U.S. patent application number 15/185990 was filed with the patent office on 2016-12-22 for novel peptides and combination of peptides for use in immunotherapy and methods for generating scaffolds for the use against pancreatic cancer and other cancers.
This patent application is currently assigned to IMMATICS BIOTECHNOLOGIES GMBH. The applicant listed for this patent is IMMATICS BIOTECHNOLOGIES GMBH. Invention is credited to Jens FRITSCHE, Andrea MAHR, Oliver SCHOOR, Harpreet SINGH, Toni WEINSCHENK.
Application Number | 20160368965 15/185990 |
Document ID | / |
Family ID | 53784167 |
Filed Date | 2016-12-22 |
United States Patent
Application |
20160368965 |
Kind Code |
A1 |
MAHR; Andrea ; et
al. |
December 22, 2016 |
Novel peptides and combination of peptides for use in immunotherapy
and methods for generating scaffolds for the use against pancreatic
cancer and other cancers
Abstract
The present invention relates to peptides, proteins, nucleic
acids and cells for use in immunotherapeutic methods. In
particular, the present invention relates to the immunotherapy of
cancer. The present invention furthermore relates to
tumor-associated T-cell peptide epitopes, alone or in combination
with other tumor-associated peptides that can for example serve as
active pharmaceutical ingredients of vaccine compositions that
stimulate anti-tumor immune responses, or to stimulate T cells ex
vivo and transfer into patients. Peptides bound to molecules of the
major histocompatibility complex (MHC), or peptides as such, can
also be targets of antibodies, soluble T-cell receptors, and other
binding molecules.
Inventors: |
MAHR; Andrea; (Tuebingen,
DE) ; WEINSCHENK; Toni; (Aichwald, DE) ;
SCHOOR; Oliver; (Tuebingen, DE) ; FRITSCHE; Jens;
(Dusslingen, DE) ; SINGH; Harpreet; (Muenchen,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IMMATICS BIOTECHNOLOGIES GMBH |
TUEBINGEN |
|
DE |
|
|
Assignee: |
IMMATICS BIOTECHNOLOGIES
GMBH
TUEBINGEN
DE
|
Family ID: |
53784167 |
Appl. No.: |
15/185990 |
Filed: |
June 17, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62182026 |
Jun 19, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/72 20130101;
C07K 14/70503 20130101; A61K 51/1057 20130101; A61K 39/0011
20130101; A61K 2039/572 20130101; A61P 35/00 20180101; C07K 16/2833
20130101; C12P 21/02 20130101; C07K 14/4748 20130101; C12Q 1/6881
20130101; G01N 2333/70539 20130101; C07K 14/70539 20130101; C07K
2317/73 20130101; G01N 33/56977 20130101; C12N 15/115 20130101;
A61K 2039/5158 20130101; C07K 2319/55 20130101; A61K 51/1027
20130101; C07K 16/303 20130101; C12Q 2600/158 20130101; C12N 5/0636
20130101; C12Q 2600/156 20130101; C12N 2310/16 20130101; C07K
2319/00 20130101; C07K 2319/30 20130101 |
International
Class: |
C07K 14/74 20060101
C07K014/74; C07K 16/28 20060101 C07K016/28; A61K 51/10 20060101
A61K051/10; C12N 15/115 20060101 C12N015/115; C12Q 1/68 20060101
C12Q001/68; C07K 14/705 20060101 C07K014/705; C12N 5/0783 20060101
C12N005/0783; C12P 21/02 20060101 C12P021/02; G01N 33/569 20060101
G01N033/569; C07K 16/30 20060101 C07K016/30; A61K 39/00 20060101
A61K039/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 19, 2015 |
GB |
1510771.7 |
Claims
1. A peptide comprising an amino acid sequence selected from the
group consisting of SEQ ID No. 1 to SEQ ID No. 161, and variant
sequences thereof which are at least 88% homologous to SEQ ID No. 1
to SEQ ID No. 161, wherein said variant binds to molecule(s) of the
major histocompatibility complex (MHC) and/or induces T cells
cross-reacting with said variant peptide; and a pharmaceutical
acceptable salt thereof, wherein said peptide is not a full-length
polypeptide.
2. The peptide or variant according to claim 1, wherein said
peptide has the ability to bind to a MHC class-I or -II molecule,
and wherein said peptide, when bound to said MHC, is capable of
being recognized by CD4 and/or CD8 T cells.
3. The peptide or variant thereof according to claim 1, wherein the
amino acid sequence thereof comprises a continuous stretch of amino
acids according to the group of SEQ ID No. 1 to SEQ ID No. 161.
4. The peptide or variant thereof according to claim 1, wherein
said peptide or variant thereof has an overall length of from 8 to
100, optionally from 8 to 30, and optionally from 8 to 16 amino
acids, and optionally wherein the peptide consists or consists
essentially of an amino acid sequence according to the group of SEQ
ID No. 1 to SEQ ID No. 161.
5. The peptide comprising an amino acid sequence selected from the
group consisting of SEQ ID No. 1 to SEQ ID No. 161, and variant
sequences thereof which are at least 88% homologous to SEQ ID No. 1
to SEQ ID No. 161, wherein said variant binds to molecule(s) of the
major histocompatibility complex (MHC) and/or induces T cells
cross-reacting with said variant peptide; and a pharmaceutical
acceptable salt thereof, wherein said peptide is not a full-length
polypeptide, wherein said peptide is modified and/or includes
non-peptide bonds.
6. The peptide or variant thereof according to claim 1, wherein
said peptide is part of a fusion protein, optionally comprising
N-terminal amino acids of the HLA-DR antigen-associated invariant
chain (Ii).
7. A nucleic acid, encoding a peptide or variant thereof according
to claim 1, optionally linked to a heterologous promoter
sequence.
8. An expression vector capable of expressing the nucleic acid
according to claim 7.
9. A recombinant host cell comprising the peptide or variant
according to claim 1, a nucleic acid encoding said peptide or
variant or an expression vector capable of expressing said nucleic
acid, wherein said host cell preferably is an antigen presenting
cell such as a dendritic cell.
10. The peptide or variant thereof according to claim 1, a nucleic
acid encoding said peptide or variant or an expression vector
capable of expressing said nucleic acid, or a host cell comprising
said peptide or variant, capable of being used in medicine.
11. A method for producing the peptide or variant thereof according
to claim 1, the method comprising culturing a host cell that
presents the peptide or variant, or expresses a nucleic acid
encoding said peptide or variant or comprises an expression vector
capable of expressing said nucleic acid, and isolating the peptide
or variant thereof from the host cell or its culture medium.
12. An in vitro method for producing activated T lymphocytes, the
method comprising contacting in vitro T cells with antigen loaded
human class I or II MHC molecules expressed on the surface of a
suitable antigen-presenting cell or an artificial construct
mimicking an antigen-presenting cell for a period of time
sufficient to activate said T cells in an antigen specific manner,
wherein said antigen is a peptide according to claim 1.
13. An activated T cell, optionally an activated T lymphocyte,
produced by the method according to claim 12, that selectively
recognizes a cell which presents a polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID No. 1 to
SEQ ID No. 161, and variant sequences thereof which are at least
88% homologous to SEQ ID No. 1 to SEQ ID No. 161.
14. A method for killing target cells in a patient which target
cells present a polypeptide comprising an amino acid sequence
selected from the group consisting of SEQ ID No. 1 to SEQ ID No.
161, and variant sequences thereof which are at least 88%
homologous to SEQ ID No. 1 to SEQ ID No. 161, the method comprising
administering to the patient an effective number of activated T
cells as defined in claim 13.
15. An antibody, optionally a soluble or membrane-bound antibody,
that specifically recognizes the peptide or variant thereof
according to claim 1, optionally the peptide or variant are bound
to an MHC molecule, and wherein said antibody optionally carries a
further effector function, optionally an immune stimulating domain
or toxin.
16. A peptide or variant according to claim 1, a nucleic acid
encoding said peptide or variant or an expression vector capable of
expressing said nucleic acid, or a host cell comprising said
peptide or variant, an activated T lymphocyte that selectively
recognizes a cell which presents said peptide or variant or the
antibody optionally a soluble or membrane-bound antibody, that
specifically recognizes the peptide or variant capable of being
used in the treatment of cancer or in the manufacture of a
medicament against cancer, or in a diagnostic method for detection
of cancerous cells.
17. The peptide or variant according to claim 16, wherein said
cancer is selected from the group of lung cancer, kidney cancer,
brain cancer, stomach cancer, colon or rectal cancer, liver cancer,
prostate cancer, leukemia, breast cancer, Merkel cell carcinoma
(MCC), melanoma, ovarian cancer, esophageal cancer, urinary bladder
cancer, endometrial cancer, gall bladder cancer, and bile duct
cancer, and other tumors that show an overexpression of a protein
comprising a peptide sequence of any of SEQ ID No. 1 to SEQ ID No.
161.
18. A kit comprising: (a) a container comprising a pharmaceutical
composition containing the peptide(s) or the variant according to
claim 1, a nucleic acid encoding said peptide or variant or an
expression vector capable of expressing said nucleic acid, or a
host cell comprising said peptide or variant, an activated T
lymphocyte that selectively recognizes a cell which presents said
peptide or variant or the antibody optionally a soluble or
membrane-bound antibody, that specifically recognizes the peptide
or variant, in solution or in lyophilized form; (b) optionally, a
second container containing a diluent or reconstituting solution
for the lyophilized formulation; (c) optionally, at least one more
peptide selected from the group consisting of SEQ ID No. 1 to SEQ
ID No. 178, and (d) optionally, instructions for (i) use of the
solution or (ii) reconstitution and/or use of the lyophilized
formulation.
19. The kit according to claim 18, further comprising one or more
of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or
(v) a syringe.
20. The kit according to claim 18, wherein said peptide is selected
from the group consisting of SEQ ID No. 1 to SEQ ID No. 161.
21. A method for producing a personalized anti-cancer vaccine for
the use as a compound-based and/or cellular therapy for an
individual patient, said method comprising: a) identifying
tumor-associated peptides (TUMAPs) presented by a tumor sample from
the individual patient; b) comparing the peptides as identified in
a) with a warehouse of peptides that have been pre-screened for
immunogenicity and/or over-presentation in tumors as compared to
normal tissues c) selecting at least one peptide from the warehouse
that matches a TUMAP identified in the patient; and d)
manufacturing or formulating the personalized vaccine or
compound-based or cellular therapy based on c).
22. The method according to claim 21, wherein said TUMAPs are
identified by: a1) comparing expression data from the tumor sample
to expression data from a sample of normal tissue corresponding to
a tissue type of the tumor sample to identify proteins that are
over-expressed or aberrantly expressed in the tumor sample; and a2)
correlating the expression data with sequences of MHC ligands bound
to MHC class I and/or class II molecules in the tumor sample to
identify MHC ligands derived from proteins over-expressed or
aberrantly expressed by the tumor.
23. The method according to claim 21, wherein the sequences of MHC
ligands are identified by eluting bound peptides from MHC molecules
isolated from the tumor sample, and sequencing the eluted
ligands.
24. The method according to claim 21, wherein the normal tissue
corresponding to the tissue type of the tumor sample is obtained
from the same patient.
25. The method according to claim 21, wherein the peptides included
in the warehouse are identified based on the following steps: aa.
Performing genome-wide messenger ribonucleic acid (mRNA) expression
analysis by highly parallel methods, such as microarrays or
sequencing-based expression profiling, comprising identify genes
that over-expressed in a malignant tissue, compared with a normal
tissue or tissues; ab. Selecting peptides encoded by selectively
expressed or over-expressed genes as detected in aa, and ac.
Determining an induction of in vivo T-cell responses by the
peptides as selected comprising in vitro immunogenicity assays
using human T cells from healthy donors or said patient; or ba.
Identifying HLA ligands from said tumor sample using mass
spectrometry; bb. Performing genome-wide messenger ribonucleic acid
(mRNA) expression analysis by highly parallel methods, optionally
microarrays or sequencing-based expression profiling, comprising
identify genes that over-expressed in a malignant tissue, compared
with a normal tissue or tissues; bc. Comparing the identified HLA
ligands to said gene expression data; bd. Selecting peptides
encoded by selectively expressed or over-expressed genes as
detected in bc; be. Re-detecting of selected TUMAPs from bd on
tumor tissue and lack of or infrequent detection on healthy tissues
and confirming the relevance of over-expression at the mRNA level;
and bf. Determining an induction of in vivo T-cell responses by the
peptides as selected comprising in vitro immunogenicity assays
using human T cells from healthy donors or said patient.
26. The method according to claim 21, wherein the immunogenicity of
the peptides included in the warehouse is determined by a method
comprising in vitro immunogenicity assays, patient immunomonitoring
for individual HLA binding, MHC multimer staining, ELISPOT assays
and/or intracellular cytokine staining.
27. The method according to claim 21, wherein said warehouse
comprises a plurality of peptides selected from the group
consisting of SEQ ID No. 1 to SEQ ID No. 178.
28. The method according to claim 21, further comprising
identifying at least one mutation that is unique to the tumor
sample relative to normal corresponding tissue from the individual
patient, and selecting a peptide that correlates with the mutation
for inclusion in the vaccine or for the generation of cellular
therapies.
29. The method according to claim 28, wherein said at least one
mutation is identified by whole genome sequencing.
30. A T-cell receptor, optionally soluble or membrane-bound, that
is reactive with an HLA ligand, wherein said ligand has at least
75% identity to an amino acid sequence selected from the group
consisting of SEQ ID No. 1 to SEQ ID No. 161.
31. The T-cell receptor according to claim 30, wherein said amino
acid sequence is at least 88% identical to SEQ ID No. 1 to SEQ ID
No. 161.
32. The T-cell receptor according to claim 30, wherein said amino
acid sequence consists any of SEQ ID No. 1 to SEQ ID No. 161.
33. The T-cell receptor according to claim 30, wherein said T-cell
receptor is provided as a soluble molecule and optionally carries a
further effector function optionally an immune stimulating domain
or toxin.
34. A nucleic acid, encoding for a TCR according to claim 30,
optionally linked to a heterologous promoter sequence.
35. An expression vector expressing the nucleic acid according to
claim 34.
36. A recombinant host cell comprising the nucleic acid according
to claim 34 or a nucleic acid encoding an antibody optionally a
soluble or membrane-bound antibody, that specifically recognizes
the peptide or variant or an expression vector comprising said
nucleic acid, wherein said host cell optionally is a T cell or NK
cell.
37. A method for producing the T cell receptor according to claim
30, said method comprising culturing a host cell, and isolating
said T cell receptor from said host cell and/or its culture
medium.
38. A pharmaceutical composition comprising at least one active
ingredient selected from the group consisting of a) a peptide
selected from the group consisting of SEQ ID No. 1 to SEQ ID No.
161; b) a T-cell receptor reactive with a peptide and/or the
peptide-MHC complex according to a); c) a fusion protein comprising
a peptide according to a), and the N-terminal amino acids 1 to 80
of the HLA-DR antigen-associated invariant chain (Ii); d) a nucleic
acid encoding for any of a) to c) or an expression vector
comprising said nucleic acid, e) a host cell comprising the
expression vector of d, f) an activated T-lymphocyte, obtained by a
method comprising contacting in vitro T cells with a peptide
according to a) expressed on the surface of a suitable antigen
presenting cell for a period of time sufficient to activate said T
cell in an antigen specific manner, as well as a method to transfer
these activated T cells into the autologous or other patients; g)
an antibody, or soluble T-cell receptor, reactive to a peptide
and/or the peptide--MHC complex according to a) and/or a cell
presenting a peptide according to a), and potentially modified by
fusion with for example immune-activating domains or toxins, h) an
aptamer recognizing a peptide selected from the group consisting of
SEQ ID No. 1 to SEQ ID No. 161, and/or a complex of a peptide
selected from the group consisting of SEQ ID No. 1 to SEQ ID No.
