtgagagg ggcgcgcagc agcagctcct caacgccgca acgcgccggc ccaactgcag
60gaaggtctgt gctctggagc cagggtaaat ggttataaaa ttatacacca tggccctcct
120aaagacactc taggaaaacc atgtcatcct gatcttaaaa cacctgcaag
aaagagcaca 180gtacttcacc attaataaag tagatatttc atcctgctca
gaaaaccaac atttccagca 240atggctttac taccggtgtt gtttctggtt
actgtgctgc ttccatcttt acctgcagaa 300ggaaaggatc ccgcttttac
tgctttgtta accacccagt tgcaagtgca aagggagatt 360gtaaataaac
acaatgaact aaggaaagca gtctctccac ctgccagtaa catgctaaag
420atggaatgga gcagagaggt aacaacgaat gcccaaaggt gggcaaacaa
gtgcacttta 480caacatagtg atccagagga ccgcaaaacc agtacaagat
gtggtgagaa tctctatatg 540tcaagtgacc ctacttcctg gtcttctgca
atccaaagct ggtatgacga gatcctagat 600tttgtctatg gtgtaggacc
aaagagtccc aatgcagttg ttggacatta tactcagctt 660gtttggtact
cgacttacca ggtaggctgt ggaattgcct actgtcccaa tcaagatagt
720ctaaaatact actatgtttg ccaatattgt cctgctggta ataatatgaa
tagaaagaat 780accccgtacc aacaaggaac accttgtgcc ggttgccctg
atgactgtga caaaggacta 840tgcaccaata gttgccagta tcaagatctc
ctaagtaact gtgattcctt gaagaataca 900gctggctgtg aacatgagtt
actcaaggaa aagtgcaagg ctacttgcct atgtgagaac 960aaaatttact
gatttaccta gtgagcattg tgcaagactg catggataag ggctgcatca
1020tttaattgcg acataccagt ggaaattgta tgtatgttag tgacaaattt
gatttcaaag 1080agcaatgcat cttctccccc agatcatcac agaaatcact
ttcaggcaat gatttacaaa 1140agtagcatag tagatgatga caactgtgaa
ctctgacata aatttagtgc tttataacga 1200actgaatcag gttgaggatt
ttgaaaactg tataaccata ggatttaggt cactaggact 1260ttggatcaaa
atggtgcatt acgtatttcc tgaaacatgc taaagaagaa gactgtaaca
1320tcattgccat tcctactacc tgagttttta cttgcataaa caataaattc
aaagctttac 1380atctgcaaaa aaaaaaaaaa aaaaaa 140641243PRTHomo
sapiens 41Met Ala Leu Leu Pro Val Leu Phe Leu Val Thr Val Leu Leu
Pro Ser 1 5 10 15 Leu Pro Ala Glu Gly Lys Asp Pro Ala Phe Thr Ala
Leu Leu Thr Thr 20 25 30 Gln Leu Gln Val Gln Arg Glu Ile Val Asn
Lys His Asn Glu Leu Arg 35 40 45 Lys Ala Val Ser Pro Pro Ala Ser
Asn Met Leu Lys Met Glu Trp Ser 50 55 60 Arg Glu Val Thr Thr Asn
Ala Gln Arg Trp Ala Asn Lys Cys Thr Leu 65 70 75 80 Gln His Ser Asp
Pro Glu Asp Arg Lys Thr Ser Thr Arg Cys Gly Glu 85 90 95 Asn Leu
Tyr Met Ser Ser Asp Pro Thr Ser Trp Ser Ser Ala Ile Gln 100 105 110
Ser Trp Tyr Asp Glu Ile Leu Asp Phe Val Tyr Gly Val Gly Pro Lys 115
120 125 Ser Pro Asn Ala Val Val Gly His Tyr Thr Gln Leu Val Trp Tyr
Ser 130 135 140 Thr Tyr Gln Val Gly Cys Gly Ile Ala Tyr Cys Pro Asn
Gln Asp Ser 145 150 155 160 Leu Lys Tyr Tyr Tyr Val Cys Gln Tyr Cys
Pro Ala Gly Asn Asn Met 165 170 175 Asn Arg Lys Asn Thr Pro Tyr Gln
Gln Gly Thr Pro Cys Ala Gly Cys 180 185 190 Pro Asp Asp Cys Asp Lys
Gly Leu Cys Thr Asn Ser Cys Gln Tyr Gln 195 200 205 Asp Leu Leu Ser
Asn Cys Asp Ser Leu Lys Asn Thr Ala Gly Cys Glu 210 215 220 His Glu
Leu Leu Lys Glu Lys Cys Lys Ala Thr Cys Leu Cys Glu Asn 225 230 235
240 Lys Ile Tyr 4221DNAArtificial SequenceDescription of the
artificial sequence Oligonucleotide 42tctagcactg tctcgatcaa g
214321DNAArtificial SequenceDescription of the artificial sequence
Oligonucleotide 43tgtcctcttg gtacatctga c 214421DNAArtificial
SequenceDescription of the artificial sequence Oligonucleotide
44ctgtgtcagc atccaaggag c 214521DNAArtificial SequenceDescription
of the artificial sequence Oligonucleotide 45ttcacctttg ccagcatgta
g 214621DNAArtificial SequenceDescription of the artificial
sequence Oligonucleotide 46cttgctctga gtcatcagat g
214721DNAArtificial SequenceDescription of the artificial sequence
Oligonucleotide 47cacagaatat gagccataca g 214822DNAArtificial
SequenceDescription of the artificial sequence Oligonucleotide
48ggtgtcactt ctgtgccttc ct 224921DNAArtificial SequenceDescription
of the artificial sequence Oligonucleotide 49cggcaccagt tccaacaata
g 215018DNAArtificial SequenceDescription of the artificial
sequence Oligonucleotide 50caaaggttct ccaaatgt 185121DNAArtificial
SequenceDescription of the artificial sequence Oligonucleotide
51tagcgcctca actgtcgttg g 215223DNAArtificial SequenceDescription
of the artificial sequence Oligonucleotide 52cgtgagcgct tcgagatgtt
ccg 235323DNAArtificial SequenceDescription of the artificial
sequence Oligonucleotide 53cctaaccagc tgcccaactg tag
23541550DNAHomo sapiens 54atgaatgaaa gtcctgatcc gactgacctg
gcgggagtca tcattgagct cggccccaat 60gacagtccac agacaagtga atttaaagga
gcaaccgagg aggcacctgc gaaagaaagc 120ccacacacaa gtgaatttaa
aggagcagcc cgggtgtcac ctatcagtga aagtgtgtta 180gcacgacttt
ccaagtttga agttgaagat gctgaaaatg ttgcttcata tgacagcaag
240attaagaaaa ttgtgcattc aattgtatca tcctttgcat ttggactatt
tggagttttc 300ctggtcttac tggatgtcac tctcatcctt gccgacctaa
ttttcactga cagcaaactt 360tatattcctt tggagtatcg ttctatttct
ctagctattg ccttattttt tctcatggat 420gttcttcttc gagtatttgt
agaaaggaga cagcagtatt tttctgactt atttaacatt 480ttagatactg
ccattattgt gattcttctg ctggttgatg tcgtttacat tttttttgac
540attaagttgc ttaggaatat tcccagatgg acacatttac ttcgacttct
acgacttatt 600attctgttaa gaatttttca tctgtttcat caaaaaagac
aacttgaaaa gctgataaga 660aggcgggttt cagaaaacaa aaggcgatac
acaagggatg gatttgacct agacctcact 720tacgttacag aacgtattat
tgctatgtca tttccatctt ctggaaggca gtctttctat 780agaaatccaa
tcaaggaagt tgtgcggttt ctagataaga aacaccgaaa ccactatcga
840gtctacaatc tatgcagtga aagagcttac gatcctaagc acttccataa
tagggtcgtt 900agaatcatga ttgatgatca taatgtcccc actctacatc
agatggtggt tttcaccaag 960gaagtaaatg agtggatggc tcaagatctt
gaaaacatcg tagcgattca ctgtaaagga 1020ggcacagata gaacaggaac
tatggtttgt gccttcctta ttgcctctga aatatgttca 1080actgcaaagg
aaagcctgta ttattttgga gaaaggcgaa cagataaaac ccacagcgaa
1140aaatttcagg gagtagaaac tccttctcag gttatgtacg tgatctaaaa
atccaaatag 1200aaatggagaa aaaggttgtc ttttccacta tttcattagg
aaaatgttcg gtacttgata 1260acattacaac agacaaaata ttaattgatg
tattcgacgg tccacctctg tatgatgatg 1320tgaaagtgca gtttttctat
tcgaatcttc ctacatacta tgacaattgc tcattttact 1380tctggttgca
cacatctttt attgaaaata acaggcttta tctaccaaaa aatgaattgg
1440ataatctaca taaacaaaaa gcacggagaa tttatccatc agattttgcc
gtggagatac 1500tttttggcga gaaaatgact tccagtgatg ttgtagctgg
atccgattaa 1550551407DNAHomo sapiens 55atgaatgaaa gtcctgatcc
gactgacctg gcgggagtca tcattgagct cggccccaat 60gacagtccac agacaagtga
atttaaagga gcaaccgagg aggcacctgc gaaagaaagc 120ccacacacaa
gtgaatttaa aggagcagcc cgggtgtcac ctatcagtga aagtgtgtta
180gcacgacttt ccaagtttga agttgaagat gctgaaaatg ttgcttcata
tgacagcaag 240attaagaaaa ttgtgcattc aattgtatca tcctttgcat
ttggactatt tggagttttc 300ctggtcttac tggatgtcac tctcatcctt
gccgacctaa ttttcactga cagcaaactt 360tatattcctt tggagtatcg
ttctatttct ctagctattg ccttattttt tctcatggat 420gttcttcttc
gagtatttgt agaaaggaga cagcagtatt tttctgactt atttaacatt
480ttagatactg ccattattgt gattcttctg ctggttgatg tcgtttacat
tttttttgac 540attaagttgc ttaggaatat tcccagatgg acacatttac
ttcgacttct acgacttatt 600attctgttaa gaatttttca tctgtttcat
caaaaaagac aacttgaaaa gctgataaga 660aggcgggttt cagaaaacaa
aaggcgatac acaagggatg gatttgacct agacctcact 720tacgttacag
aacgtattat tgctatgtca tttccatctt ctggaaggca gtctttctat
780agaaatccaa tcaaggaagt tgtgcggttt ctagataaga aacaccgaaa
ccactatcga 840gtctacaatc tatgcagtga aagagcttac gatcctaagc
acttccataa tagggtcgtt 900agaatcatga ttgatgatca taatgtcccc
actctacatc agatggtggt tttcaccaag 960gaagtaaatg agtggatggc
tcaagatctt gaaaacatcg tagcgattca ctgtaaagga 1020ggcacaggtt
atgtacgtga tctaaaaatc caaatagaaa tggagaaaaa ggttgtcttt
1080tccactattt cattaggaaa atgttcggta cttgataaca ttacaacaga
caaaatatta 1140attgatgtat tcgacggtcc acctctgtat gatgatgtga
aagtgcagtt tttctattcg 1200aatcttccta catactatga caattgctca
ttttacttct ggttgcacac atcttttatt 1260gaaaataaca ggctttatct
accaaaaaat gaattggata atctacataa acaaaaagca 1320cggagaattt
atccatcaga ttttgccgtg gagatacttt ttggcgagaa aatgacttcc
1380agtgatgttg tagctggatc cgattaa 1407561413DNAHomo sapiens
56atgaatgaaa gtcctgatcc gactgacctg gcgggagtca tcattgagct cggccccaat
60gacagtccac agacaagtga atttaaagga gcaaccgagg aggcacctgc gaaagaaagt
120gtgttagcac gactttccaa gtttgaagtt gaagatgctg aaaatgttgc
ttcatatgac 180agcaagatta agaaaattgt gcattcaatt gtatcatcct
ttgcatttgg actatttgga 240gttttcctgg tcttactgga tgtcactctc
atccttgccg acctaatttt cactgacagc 300aaactttata ttcctttgga
gtatcgttct atttctctag ctattgcctt attttttctc 360atggatgttc
ttcttcgagt atttgtagaa aggagacagc agtatttttc tgacttattt
420aacattttag atactgccat tattgtgatt cttctgctgg ttgatgtcgt
ttacattttt 480tttgacatta agttgcttag gaatattccc agatggacac
atttacttcg acttctacga 540cttattattc tgttaagaat ttttcatctg
tttcatcaaa aaagacaact tgaaaagctg 600ataagaaggc gggtttcaga
aaacaaaagg cgatacacaa gggatggatt tgacctagac 660ctcacttacg
ttacagaacg tattattgct atgtcatttc catcttctgg aaggcagtct
720ttctatagaa atccaatcaa ggaagttgtg cggtttctag ataagaaaca
ccgaaaccac 780tatcgagtct acaatctatg cagtgaaaga gcttacgatc
ctaagcactt ccataatagg 840gtcgttagaa tcatgattga tgatcataat
gtccccactc tacatcagat ggtggttttc 900accaaggaag taaatgagtg
gatggctcaa gatcttgaaa acatcgtagc gattcactgt 960aaaggaggca
cagatagaac aggaactatg gtttgtgcct tccttattgc ctctgaaata
1020tgttcaactg caaaggaaag cctgtattat tttggagaaa ggcgaacaga
taaaacccac 1080agcgaaaaat ttcagggagt agaaactcct tctgtacttg
ataacattac aacagacaaa 1140atattaattg atgtattcga cggtccacct
ctgtatgatg atgtgaaagt gcagtttttc 1200tattcgaatc ttcctacata
ctatgacaat tgctcatttt acttctggtt gcacacatct 1260tttattgaaa
ataacaggct ttatctacca aaaaatgaat tggataatct acataaacaa
1320aaagcacgga gaatttatcc atcagatttt gccgtggaga tactttttgg
cgagaaaatg 1380acttccagtg atgttgtagc tggatccgat taa
1413571353DNAHomo sapiens 57atgaatgaaa gtcctgatcc gactgacctg