178 with a MHC molecule, i) a conjugated or labelled peptide or
scaffold according to any of a) to h) and a pharmaceutically
acceptable carrier, and optionally, pharmaceutically acceptable
excipients and/or stabilizers.
39. An aptamer that specifically recognizes the peptide or variant
thereof according to claim 1, optionally the peptide or variant
thereof being bound to an MHC molecule.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from GB 1510771.7, filed
Jun. 19, 2015 and U.S. Provisional Application No. 62/182,026,
filed Jun. 19, 2015, both of which are incorporated herein by
reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to peptides, proteins, nucleic
acids and cells for use in immunotherapeutic methods. In
particular, the present invention relates to the immunotherapy of
cancer. The present invention furthermore relates to
tumor-associated T-cell peptide epitopes, alone or in combination
with other tumor-associated peptides that can for example serve as
active pharmaceutical ingredients of vaccine compositions that
stimulate anti-tumor immune responses, or to stimulate T cells ex
vivo and transfer into patients. Peptides bound to molecules of the
major histocompatibility complex (MHC), or peptides as such, can
also be targets of antibodies, soluble T-cell receptors, and other
binding molecules.
[0003] The present invention relates to several novel peptide
sequences and their variants derived from HLA class I molecules of
human tumor cells that can be used in vaccine compositions for
eliciting anti-tumor immune responses, or as targets for the
development of pharmaceutically/immunologically active compounds
and cells.
BACKGROUND OF THE INVENTION
[0004] Pancreatic cancer is one of the most aggressive and deadly
cancers in the world. In 2012, it was the 12.sup.th most common
cancer in men with 178,000 cases and the 11.sup.th most common
cancer in women with 160,000 cases worldwide. In the same year,
330,000 deaths were reported, making pancreatic cancer the seventh
most common cause of death from cancer (World Cancer Report,
2014).
[0005] Pancreatic cancer is not one single cancer entity, but
several distinct subtypes have to be distinguished. Exocrine tumors
account for approximately 95% of all pancreatic cancers and include
ductal and acinary adenocarcinomas, intraductal papillary mucinous
neoplasms (IPMN), solid pseudopapillary neoplasms, mucinous cystic
adenomas and serous cystadenomas. The remaining 5% of all
pancreatic cancers belong to the subgroup of pancreatic
neuroendocrine tumors (World Cancer Report, 2014).
[0006] Infiltrating ductal adenocarcinoma represents the most
aggressive form of pancreatic cancer and due to its high frequency
(90% of all pancreatic cancers), epidemiologic data mainly reflect
this specific subtype (World Cancer Report, 2014).
[0007] In 2012, 68% of all new cases occurred in developed
countries, with highest incidence rates in central and Eastern
Europe, North America, Argentina, Uruguay and Australia. In
contrast, most countries in Africa and East Asia display low
incidence rates. Globally, incidence rates appear to be rather
stable over time in both genders (World Cancer Report, 2014).
[0008] Due to a lack of specific symptoms, pancreatic cancer is
typically diagnosed at an advanced and often already metastatic
stage. The prognosis upon diagnosis is very poor, with a 5 years
survival rate of 5% and a mortality-to-incidence ratio of 0.98
(World Cancer Report, 2014).
[0009] Several factors have been reported to increase the risk to
develop pancreatic cancer, including older age, as most patients
are older than 65 years at diagnosis, and race, as in the USA the
Black population has a 1.5-fold increased risk compared to the
White population. Further risk factors are cigarette smoking, body
fatness, diabetes, non-0 AB0 blood type, pancreatitis and a
familial history of pancreatic cancer (World Cancer Report,
2014).
[0010] Up to 10% of all pancreatic cancer cases are thought to have
a familial basis. Germline mutations in the following genes are
associated with an increased risk to develop pancreatic cancer:
p16/CDKN2A, BRCA2, PALB2, PRSS1, STK11, ATM and DNA mismatch repair
genes. Additionally, the sporadic cases of pancreatic cancer are
also characterized by mutations in different oncogenes and tumor
suppressor genes. The most common mutations in ductal
adenocarcinoma occur within the oncogenes KRAS (95%) and AIB1 (up
to 60%) and the tumor suppressor genes TP53 (75%), p16/CDKN2A (95%)
and SMAD4 (55%) (World Cancer Report, 2014).
[0011] Therapeutic options for pancreatic cancer patients are very
limited. One major problem for effective treatment is the typically
advanced tumor stage at diagnosis. Additionally, pancreatic cancer
is rather resistant to chemotherapeutics, which might be caused by
the dense and hypovascular desmoplastic tumor stroma.
[0012] According to the guidelines released by the German Cancer
Society, the German Cancer Aid and the Association of the
Scientific Medical Societies in Germany, resection of the tumor is
the only available curative treatment option. Resection is
recommended if the tumor is restricted to the pancreas or if
metastases are limited to adjacent organs. Resection is not
recommended if the tumor has spread to distant sites. Resection is
followed by adjuvant chemotherapy with gemcitabine or
5-fluorouracil+/-leucovorin for six months (S3-Leitlinie Exokrines
Pankreaskarzinom, 2013).
[0013] Patients with inoperable tumors in advanced stage can be
treated with a combination of chemotherapy with
radiation-chemotherapy (S3-Leitlinie Exokrines Pankreaskarzinom,
2013).
[0014] The standard regimen for palliative chemotherapy is
gemcitabine, either as monotherapy or in combination with the EGF
receptor tyrosine kinase inhibitor erlotinib. Alternative options
are a combination of 5-fluorouracil, leucovorin, irinotecan and
oxaliplatin, also known as FOLFIRINOX protocol or the combination
of gemcitabine with nab-paclitaxel, which was shown to have
superior effects compared to gemcitabine monotherapy in the MPACT
study (Von Hoff et al., 2013; S3-Leitlinie Exokrines
Pankreaskarzinom, 2013).
[0015] The high mortality-to-incidence ratio reflects the urgent
need to implement more effective therapeutic strategies in
pancreatic cancer.
[0016] Targeted therapies, which have already been shown to be
efficient in several other cancer entities, represent an
interesting option. Therefore, several studies have been performed
to evaluate the benefit of targeted therapies in advanced
pancreatic cancers, unfortunately with very limited success (Walker
and Ko, 2014). Nevertheless, the genetic diversity of pancreatic
cancer might offer the possibility of personalized therapy, as
invasive ductal adenocarcinoma with bi-allelic inactivation of
BRCA2 or PALB2 was shown to be more sensitive to poly (ADP-ribose)
polymerase inhibitors and mitomycin C treatment (World Cancer
Report, 2014).
[0017] Targeting the tumor stroma constitutes an alternative
approach to develop new treatments for pancreatic cancer. The
typically dense and hypovascular stroma might function as barrier
for chemotherapeutics and was shown to deliver signals that promote
tumor proliferation, invasion and cancer stem cell maintenance.
Thus, different preclinical and clinical studies were designed to
analyze the effect of stromal depletion and inactivation (Rucki and
Zheng, 2014).
[0018] Vaccination strategies are investigated as further
innovative and promising alternative for the treatment of
pancreatic cancer. Peptide-based vaccines targeting KRAS mutations,
reactive telomerase, gastrin, survivin, CEA and MUC1 have already
been evaluated in clinical trials, partially with promising
results. Furthermore, clinical trials for dendritic cell-based
vaccines, allogeneic GM-CSF-secreting vaccines and algenpantucel-L
in pancreatic cancer patients also revealed beneficial effects of
immunotherapy. Additional clinical trials further investigating the
efficiency of different vaccination protocols are currently ongoing
(Salman et al., 2013).
[0019] Considering the severe side-effects and expense associated
with treating cancer, there is a need to identify factors that can
be used in the treatment of cancer in general and pancreatic cancer
in particular. There is also a need to identify factors
representing biomarkers for cancer in general and pancreatic cancer
in particular, leading to better diagnosis of cancer, assessment of
prognosis, and prediction of treatment success.
[0020] Immunotherapy of cancer represents an option of specific
targeting of cancer cells while minimizing side effects. Cancer
immunotherapy makes use of the existence of tumor associated
antigens.
[0021] The current classification of tumor associated antigens
(TAAs) comprises the following major groups:
[0022] a) Cancer-testis antigens: The first TAAs ever identified
that can be recognized by T cells belong to this class, which was
originally called cancer-testis (CT) antigens because of the
expression of its members in histologically different human tumors
and, among normal tissues, only in spermatocytes/spermatogonia of
testis and, occasionally, in placenta. Since the cells of testis do
not express class I and II HLA molecules, these antigens cannot be
recognized by T cells in normal tissues and can therefore be
considered as immunologically tumor-specific. Well-known examples
for CT antigens are the MAGE family members and NY-ESO-1.
[0023] b) Differentiation antigens: These TAAs are shared between
tumors and the normal tissue from which the tumor arose. Most of
the known differentiation antigens are found in melanomas and
normal melanocytes. Many of these melanocyte lineage-related
proteins are involved in biosynthesis of melanin and are therefore
not tumor specific but nevertheless are widely used for cancer
immunotherapy. Examples include, but are not limited to, tyrosinase
and Melan-A/MART-1 for melanoma or PSA for prostate cancer.
[0024] c) Over-expressed TAAs: Genes encoding widely expressed TAAs
have been detected in histologically different types of tumors as
well as in many normal tissues, generally with lower expression
levels. It is possible that many of the epitopes processed and
potentially presented by normal tissues are below the threshold
level for T-cell recognition, while their over-expression in tumor
cells can trigger an anticancer response by breaking previously
established tolerance. Prominent examples for this class of TAAs
are Her-2/neu, survivin, telomerase, or WT1.
[0025] d) Tumor-specific antigens: These unique TAAs arise from
mutations of normal genes (such as .beta.-catenin, CDK4, etc.).
Some of these molecular changes are associated with neoplastic
transformation and/or progression. Tumor-specific antigens are
generally able to induce strong immune responses without bearing
the risk for autoimmune reactions against normal tissues. On the
other hand, these TAAs are in most cases only relevant to the exact
tumor on which they were identified and are usually not shared
between many individual tumors. Tumor-specificity (or -association)
of a peptide may also arise if the peptide originates from a tumor-
(-associated) exon in case of proteins with tumor-specific
(-associated) isoforms.
[0026] e) TAAs arising from abnormal post-translational
modifications: Such TAAs may arise from proteins which are neither
specific nor overexpressed in tumors but nevertheless become tumor
associated by posttranslational processes primarily active in
tumors. Examples for this class arise from altered glycosylation
patterns leading to novel epitopes in tumors as for MUC1 or events
like protein splicing during degradation which may or may not be
tumor specific.
[0027] f) Oncoviral proteins: These TAAs are viral proteins that
may play a critical role in the oncogenic process and, because they
are foreign (not of human origin), they can evoke a T-cell
response. Examples of such proteins are the human papilloma type 16
virus proteins, E6 and E7, which are expressed in cervical
carcinoma.
[0028] T-cell based immunotherapy targets peptide epitopes derived
from tumor-associated or tumor-specific proteins, which are
presented by molecules of the major histocompatibility complex
(MHC). The antigens that are recognized by the tumor specific T
lymphocytes, that is, the epitopes thereof, can be molecules
derived from all protein classes, such as enzymes, receptors,
transcription factors, etc. which are expressed and, as compared to
unaltered cells of the same origin, usually up-regulated in cells
of the respective tumor.
[0029] There are two classes of MHC-molecules, MHC class I and MHC
class II. MHC class I molecules are composed of an alpha heavy
chain and beta-2-microglobulin, MHC class II molecules of an alpha
and a beta chain. Their three-dimensional conformation results in a
binding groove, which is used for non-covalent interaction with
peptides.
[0030] MHC class I molecules can be found on most nucleated cells.
They present peptides that result from proteolytic cleavage of
predominantly endogenous proteins, defective ribosomal products
(DRIPs) and larger peptides. However, peptides derived from
endosomal compartments or exogenous sources are also frequently
found on MHC class I molecules. This non-classical way of class I
presentation is referred to as cross-presentation in the literature
(Brossart and Bevan, 1997; Rock et al., 1990). MHC class II
molecules can be found predominantly on professional antigen
presenting cells (APCs), and primarily present peptides of
exogenous or transmembrane proteins that are taken up by APCs e.g.
during endocytosis, and are subsequently processed.
[0031] Complexes of peptide and MHC class I are recognized by
CD8-positive T cells bearing the appropriate T-cell receptor (TCR),
whereas complexes of peptide and MHC class II molecules are
recognized by CD4-positive-Helper-T cells bearing the appropriate
TCR. It is well known that the TCR, the peptide and the MHC are
thereby present in a stoichiometric amount of 1:1:1.
[0032] CD4-positive helper T cells play an important role in
inducing and sustaining effective responses by CD8-positive
cytotoxic T cells. The identification of CD4-positive T-cell
epitopes derived from tumor associated antigens (TAA) is of great
importance for the development of pharmaceutical products for
triggering anti-tumor immune responses (Gnjatic et al., 2003). At
the tumor site, T helper cells, support a cytotoxic T cell- (CTL-)
friendly cytokine milieu (Mortara et al., 2006) and attract
effector cells, e.g. CTLs, natural killer (NK) cells, macrophages,
and granulocytes (Hwang et al., 2007).
[0033] In the absence of inflammation, expression of MHC class II
molecules is mainly restricted to cells of the immune system,
especially professional antigen-presenting cells (APC), e.g.,
monocytes, monocyte-derived cells, macrophages, dendritic cells. In
cancer patients, cells of the tumor have been found to express MHC
class II molecules (Dengjel et al., 2006).
[0034] Elongated peptides of the invention can act as MHC class II
active epitopes.
[0035] T-helper cells, activated by MHC class II epitopes, play an
important role in orchestrating the effector function of CTLs in
anti-tumor immunity. T-helper cell epitopes that trigger a T-helper
cell response of the TH1 type support effector functions of
CD8-positive killer T cells, which include cytotoxic functions
directed against tumor cells displaying tumor-associated
peptide/MHC complexes on their cell surfaces. In this way
tumor-associated T-helper cell peptide epitopes, alone or in
combination with other tumor-associated peptides, can serve as
active pharmaceutical ingredients of vaccine compositions that
stimulate anti-tumor immune responses.
[0036] It was shown in mammalian animal models, e.g., mice, that
even in the absence of CD8-positive T lymphocytes, CD4-positive T
cells are sufficient for inhibiting manifestation of tumors via
inhibition of angiogenesis by secretion of interferon-gamma
(IFN.gamma.) (Beatty and Paterson, 2001; Mumberg et al., 1999).
There is evidence for CD4 T cells as direct anti-tumor effectors
(Braumuller et al., 2013; Tran et al., 2014).
[0037] Since the constitutive expression of HLA class II molecules
is usually limited to immune cells, the possibility of isolating
class II peptides directly from primary tumors was previously not
considered possible. However, Dengjel et al. were successful in
identifying a number of MHC Class II epitopes directly from tumors
(WO 2007/028574, EP 1 760 088 B1).