gcgggagtca tcattgagct cggccccaat 60gacagtccac agacaagtga atttaaagga
gcaaccgagg aggcacctgc gaaagaaagt 120gtgttagcac gactttccaa
gtttgaagtt gaagatgctg aaaatgttgc ttcatatgac 180agcaagatta
agaaaattgt gcattcaatt gtatcatcct ttgcatttgg actatttgga
240gttttcctgg tcttactgga tgtcactctc atccttgccg acctaatttt
cactgacagc 300aaactttata ttcctttgga gtatcgttct atttctctag
ctattgcctt attttttctc 360atggatgttc ttcttcgagt atttgtagaa
aggagacagc agtatttttc tgacttattt 420aacattttag atactgccat
tattgtgatt cttctgctgg ttgatgtcgt ttacattttt 480tttgacatta
agttgcttag gaatattccc agatggacac atttacttcg acttctacga
540cttattattc tgttaagaat ttttcatctg tttcatcaaa aaagacaact
tgaaaagctg 600ataagaaggc gggtttcaga aaacaaaagg cgatacacaa
gggatggatt tgacctagac 660ctcacttacg ttacagaacg tattattgct
atgtcatttc catcttctgg aaggcagtct 720ttctatagaa atccaatcaa
ggaagttgtg cggtttctag ataagaaaca ccgaaaccac 780tatcgagtct
acaatctatg cagtgaaaga gcttacgatc ctaagcactt ccataatagg
840gtcgttagaa tcatgattga tgatcataat gtccccactc tacatcagat
ggtggttttc 900accaaggaag taaatgagtg gatggctcaa gatcttgaaa
acatcgtagc gattcactgt 960aaaggaggca caggttatgt acgtgatcta
aaaatccaaa tagaaatgga gaaaaaggtt 1020gtcttttcca ctatttcatt
aggaaaatgt tcggtacttg ataacattac aacagacaaa 1080atattaattg
atgtattcga cggtccacct ctgtatgatg atgtgaaagt gcagtttttc
1140tattcgaatc ttcctacata ctatgacaat tgctcatttt acttctggtt
gcacacatct 1200tttattgaaa ataacaggct ttatctacca aaaaatgaat
tggataatct acataaacaa 1260aaagcacgga gaatttatcc atcagatttt
gccgtggaga tactttttgg cgagaaaatg 1320acttccagtg atgttgtagc
tggatccgat taa 135358395PRTHomo sapiens 58Met Asn Glu Ser Pro Asp
Pro Thr Asp Leu Ala Gly Val Ile Ile Glu 1 5 10 15 Leu Gly Pro Asn
Asp Ser Pro Gln Thr Ser Glu Phe Lys Gly Ala Thr 20 25 30 Glu Glu
Ala Pro Ala Lys Glu Ser Pro His Thr Ser Glu Phe Lys Gly 35 40 45
Ala Ala Arg Val Ser Pro Ile Ser Glu Ser Val Leu Ala Arg Leu Ser 50
55 60 Lys Phe Glu Val Glu Asp Ala Glu Asn Val Ala Ser Tyr Asp Ser
Lys 65 70 75 80 Ile Lys Lys Ile Val His Ser Ile Val Ser Ser Phe Ala
Phe Gly Leu 85 90 95 Phe Gly Val Phe Leu Val Leu Leu Asp Val Thr
Leu Ile Leu Ala Asp 100 105 110 Leu Ile Phe Thr Asp Ser Lys Leu Tyr
Ile Pro Leu Glu Tyr Arg Ser 115 120 125 Ile Ser Leu Ala Ile Ala Leu
Phe Phe Leu Met Asp Val Leu Leu Arg 130 135 140 Val Phe Val Glu Arg
Arg Gln Gln Tyr Phe Ser Asp Leu Phe Asn Ile 145 150 155 160 Leu Asp
Thr Ala Ile Ile Val Ile Leu Leu Leu Val Asp Val Val Tyr 165 170 175
Ile Phe Phe Asp Ile Lys Leu Leu Arg Asn Ile Pro Arg Trp Thr His 180
185 190 Leu Leu Arg Leu Leu Arg Leu Ile Ile Leu Leu Arg Ile Phe His
Leu 195 200 205 Phe His Gln Lys Arg Gln Leu Glu Lys Leu Ile Arg Arg
Arg Val Ser 210 215 220 Glu Asn Lys Arg Arg Tyr Thr Arg Asp Gly Phe
Asp Leu Asp Leu Thr 225 230 235 240 Tyr Val Thr Glu Arg Ile Ile Ala
Met Ser Phe Pro Ser Ser Gly Arg 245 250 255 Gln Ser Phe Tyr Arg Asn
Pro Ile Lys Glu Val Val Arg Phe Leu Asp 260 265 270 Lys Lys His Arg
Asn His Tyr Arg Val Tyr Asn Leu Cys Ser Glu Arg 275 280 285 Ala Tyr
Asp Pro Lys His Phe His Asn Arg Val Val Arg Ile Met Ile 290 295 300
Asp Asp His Asn Val Pro Thr Leu His Gln Met Val Val Phe Thr Lys 305
310 315 320 Glu Val Asn Glu Trp Met Ala Gln Asp Leu Glu Asn Ile Val
Ala Ile 325 330 335 His Cys Lys Gly Gly Thr Asp Arg Thr Gly Thr Met
Val Cys Ala Phe 340 345 350 Leu Ile Ala Ser Glu Ile Cys Ser Thr Ala
Lys Glu Ser Leu Tyr Tyr 355 360 365 Phe Gly Glu Arg Arg Thr Asp Lys
Thr His Ser Glu Lys Phe Gln Gly 370 375 380 Val Glu Thr Pro Ser Gln
Val Met Tyr Val Ile 385 390 395 59468PRTHomo sapiens 59Met Asn Glu
Ser Pro Asp Pro Thr Asp Leu Ala Gly Val Ile Ile Glu 1 5 10 15 Leu
Gly Pro Asn Asp Ser Pro Gln Thr Ser Glu Phe Lys Gly Ala Thr 20 25
30 Glu Glu Ala Pro Ala Lys Glu Ser Pro His Thr Ser Glu Phe Lys Gly
35 40 45 Ala Ala Arg Val Ser Pro Ile Ser Glu Ser Val Leu Ala Arg
Leu Ser 50 55 60 Lys Phe Glu Val Glu Asp Ala Glu Asn Val Ala Ser
Tyr Asp Ser Lys 65 70 75 80 Ile Lys Lys Ile Val His Ser Ile Val Ser
Ser Phe Ala Phe Gly Leu 85 90 95 Phe Gly Val Phe Leu Val Leu Leu
Asp Val Thr Leu Ile Leu Ala Asp 100 105 110 Leu Ile Phe Thr Asp Ser
Lys Leu Tyr Ile Pro Leu Glu Tyr Arg Ser 115 120 125 Ile Ser Leu Ala
Ile Ala Leu Phe Phe Leu Met Asp Val Leu Leu Arg 130 135 140 Val Phe
Val Glu Arg Arg Gln Gln Tyr Phe Ser Asp Leu Phe Asn Ile 145 150 155
160 Leu Asp Thr Ala Ile Ile Val Ile Leu Leu Leu Val Asp Val Val Tyr
165 170 175 Ile Phe Phe Asp Ile Lys Leu Leu Arg Asn Ile Pro Arg Trp
Thr His 180 185 190 Leu Leu Arg Leu Leu Arg Leu Ile Ile Leu Leu Arg
Ile Phe His Leu 195 200 205 Phe His Gln Lys Arg Gln Leu Glu Lys Leu
Ile Arg Arg Arg Val Ser 210 215 220 Glu Asn Lys Arg Arg Tyr Thr Arg
Asp Gly Phe Asp Leu Asp Leu Thr 225 230 235 240 Tyr Val Thr Glu Arg
Ile Ile Ala Met Ser Phe Pro Ser Ser Gly Arg 245 250 255 Gln Ser Phe
Tyr Arg Asn Pro Ile Lys Glu Val Val Arg Phe Leu Asp 260 265 270 Lys
Lys His Arg Asn His Tyr Arg Val Tyr Asn Leu Cys Ser Glu Arg 275 280
285 Ala Tyr Asp Pro Lys His Phe His Asn Arg Val Val Arg Ile Met Ile
290 295 300 Asp Asp His Asn Val Pro Thr Leu His Gln Met Val Val Phe
Thr Lys 305 310 315 320 Glu Val Asn Glu Trp Met Ala Gln Asp Leu Glu
Asn Ile Val Ala Ile 325 330 335 His Cys Lys Gly Gly Thr Gly Tyr Val
Arg Asp Leu Lys Ile Gln Ile 340 345 350 Glu Met Glu Lys Lys Val Val
Phe Ser Thr Ile Ser Leu Gly Lys Cys 355 360 365 Ser Val Leu Asp Asn
Ile Thr Thr Asp Lys Ile Leu Ile Asp Val Phe 370 375 380 Asp Gly Pro
Pro Leu Tyr Asp Asp Val Lys Val Gln Phe Phe Tyr Ser 385 390 395 400
Asn Leu Pro Thr Tyr Tyr Asp Asn Cys Ser Phe Tyr Phe Trp Leu His 405
410 415 Thr Ser Phe Ile Glu Asn Asn Arg Leu Tyr Leu Pro Lys Asn Glu
Leu 420 425 430 Asp Asn Leu His Lys Gln Lys Ala Arg Arg Ile Tyr Pro
Ser Asp Phe 435 440 445 Ala Val Glu Ile Leu Phe Gly Glu Lys Met Thr
Ser Ser Asp Val Val 450 455 460 Ala Gly Ser Asp 465 60470PRTHomo
sapiens 60Met Asn Glu Ser Pro Asp Pro Thr Asp
Leu Ala Gly Val Ile Ile Glu 1 5 10 15 Leu Gly Pro Asn Asp Ser Pro
Gln Thr Ser Glu Phe Lys Gly Ala Thr 20 25 30 Glu Glu Ala Pro Ala
Lys Glu Ser Val Leu Ala Arg Leu Ser Lys Phe 35 40 45 Glu Val Glu
Asp Ala Glu Asn Val Ala Ser Tyr Asp Ser Lys Ile Lys 50 55 60 Lys
Ile Val His Ser Ile Val Ser Ser Phe Ala Phe Gly Leu Phe Gly 65 70
75 80 Val Phe Leu Val Leu Leu Asp Val Thr Leu Ile Leu Ala Asp Leu
Ile 85 90 95 Phe Thr Asp Ser Lys Leu Tyr Ile Pro Leu Glu Tyr Arg
Ser Ile Ser 100 105 110 Leu Ala Ile Ala Leu Phe Phe Leu Met Asp Val
Leu Leu Arg Val Phe 115 120 125 Val Glu Arg Arg Gln Gln Tyr Phe Ser
Asp Leu Phe Asn Ile Leu Asp 130 135 140 Thr Ala Ile Ile Val Ile Leu
Leu Leu Val Asp Val Val Tyr Ile Phe 145 150 155 160 Phe Asp Ile Lys
Leu Leu Arg Asn Ile Pro Arg Trp Thr His Leu Leu 165 170 175 Arg Leu
Leu Arg Leu Ile Ile Leu Leu Arg Ile Phe His Leu Phe His 180 185 190
Gln Lys Arg Gln Leu Glu Lys Leu Ile Arg Arg Arg Val Ser Glu Asn 195
200 205 Lys Arg Arg Tyr Thr Arg Asp Gly Phe Asp Leu Asp Leu Thr Tyr
Val 210 215 220 Thr Glu Arg Ile Ile Ala Met Ser Phe Pro Ser Ser Gly
Arg Gln Ser 225 230 235 240 Phe Tyr Arg Asn Pro Ile Lys Glu Val Val
Arg Phe Leu Asp Lys Lys 245 250 255 His Arg Asn His Tyr Arg Val Tyr
Asn Leu Cys Ser Glu Arg Ala Tyr 260 265 270 Asp Pro Lys His Phe His
Asn Arg Val Val Arg Ile Met Ile Asp Asp 275 280 285 His Asn Val Pro
Thr Leu His Gln Met Val Val Phe Thr Lys Glu Val 290 295 300 Asn Glu
Trp Met Ala Gln Asp Leu Glu Asn Ile Val Ala Ile His Cys 305 310 315
320 Lys Gly Gly Thr Asp Arg Thr Gly Thr Met Val Cys Ala Phe Leu Ile
325 330 335 Ala Ser Glu Ile Cys Ser Thr Ala Lys Glu Ser Leu Tyr Tyr
Phe Gly 340 345 350 Glu Arg Arg Thr Asp Lys Thr His Ser Glu Lys Phe
Gln Gly Val Glu 355 360 365 Thr Pro Ser Val Leu Asp Asn Ile Thr Thr
Asp Lys Ile Leu Ile Asp 370 375 380 Val Phe Asp Gly Pro Pro Leu Tyr
Asp Asp Val Lys Val Gln Phe Phe 385 390 395 400 Tyr Ser Asn Leu Pro
Thr Tyr Tyr Asp Asn Cys Ser Phe Tyr Phe Trp 405 410 415 Leu His Thr
Ser Phe Ile Glu Asn Asn Arg Leu Tyr Leu Pro Lys Asn 420 425 430 Glu
Leu Asp Asn Leu His Lys Gln Lys Ala Arg Arg Ile Tyr Pro Ser 435 440
445 Asp Phe Ala Val Glu Ile Leu Phe Gly Glu Lys Met Thr Ser Ser Asp
450 455 460 Val Val Ala Gly Ser Asp 465 470 61450PRTHomo sapiens
61Met Asn Glu Ser Pro Asp Pro Thr Asp Leu Ala Gly Val Ile Ile Glu 1
5 10 15 Leu Gly Pro Asn Asp Ser Pro Gln Thr Ser Glu Phe Lys Gly Ala
Thr 20 25 30 Glu Glu Ala Pro Ala Lys Glu Ser Val Leu Ala Arg Leu
Ser Lys Phe 35 40 45 Glu Val Glu Asp Ala Glu Asn Val Ala Ser Tyr
Asp Ser Lys Ile Lys 50 55 60 Lys Ile Val His Ser Ile Val Ser Ser
Phe Ala Phe Gly Leu Phe Gly 65 70 75 80 Val Phe Leu Val Leu Leu Asp
Val Thr Leu Ile Leu Ala Asp Leu Ile 85 90 95 Phe Thr Asp Ser Lys
Leu Tyr Ile Pro Leu Glu Tyr Arg Ser Ile Ser 100 105 110 Leu Ala Ile
Ala Leu Phe Phe Leu Met Asp Val Leu Leu Arg Val Phe 115 120 125 Val
Glu Arg Arg Gln Gln Tyr Phe Ser Asp Leu Phe Asn Ile Leu Asp 130 135
140 Thr Ala Ile Ile Val Ile Leu Leu Leu Val Asp Val Val Tyr Ile Phe
145 150 155 160 Phe Asp Ile Lys Leu Leu Arg Asn Ile Pro Arg Trp Thr
His Leu Leu 165 170 175 Arg Leu Leu Arg Leu Ile Ile Leu Leu Arg Ile
Phe His Leu Phe His 180 185 190 Gln Lys Arg Gln Leu Glu Lys Leu Ile
Arg Arg Arg Val Ser Glu Asn 195 200 205 Lys Arg Arg Tyr Thr Arg Asp
Gly Phe Asp Leu Asp Leu Thr Tyr Val 210 215 220 Thr Glu Arg Ile Ile
Ala Met Ser Phe Pro Ser Ser Gly Arg Gln Ser 225 230 235 240 Phe Tyr
Arg Asn Pro Ile Lys Glu Val Val Arg Phe Leu Asp Lys Lys 245 250 255
His Arg Asn His Tyr Arg Val Tyr Asn Leu Cys Ser Glu Arg Ala Tyr 260