[0038] Since both types of response, CD8 and CD4 dependent,
contribute jointly and synergistically to the anti-tumor effect,
the identification and characterization of tumor-associated
antigens recognized by either CD8+ T cells (ligand: MHC class I
molecule+peptide epitope) or by CD4-positive T-helper cells
(ligand: MHC class II molecule+peptide epitope) is important in the
development of tumor vaccines.
[0039] For an MHC class I peptide to trigger (elicit) a cellular
immune response, it also must bind to an MHC-molecule. This process
is dependent on the allele of the MHC-molecule and specific
polymorphisms of the amino acid sequence of the peptide.
MHC-Class-I-binding peptides are usually 8-12 amino acid residues
in length and usually contain two conserved residues ("anchors") in
their sequence that interact with the corresponding binding groove
of the MHC-molecule. In this way each MHC allele has a "binding
motif" determining which peptides can bind specifically to the
binding groove.
[0040] In the MHC class I dependent immune reaction, peptides not
only have to be able to bind to certain MHC class I molecules
expressed by tumor cells, they subsequently also have to be
recognized by T cells bearing specific T cell receptors (TCR).
[0041] For proteins to be recognized by T-lymphocytes as
tumor-specific or -associated antigens, and to be used in a
therapy, particular prerequisites must be fulfilled. The antigen
should be expressed mainly by tumor cells and not, or in comparably
small amounts, by normal healthy tissues. In a preferred
embodiment, the peptide should be over-presented by tumor cells as
compared to normal healthy tissues. It is furthermore desirable
that the respective antigen is not only present in a type of tumor,
but also in high concentrations (i.e. copy numbers of the
respective peptide per cell). Tumor-specific and tumor-associated
antigens are often derived from proteins directly involved in
transformation of a normal cell to a tumor cell due to their
function, e.g. in cell cycle control or suppression of apoptosis.
Additionally, downstream targets of the proteins directly causative
for a transformation may be up-regulated and thus may be indirectly
tumor-associated. Such indirect tumor-associated antigens may also
be targets of a vaccination approach (Singh-Jasuja et al., 2004).
It is essential that epitopes are present in the amino acid
sequence of the antigen, in order to ensure that such a peptide
("immunogenic peptide"), being derived from a tumor associated
antigen, leads to an in vitro or in vivo T-cell-response.
[0042] Basically, any peptide able to bind an MHC molecule may
function as a T-cell epitope. A prerequisite for the induction of
an in vitro or in vivo T-cell-response is the presence of a T cell
having a corresponding TCR and the absence of immunological
tolerance for this particular epitope.
[0043] Therefore, TAAs are a starting point for the development of
a T cell based therapy including but not limited to tumor vaccines.
The methods for identifying and characterizing the TAAs are usually
based on the use of T-cells that can be isolated from patients or
healthy subjects, or they are based on the generation of
differential transcription profiles or differential peptide
expression patterns between tumors and normal tissues. However, the
identification of genes over-expressed in tumor tissues or human
tumor cell lines, or selectively expressed in such tissues or cell
lines, does not provide precise information as to the use of the
antigens being transcribed from these genes in an immune therapy.
This is because only an individual subpopulation of epitopes of
these antigens are suitable for such an application since a T cell
with a corresponding TCR has to be present and the immunological
tolerance for this particular epitope needs to be absent or
minimal. In a very preferred embodiment of the invention it is
therefore important to select only those over- or selectively
presented peptides against which a functional and/or a
proliferating T cell can be found. Such a functional T cell is
defined as a T cell, which upon stimulation with a specific antigen
can be clonally expanded and is able to execute effector functions
("effector T cell").
[0044] In case of targeting peptide-MHC by specific TCRs (e.g.
soluble TCRs) and antibodies or other binding molecules (scaffolds)
according to the invention, the immunogenicity of the underlying
peptides is secondary. In these cases, the presentation is the
determining factor.
SUMMARY OF THE INVENTION
[0045] In a first aspect of the present invention, the present
invention relates to a peptide comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:
161 or a variant sequence thereof which is at least 77%, preferably
at least 88%, homologous (preferably at least 77% or at least 88%
identical) to SEQ ID NO: 1 to SEQ ID NO: 161, wherein said variant
binds to MHC and/or induces T cells cross-reacting with said
peptide, or a pharmaceutical acceptable salt thereof, wherein said
peptide is not the underlying full-length polypeptide.
[0046] The present invention further relates to a peptide of the
present invention comprising a sequence that is selected from the
group consisting of SEQ ID NO: 1 to SEQ ID NO: 161 or a variant
thereof, which is at least 77%, preferably at least 88%, homologous
(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1
to SEQ ID NO: 161, wherein said peptide or variant thereof has an
overall length of between 8 and 100, preferably between 8 and 30,
and most preferred of between 8 and 14 amino acids.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIGS. 1A-1AF depict embodiments as described herein.
[0048] FIGS. 2A-2C depict embodiments as described herein.
[0049] FIGS. 3A-3D depict embodiments as described herein.
[0050] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
[0051] The following tables show the peptides according to the
present invention, their respective SEQ ID NOs, and the prospective
source (underlying) genes for these peptides. All peptides in Table
1 and Table 2 bind to HLA-A*02. The peptides in Table 2 have been
disclosed before in large listings as results of high-throughput
screenings with high error rates or calculated using algorithms,
but have not been associated with cancer at all before. The
peptides in Table 3 are additional peptides that may be useful in
combination with the other peptides of the invention. The peptides
in Table 4 are furthermore useful in the diagnosis and/or treatment
of various other malignancies that involve an over-expression or
over-presentation of the respective underlying polypeptide.
TABLE-US-00001 TABLE 1 Peptides according to the present invention
SEQ Official ID gene NO. Sequence Gene ID(s) symbol(s) 1 FVDTRTLL
1278 COL1A2 2 FGYDGDFYRA 1278 COL1A2 3 ILIGETIKI 5742,5743 PTGS1,
PTGS2 4 ALDPAAQAFLL 84919 PPP1R15B 5 ALLTGIISKA 23165 NUP205 6
ALTGIPLPLI 1017 CDK2 7 ALVDIVRSL 3995 FADS3 8 ALYTGSALDFV 1293
COL6A3 9 QIIDAINKV 1293 COL6A3 10 VLLDKIKNL 1293 COL6A3 11
ALYYNPHLL 10527 IPO7 12 AQYKFVYQV 5784 PTPN14 13 FIDSSNPGL 92126
DSEL 14 FIIDNPQDLKV 5362 PLXNA2 15 FILANEHNV 3843 IPO5 16 GLIDYDTGI
667 DST 17 GLIDYDTGIRL 667 DST 18 ALFVRLLAL 7045 TGFBI 19
ALWHDAENQTVV 23279 NUP160 20 GLIDIENPNRV 11333 PDAP1 21 GLVDGRDLVIV
9943 OXSR1 22 ILSTEIFGV 79703 C11orf80 23 KLDSSGGAVQL 23677 SH3BP4
24 KLSENAGIQSL 26064 RAI14 25 LINPNIATV 790 CAD 26 SLYTALTEA 4124
MAN2A1 27 TLLAHPVTL 27063 ANKRD1 28 VLDEFYSSL 11321 GPN1 29
YILPFSEVL 2132 EXT2 30 YIYKDTIQV 346389 MACC1 31 YLDSMYIML 8754
ADAM9 32 YVDDGLISL 5315 PKM 33 FLADPDTVNHL 57231 SNX14 34
FLEDDDIAAV 9945 GFPT2 35 FLFPSQYVDV 9871 SEC24D 36 FLGDLSHLL 10945
KDELR1 37 FLNPDEVHAI 81610 FAM83D 38 FLTEAALGDA 7980 TFPI2 39
FLTPSIFII 79971 WLS 40 GLAPQIHDL 128239 IQGAP3 41 GLLAGNEKLTM 3880
KRT19 42 ILSDMRSQYEV 3880 KRT19 43 HLGVKVFSV 1291 COL6A1 44
ILAQVGFSV 55117 SLC6A15 45 ILYSDDGQKWTV 131566 DCBLD2 46 TMVEHNYYV
131566 DCBLD2 47 LIYKDLVSV 85016 C11orf70 48 LLDENGVLKL 1022 CDK7
49 LLDGFPRTV 204 AK2 50 LLFGSDGYYV 10897 YIF1A 51 LLGPAGARA 255738
PCSK9 52 LLSDPIPEV 57521 RPTOR 53 LLWDPSTGKQV 54475 NLE1 54
LTQPGPIASA 6374 CXCL5 55 NLAPAPLNA 7035 TFPI 56 NLIGVTAEL 80210
ARMC9 57 RLSELGITQA 79801 SHCBP1 58 RQYPWGVVQV 151011, SEPT10,
SEPT8, 23176, SEPT11 55752 59 SLSESFFMV 54434 SSH1 60 SLWEDYPHV
9697 TRAM2 61 SMYDGLLQA 51393 TRPV2 62 SVFPGARLL 10498 CARM1 63
SVTGIIVGV 57722 IGDCC4 64 TLFSEPKFAQV 84886 C1orf198 65 TLNEKLTAL
55845 BRK1 66 TVDDPYATFV 1072 CFL1 67 VIWGTDVNV 4173 MCM4 68
VLFDVTGQV 9961 MVP 69 VLFSGSLRL 115908 CTHRC1 70 VLGVIWGV
100527943, TGIF2-C20orf24, 55969 C20orf24 71 VLLPEGGITAI 9904 RBM19
72 VMASPGGLSAV 54443 ANLN 73 VMVDGKPVNL 5879,5881 RAC1, RAC3 74
YIDKDLEYV 29102 DROSHA 75 FSFVDLRLL 1277 COL1A1 76 LVSESSDVLPK
100129958, KRT8P44, KRT8 3856 77 RLFPGSSFL 90993 CREB3L1 78
SLQDTEEKSRS 2641 GCG 79 VVYEGQLISI 2335 FN1 80 LLPGTEYVVSV 2335 FN1
81 VVYDDSTGLIRL 2898,2899 GRIK2, GRIK3 82 ALIAEGIAL 1778 DYNC1H1 83
ALSKEIYVI 515 ATP5F1 84 FILPIGATV 6509,6510 SLC1A4, SLC1A5 85
FLSDGTIISV 84916 CIRH1A 86 GLGDFIFYSV 5663,5664 PSEN1, PSEN2 87
GLLPALVAL 113278 SLC52A3 88 IIDDTIFNL 257641,4864 NPC1 89
KLADIQIEQL 5201 PFDN 1 90 KLLTPITTL 1293 COL6A3 91 LLFNDVQTL 5339
PLEC 92 YLTNEGIAHL 5339 PLEC 93 SIDSEPALV 23420, NOMO1, NOMO2,
283820, NOMO3 408050 94 VMMEEFVQL 9875 URB1 95 ALADDDFLTV 4173 MCM4
96 ALAPATGGGSLLL 80830 APOL6 97 ALDDMISTL 7203 CCT3 98 ALDQKVRSV
4130 MAP1A 99 ALESFLKQV 5591 PRKDC 100 ALFGAGPASI 1806 DPYD 101
ALVEENGIFEL 11187 PKP3 102 ALYPGTDYTV 64420 SUSD1 103 AVAAVLTQV
10280 SIGMAR1 104 FLQPDLDSL 10514 MYBBP1A 105 FLSEVFHQA 5055
SERPINB2 106 FVWSGTAEA 23326 USP22 107 FVYGGPQVQL 91039 DPP9 108
IADGGFTEL 1107,1108, CHD3, CHD4, 26038 CHD5 109 ILASVILNV 644538
SMIM10 110 ILLTGTPAL 84083 ZRANB3 111 LLLAAARLAAA 2923 PDIA3 112
LLSDVRFVL 53339 BTBD1 113 LMMSEDRISL 9945 GFPT2 114 SLFPHNPQFI
80135 RPF1 115 SLMDPNKFLLL 197131 UBR1 116 SMMDPNHFL 23304 UBR2 117
SVDGVIKEV 10577 NPC2 118 TLWYRPPEL 100422910, MIR2861, CDK9, 1025,
CDK12, CDK13
51755,8621 119 VLGDDPQLMKV 10629 TAF6L 120 VLVNDFFLV 3646 EIF3E 121
YLDEDTIYHL 4144 MAT2A
TABLE-US-00002 TABLE 2 Additional peptides according to the present
invention with no prior known cancer association SEQ ID Official
No. Sequence Gene ID(s) Gene Symbol(s) 122 MQAPRAALVFA 201799
TMEM154 123 KISTITPQI 996 CDC27 124 ALFEESGLIRI 1951, CELSR3,
SLC26A6 65010 125 ALLGKLDAINV 5876 RABGGTB 126 ALLSLDPAAV 5591
PRKDC 127 ALSDLALHFL 10575 CCT4 128 ALYDVRTILL 11065 UBE2C 129
ALYEKDNTYL 80279 CDK5RAP3 130 FLFGEEPSKL 23141 ANKLE2 131
FLIEEQKIVV 6164 RPL34 132 FLWAGGRASYGV 3192 HNRNPU 133 ILDDVSLTHL
5245 PHB 134 ILLAEGRLVNL 191 AHCY 135 KLDDTYIKA 7266 DNAJC7 136
KLFPGFEIETV 440 ASNS 137 KLGPEGELL 6510 SLC1A5 138 NIFPNPEATFV
11198 SUPT16H 139 SIDRNPPQL 6773 STAT2 140 SLLNPPETLNL 890 CCNA2
141 SLTEQVHSL 79598 CEP97 142 SLYGYLRGA 9790 BMS1 143 TADPLDYRL
4928 NUP98 144 TAVALLRLL 9761 MLEC 145 TTFPRPVTV 4841 NONO 146
VLISGVVHEI 51360 MBTPS2 147 YAFPKAVSV 9123 SLC16A3 148 YLHNQGIGV
701 BUB1B 149 ILGTEDLIVEV 79719 AAGAB 150 ALFQPHLINV 10097 ACTR2
151 ALLDIIRSL 9415 FADS2 152 ALLEPEFILKA 7011 TEP1 153 ALPKEDPTAV
22820 COPG1 154 KVADLVLML 399761, BMS1P5, 642517,9790 AGAP9, BMS1
155 LLLDPDTAVLKL 2932 GSK3B 156 LLLPPPPCPA 2519 FUCA2 157
MLLEIPYMAA 728689,8663 EIF3CL, EIF3C 158 SLIEKYFSV 3838,645680
KPNA2 159 SLLDLHTKV 27340 UTP20 160 VLLPDERTISL 1477 CSTF1 161
YLPDIIKDQKA 5496 PPM1G
TABLE-US-00003 TABLE 3 Peptides useful for e.g. personalized cancer
therapies SEQ ID Official No. Sequence Gene ID(s) Gene Symbol(s)
162 NADPQAVTM 10916 MAGED2 163 VMAPRTLVL 100507703, HLA-A 3105 164
YLGRLAHEV 23521,387841, RPL13A, 728658 RPL13AP20, RPL13AP5 165
YLLSYIQSI 64151 NCAPG 166 SLFPGQVVI 23649 POLA2 167 MLFGHPLLVSV
8237 USP11 168 SEWGSPHAAVP 5539 PPY 169 FMLPDPQNI 116461 TSEN15 170
ILAPAGSLPKI 29914 UBIAD1 171 LLLDVTPLSL 100287551,3306, HSPA8P8,
3312,3313 HSPA2, HSPA8, HSPA9 172 TMMSRPPVL 57708,79971 MIER1, WLS
173 SLAGDVALQQL 9918 NCAPD2 174 TLDPRSFLL 2149 F2R 175 ALLESSLRQA
595 CCND1 176 YLMPGFIHL 168400,55510 DDX53, DDX43 177 SLYKGLLSV
25788 RAD54B 178 KIQEILTQV 10643 IGF2BP3
[0052] The present invention furthermore generally relates to the
peptides according to the present invention for use in the
treatment of proliferative diseases, such as, for example, lung
cancer, kidney cancer, brain cancer, stomach cancer, colon or
rectal cancer, liver cancer, prostate cancer, leukemia, breast
cancer, Merkel cell carcinoma (MCC), melanoma, ovarian cancer,
esophageal cancer, urinary bladder cancer, endometrial cancer, gall
bladder cancer, and bile duct cancer.