265 270 Asp Pro Lys His Phe His Asn Arg Val Val Arg Ile Met Ile Asp
Asp 275 280 285 His Asn Val Pro Thr Leu His Gln Met Val Val Phe Thr
Lys Glu Val 290 295 300 Asn Glu Trp Met Ala Gln Asp Leu Glu Asn Ile
Val Ala Ile His Cys 305 310 315 320 Lys Gly Gly Thr Gly Tyr Val Arg
Asp Leu Lys Ile Gln Ile Glu Met 325 330 335 Glu Lys Lys Val Val Phe
Ser Thr Ile Ser Leu Gly Lys Cys Ser Val 340 345 350 Leu Asp Asn Ile
Thr Thr Asp Lys Ile Leu Ile Asp Val Phe Asp Gly 355 360 365 Pro Pro
Leu Tyr Asp Asp Val Lys Val Gln Phe Phe Tyr Ser Asn Leu 370 375 380
Pro Thr Tyr Tyr Asp Asn Cys Ser Phe Tyr Phe Trp Leu His Thr Ser 385
390 395 400 Phe Ile Glu Asn Asn Arg Leu Tyr Leu Pro Lys Asn Glu Leu
Asp Asn 405 410 415 Leu His Lys Gln Lys Ala Arg Arg Ile Tyr Pro Ser
Asp Phe Ala Val 420 425 430 Glu Ile Leu Phe Gly Glu Lys Met Thr Ser
Ser Asp Val Val Ala Gly 435 440 445 Ser Asp 450 621299DNAHomo
sapiens 62cgcccttaga catggctcag atgtgcagcc acagtgagct tctgaacatt
tcttctcaga 60ctaagctctt acacacagtt gcagttgaaa gaaagaattg cttgacatgg
ccacaggagc 120aggcagcttc ctgcagacat gacagtcaac gcaaactcat
gtcactgtgg gcagacacat 180gtttgcaaag agactcagag ccaaacaagc
acactcaatg tgctttgccc aaatttaccc 240attaggtaaa tcttccctcc
tcccaagaag aaagtggaga gagcatgagt cctcacatgg 300gaacttgaag
tcagggaaat gaaggctcac caattatttg tgcatgggtt taagttttcc
360ttgaaattaa gttcaggttt gtctttgtgt gtaccaatta atgacaagag
gttagataga 420agtatgctag atggcaaaga gaaatatgtt ttgtgtcttc
aattttgcta aaaataaccc 480agaacatgga taattcattt attaattgat
tttggtaagc caagtcctat ttggagaaaa 540ttaatagttt ttctaaaaaa
gaattttctc aatatcacct ggcttgataa catttttctc 600cttcgagttc
ctttttctgg agtttaacaa acttgttctt tacaaataga ttatattgac
660tacctctcac tgatgttatg atattagttt ctattgctta ctttgtattt
ctaattttag 720gattcacaat ttagctggag aactattttt taacctgttg
cacctaaaca tgattgagct 780agaagacagt tttaccatat gcatgcattt
tctctgagtt atattttaaa atctatacat 840ttctcctaaa tatggaggaa
atcactggca tcaaatgcca gtctcagacg gaagacctaa 900agcccatttc
tggcctggag ctacttggct ttgtgaccta tggtgaggca taagtgctct
960gagtttgtgt tgcctctttt gtaaaatgag ggtttgactt aatcagtgat
tttcatagct 1020taaaattttt ttgaagaaca gaactttttt taaaaacagt
tagatgcaac catattatat 1080aaaacagaac agatacaagt agagctaact
tgctaaagaa aggatggagg ctctgaagct 1140gtgacttcat tatcccttaa
tactgctatg tcctctgtag taccttagat ttctatggga 1200catcgtttaa
aaactattgt ttatgcgaga gccttgctaa tttcctaaaa attgtggata
1260cattttttct cccatgtata attttctcac cttctattt 129963405DNAHomo
sapiens 63gcacaaggcc tgctcttact ccaaaaagat ggacccaggt ccgaaggggc
actgccactg 60tggggggcat ggccatcctc caggtcactg cgggccaccc cctggccatg
gcccagggcc 120ctgcgggcca ccccccacca tggtccaggg ccctgcgggc
caccccctgg ccatggccca 180gggccctgcg ggccaccccc ccaccatggt
ccagggccct gcgggcctcc ccctggccat 240ggcccaggtc acccaccccc
tggtccacat cactgaggaa gtagaagaaa acaggacaca 300agatggcaag
cctgagagaa ttgcccagct gacctggaat gaggcctaaa ccacaatctt
360ctcttcctaa taaacagcct cctagaggcc acattctatt ctgta
40564106PRTHomo sapiens 64Met Asp Pro Gly Pro Lys Gly His Cys His
Cys Gly Gly His Gly His 1 5 10 15 Pro Pro Gly His Cys Gly Pro Pro
Pro Gly His Gly Pro Gly Pro Cys 20 25 30 Gly Pro Pro Pro Thr Met
Val Gln Gly Pro Ala Gly His Pro Leu Ala 35 40 45 Met Ala Gln Gly
Pro Ala Gly His Pro Pro Thr Met Val Gln Gly Pro 50 55 60 Ala Gly
Leu Pro Leu Ala Met Ala Gln Val Thr His Pro Leu Val His 65 70 75 80
Ile Thr Glu Glu Val Glu Glu Asn Arg Thr Gln Asp Gly Lys Pro Glu 85
90 95 Arg Ile Ala Gln Leu Thr Trp Asn Glu Ala 100 105 6571PRTHomo
sapiens 65Met Ala Ile Leu Gln Val Thr Ala Gly His Pro Leu Ala Met
Ala Gln 1 5 10 15 Gly Pro Ala Gly His Pro Pro Pro Trp Ser Arg Ala
Leu Arg Ala Thr 20 25 30 Pro Trp Pro Trp Pro Arg Ala Leu Arg Ala
Thr Pro Pro Pro Trp Ser 35 40 45 Arg Ala Leu Arg Ala Ser Pro Trp
Pro Trp Pro Arg Ser Pro Thr Pro 50 55 60 Trp Ser Thr Ser Leu Arg
Lys 65 70 6621DNAArtificial SequenceDescription of the artificial
sequence Oligonucleotide 66agacatggct cagatgtgca g
216721DNAArtificial SequenceDescription of the artificial sequence
Oligonucleotide 67ggaaattagc aaggctctcg c 216821DNAArtificial
SequenceDescription of the artificial sequence Oligonucleotide
68tcaggtattc cctgctctta c 216921DNAArtificial SequenceDescription
of the artificial sequence Oligonucleotide 69tgggcaattc tctcaggctt
g 2170908DNAHomo sapiens 70aaaattcggc acgaggccgg gctgtggtct
agcataaagg cggagcccag aagaaggggc 60ggggtatggg agaagcctcc ccacctgccc
ccgcaaggcg gcatctgctg gtcctgctgc 120tgctcctctc taccctggtg
atcccctccg ctgcagctcc tatccatgat gctgacgccc 180aagagagctc
cttgggtctc acaggcctcc agagcctact ccaaggcttc agccgacttt
240tcctgaaagg taacctgctt cggggcatag acagcttatt ctctgccccc
atggacttcc 300ggggcctccc tgggaactac cacaaagagg agaaccagga
gcaccagctg gggaacaaca 360ccctctccag ccacctccag atcgacaaga
tgaccgacaa caagacagga gaggtgctga 420tctccgagaa tgtggtggca
tccattcaac cagcggaggg gagcttcgag ggtgatttga 480aggtacccag
gatggaggag aaggaggccc tggtacccat ccagaaggcc acggacagct
540tccacacaga actccatccc cgggtggcct tctggatcat taagctgcca
cggcggaggt 600cccaccagga tgccctggag ggcggccact ggctcagcga
gaagcgacac cgcctgcagg 660ccatccggga tggactccgc aaggggaccc
acaaggacgt cctagaagag gggaccgaga 720gctcctccca ctccaggctg
tccccccgaa agacccactt actgtacatc ctcaggccct 780ctcggcagct
gtaggggtgg ggaccgggga gcacctgcct gtagccccca tcagaccctg
840ccccaagcac catatggaaa taaagttctt tcttacatct aaaaaaaaaa
aaaaaaaaaa 900aaaaaaaa 90871242PRTHomo sapiens 71Met Gly Glu Ala
Ser Pro Pro Ala Pro Ala Arg Arg His Leu Leu Val 1 5 10 15 Leu Leu
Leu Leu Leu Ser Thr Leu Val Ile Pro Ser Ala Ala Ala Pro 20 25 30
Ile His Asp Ala Asp Ala Gln Glu Ser Ser Leu Gly Leu Thr Gly Leu 35
40 45 Gln Ser Leu Leu Gln Gly Phe Ser Arg Leu Phe Leu Lys Gly Asn
Leu 50 55 60 Leu Arg Gly Ile Asp Ser Leu Phe Ser Ala Pro Met Asp
Phe Arg Gly 65 70 75 80 Leu Pro Gly Asn Tyr His Lys Glu Glu Asn Gln
Glu His Gln Leu Gly 85 90 95 Asn Asn Thr Leu Ser Ser His Leu Gln
Ile Asp Lys Met Thr Asp Asn 100 105 110 Lys Thr Gly Glu Val Leu Ile
Ser Glu Asn Val Val Ala Ser Ile Gln 115 120 125 Pro Ala Glu Gly Ser
Phe Glu Gly Asp Leu Lys Val Pro Arg Met Glu 130 135 140 Glu Lys Glu
Ala Leu Val Pro Ile Gln Lys Ala Thr Asp Ser Phe His 145 150 155 160
Thr Glu Leu His Pro Arg Val Ala Phe Trp Ile Ile Lys Leu Pro Arg 165
170 175 Arg Arg Ser His Gln Asp Ala Leu Glu Gly Gly His Trp Leu Ser
Glu 180 185 190 Lys Arg His Arg Leu Gln Ala Ile Arg Asp Gly Leu Arg
Lys Gly Thr 195 200 205 His Lys Asp Val Leu Glu Glu Gly Thr Glu Ser
Ser Ser His Ser Arg 210 215 220 Leu Ser Pro Arg Lys Thr His Leu Leu
Tyr Ile Leu Arg Pro Ser Arg 225 230 235 240 Gln Leu
7221DNAArtificial SequenceDescription of the artificial sequence
Oligonucleotide 72ctcctatcca tgatgctgac g 217321DNAArtificial
SequenceDescription of the artificial sequence Oligonucleotide
73cctgaggatg tacagtaagt g 21742987DNAHomo sapiens 74tttcccagcg
aggtggtcat tcagagccta cacatctgtt ctgtatttta acccatggat 60gagaatattc
attcaagcca agagagttaa aactaaacat ctttgctatt gcctctacag
120acccagaaag tatctttatg tcacatcttc ttttaaagga gcatttaaag
atgaagttaa 180aaaggcagaa gaagcagtaa agattgctga atccatattg
aaagaagcac aaatcaaagt 240aaaccagtgt gacagaacct ctttatcttc
tgccaaggat gtattacaga gagctttgga 300agatgtagaa gcaaagcaaa
agaatcttaa agagaaacaa agagaattaa aaacagcaag 360aacgctctcc
ctgttctatg gagtgaacgt agaaaaccga agccaagctg gaatgttcat
420ttacagtaat aaccgtttga tcaaaatgca tgaaaaagtg ggctcacagt
tgaaactgaa 480gtccttactt ggcgcaggcg tggttggaat tgttaatata
cccttggagg tcatggaacc 540atcccataat aaacaggaat ttctcaatgt
ccaagagtat aatcatctac taaaagtcat 600gggacagtac ttggtccagt
actgtaagga caccggcatc aataatagaa atttaacatt 660gttttgcaat
gaatttggat accagaatga catcgatgtg gagaaacctt taaattcttt
720tcaatatcaa agaagacaag ccatgggtat cccattcatc atacaatgtg
atctttgtct 780taaatggaga gtcttgcctt cctctactaa ttatcaggaa
aaagaatttt ttgacatttg 840gatttgtgct aataatccca accgcttgga
aaacagttgt catcaggtag aatgtctacc 900ttccatccca ctgggcacca
tgagcacaat atcaccatca aaaaatgaga aagagaagca 960acttagagag
tcggtcataa agtatcaaaa tagactggca gaacagcagc cacagcctca
1020atttatacca gtggacgaaa tcactgtcac ttccacctgc ctaacttcag
cacataagga 1080aaataccaaa acccagaaaa tcaggctttt gggcgatgac
ttgaagcatg aatctctttc 1140atcctttgag ctttcagcga gccgtagagg
acagaaaaga aacatagaag agacagactc 1200tgatgtagag tatatttcag
aaacaaaaat tatgaaaaag tctatggagg agaaaatgaa 1260ctctcaacag
cagagaattc cagtagctct gccagaaaat gtcaaactag ctgagagatc
1320ccagagaagt cagattgcta atattaccac tgtctggaga gctcaaccaa
ctgaagggtg 1380cctgaagaat gcccaggccg cttcttggga aatgaaaagg
aagcagagtc tgaactttgt 1440agaggaatgt aaggtattga ctgaagatga
gaacacgagt gattcagata taatcctggt 1500ttcagataaa agcaacactg
atgtttcatt gaaacaagaa aaaaaggaaa ttcctctttt 1560aaaccaagaa
aaacaggagc tgtgcaatga tgttctagca atgaaaagaa gctcttcatt
1620acctagctgg aaaagcttgc tcaatgtgcc gatggaagat gtgaatctaa
gttctggaca 1680catagccaga gtttctgtga gtggcagttg taaagttgct
tcttcgccag cgtcttctca 1740aagcacacct gtcaaggaaa cagtgagaaa
actgaagtct aagttaaggg agattcttct 1800gtattttttt cctgagcatc
agctaccatc agaattggaa gaacctgcat taagttgtga 1860gctggagcag
tgcccagagc agatgaacaa aaagctgaaa atgtgtttca accagataca
1920gaatacttac atggtccaat atgaaaaaaa aataaagagg aaattgcagt
ccattatcta 1980tgattcaaat acaagaggaa tacataatga aatctctctg
gggcaatgtg aaaataaaag 2040aaaaatctct gaggataagc tgaagaatct
tcgtataaaa ctggcactat tgttgcagaa 2100actccaactg ggtggtccag
aaggtgacct ggagcagact gacacttatt tagaagcttt 2160gcttaaagaa
gataatcttc tcttccagaa caatttaaat aaagtaacta tagatgcaag