[0053] Particularly preferred are the peptides--alone or in
combination--according to the present invention selected from the
group consisting of SEQ ID NO: 1 to SEQ ID NO: 161. More preferred
are the peptides--alone or in combination--selected from the group
consisting of SEQ ID NO: 1 to SEQ ID NO: 79 (see Table 1), and
their uses in the immunotherapy of pancreatic cancer, lung cancer,
kidney cancer, brain cancer, stomach cancer, colon or rectal
cancer, liver cancer, prostate cancer, leukemia, breast cancer,
Merkel cell carcinoma (MCC), melanoma, ovarian cancer, esophageal
cancer, urinary bladder cancer, endometrial cancer, gall bladder
cancer, bile duct cancer, and preferably pancreatic cancer.
[0054] As shown in the following Table 4, many of the peptides
according to the present invention are also found on other tumor
types and can, thus, also be used in the immunotherapy of other
indications. Also refer to FIG. 1 and Example 1.
TABLE-US-00004 TABLE 4 Peptides according to the present invention
and their specific uses in other proliferative diseases, especially
in other cancerous diseases. The table shows for selected peptides
on which additional tumor types they were found and either
over-presented on more than 5% of the measured tumor samples, or
presented on more than 5% of the measured tumor samples with a
ratio of geometric means tumor vs normal tissues being larger than
3. Over-presentation is defined as higher presentation on the tumor
sample as compared to the normal sample with highest presentation.
SEQ ID No. Sequence Other relevant organs (cancer)/diseases 1
FVDTRTLL Esophagus 2 FGYDGDFYRA Pancreas, Breast, Esophagus 3
ILIGETIKI Urinary bladder 4 ALDPAAQAFLL NSCLC, Liver, Breast,
Ovary, Esophagus, Urinary bladder 5 ALLTGIISKA NSCLC, Colon,
Rectum, Liver, Esophagus 7 ALVDIVRSL Leukocytes 8 ALYTGSALDFV
NSCLC, Pancreas, Breast, Esophagus, Gallbladder, Bile duct 9
QIIDAINKV Breast, Esophagus 10 VLLDKIKNL Pancreas, Gallbladder,
Bile duct 11 ALYYNPHLL Esophagus 12 AQYKFVYQV Esophagus 13
FIDSSNPGL Kidney 14 FIIDNPQDLKV NSCLC, SCLC, Kidney, Liver,
Melanoma, Ovary, Esophagus 16 GLIDYDTGI Brain, Breast 17
GLIDYDTGIRL Brain, Melanoma 19 ALWHDAENQTVV NSCLC, SCLC, Liver,
Melanoma, Esophagus, Gallbladder, Bile duct 20 GLIDIENPNRV Urinary
bladder 22 ILSTEIFGV NSCLC, Pancreas, Leukocytes, Breast 26
SLYTALTEA Breast 28 VLDEFYSSL Colon, Rectum 29 YILPFSEVL NSCLC,
Kidney, Brain, Colon, Rectum, Esophagus, Urinary bladder 30
YIYKDTIQV NSCLC, Colon, Rectum 31 YLDSMYIML NSCLC, Stomach, Colon,
Rectum, Liver, Pancreas, Breast, Gallbladder, Bile duct 32
YVDDGLISL Stomach 34 FLEDDDIAAV Brain, Melanoma 35 FLFPSQYVDV
NSCLC, SCLC, Liver, Breast, Ovary, Esophagus 37 FLNPDEVHAI NSCLC,
Colon, Rectum, Liver, Breast, Melanoma, Ovary, Esophagus, Urinary
bladder 39 FLTPSIFII Brain, Pancreas 40 GLAPQIHDL Colon, Rectum,
Esophagus 41 GLLAGNEKLTM Colon, Rectum, Breast, Urinary bladder,
Endometrium 42 ILSDMRSQYEV Urinary bladder 45 ILYSDDGQKWTV Melanoma
46 TMVEHNYYV NSCLC, SCLC, Kidney, Pancreas, Melanoma, Ovary,
Esophagus 48 LLDENGVLKL Leukocytes 50 LLFGSDGYYV Liver, Esophagus
51 LLGPAGARA Liver, Esophagus 52 LLSDPIPEV SCLC, Melanoma, Ovary,
Esophagus 57 RLSELGITQA Esophagus 58 RQYPWGVVQV Esophagus 59
SLSESFFMV SCLC, Breast, Urinary bladder 60 SLWEDYPHV NSCLC, SCLC,
Colon, Rectum, Liver, Ovary, Urinary bladder 62 SVFPGARLL SCLC,
Leukocytes, Esophagus 63 SVTGIIVGV Brain, Esophagus 64 TLFSEPKFAQV
SCLC, Liver, Urinary bladder 67 VIWGTDVNV Brain, Esophagus 68
VLFDVTGQV Stomach 69 VLFSGSLRL NSCLC 70 VLGVIWGV NSCLC, Liver,
Ovary, Esophagus 71 VLLPEGGITAI Leukocytes 73 VMVDGKPVNL Liver,
Gallbladder, Bile duct 75 FSFVDLRLL SCLC, Esophagus, Gallbladder,
Bile duct 77 RLFPGSSFL Breast, Esophagus 79 VVYEGQLISI NSCLC, SCLC,
Pancreas, Breast, Esophagus 80 LLPGTEYVVSV SCLC, Liver 81
VVYDDSTGLIRL SCLC, Brain, Leukocytes, MCC, Ovary 82 ALIAEGIAL
Urinary bladder 83 ALSKEIYVI Leukocytes 84 FILPIGATV Kidney,
Stomach, Breast 85 FLSDGTIISV NSCLC, Colon, Rectum, Liver,
Melanoma, Ovary, Esophagus, Endometrium 86 GLGDFIFYSV Liver,
Pancreas 88 IIDDTIFNL Stomach, Urinary bladder 90 KLLTPITTL NSCLC,
SCLC, Colon, Rectum, Breast 91 LLFNDVQTL Esophagus, Urinary bladder
92 YLTNEGIAHL NSCLC, Colon, Rectum, Melanoma, Ovary, Esophagus 93
SIDSEPALV Brain, Colon, Rectum, Breast, Urinary bladder 94
VMMEEFVQL Brain, Colon, Rectum, Leukocytes, Ovary, Esophagus,
Endometrium, Gallbladder, Bile duct 95 ALADDDFLTV NSCLC, SCLC,
Stomach, Leukocytes, Melanoma, Ovary, Esophagus, Urinary bladder 96
ALAPATGGGSLLL Liver, Melanoma 97 ALDDMISTL Stomach, Urinary bladder
98 ALDQKVRSV Brain, Prostate 99 ALESFLKQV Colon, Rectum, Liver,
Breast, Urinary bladder 100 ALFGAGPASI Liver 101 ALVEENGIFEL NSCLC,
Liver, MCC, Ovary, Urinary bladder 102 ALYPGTDYTV NSCLC, SCLC,
Brain, Liver, Prostate, Gallbladder, Bile duct 103 AVAAVLTQV Liver
104 FLQPDLDSL Brain, Liver, Pancreas, Leukocytes, Urinary bladder
106 FVWSGTAEA Brain, Esophagus, Urinary bladder 107 FVYGGPQVQL
Melanoma 109 ILASVILNV Prostate 110 ILLTGTPAL SCLC, Leukocytes,
Breast 111 LLLAAARLAAA Liver, Pancreas 113 LMMSEDRISL Brain,
Melanoma 114 SLFPHNPQFI SCLC, Brain, Colon, Rectum, Liver,
Melanoma, Esophagus, Urinary bladder 115 SLMDPNKFLLL Kidney, Brain,
Colon, Rectum, Liver, Prostate, Melanoma, Urinary bladder,
Gallbladder, Bile duct 116 SMMDPNHFL Brain, Liver, MCC,
Endometrium, Gallbladder, Bile duct 117 SVDGVIKEV Stomach 118
TLWYRPPEL NSCLC, Melanoma, Esophagus 120 VLVNDFFLV Stomach, Colon,
Rectum, Liver, Ovary, Esophagus, Urinary bladder, Endometrium 121
YLDEDTIYHL Stomach 122 MQAPRAALVFA Brain, Leukocytes, Urinary
bladder, Gallbladder, Bile duct 123 KISTITPQI NSCLC, Liver,
Pancreas 124 ALFEESGLIRI NSCLC, SCLC, Colon, Rectum, Liver, MCC,
Melanoma, Ovary, Esophagus 125 ALLGKLDAINV NSCLC, SCLC, Colon,
Rectum, Liver, Ovary, Gallbladder, Bile duct 128 ALYDVRTILL NSCLC,
SCLC, Colon, Rectum 129 ALYEKDNTYL SCLC, Brain, Liver, Ovary,
Esophagus 130 FLFGEEPSKL Pancreas, Endometrium 131 FLIEEQKIVV
NSCLC, SCLC, Colon, Rectum, Liver, Melanoma, Ovary, Esophagus,
Urinary bladder, Gallbladder, Bile duct 132 FLWAGGRASYGV Liver,
Ovary, Esophagus 134 ILLAEGRLVNL Ovary 135 KLDDTYIKA Liver,
Esophagus, Urinary bladder
136 KLFPGFEIETV NSCLC, SCLC, Liver, Ovary, Esophagus 137 KLGPEGELL
Colon, Rectum, Liver, Breast, Esophagus, Urinary bladder 138
NIFPNPEATFV NSCLC, SCLC, Brain, Melanoma 142 SLYGYLRGA NSCLC,
Colon, Rectum, Liver, Pancreas, Prostate, Breast, Ovary, Esophagus,
Urinary bladder 143 TADPLDYRL SCLC, Endometrium 144 TAVALLRLL SCLC,
Leukocytes 145 TTFPRPVTV SCLC, Colon, Rectum, Leukocytes 146
VLISGVVHEI Brain, Liver, Melanoma, Ovary 147 YAFPKAVSV NSCLC, SCLC,
Kidney, Stomach, Leukocytes, Ovary, Esophagus 148 YLHNQGIGV SCLC,
Colon, Rectum, Liver, Esophagus 149 ILGTEDLIVEV NSCLC, SCLC, Liver,
Leukocytes, Melanoma, Ovary, Esophagus, Gallbladder, Bile duct 150
ALFQPHLINV NSCLC, SCLC, Liver, Leukocytes, Breast, Melanoma, Ovary,
Urinary bladder 151 ALLDIIRSL NSCLC, Brain, Colon, Rectum,
Prostate, Urinary bladder 152 ALLEPEFILKA Colon, Rectum,
Leukocytes, Urinary bladder 154 KVADLVLML NSCLC, Colon, Rectum,
Leukocytes, Ovary, Esophagus, Urinary bladder 155 LLLDPDTAVLKL
Liver, Melanoma 156 LLLPPPPCPA Pancreas, Urinary bladder 157
MLLEIPYMAA Colon, Rectum, Melanoma, Ovary, Urinary bladder 158
SLIEKYFSV NSCLC, SCLC, Colon, Rectum, Liver, Melanoma, Ovary,
Esophagus 159 SLLDLHTKV Brain, Colon, Rectum, Liver, Leukocytes 160
VLLPDERTISL NSCLC, SCLC, Liver, Leukocytes, Ovary, Urinary bladder
161 YLPDIIKDQKA Brain, Liver, Leukocytes, Melanoma 162 NADPQAVTM
SCLC, Kidney, Ovary, Endometrium 163 VMAPRTLVL SCLC 165 YLLSYIQSI
SCLC, Colon, Rectum, Liver, Melanoma, Ovary, Esophagus, Endometrium
166 SLFPGQVVI Brain, Urinary bladder, Endometrium 167 MLFGHPLLVSV
NSCLC, SCLC, Brain, Liver, Pancreas, Prostate, Ovary 169 FMLPDPQNI
NSCLC, SCLC, Brain, Liver, Breast, Melanoma, Esophagus, Urinary
bladder 170 ILAPAGSLPKI Urinary bladder 171 LLLDVTPLSL Leukocytes,
Urinary bladder 172 TMMSRPPVL Brain 174 TLDPRSFLL Stomach, Liver
175 ALLESSLRQA Kidney, Breast, Urinary bladder 176 YLMPGFIHL Liver,
Leukocytes
[0055] Table 4B: Peptides according to the present invention and
their specific uses in other proliferative diseases, especially in
other cancerous diseases (amendment of Table 4). The table shows,
like Table 4, for selected peptides on which additional tumor types
they were found showing over-presentation (including specific
presentation) on more than 5% of the measured tumor samples, or
presentation on more than 5% of the measured tumor samples with a
ratio of geometric means tumor vs normal tissues being larger than
3. Over-presentation is defined as higher presentation on the tumor
sample as compared to the normal sample with highest presentation.
Normal tissues against which over-presentation was tested were:
adipose tissue, adrenal gland, blood cells, blood vessel, bone
marrow, brain, esophagus, eye, gallbladder, heart, kidney, large
intestine, liver, lung, lymph node, nerve, pancreas, parathyroid
gland, peritoneum, pituitary, pleura, salivary gland, skeletal
muscle, skin, small intestine, spleen, stomach, thyroid gland,
trachea, ureter, urinary bladder.
TABLE-US-00005 SEQ ID NO. Sequence Additional Entities 1 FVDTRTLL
Melanoma, Urinary Bladder Cancer 3 ILIGETIKI OC, AML 4 ALDPAAQAFLL
SCLC, GC, CRC, CLL, Uterine Cancer, Gallbladder Cancer, Bile Duct
Cancer, AML, NHL 5 ALLTGIISKA Melanoma, Urinary Bladder Cancer,
Uterine Cancer 6 ALTGIPLPLI NSCLC, SCLC, CLL, Melanoma, Urinary
Bladder Cancer, Uterine Cancer, NHL 9 QIIDAINKV Melanoma, NHL, GC,
NSCLC 11 ALYYNPHLL Brain Cancer 12 AQYKFVYQV RCC, Melanoma, Urinary
Bladder Cancer, Uterine Cancer 14 FIIDNPQDLKV Brain Cancer, Urinary
Bladder Cancer, Uterine Cancer 15 FILANEHNV Urinary Bladder Cancer,
Uterine Cancer 16 GLIDYDTGI Melanoma 18 ALFVRLLAL Melanoma 19
ALWHDAENQT Brain Cancer, Urinary Bladder Cancer, Uterine VV Cancer
20 GLIDIENPNRV Esophageal Cancer 21 GLVDGRDLVIV NSCLC, Melanoma,
Gallbladder Cancer, Bile Duct Cancer, AML, NHL 22 ILSTEIFGV
Melanoma, Gallbladder Cancer, Bile Duct Cancer 23 KLDSSGGAVQL SCLC,
Melanoma 25 LINPNIATV Melanoma 28 VLDEFYSSL Melanoma 29 YILPFSEVL
BRCA, Melanoma, Uterine Cancer, AML, NHL 30 YIYKDTIQV RCC, Urinary
Bladder Cancer, Gallbladder Cancer, Bile Duct Cancer, AML 31
YLDSMYIML Melanoma, Esophageal Cancer, Urinary Bladder Cancer 32
YVDDGLISL Melanoma, AML 34 FLEDDDIAAV CRC 37 FLNPDEVHAI SCLC,
Uterine Cancer, NHL 38 FLTEAALGDA RCC, Urinary Bladder Cancer,
Uterine Cancer 39 FLTPSIFII Uterine Cancer 41 GLLAGNEKLTM GC,
Esophageal Cancer 42 ILSDMRSQYEV BRCA, Uterine Cancer, Gallbladder
Cancer, Bile Duct Cancer 44 ILAQVGFSV Melanoma 46 TMVEHNYYV Urinary
Bladder Cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct
Cancer 47 LIYKDLVSV OC 50 LLFGSDGYYV Uterine Cancer, Gallbladder
Cancer, Bile Duct Cancer 52 LLSDPIPEV Urinary Bladder Cancer, AML,
NHL 55 NLAPAPLNA Melanoma 56 NLIGVTAEL Melanoma, Uterine Cancer 57
RLSELGITQA Melanoma, Urinary Bladder Cancer, Uterine Cancer, AML,
NHL, OC 58 RQYPWGVVQV Melanoma 59 SLSESFFMV NHL 60 SLWEDYPHV BRCA,
Melanoma, Esophageal Cancer, Uterine Cancer 61 SMYDGLLQA Melanoma
65 TLNEKLTAL Melanoma, Urinary Bladder Cancer, AML 66 TVDDPYATFV
Melanoma 67 VIWGTDVNV Melanoma, Urinary Bladder Cancer, AML 68
VLFDVTGQV Melanoma 69 VLFSGSLRL BRCA, Esophageal Cancer,
Gallbladder Cancer, Bile Duct Cancer 70 VLGVIWGV Brain Cancer,
BRCA, Urinary Bladder Cancer, Uterine Cancer 71 VLLPEGGITAI Brain
Cancer, Urinary Bladder Cancer 74 YIDKDLEYV Urinary Bladder Cancer,
Uterine Cancer 75 FSFVDLRLL RCC, BRCA, Melanoma, NHL 77 RLFPGSSFL
GC 79 VVYEGQLISI Gallbladder Cancer, Bile Duct Cancer, NHL 80
LLPGTEYVVSV BRCA, Gallbladder Cancer, Bile Duct Cancer 82 ALIAEGIAL
BRCA, Uterine Cancer 83 ALSKEIYVI Brain Cancer AML, CLL, CRC, HCC,
Melanoma, NHL, OC, 84 FILPIGATV Esophageal Cancer, NSCLC, Urinary
Bladder Cancer, Uterine Cancer 86 GLGDFIFYSV NSCLC, BRCA,
Esophageal Cancer, Urinary Bladder Cancer 87 GLLPALVAL Brain
Cancer, Melanoma 88 IIDDTIFNL Melanoma 89 KLADIQIEQL Urinary
Bladder Cancer, OC 90 KLLTPITTL Melanoma, Gallbladder Cancer, Bile
Duct Cancer 91 LLFNDVQTL CLL, Uterine Cancer, NHL 92 YLTNEGIAHL
Urinary Bladder Cancer 93 SIDSEPALV Melanoma, AML 94 VMMEEFVQL
NSCLC, SCLC, Melanoma, Urinary Bladder Cancer 95 ALADDDFLTV RCC,
BRCA, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 96
ALAPATGGGSL NSCLC, Gallbladder Cancer, Bile Duct Cancer, NHL LL 97
ALDDMISTL Melanoma 99 ALESFLKQV NSCLC, RCC, Brain Cancer, CLL,
Melanoma, OC, Esophageal Cancer, AML, NHL 100 ALFGAGPASI Urinary
Bladder Cancer 101 ALVEENGIFEL Uterine Cancer 102 ALYPGTDYTV AML
103 AVAAVLTQV Esophageal Cancer, Urinary Bladder Cancer, Uterine
Cancer, Gallbladder Cancer, Bile Duct Cancer, AML 104 FLQPDLDSL
SCLC, Uterine Cancer 106 FVWSGTAEA Melanoma, Uterine Cancer, AML,
NHL 107 FVYGGPQVQL CLL, Urinary Bladder Cancer, NHL 108 IADGGFTEL
AML 109 ILASVILNV Urinary Bladder Cancer 110 ILLTGTPAL Uterine
Cancer 111 LLLAAARLAAA AML, PrC, BRCA, CRC, Gallbladder Cancer,
Bile Duct Cancer, Melanoma, NHL, OC, Brain Cancer, NSCLC, RCC,
SCLC, Urinary Bladder Cancer, Uterine Cancer 113 LMMSEDRISL NSCLC,
Urinary Bladder Cancer 114 SLFPHNPQFI NSCLC, CLL, AML, NHL 116
SMMDPNHFL NSCLC, Melanoma 117 SVDGVIKEV Melanoma, AML 118 TLWYRPPEL
CLL, Urinary Bladder Cancer, Uterine Cancer 120 VLVNDFFLV BRCA,
Melanoma, Gallbladder Cancer, Bile Duct Cancer, AML 121 YLDEDTIYHL
Melanoma 123 KISTITPQI Brain Cancer, Melanoma, Urinary Bladder
Cancer, Uterine Cancer, AML, NHL 124 ALFEESGLIRI BRCA, NHL 125
ALLGKLDAINV NHL 126 ALLSLDPAAV Brain Cancer, Urinary Bladder
Cancer, AML 127 ALSDLALHFL CLL, BRCA, Melanoma, Urinary Bladder
Cancer, AML, NHL 128 ALYDVRTILL BRCA, Urinary Bladder Cancer, AML
129 ALYEKDNTYL NSCLC, BRCA, Urinary Bladder Cancer, Uterine Cancer,
Gallbladder Cancer, Bile Duct Cancer, NHL 130 FLFGEEPSKL RCC, CLL,
Melanoma, Esophageal Cancer, Urinary Bladder Cancer, AML 131
FLIEEQKIVV AML, NHL 132 FLWAGGRASY Brain Cancer, Melanoma, Uterine
Cancer, AML GV 133 ILDDVSLTHL Melanoma 134 ILLAEGRLVNL NSCLC,
Melanoma 135 KLDDTYIKA Melanoma, Uterine Cancer 137 KLGPEGELL
Melanoma, AML 138 NIFPNPEATFV BRCA, Urinary Bladder Cancer, AML,
NHL, OC 139 SIDRNPPQL Melanoma, AML 140 SLLNPPETLNL AML 142
SLYGYLRGA CLL, Melanoma, Gallbladder Cancer, Bile Duct Cancer, AML
143 TADPLDYRL Melanoma, AML 144 TAVALLRLL BRCA, Gallbladder Cancer,
Bile Duct Cancer
145 TTFPRPVTV HCC, Gallbladder Cancer, Bile Duct Cancer 146
VLISGVVHEI CRC, Uterine Cancer 147 YAFPKAVSV Gallbladder Cancer,
Bile Duct Cancer 148 YLHNQGIGV Urinary Bladder Cancer, Uterine
Cancer, AML, NHL, OC 149 ILGTEDLIVEV PrC, BRCA, CRC, MCC, GC,
Urinary Bladder Cancer, Uterine Cancer 151 ALLDIIRSL BRCA, Uterine
Cancer, AML 152 ALLEPEFILKA NSCLC, Brain Cancer, Gallbladder
Cancer, Bile Duct Cancer 154 KVADLVLML Gallbladder Cancer, Bile
Duct Cancer 155 LLLDPDTAVLKL SCLC, CLL, BRCA 156 LLLPPPPCPA
Melanoma, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 157
MLLEIPYMAA Uterine Cancer 158 SLIEKYFSV CLL, BRCA, Urinary Bladder
Cancer, Uterine Cancer, AML, NHL 159 SLLDLHTKV NSCLC, Melanoma,
Urinary Bladder Cancer, Uterine Cancer 160 VLLPDERTISL BRCA, CRC,
Gallbladder Cancer, Bile Duct Cancer, Melanoma, Brain Cancer, GC,
RCC, Uterine Cancer 161 YLPDIIKDQKA Uterine Cancer NSCLC =
non-small cell lung cancer, SCLC = small cell lung cancer, RCC =
kidney cancer, CRC = colon or rectum cancer, GC = stomach cancer,
HCC = liver cancer, PrC = prostate cancer, BRCA = breast cancer,
MCC = Merkel cell carcinoma, OC = ovarian cancer, NHL = non-Hodgkin
lymphoma, AML = acute myeloid leukemia, CLL = chronic lymphocytic
leukemia.
[0056] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 4, 5, 8, 14, 19, 22, 29, 30, 31,
35, 37, 46, 60, 69, 70, 79, 85, 90, 92, 95, 101, 102, 118, 123,
124, 125, 128, 131, 136, 138, 142, 147, 149, 150, 151, 154, 158,
160, 167, 6, 9, 21, 84, 85, 94, 96, 99, 111, 113, 114, 116, 129,
134, 152, 159, and 169 for the--in one preferred embodiment
combined--treatment of non-small cell lung cancer (NSCLC).
[0057] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 14, 19, 35, 46, 52, 59, 60, 62,
64, 75, 79, 80, 81, 90, 95, 102, 110, 114, 124, 125, 128, 129, 131,
136, 138, 143, 144, 145, 147, 148, 149, 150, 158, 160, 162, 163,
165, 167, 169, 4, 6, 23, 37, 94, 104, and 155 for the--in one
preferred embodiment combined--treatment of small cell lung cancer
(SCLC).
[0058] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 13, 14, 29, 46, 84, 115, 147,
162, 175, 12, 30, 38, 75, 95, 99, 111, 130, and 160 for the--in one
preferred embodiment combined--treatment of kidney cancer.
[0059] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 16, 17, 29, 34, 39, 63, 67, 81,
93, 94, 98, 102, 104, 106, 113, 114, 115, 116, 122, 129, 138, 146,
151, 159, 161, 166, 167, 169, 172, 11, 14, 19, 70, 71, 83, 87, 99,
112, 123, 126, 132, 152, and 160 for the--in one preferred
embodiment combined--treatment of brain cancer.
[0060] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 31, 32, 68, 84, 88, 95, 97, 117,
120, 121, 147, 174, 4, 9, 41, 77, 149, and 160 for the--in one
preferred embodiment combined--treatment of stomach cancer.
[0061] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 5, 28, 29, 30, 31, 37, 40, 41,
60, 85, 90, 92, 93, 94, 99, 114, 115, 120, 124, 125, 128, 131, 137,
142, 145, 148, 151, 152, 154, 157, 158, 159, 165, 4, 34, 84, 111,
146, 149, and 160 for the--in one preferred embodiment
combined--treatment of colon and rectal cancer.
[0062] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 4, 5, 14, 19, 31, 35, 37, 48,
50, 51, 60, 64, 70, 73, 80, 85, 86, 96, 99, 100, 101, 102, 103,
104, 111, 114, 115, 116, 120, 123, 124, 125, 129, 131, 132, 135,
136, 137, 142, 145, 146, 148, 149, 150, 155, 158, 159, 160, 161,
165, 167, 169, 174, and 176 for the--in one preferred embodiment
combined--treatment of liver cancer.
[0063] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 2, 8, 10, 22, 31, 39, 46, 79,
86, 104, 111, 123, 130, 142, 156, and 167 for the--in one preferred
embodiment combined--treatment of pancreatic cancer.
[0064] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 98, 102, 109, 111, 115, 142,
148, 151, and 167 for the--in one preferred embodiment
combined--treatment of prostate cancer.
[0065] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 7, 22, 48, 62, 71, 81, 83, 94,
95, 104, 110, 122, 144, 145, 147, 149, 150, 152, 154, 159, 160,
161, 171, and 176 for the--in one preferred embodiment
combined--treatment of leukemia.
[0066] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 3, 4, 21, 29, 30, 32, 52, 57,
65, 67, 84, 93, 99, 102, 103, 106, 108, 111, 114, 117, 120, 123,
126, 127, 128, 139, 140, 142, 143, 148, 151, and 158 for the--in
one preferred embodiment combined--treatment of AML.
[0067] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 4, 6, 84, 91, 99, 107, 114, 118,
127, 130, 142, 155, and 158 for the--in one preferred embodiment
combined--treatment of CLL.
[0068] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 2, 4, 8, 9, 16, 22, 26, 31, 35,
37, 41, 59, 77, 79, 84, 90, 93, 99, 110, 137, 142, 150, 169, 175,
29, 42, 60, 69, 70, 75, 80, 82, 86, 95, 111, 120, 124, 127, 128,
129, 138, 144, 149, 151, 155, 158, and 160 for the--in one
preferred embodiment combined--treatment of breast cancer.
[0069] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 149, 81, 101, 116, and 124 for
the--in one preferred embodiment combined--treatment of Merkel cell
carcinoma (MCC).
[0070] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 14, 17, 19, 34, 37, 45, 46, 52,
85, 92, 95, 96, 107, 113, 114, 115, 118, 124, 131, 138, 146, 149,
150, 155, 157, 158, 161, 165, 169, 1, 5, 6, 9, 12, 16, 18, 21, 22,
23, 25, 28, 29, 31, 32, 44, 55, 56, 57, 58, 60, 61, 65, 66, 67, 68,
75, 84, 87, 88, 90, 93, 94, 97, 99, 106, 111, 116, 117, 120, 121,
123, 127, 128, 129, 130, 132, 133, 134, 135, 137, 139, 142, 143,
156, 159, and 160 for the--in one preferred embodiment
combined--treatment of melanoma.
[0071] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 4, 14, 35, 37, 46, 52, 60, 70,
81, 85, 92, 94, 95, 101, 120, 124, 125, 129, 131, 132, 134, 136,
142, 146, 147, 149, 150, 154, 157, 158, 160, 162, 165, 167, 3, 47,
57, 84, 89, 99, 111, 138, and 148 for the--in one preferred
embodiment combined--treatment of ovarian cancer.
[0072] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 1, 2, 4, 5, 8, 9, 11, 12, 14,
19, 29, 35, 37, 40, 46, 50, 51, 52, 57, 58, 62, 63, 67, 70, 75, 77,
79, 85, 91, 92, 94, 95, 106, 114, 118, 120, 124, 129, 131, 132,
135, 136, 137, 142, 147, 148, 149, 154, 158, 165, 169, 1, 2, 4, 5,
8, 9, 11, 12, 14, 19, 29, 35, 37, 40, 46, 50, 51, 52, 57, 58, 62,
63, 67, 70, 75, 77, 79, 85, 91, 92, 94, 95, 106, 114, 118, 120,
124, 129, 131, 132, 135, 136, 137, 142, 147, 148, 149, 154, 158,
165, and 169 for the--in one preferred embodiment
combined--treatment of esophageal cancer.