2220acatagactc cctttagaaa aaaatgaaaa gacttcggaa aattaagtca
gagatggtat 2280taccttttaa aaaatgctaa taagaaaatt ggaagattct
tttaaaaatt tttctttttt 2340gttgttgtta ctgtaaagtc tattctgttt
aacaataaga aataagaaat aatttttttc 2400aaataagaaa attgtgtact
ctagaaatgg agaccgattt acaatttatg tattccctaa 2460tccaattatc
taaatcttcc ttttctttca gaaatattaa taatatctag agttctctaa
2520ttttcatgtg agctactgaa aaaaatgaaa atgtcactca agcttaactt
ttgttattcc 2580ttaaaagatt gttattgtaa ttttgttatt ccttaaaaac
atttaaaagc agattttttc 2640aaaatcgata tgtgaaggac tacagaatca
cctcctcttg aagatattga aaaagaaaga 2700cattatgccc tttctccact
atagccaaca ctcagtcaag cagaaaatac aaatcccccc 2760aaaactttga
gacatagctt atataatttt attatttagt catagtaaaa gaataaatct
2820cctaagcata atatgtatac atattacaca tatgtaaaaa ttgttgtttt
acatttacat 2880atacgtaaag aagtatgttt ttacactttt cttgataagt
gttttttttt tgtttagaaa 2940tgtctgaaac tttagacaaa aacagtaaaa
catttaatat tcatttg 298775735PRTHomo sapiens 75Met Arg Ile Phe Ile
Gln Ala Lys Arg Val Lys Thr Lys His Leu Cys 1 5 10 15 Tyr Cys Leu
Tyr Arg Pro Arg Lys Tyr Leu Tyr Val Thr Ser Ser Phe 20 25 30 Lys
Gly Ala Phe Lys Asp Glu Val Lys Lys Ala Glu Glu Ala Val Lys 35 40
45 Ile Ala Glu Ser Ile Leu Lys Glu Ala Gln Ile Lys Val Asn Gln Cys
50 55 60 Asp Arg Thr Ser Leu Ser Ser Ala Lys Asp Val Leu Gln Arg
Ala Leu 65 70 75 80 Glu Asp Val Glu Ala Lys Gln Lys Asn Leu Lys Glu
Lys Gln Arg Glu 85 90 95 Leu Lys Thr Ala Arg Thr Leu Ser Leu Phe
Tyr Gly Val Asn Val Glu 100 105 110 Asn Arg Ser Gln Ala Gly Met Phe
Ile Tyr Ser Asn Asn Arg Leu Ile 115 120 125 Lys Met His Glu Lys Val
Gly Ser Gln Leu Lys Leu Lys Ser Leu Leu 130 135 140 Gly Ala Gly Val
Val Gly Ile Val Asn Ile Pro Leu Glu Val Met Glu 145 150 155 160 Pro
Ser His Asn Lys Gln Glu Phe Leu Asn Val Gln Glu Tyr Asn His 165 170
175 Leu Leu Lys Val Met Gly Gln Tyr Leu Val Gln Tyr Cys Lys Asp Thr
180 185 190 Gly Ile Asn Asn Arg Asn Leu Thr Leu Phe Cys Asn Glu Phe
Gly Tyr 195 200 205 Gln Asn Asp Ile Asp Val Glu Lys Pro Leu Asn Ser
Phe Gln Tyr Gln 210 215 220 Arg Arg Gln Ala Met Gly Ile Pro Phe Ile
Ile Gln Cys Asp Leu Cys 225 230 235 240 Leu Lys Trp Arg Val Leu Pro
Ser Ser Thr Asn Tyr Gln Glu Lys Glu 245 250 255 Phe Phe Asp Ile Trp
Ile Cys Ala Asn Asn Pro Asn Arg Leu Glu Asn 260 265 270 Ser Cys His
Gln Val Glu Cys Leu Pro Ser Ile Pro Leu Gly Thr Met 275 280 285 Ser
Thr Ile Ser Pro Ser Lys Asn Glu Lys Glu Lys Gln Leu Arg Glu 290 295
300 Ser Val Ile Lys Tyr Gln Asn Arg Leu Ala Glu Gln Gln Pro Gln Pro
305 310 315 320 Gln Phe Ile Pro Val Asp Glu Ile Thr Val Thr Ser Thr
Cys Leu Thr 325 330 335 Ser Ala His Lys Glu Asn Thr Lys Thr Gln Lys
Ile Arg Leu Leu Gly 340 345 350 Asp Asp Leu Lys His Glu Ser Leu Ser
Ser Phe Glu Leu Ser Ala Ser 355 360 365 Arg Arg Gly Gln Lys Arg Asn
Ile Glu Glu Thr Asp Ser Asp Val Glu 370 375 380 Tyr Ile Ser Glu Thr
Lys Ile Met Lys Lys Ser Met Glu Glu Lys Met 385 390 395 400 Asn Ser
Gln Gln Gln Arg Ile Pro Val Ala Leu Pro Glu Asn Val Lys 405 410 415
Leu Ala Glu Arg Ser Gln Arg Ser Gln Ile Ala Asn Ile Thr Thr Val 420
425 430 Trp Arg Ala Gln Pro Thr Glu Gly Cys Leu Lys Asn Ala Gln Ala
Ala 435 440 445 Ser Trp Glu Met Lys Arg Lys Gln Ser Leu Asn Phe Val
Glu Glu Cys 450 455 460 Lys Val Leu Thr Glu Asp Glu Asn Thr Ser Asp
Ser Asp Ile Ile Leu 465 470 475 480 Val Ser Asp Lys Ser Asn Thr Asp
Val Ser Leu Lys Gln Glu Lys Lys 485 490 495 Glu Ile Pro Leu Leu Asn
Gln Glu Lys Gln Glu Leu Cys Asn Asp Val 500 505 510 Leu Ala Met Lys
Arg Ser Ser Ser Leu Pro Ser Trp Lys Ser Leu Leu 515 520 525 Asn Val
Pro Met Glu Asp Val Asn Leu Ser Ser Gly His Ile Ala Arg 530 535 540
Val Ser Val Ser Gly Ser Cys Lys Val Ala Ser Ser Pro Ala Ser Ser 545
550 555 560 Gln Ser Thr Pro Val Lys Glu Thr Val Arg Lys Leu Lys Ser
Lys Leu 565 570 575 Arg Glu Ile Leu Leu Tyr Phe Phe Pro Glu His Gln
Leu Pro Ser Glu 580 585 590 Leu Glu Glu Pro Ala Leu Ser Cys Glu Leu
Glu Gln Cys Pro Glu Gln 595 600 605 Met Asn Lys Lys Leu Lys Met Cys
Phe Asn Gln Ile Gln Asn Thr Tyr 610 615 620 Met Val Gln Tyr Glu Lys
Lys Ile Lys Arg Lys Leu Gln Ser Ile Ile 625 630 635 640 Tyr Asp Ser
Asn Thr Arg Gly Ile His Asn Glu Ile Ser Leu Gly Gln 645 650 655 Cys
Glu Asn Lys Arg Lys Ile Ser Glu Asp Lys Leu Lys Asn Leu Arg 660 665
670 Ile Lys Leu Ala Leu Leu Leu Gln Lys Leu Gln Leu Gly Gly Pro Glu
675 680 685 Gly Asp Leu Glu Gln Thr Asp Thr Tyr Leu Glu Ala Leu Leu
Lys Glu 690 695 700 Asp Asn Leu Leu Phe Gln Asn Asn Leu Asn Lys Val
Thr Ile Asp Ala 705 710 715 720 Arg His Arg Leu Pro Leu Glu Lys Asn
Glu Lys Thr Ser Glu Asn 725 730 735 7621DNAArtificial
SequenceDescription of the artificial sequence Oligonucleotide
76ctgagtatca gctaccatca g 217721DNAArtificial SequenceDescription
of the artificial sequence Oligonucleotide 77tctgtagtcc ttcacatatc