[0073] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 3, 4, 20, 29, 37, 41, 42, 59,
60, 64, 82, 88, 91, 93, 95, 97, 99, 101, 104, 106, 114, 115, 120,
122, 131, 135, 137, 142, 150, 151, 152, 154, 156, 157, 160, 166,
169, 170, 171, 175, 1, 5, 6, 12, 14, 15, 19, 30, 31, 38, 46, 52,
57, 65, 67, 70, 71, 74, 84, 86, 89, 92, 94, 100, 103, 107, 109,
111, 113, 118, 123, 126, 127, 128, 129, 130, 138, 148, 149, 158,
and 159 for the--in one preferred embodiment combined--treatment of
urinary bladder cancer.
[0074] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 41, 85, 94, 116, 120, 130, 143,
162, 165, and 166 for the--in one preferred embodiment
combined--treatment of endometrial cancer.
[0075] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 8, 10, 19, 31, 73, 75, 94, 102,
115, 116, 122, 125, 131, 149, 4, 21, 22, 30, 46, 50, 69, 70, 80,
90, 95, 96, 103, 111, 120, 129, 142, 144, 145, 147, 152, 154, 156,
and 160 for the--in one preferred embodiment combined--treatment of
gall bladder and bile duct cancer.
[0076] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 4, 5, 6, 12, 14, 15, 19, 29, 37,
38, 39, 42, 46, 50, 56, 57, 60, 70, 74, 82, 84, 91, 95, 101, 103,
104, 106, 110, 111, 118, 123, 129, 132, 135, 146, 148, 149, 151,
156, 157, 158, 159, 160, and 161 for the--in one preferred
embodiment combined--treatment of uterine cancer.
[0077] Thus, another aspect of the present invention relates to the
use of the peptides according to the present invention for
the--preferably combined--treatment of a proliferative disease
selected from the group of pancreatic cancer, lung cancer, kidney
cancer, brain cancer, stomach cancer, colon or rectal cancer, liver
cancer, prostate cancer, leukemia, breast cancer, Merkel cell
carcinoma (MCC), melanoma, ovarian cancer, esophageal cancer,
urinary bladder cancer, endometrial cancer, gall bladder cancer,
and bile duct cancer.
[0078] The present invention furthermore relates to peptides
according to the present invention that have the ability to bind to
a molecule of the human major histocompatibility complex (MHC)
Class-I or--in an elongated form, such as a length-variant--MHC
class-II.
[0079] The present invention further relates to the peptides
according to the present invention wherein said peptides (each)
consist or consist essentially of an amino acid sequence according
to SEQ ID NO: 1 to SEQ ID NO: 161.
[0080] The present invention further relates to the peptides
according to the present invention, wherein said peptide is
modified and/or includes non-peptide bonds.
[0081] The present invention further relates to the peptides
according to the present invention, wherein said peptide is part of
a fusion protein, in particular fused to the N-terminal amino acids
of the HLA-DR antigen-associated invariant chain (Ii), or fused to
(or into the sequence of) an antibody, such as, for example, an
antibody that is specific for dendritic cells.
[0082] The present invention further relates to a nucleic acid,
encoding the peptides according to the present invention. The
present invention further relates to the nucleic acid according to
the present invention that is DNA, cDNA, PNA, RNA or combinations
thereof.
[0083] The present invention further relates to an expression
vector capable of expressing and/or expressing a nucleic acid
according to the present invention.
[0084] The present invention further relates to a peptide according
to the present invention, a nucleic acid according to the present
invention or an expression vector according to the present
invention for use in the treatment of diseases and in medicine, in
particular in the treatment of cancer.
[0085] The present invention further relates to antibodies that are
specific against the peptides according to the present invention or
complexes of said peptides according to the present invention with
MHC, and methods of making these.
[0086] The present invention further relates to T-cell receptors
(TCRs), in particular soluble TCR (sTCRs) and cloned TCRs
engineered into autologous or allogeneic T cells, and methods of
making these, as well as NK cells or other cells bearing said TCR
or cross-reacting with said TCRs.
[0087] The antibodies and TCRs are additional embodiments of the
immunotherapeutic use of the peptides according to the invention at
hand.
[0088] The present invention further relates to a host cell
comprising a nucleic acid according to the present invention or an
expression vector as described before. The present invention
further relates to the host cell according to the present invention
that is an antigen presenting cell, and preferably is a dendritic
cell.
[0089] The present invention further relates to a method for
producing a peptide according to the present invention, said method
comprising culturing the host cell according to the present
invention, and isolating the peptide from said host cell or its
culture medium.
[0090] The present invention further relates to said method
according to the present invention, wherein the antigen is loaded
onto class I or II MHC molecules expressed on the surface of a
suitable antigen-presenting cell or artificial antigen-presenting
cell by contacting a sufficient amount of the antigen with an
antigen-presenting cell.
[0091] The present invention further relates to the method
according to the present invention, wherein the antigen-presenting
cell comprises an expression vector capable of expressing or
expressing said peptide containing SEQ ID No. 1 to SEQ ID No.: 161,
preferably containing SEQ ID No. 1 to SEQ ID No. 79, or a variant
amino acid sequence.
[0092] The present invention further relates to activated T cells,
produced by the method according to the present invention, wherein
said T cell selectively recognizes a cell which expresses a
polypeptide comprising an amino acid sequence according to the
present invention.
[0093] The present invention further relates to a method of killing
target cells in a patient which target cells aberrantly express a
polypeptide comprising any amino acid sequence according to the
present invention, the method comprising administering to the
patient an effective number of T cells as produced according to the
present invention.
[0094] The present invention further relates to the use of any
peptide as described, the nucleic acid according to the present
invention, the expression vector according to the present
invention, the cell according to the present invention, the
activated T lymphocyte, the T cell receptor or the antibody or
other peptide- and/or peptide-MHC-binding molecules according to
the present invention as a medicament or in the manufacture of a
medicament. Preferably, said medicament is active against
cancer.
[0095] Preferably, said medicament is a cellular therapy, a vaccine
or a protein based on a soluble TCR or antibody.
[0096] The present invention further relates to a use according to
the present invention, wherein said cancer cells are pancreatic
cancer, lung cancer, kidney cancer, brain cancer, stomach cancer,
colon or rectal cancer, liver cancer, prostate cancer, leukemia,
breast cancer, Merkel cell carcinoma (MCC), melanoma, ovarian
cancer, esophageal cancer, urinary bladder cancer, endometrial
cancer, gall bladder cancer, bile duct cancer, and preferably
pancreatic cancer cells.
[0097] The present invention further relates to biomarkers based on
the peptides according to the present invention, herein called
"targets" that can be used in the diagnosis of cancer, preferably
pancreatic cancer. The marker can be over-presentation of the
peptide(s) themselves, or over-expression of the corresponding
gene(s). The markers may also be used to predict the probability of
success of a treatment, preferably an immunotherapy, and most
preferred an immunotherapy targeting the same target that is
identified by the biomarker. For example, an antibody or soluble
TCR can be used to stain sections of the tumor to detect the
presence of a peptide of interest in complex with MHC.
[0098] Optionally, the antibody carries a further effector function
such as an immune stimulating domain or toxin.
[0099] The present invention also relates to the use of these novel
targets in the context of cancer treatment.
[0100] AAGAB encodes a protein that interacts with the
gamma-adaptin and alpha-adaptin subunits of complexes involved in
clathrin-coated vesicle trafficking. Mutations in this gene are
associated with type I punctate palmoplantar keratoderma (RefSeq,
2002). AAGAB is a target of miR-205, which is over-expressed in
cervical cancer (Xie et al., 2012). Knock-down of AAGAB leads to
increased cell division and proliferation (Pohler et al.,
2012).
[0101] ACTR2 encodes ARP2 actin-related protein 2 homolog, a major
constituent of the ARP2/3 complex. This complex is essential for
cell shape and motility through lamellipodial actin assembly and
protrusion (RefSeq, 2002). ARP2/3 in complex with other proteins
was shown to play a critical role in cancer cell invasion and
migration (Nurnberg et al., 2011; Feldner and Brandt, 2002;
Frugtniet et al., 2015; Kurisu and Takenawa, 2010; Kirkbride et
al., 2011). The ARP2/3 complex with WASP/WAVE protein family
members contributes to cell invasion and migration in breast cancer
(Frugtniet et al., 2015). The ARP2/3 complex with ArgBP2 is endowed
with an anti-tumoral function, when the adhesion and migration of
pancreatic cancer cells is regulated (Roignot and Soubeyran,
2009).
[0102] ADAM9 encodes one member of the ADAM (a disintegrin and
metalloprotease domain) family (member 9). Members of this family
take part in the cell-cell and cell-matrix interactions (RefSeq,
2002). ADAM9 gene silencing reduces esophageal squamous cell
carcinoma (ESCC) cancer proliferation (Liu et al., 2015b). ADAM9
plays an important role in melanoma proliferation and invasion
(Ebrahimi et al., 2014). ADAM9 was shown to be up-regulated in
osteosarcoma cells, muscle invasive (MI) bladder cancer cells,
non-small cell lung cancer, pancreatic cancer, colon cancer, oral
squamous cell carcinoma, cervical cancer, prostate cancer, renal
cancer, gastric cancer, lymph node cancer, and breast cancer
(Shaker et al., 2011; Vincent-Chong et al., 2013; Li et al., 2013;
Ebrahimi et al., 2014; Zhang et al., 2014a; Jia et al., 2014;
O'Shea et al., 2003; Jiang et al., 2014a; Zubel et al., 2009).
ADAM9 has been implicated in lung cancer metastasis to the brain
(Sher et al., 2014; Lin et al., 2014a; Shintani et al., 2004).
[0103] AGAP9 encodes ArfGAP with GTPase domain, Ankyrin repeat and
PH domain 9 and is located on chromosome 10811.22 (RefSeq,
2002).
[0104] AHCY encodes adenosylhomocysteinase. It regulates the
intracellular S-adenosylhomocysteine (SAH) concentration thought to
be important for transmethylation reactions (RefSeq, 2002). AHCY
down-regulation contributes to tumorigenesis (Leal et al., 2008).
AHCY can promote apoptosis. It inhibits migration and adhesion of
esophageal squamous cell carcinoma cells suggesting a role in
carcinogenesis of the esophagus (Li et al., 2014b). AHCY protein
expression is up-regulated in colon cancer (Kim et al., 2009;
Watanabe et al., 2008; Fan et al., 2011). AHCY may be a potential
biomarker in ovarian cancer (Peters et al., 2005).
[0105] AK2 encodes adenylate kinase 2. AK2 is localized in the
mitochondrial intermembrane space and may play a role in apoptosis
(RefSeq, 2002). AK2 mediates a novel intrinsic apoptotic pathway
that may be involved in tumorigenesis (Lee et al., 2007).
[0106] ANKLE2 encodes Ankyrin repeat and LEM domain containing 2.
ANKLE2 is a member of the LEM family of inner nuclear membrane
proteins. The encoded protein functions as a mitotic regulator
through post-mitotic formation of the nuclear envelope (RefSeq,
2002).
[0107] ANKRD1 encodes Ankyrin repeat domain-1. It is localized to
the nucleus of endothelial cells and is induced by IL-1 and
TNF-alpha stimulation. Interactions between this protein and the
sarcometric proteins myopalladin and titin suggest that it may also
be involved in the myofibrillar stretch-sensor system (RefSeq,
2002). The ectopic expression of ANKRD1 leads to reduced colony
formation and to enhanced apoptotic cell death in hepatoma cells
(Park et al., 2005). High expression of ANKRD1 in ovarian carcinoma
is associated with poor survival (Lei et al., 2015).
[0108] ANLN encodes an actin-binding protein that plays a role in
cell growth and migration, and in cytokinesis. ANLN is thought to
regulate actin cytoskeletal dynamics in podocytes, components of
the glomerulus. Mutations in this gene are associated with focal
segmental glomerulosclerosis 8 (RefSeq, 2002). ANLN was found to be
highly expressed in breast cancer tissues as well as head and neck
squamous cell carcinomas. Knock-down of ANLN remarkably inhibited
the proliferation rate, colony formation ability and migration of
breast cancer cells (Zhou et al., 2015b). ANLN is over-expressed in
proliferative gastric tumors, pancreatic carcinoma and
hormone-refractory prostate cancers (Pandi et al., 2014; Tamura et
al., 2007; Shimizu et al., 2007; Olakowski et al., 2009). ANLN is a
biomarker for hepatocellular carcinoma (Kim et al., 2013a). ANLN
expression is a marker of favorable prognosis in patients with
renal cell carcinoma (Ronkainen et al., 2011).
[0109] APOL6 encodes apolipoprotein L, 6. APOL6 is a member of the
apolipoprotein L gene family. The encoded protein is found in the
cytoplasm, where it may affect the movement of lipids or allow the
binding of lipids to organelles (RefSeq, 2002). APOL6 induces
mitochondria-mediated apoptosis in cancer cells (Liu et al.,
2005).
[0110] ARMC9 (also called KU-MEL-1) encodes an armadillo
repeat-containing protein that was a previously isolated melanoma
antigen preferentially expressed in melanocytes. It is associated
with Vogt-Koyanagi-Harada disease (Otani et al., 2006). ARMC9 is
strongly expressed in melanoma cell lines and tissue samples.
Antigens against ARMC9 were detected in the sera of patients
treated against brain, colon and esophageal cancer (Kiniwa et al.,
2001).
[0111] ASNS encodes asparagine synthetase. The ASNS gene
complements a mutation in the temperature-sensitive hamster mutant
ts11, which blocks progression through the G1 phase of the cell
cycle at non-permissive temperature (RefSeq, 2002). ASNS expression
is induced by glucose deprivation and protects pancreatic cancer
cells from apoptosis (Cui et al., 2007). ASNS is associated with
drug resistance in leukemia and uterine cancer (Lin et al., 2012;
Zhang et al., 2013a). Knock-down of ASNS in A375 cells
down-regulates the expression levels of CDK4, CDK6, and cyclin D1
and up-regulates the expression of p21 (Li et al., 2015a).
Down-regulation of ASNS induces cell cycle arrest and inhibits cell
proliferation of breast cancer (Yang et al., 2014a). ASNS is highly
expressed in gliomas (Panosyan et al., 2014). ASNS is a potential
biomarker in ovarian cancer (Lorenzi et al., 2006; Lorenzi et al.,
2008; Lorenzi and Weinstein, 2009).
[0112] ATP5F1 encodes ATP synthase, H+ transporting, mitochondrial
F0 complex, subunit B1, a subunit of mitochondrial ATP synthase
(RefSeq, 2002). ATP5F1 is up-regulated in hepatitis B
virus-associated hepatocellular carcinoma (Lee et al., 2008a).
[0113] BMS1 encodes BMS1 ribosome biogenesis factor and is located
on chromosome 10811.21. A similar protein in yeast functions in
35S-rRNA processing, which includes a series of cleavage steps
critical for formation of 40S ribosomes (RefSeq, 2002;
Perez-Fernandez et al., 2011).
[0114] BMS1P5 encodes BMS1 ribosome biogenesis factor pseudogene 5
and is located on chromosome 10q11.22 (RefSeq, 2002).
[0115] BRK1 (also called C3orf10 or HSPC300) encodes the smallest
subunit of the Wave complex and is an important regulator of the
Wave/Scar pathway involved in actin cytoskeleton dynamics during
embryonic development and cell transformation (Derivery et al.,
2008; Escobar et al., 2010). BRK1 has oncogenic potential in
different cancer types including lung cancer and renal cell
carcinomas (Cascon et al., 2007; Cai et al., 2009; Escobar et al.,
2010). BRK1 is regulated by the transcription factors Sp1 and
NRF-1. It is involved in the Wave/Scar pathway following Arp2/3
regulation and required for cell proliferation and transformation
(Li et al., 2014a; van't Veer et al., 2006; Escobar et al., 2010;
Wang et al., 2013c).
[0116] BTBD1 encodes BTB (POZ) domain containing 1. The C-terminus
of the protein binds topoisomerase I. The N-terminus contains
proline rich region and a BTB/POZ domain, both of which are
typically involved in protein-protein interactions (RefSeq,
2002).
[0117] BUB1B encodes a kinase involved in spindle checkpoint
function. The protein is localized to the kinetochore and plays a
role in the inhibition of the anaphase-promoting complex/cyclosome
(APC/C), delaying the onset of anaphase and ensuring proper
chromosome segregation. Impaired spindle checkpoint has been found
in many forms of cancer (RefSeq, 2002). BUB1B is a tumor inhibitory
protein. BUB1B regulates the spindle assembly checkpoint. BUB1B is
inactivated or down-regulated in tumors. Mutations in BUB1B are
also linked to tumor development (Aylon and Oren, 2011; Fagin,
2002; Malumbres and Barbacid, 2007; Rao et al., 2009). BUB1B is
associated with gastric carcinogenesis through oncogenic activation
(Resende et al., 2010). BUB1B mutation is one of the causes for
colorectal cancer (Karess et al., 2013; Grady, 2004).
[0118] C11orf70 encodes a protein with uncharacterized function,
but is linked to the binding of a mutated protein that causes
amyotrophic lateral sclerosis (Wang et al., 2015i). C11orf70 is
down-regulated in testicular germ cell tumors in comparison to
normal testis tissue (Gonzalez-Exposito et al., 2015; Alagaratnam
et al., 2009). The genetic region of C11orf70 displays DNA copy
number aberrations in oral squamous cell carcinomas, which is
associated with oral cancer-specific mortality (Chen et al.,
2015a).
[0119] C11 orf80 encodes chromosome 11 open reading frame 80 and is
located on chromosome 11q13.2 (RefSeq, 2002).
[0120] C1orf198 encodes chromosome 1 open reading frame 198 and is
located on chromosome 1q42.2 (RefSeq, 2002).
[0121] C20orf24 encodes chromosome 20 open reading frame 24 and is
located on chromosome 20q11.23 (RefSeq, 2002). C20orf24 plays an
important role in chromosomal instability-related progression from
adenoma to carcinoma. C20orf24 is significantly over-expressed in
carcinomas compared with adenomas. C20orf24 may serve as a highly
specific biomarker for colorectal cancer (Carvalho et al.,
2009).
[0122] CAD encodes for trifunctional protein carbamoylphosphate
synthetase 2, aspartate transcarbamylase, and dihydroorotase, which
catalyzes the first three reactions of the pyrimidine biosynthesis
pathway (RefSeq, 2002). CAD activity is increased in different
cancer types, including hepatomas, sarcomas and kidney
adenocarcinomas and is very frequently associated with the
amplification of the CAD gene (Smith et al., 1990; Aoki and Weber,
1981; Smith et al., 1997). CAD is a target of different oncogenes
and tumorigenesis regulating pathways like MAPK, mTORC1 and c-Myc
(Mac and Farnham, 2000; Graves et al., 2000; Sharma et al., 2014).
CAD promotes androgen receptor translocation into the nucleus and
stimulates its transcriptional activity in prostate tumor cells.
After radical prostatectomy a higher CAD mRNA level is associated
with local tumor extension and cancer relapse (Morin et al.,
2012).
[0123] CARM1 encodes coactivator-associated arginine
methyltransferase 1. CARM1 belongs to the protein arginine
methyltransferase (PRMT) family. The encoded enzyme catalyzes the
methylation of guanidine nitrogens of arginyl residues of proteins.
The enzyme is involved in gene expression (RefSeq, 2002). CARM1 has
shown to be dysregulated in colorectal and prostate cancer,
melanoma and breast cancer. CARM1 is over-expressed not only in
prostate tumors, but also in prostatic intraepithelial neoplasia
(PIN). CARM1 is significantly over-expressed in non-small cell lung
carcinomas (NSCLC). CARM1 expression is elevated in adenomas and
aberrant in carcinomas during hepatocellular carcinogenesis (Limm
et al., 2013; Osada et al., 2013; Elakoum et al., 2014; Baldwin et
al., 2014). CARM1 methylates chromatin remodeling factor BAF155 to
enhance tumor progression and metastasis (Wang et al., 2014a;
Stefansson and Esteller, 2014).
[0124] CCNA2 encodes cyclin A2, a member of the highly conserved
cyclin family. CCNA2 binds and activates CDC2 or CDK2 kinases, and
thus promotes both cell cycle G1/S and G2/M transitions (RefSeq,
2002). Over-expression of CCNA2 inhibits the proliferation of
hepatocellular carcinoma cells. Over-expression of CCNA2 in
endometrial adenocarcinoma cells decreases cell growth and
increases apoptosis. CCNA2 expression in melanoma cells reduces
tumor growth and metastasis and concomitantly increases apoptosis
in tumors (Lau, 2011). CCNA2 can promote cancer cell proliferation,
invasion, adhesion, differentiation, survival and metastasis. It
plays an important role in angiogenesis and extracellular matrix
production. CCNA2 promotes tumor growth and increases tumor
vascularization when over-expressed in gastric adenocarcinoma
cells. Silencing of CCNA2 expression decreases tumor growth in
pancreatic cancer cells.
[0125] CCNA2 can promote the proliferation of prostate cancer cells
(Lau, 2011; Chen and Du, 2007). CCNA2 over-expression induces
epithelial-mesenchymal transition, leading to laryngeal tumor
invasion and metastasis (Liu et al., 2015e). CCNA2 is dysregulated
in colorectal cancer (Chang et al., 2014). CCNA2 is over-expressed
in prostate cancer, gliomas, pancreatic cancer, and breast cancer.
CCNA2 is associated with increased aggressiveness, vascularization,
and estrogen independence in breast cancer, suggesting a major role
of CCNA2 in breast cancer progression (Zuo et al., 2010).
[0126] CCND1 encodes cyclin D1. It belongs to the highly conserved
cyclin family, whose members are characterized by a dramatic
periodicity in protein abundance throughout the cell cycle.
Mutations, amplifications and over-expression of CCND1, which
alters cell cycle progression, are observed frequently in a variety
of tumors and may contribute to tumorigenesis (RefSeq, 2002). CCND1
is amplified and over-expressed in cases of lymph node metastasis
in oral squamous cell carcinoma, gastrointestinal stromal tumor,
non-small cell lung cancer, pituitary tumors and breast cancer
(Noorlag et al., 2015; Dworakowska, 2005; Gautschi et al., 2007;
Lambros et al., 2007; Yang et al., 2008; Yu and Melmed, 2001).
CCND1 is over-expressed in mantle cell lymphoma, pancreatic
neuroendocrine tumors, parathyroid adenoma, and Ewing sarcoma
(Navarro et al., 2011; Sander, 2011; Capurso et al., 2012; Delas et
al., 2013; Setoodeh et al., 2013; Sanchez et al., 2008; Westin et
al., 2009). CCND1 can increase colorectal cancer risk (Yang et al.,
2012b; Andersen et al., 2013). CCND1 genetic alterations can cause
bladder cancer (Zhang et al., 2003; Baffa et al., 2006).
[0127] CCT3 encodes chaperonin containing TCP1, subunit 3 (gamma),
a molecular chaperone (RefSeq, 2002). CCT3 is elevated in
hepatocellular carcinoma (Midorikawa et al., 2002; Skawran et al.,
2008). CCT3 is a potentially novel biomarker for ovarian cancer
(Peters et al., 2005).
[0128] CCT4 encodes chaperonin containing TCP1, subunit 4. CCT4
assists the folding of newly translated polypeptide substrates
through multiple rounds of ATP-driven release and rebinding of
partially folded intermediate forms (RefSeq, 2002). CCT4
deregulation causes esophageal squamous cell carcinoma and lung
adenocarcinoma (Wang et al., 2015j; Tano et al., 2010). CCT4 is
upregulated in gastric cancers (Malta-Vacas et al., 2009).
[0129] CDC27 encodes cell division cycle 27. The protein encoded by
this gene is a component of the anaphase-promoting complex (APC).
The protein may be involved in controlling the timing of mitosis
(RefSeq, 2002). CDC27 confers increased radio-resistance of triple
negative breast cancer cells and squamous cell cervix carcinoma,
when it is down-regulated (Rajkumar et al., 2005; Ren et al.,
2015). CDC27 plays a crucial role in the progression of
hepatocellular carcinoma, and also correlates with poor prognosis
in esophageal squamous cell carcinoma and pancreatic cancer (Ahn et
al., 2014; Wang et al., 2015h). CDC27 polymorphisms may contribute
to the susceptibility of breast cancer through influencing the
mitotic progression of cells (Guo et al., 2015). CDC27 mutation is
involved in prostate cancer (Lindberg et al., 2013). CDC27 mutation
and down-regulation is involved in several breast and colon
carcinoma cell lines (Fan et al., 2004; Roy et al., 2010; Pawar et
al., 2010).
[0130] CDK12 encodes cyclin dependent kinase 12 and is located on
chromosome 17q12 (RefSeq, 2002). CDK12 mutations were identified in
a variety of tumors, including ovarian, breast, prostate, and
intestinal tumors (Vrabel et al., 2014).
[0131] CDK13 encodes cyclin dependent kinase 13, a member of the
cyclin dependent serine/threonine protein kinase family. Members of
this family are known for their essential roles as master switches
in cell cycle control. They may play a role in mRNA processing and
may be involved in regulation of hematopoiesis (RefSeq, 2002).
CDK13 is associated with pancreatic cancer and skin cancer (Ansari
et al., 2015; Nelson et al., 1999; Chandramouli et al., 2007).
CDK13 is amplified in hepatocellular carcinoma (Kim et al.,
2012b).
[0132] CDK2 encodes cyclin dependent kinase 2, a serine/threonine
protein kinase that participates in cell cycle regulation. Activity
of this protein is especially critical during the G1 to S phase
transition (RefSeq, 2002). CDK2 over-expression indicates the
abnormal regulation of cell cycle, which would be directly related
to hyper-proliferation in cancer cells (Chohan et al., 2015). CDK2
is associated with leukemia, colorectal carcinoma, melanoma, human
papillomavirus-associated cervical neoplasia, lung cancer, breast
cancer and prostate cancer (Foster et al., 2001; Zajac-Kaye, 2001;
Raso et al., 2013; He et al., 2013; Duensing and Munger, 2002; Hu
and Zuckerman, 2014; Agarwal, 2000). CDK2 is highly-expressed in
mantle cell lymphoma (Rummel et al., 2004).
[0133] CDK5RAP3 encodes CDK5 regulatory subunit associated protein
3. CDK5RAP3 plays a role in signaling pathways governing
transcriptional regulation and cell cycle progression. It may have
a function in tumorigenesis and metastasis (RefSeq, 2002). CDK5RAP3
is over-expressed in hepatocellular carcinoma and promotes
metastasis (Mak et al., 2011; Mak et al., 2012).
[0134] CDK7 encodes cyclin dependent kinase 7, a member of the
cyclin dependent protein kinase family. It is an essential
component of the transcription factor TFIIH, which is involved in
transcription initiation and DNA repair. This protein is thought to
serve as a direct link between the regulation of transcription and
the cell cycle (RefSeq, 2002). CDK7 genetic polymorphisms
predispose individuals to breast cancer by gene-environment or
gene-gene interactions (Yoo and Kang, 2003). CDK7 is associated
with an increased risk for pancreatic cancer (Efthimiou et al.,
2001). CDK7 has been associated with breast cancer (Cance and Liu,
1995).
[0135] CDK9 encodes cyclin dependent kinase 9, a member of the
cyclin dependent protein kinase family. This protein forms a
complex with and is regulated by its regulatory subunit cyclin T or
cyclin K (RefSeq, 2002). CDK9 appears to be involved in the
differentiation program of several cell types, such as muscle
cells, monocytes and neurons. CDK9 seems to have an anti-apoptotic
function in monocytes. Involvement of CDK9 in several physiological
processes in the cell may lead to the onset of cancer (De and
Giordano, 2002).
[0136] CELSR3 encodes cadherin, EGF LAG seven-pass G-type receptor
3. The encoded protein may be involved in the regulation of contact
dependent neurite growth and may play a role in tumor formation
(RefSeq, 2002). Microarray screening revealed that CELSR3
hyper-methylated in primary oral squamous cell carcinoma compared
to normal oral mucosa (Khor et al., 2014). CELSR3 is associated
with ovarian cancer and brain tumors (Asad et al., 2014; Katoh and
Katoh, 2007). CELSR3 is up-regulated in pancreatic and hepatic
tumor stellate cells (Erkan et al., 2010).
[0137] CEP97 encodes centrosomal protein 97 kDa and is located on
chromosome 3q12.3 (RefSeq, 2002). CEP97 is associated with breast
cancer (Rappa et al., 2014).
[0138] CFL1 encodes cofilin 1. It is involved in the translocation
of the actin-cofilin complex from cytoplasm to nucleus (RefSeq,
2002). CFL1 mutation is associated with multiple endocrine
neoplasia type 4 and glioblastoma multiforme (Solomon et al., 2008;
Georgitsi, 2010). CFL1 is over-expressed in lymphoma, leukemia,
neuroblastoma, ovarian, prostate, breast and lung cancers and
mesothelioma (Rana et al., 2008). CFL1 is down-regulated in
testicular germ cell tumors (von Eyben, 2004).
[0139] CHD3 encodes chromodomain helicase DNA binding protein 3.
The protein is one of the components of a histone deactelylase
complex referred to as the Mi-2/NuRD complex which participates in
the remodeling of chromatin by deacetylating histones (RefSeq,
2002). CHD3 is up-regulated in pancreatic intraepithelial neoplasia
and pancreatic carcinoma (Wang et al., 2011). CHD3 mutation is
associated with gastric and colorectal cancer (Kim et al., 2011a).
CHD3 is over-expressed in acute myeloid leukemia (Camos et al.,
2006).
[0140] CHD4 encodes chromodomain helicase DNA binding protein 4. It
represents the main component of the nucleosome remodeling and
deacetylase complex and plays an important role in epigenetic
transcriptional repression. Somatic mutations in this gene are
associated with serous endometrial tumors (RefSeq, 2002). CHD4 is a
novel therapeutic target for acute myeloid leukemia (Sperlazza et
al., 2015). CHD4 epigenetically controls gene regulation and DNA
damage responses in EpCAM+ liver cancer stem cells (Nio et al.,
2015). CHD4 modulates therapeutic response in BRCA2 mutant cancer
cells (Guillemette et al., 2015). CHD4 is associated with
glioblastoma and colon cancer (Cai et al., 2014; Chudnovsky et al.,
2014).
[0141] CHD5 encodes chromodomain helicase DNA binding protein 5.
CHD5 is a potential tumor suppressor that may play a role in the
development of neuroblastoma (RefSeq, 2002). CHD5 functions as a
tumor suppressor gene in gliomas and a variety of other tumor
types, including breast, colon, lung, ovary, and prostate cancer
(Kolla et al., 2014).