g 217821DNAArtificial SeqeunceDescription of the artificial
sequence Oligonucleotide 78ttttgtctat ggtgtaggac c
217921DNAArtificial SequenceDescription of the artificial sequence
Oligonucleotide 79ggaatggcaa tgatgttaca g 218020PRTHomo sapiens
80Met Ser Thr Val Lys Glu Gln Leu Ile Glu Lys Leu Ile Glu Asp Asp 1
5 10 15 Glu Asn Ser Gln 20 8114PRTHomo sapiens 81Phe Thr Asp Ser
Lys Leu Tyr Ile Pro Leu Glu Tyr Arg Ser 1 5 10 8213PRTHomo sapiens
82Phe Asp Ile Lys Leu Leu Arg Asn Ile Pro Arg Trp Thr 1 5 10
8315PRTHomo sapiens 83Gly Val Ala Gly Gln Asp Tyr Trp Ala Val Leu
Ser Gly Lys Gly 1 5 10 15 8410PRTHomo sapiens 84Ser Arg Glu Val Thr
Thr Asn Ala Gln Arg 1 5 10 85216DNAHomo sapiens 85tgctcttact
ccaaaaagat ggacccaggg ccctgcgggc ctccccctgg ccatggccca 60ggtcacccac
cccctggtcc acatcactga ggaagtagaa gaaaacagga cacaagatgg
120caagcctgag agaattgccc agctgacctg gaaggaggcc taaaccgcaa
tattctcttc 180ctaataaaca gcctcctaga ggccacattc tattct
21686227DNAHomo sapiens 86tgctcttact ccaaaaagat ggacccaggt
ccgaaggggc actgccactg tggggggcat 60ggccatcctc caggtcaccc accccctggt
ccacatcact gaggaagtag aagaaaacag 120gacacaagat ggcaagcctg
agagaattgc ccagctgacc tggaatgagg cctaaaccac 180aatcttctct
tcctaataaa cagcctccta gaggccacat tctattc 22787261DNAHomo sapiens
87tgctcttact ccaaaaagat ggacccaggt ccgaaggggc actgccactg tggggggcat
60ggccatcctc caggtcactg cgggcctccc cctggccatg gcccaggtca cccaccccct
120ggtccacatc actgaggaag tagaagaaaa caggacacaa gatggcaagc
ctgagagaat 180tgcccagctg acctggaatg aggcctaaac cacaatcttc
tcttcctaat aaacagcctc 240ctagaggcca cattctattc t 26188327DNAHomo
sapiens 88tgctcttact ccaaaaagat ggacccaggt ccgaaggggc actgccactg
tggggggcat 60ggccatcctc caggtcactg cgggccaccc ccccaccatg gtccagggcc
ctgcgggcca 120cccccccacc atggtccagg gccctgcggg cctccccctg
gccatggccc aggtcaccca 180ccccctggtc cacatcactg aggaagtaga
agaaaacagg acacaagatg gcaagcctga 240gagaattgcc cagctgacct
ggaatgaggc ctaaaccaca atcttctctt cctaataaac 300agcctcctag
aggccacatt ctattct 3278931PRTHomo sapiens 89Leu Leu Leu Gln Lys Asp
Gly Pro Arg Ala Leu Arg Ala Ser Pro Trp 1 5 10 15 Pro Trp Pro Arg
Ser Pro Thr Pro Trp Ser Thr Ser Leu Arg Lys 20 25 30 9023PRTHomo
sapiens 90Met Asp Pro Gly Pro Cys Gly Pro Pro Pro Gly His Gly Pro
Gly His 1 5 10 15 Pro Pro Pro Gly Pro His His 20 9136PRTHomo
sapiens 91Met Ala Gln Val Thr His Pro Leu Val His Ile Thr Glu Glu
Val Glu 1 5 10 15 Glu Asn Arg Thr Gln Asp Gly Lys Pro Glu Arg Ile
Ala Gln Leu Thr 20 25 30 Trp Lys Glu Ala 35 9234PRTHomo sapiens
92Leu Leu Gln Lys Asp Gly Pro Arg Ser Glu Gly Ala Leu Pro Leu Trp 1
5 10 15 Gly Ala Trp Pro Ser Ser Arg Ser Pro Thr Pro Trp Ser Thr Ser
Leu 20 25 30 Arg Lys 9327PRTHomo sapiens 93Met Asp Pro Gly Pro Lys
Gly His Cys His Cys Gly Gly His Gly His 1 5 10 15 Pro Pro Gly His
Pro Pro Pro Gly Pro His His 20 25 9438PRTHomo sapiens 94Met Ala Ile
Leu Gln Val Thr His Pro Leu Val His Ile Thr Glu Glu 1 5 10 15 Val
Glu Glu Asn Arg Thr Gln Asp Gly Lys Pro Glu Arg Ile Ala Gln 20 25
30 Leu Thr Trp Asn Glu Ala 35 9546PRTHomo sapiens 95Leu Leu Leu Gln
Lys Asp Gly Pro Arg Ser Glu Gly Ala Leu Pro Leu 1 5 10 15 Trp Gly
Ala Trp Pro Ser Ser Arg Ser Leu Arg Ala Ser Pro Trp Pro 20 25 30
Trp Pro Arg Ser Pro Thr Pro Trp Ser Thr Ser Leu Arg Lys 35 40 45
9638PRTHomo sapiens 96Met Asp Pro Gly Pro Lys Gly His Cys His Cys
Gly Gly His Gly His 1 5 10 15 Pro Pro Gly His Cys Gly Pro Pro Pro
Gly His Gly Pro Gly His Pro 20 25 30 Pro Pro Gly Pro His His 35
9749PRTHomo sapiens 97Met Ala Ile Leu Gln Val Thr Ala Gly Leu Pro
Leu Ala Met Ala Gln 1 5 10 15 Val Thr His Pro Leu Val His Ile Thr
Glu Glu Val Glu Glu Asn Arg 20 25 30 Thr Gln Asp Gly Lys Pro Glu
Arg Ile Ala Gln Leu Thr Trp Asn Glu 35 40 45 Ala 9868PRTHomo
sapiens 98Leu Leu Leu Gln Lys Asp Gly Pro Arg Ser Glu Gly Ala Leu
Pro Leu 1 5 10 15 Trp Gly Ala Trp Pro Ser Ser Arg Ser Leu Arg Ala
Thr Pro Pro Pro 20 25 30 Trp Ser Arg Ala Leu Arg Ala Thr Pro Pro
Pro Trp Ser Arg Ala Leu 35 40 45 Arg Ala Ser Pro Trp Pro Trp Pro
Arg Ser Pro Thr Pro Trp Ser Thr 50 55 60 Ser Leu Arg Lys 65
9960PRTHomo sapiens 99Met Asp Pro Gly Pro Lys Gly His Cys His Cys
Gly Gly His Gly His 1 5 10 15 Pro Pro Gly His Cys Gly Pro Pro Pro
His His Gly Pro Gly Pro Cys 20 25 30 Gly Pro Pro Pro His His Gly
Pro Gly Pro Cys Gly Pro Pro Pro Gly 35 40 45 His Gly Pro Gly His
Pro Pro Pro Gly Pro His His 50 55 60 10071PRTHomo sapiens 100Met
Ala Ile Leu Gln Val Thr Ala Gly His Pro Pro Thr Met Val Gln 1 5 10
15 Gly Pro Ala Gly His Pro Pro Thr Met Val Gln Gly Pro Ala Gly Leu
20 25 30 Pro Leu Ala Met Ala Gln Val Thr His Pro Leu Val His Ile
Thr Glu 35 40 45 Glu Val Glu Glu Asn Arg Thr Gln Asp Gly Lys Pro
Glu Arg Ile Ala 50 55 60 Gln Leu Thr Trp Asn Glu Ala 65 70
* * * * *
References