[0142] CIRH1A (also called Cirhin) encodes cirrhosis autosomal
recessive 1 A, a WD40-repeat-containing protein localized in the
nucleolus. It causes North American Indian childhood cirrhosis
(NAIC) (RefSeq, 2002). CIRH1A can up-regulate a canonical NF-kappaB
element and might participate in the regulation of other genes
containing NF-kappaB elements. This suggests that CIRH1A can
influence the cancer-related NF-kappaB pathway (Yu et al.,
2009).
[0143] COL1A1 encodes collagen, type 1, alpha 1. Type 1 is a fibril
forming collagen found in most connective tissues and is abundant
in bone, cornea, dermis, and tendon. Reciprocal translocations
between chromosomes 17 and 22, where this gene and the gene for
platelet derived growth factor beta are located, are associated
with a particular type of skin tumor called dermatofibrosarcoma
protuberans, resulting from unregulated expression of the growth
factor (RefSeq, 2002). COL1A1 is differentially expressed in
gastric cancer (Yasui et al., 2004). COL1A1 is associated with
pigmented dermatofibrosarcoma protuberans (Zhang et al.,
2013c).
[0144] COL1A2 encodes collagen, type 1, alpha 2. Type 1 is a fibril
forming collagen found in most connective tissues and is abundant
in bone, cornea, dermis and tendon (RefSeq, 2002). COL1A2 is
associated with gastric cancer (Yasui et al., 2004; Yasui et al.,
2005).
[0145] COL6A1 encodes collagen, type 6, alpha 1. Collagen VI is a
major structural component of microfibrils. Mutations in the genes
that code for the collagen VI subunits result in the autosomal
dominant disorder Bethlem myopathy (RefSeq, 2002). COL6A1 is
up-regulated in the reactive stroma of castration-resistant
prostate cancer and promotes tumor growth (Zhu et al., 2015c).
COL6A1 is over-expressed in CD166--pancreatic cancer cells that
show stronger invasive and migratory activities than those of
CD166+ cancer cells (Fujiwara et al., 2014). COL6A1 is highly
expressed in bone metastasis (Blanco et al., 2012). COL6A1 was
found to be up-regulated in cervical and ovarian cancer (Zhao et
al., 2011; Parker et al., 2009). COL6A1 is differentially expressed
in astrocytomas and glioblastomas (Fujita et al., 2008).
[0146] COL6A3 encodes collagen, type VI, alpha 3, one of the three
alpha chains of type VI collagen, a beaded filament collagen found
in most connective tissues, and important in organizing matrix
components (RefSeq, 2002). COL6A3 encodes the alpha-3 chain of type
VI collagen, a beaded filament collagen found in most connective
tissues, playing an important role in the organization of matrix
components (RefSeq, 2002). COL6A3 is alternatively spliced in
colon, bladder and prostate cancer. The long isoform of COL6A3 is
expressed almost exclusively in cancer samples and could
potentially serve as a new cancer marker (Thorsen et al., 2008).
COL6A3 is highly expressed in pancreatic ductal adenocarcinoma
tissue and undergoes tumor-specific alternative splicing (Kang et
al., 2014). COL6A3 has been demonstrated to correlate with
high-grade ovarian cancer and contributes to cisplatin resistance.
COL6A3 was observed to be frequently over-expressed in gastric
cancer tissues (Xie et al., 2014). COL6A3 mutation(s) significantly
predicted a better overall survival in patients with colorectal
carcinoma independent of tumor differentiation and TNM staging (Yu
et al., 2015b). COL6A3 expression was reported to be increased in
pancreatic cancer, colon cancer, gastric cancer, mucoepidermoid
carcinomas and ovarian cancer. Cancer associated transcript
variants including exons 3, 4 and 6 were detected in colon cancer,
bladder cancer, prostate cancer and pancreatic cancer (Arafat et
al., 2011; Smith et al., 2009; Yang et al., 2007; Xie et al., 2014;
Leivo et al., 2005; Sherman-Baust et al., 2003; Gardina et al.,
2006; Thorsen et al., 2008). In ovarian cancer COL6A3 levels
correlated with higher tumor grade and in pancreatic cancer COL6A3
was shown to represent a suitable diagnostic serum biomarker
(Sherman-Baust et al., 2003; Kang et al., 2014).
[0147] COPG1 (also called COPG) encodes for the gamma subunit of
the coatomer protein complex (COPI) that mediates retrograde
transport from the Golgi back to the ER and intra-Golgi transport.
COPG1 binds to ARF-GAP (Waters et al., 1991; Watson et al., 2004).
COPG1 correlates with the age of the patients as well as a higher
grade of malignancy and the grade of gliosarcomas (Coppola et al.,
2014). COPG1 was found abundantly expressed in lung cancer and lung
cancer-related endothelial cells (Park et al., 2008).
[0148] CREB3L1 encodes cAMP responsive element binding protein
3-like 1. In response to ER stress, CREB3L1 is cleaved and the
released cytoplasmic transcription factor domain translocates to
the nucleus. There it activates the transcription of target genes
by binding to box-B elements (RefSeq, 2002). CREB3L1 mutations are
frequently found in sclerosing epithelioid fibrosarcoma (SEF)
(Prieto-Granada et al., 2015). CREB3L1 is induced by ER stress in
human glioma cell lines and contributes to the unfolded protein
response, extracellular matrix production and cell migration
(Vellanki et al., 2013). CREB3L1 is epigenetically silenced in
bladder cancer, facilitating tumor cell spreading and migration
(Rose et al., 2014). CREB3L1 plays an important role in suppressing
tumorigenesis in breast cancer. Loss of expression is required for
the development of a metastatic phenotype (Mellor et al.,
2013).
[0149] CSTF1 encodes cleavage stimulation factor, 3' pre-RNA,
subunit 1, 50 kDa. It is involved in the polyadenylation and 3' end
cleavage of pre-mRNAs (RefSeq, 2002). CSTF1 variation was found to
be associated with breast cancer risk in BRCA2 mutation carriers
(Blanco et al., 2015).
[0150] CTHRC1 encodes collagen triple helix repeat containing 1.
CTHRC1 may play a role in the cellular response to arterial injury
through involvement in vascular remodeling. Mutations at this locus
have been associated with Barrett esophagus and esophageal
adenocarcinoma (RefSeq, 2002). CTHRC1 shows increased expression in
gastric cancer and ductal carcinoma of the breast (Kim et al.,
2013b; Yu et al., 2015a; Song et al., 2015). CTHRC1 is up-regulated
in colorectal cancer (Yan et al., 2015a; Yan et al., 2015b). CTHRC1
expression is highly correlated with hepatocellular carcinoma
progression in patients infected with hepatitis B virus. CTHRC1
enhances colony formation, migration and invasion of hepatoma cells
(Tameda et al., 2014; Zhang et al., 2015b). CTHRC1 is
over-expressed in non-small cell lung cancer. Over-expression is
associated with tumor aggressiveness and poor prognosis (Ke et al.,
2014b). CTHRC1 is up-regulated in esophageal squamous cell
carcinoma and Barrett's adenocarcinoma (Timme et al., 2014). CTHRC1
promotes cell adhesion and survival in melanoma (Ip et al.,
2011).
[0151] CXCL5 encodes chemokines C-X-C motif ligand 5. This protein
is proposed to bind the G-protein coupled receptor chemokine C-X-C
motif receptor 2 to recruit neutrophils, to promote angiogenesis
and to remodel connective tissues. This protein is thought to play
a role in cancer cell proliferation, migration, and invasion
(RefSeq, 2002). CXCL5 plays a crucial role in survival, growth and
metastasis of renal cell carcinoma (Parihar and Tunuguntla, 2014).
CXCL5 is involved in the transition of chronic inflammation to
esophageal and gastric cancer (Verbeke et al., 2012). CXCL5 is
associated with acute myelogenous leukemia (Kittang et al.,
2010).
[0152] DCBLD2 encodes discoidin, CUB and LCCL domain-containing
protein 2 also referred to as endothelial and smooth muscle
cell-derived neuropilin-like protein, a transmembrane co-receptor
protein (RefSeq, 2002). DCBLD2 is up-regulated in glioblastomas and
head and neck cancers (HNCs) and is required for EGFR-stimulated
tumorigenesis (Feng et al., 2014). Furthermore, DCBLD2 is
up-regulated in highly metastatic lung cancer sublines and tissue
samples (Koshikawa et al., 2002). In contrast, the expression of
DCBLD2 is silenced by hypermethylation of its promoter in gastric
cancer (Kim et al., 2008).
[0153] DDX43 encodes DEAD (Asp-Glu-Ala-Asp) box polypeptide 43.
DDX43 is an ATP dependent RNA helicase and displays tumor specific
expression (RefSeq, 2002). DDX43 is over-expressed in uveal
melanoma cells and in acute and chronic myeloid leukemia (Chen et
al., 2011a; Lin et al., 2014b; Ambrosini et al., 2014). DDX43 is a
biomarker for breast cancer prognosis (Wiese and Pajeva, 2014).
DDX43 is expressed on glioma cell lines (Akiyama et al., 2014).
[0154] DDX53 encodes DEAD (Asp-Glu-Ala-Asp) box polypeptide 53.
DDX53 contains several domains found in members of the DEAD box
helicase protein family (RefSeq, 2002). Cancer/testis antigen DDX53
exerts negative regulation on p53 expression through HDAC2 and
confers resistance to anti-cancer drugs (Kim et al., 2010b).
miR-200b and cancer/testis antigen DDX53 form a feedback loop to
regulate the invasion and tumorigenic and angiogenic responses of a
cancer cell line to microtubule-targeting drugs (Kim et al.,
2013c). miR-217 and DDX53 form a feedback loop to regulate the
response to anti-cancer drugs through EGFR and HER2 (Kim et al.,
2016). DDX53 is one of several genes with an abnormal DNA
hypo-methylation status in uterine leiomyoma (Maekawa et al.,
2011). In cell lines derived from 21B-cell and 4 T-cell
malignancies, a broad mRNA expression profile was observed for
DDX53 (Liggins et al., 2010).
[0155] DNAJC7 encodes DnaJ (Hsp40) homolog, subfamily C, member 7,
a member of the DNAJ heat shock protein (HSP) 40 family of
proteins. This protein binds the chaperone proteins HSP70 and HSP90
in an ATP dependent manner and may function as a co-chaperone
(RefSeq, 2002). DNAJC7 enhances p53 stability and activity through
blocking the complex formation between p53 and MDM2 (Kubo et al.,
2013).
[0156] DPP9 encodes dipeptidyl peptidase 9. DPP9 appears to be
involved in the regulation of the activity of its substrates and
has been linked to a variety of diseases including type 2 diabetes,
obesity and cancer (RefSeq, 2002). DPP9 plays a potential role in
breast and ovarian cancer (Wilson and Abbott, 2012). DPP9 plays an
important signaling role in the regulation of cell survival and
proliferation pathways (Yao et al., 2011). DPP9 mRNA levels are
elevated in testicular tumors (Yu et al., 2010). DPP9 is
over-expressed in meningiomas (Stremenova et al., 2010).
[0157] DPYD (also known as DPD) encodes dihydropyrimidine
dehydrogenase, a pyrimidine catabolic enzyme and the initial and
rate-limiting factor in the pathway of uracil and thymidine
catabolism. Mutations in this gene result in dihydropyrimidine
dehydrogenase deficiency, an error in pyrimidine metabolism
associated with thymine-uraciluria and an increased risk of
toxicity in cancer patients receiving 5-fluorouracil chemotherapy
(RefSeq, 2002). The DPYD expression level can be used as a
predictive factor for the efficacy of chemotherapy in gastric
cancer (Wan et al., 2016). Statistically significant associations
were found between DPYD variants and increased incidence of grade 3
or greater fluorouracil-related adverse events in patients treated
with adjuvant fluorouracil-based combination chemotherapy
(Cavalcante et al., 2015; Lee et al., 2016; Boige et al., 2016).
There is a correlation between DPYD polymorphism and KRAS wild type
expression in colorectal cancer (Kleist et al., 2015). The
up-regulation of DPYD gene expression leads to fluoropyrimidine
toxicity in colorectal cancer (Chai et al., 2015; Falvella et al.,
2015; van Staveren et al., 2015; Nakamura et al., 2015; Chen et
al., 2015c; Hu et al., 2015b). Polymorphic expression of DPYD may
be important in determining the treatment response in patients with
head and neck cancer, pancreatic cancer, esophageal squamous cell
carcinoma, digestive cancer, gastric cancer, hepatocellular
carcinoma, and colorectal cancer (Kim et al., 2015; Toffoli et al.,
2015; Ishizuka et al., 2015; Baba et al., 2015; Launay et al.,
2016; Kikuchi et al., 2015; Li et al., 2016; Shimamoto et al.,
2016; Bai et al., 2015; Dhawan et al., 2016).
[0158] DROSHA, one of the two critical enzymes in microRNA
biosynthesis, is over-expressed in a number of cancers including
gastrointestinal tumors, breast cancer and cervical cancer and
appears to enhance proliferation, colony formation and migration of
tumor cells (Avery-Kiejda et al., 2014; Havens et al., 2014; Zhou
et al., 2013).
[0159] DSEL encodes dermatan sulfate epimerase-like and is located
on chromosome 18q22.1 (RefSeq, 2002). DSE is an important paralog
of DSEL. DSE is an immunogenic target for immunotherapy of
hepatocellular carcinoma and colorectal carcinoma (Mizukoshi et
al., 2011; Sasatomi et al., 2002).
[0160] DST (also known as bullous pemphigoid antigen I (BPAG1))
encodes dystonin, a member of the plakin protein family of adhesion
junction plaque proteins. The full-length isoform is not defined,
however, there are several isoforms expressed in neural and muscle
tissue or in epithelial tissue, anchoring either neural
intermediate filaments to the actin cytoskeleton or
keratin-containing intermediate filaments to hemidesmosomes
(RefSeq, 2002; Bouameur et al., 2014; Li et al., 2007). DST may be
related to breast cancer metastasis (Sun et al., 2006).
Autoantibodies against DST can be found in lymphocytic leukemia and
follicular lymphomas (Aisa et al., 2005; Taintor et al., 2007). DST
is up-regulated in 5-8F cells (high tumorigenic and metastatic
ability) in comparison to 6-10B cells (tumorigenic, but lacking
metastatic ability) in nasopharyngeal carcinoma (Fang et al.,
2005). DST is highly expressed in head and neck squamous cell
carcinoma (Lin et al., 2004). There are autoantibodies against DST
in paraneoplastic pemphigus which is associated with neoplasms
(Yong and Tey, 2013; Wang et al., 2005; Preisz and Karpati, 2007;
Zhu and Zhang, 2007). DST expression in prostate cancer is strongly
inverse correlated with progression (Vanaja et al., 2003). Anti-DST
autoantibodies are a promising marker for the diagnosis of melanoma
(Shimbo et al., 2010). DST can be found in the urine of cachectic
cancer patients (Skipworth et al., 2010). DST is differentially
expressed in adenocarcinomas and squamous cell carcinomas of the
lung (McDoniels-Silvers et al., 2002). DST is distinctly
up-regulated with the onset of invasive cell growth (Herold-Mende
et al., 2001).
[0161] DYNC1H1 encodes the dynein heavy chain 1, a subunit of the
main motor protein for retrograde transport along microtubules. A
whole exome sequencing study uncovered somatic mutations within the
DYNC1H1 gene in patients with intra-ductal papillary mucinous
neoplasm of the pancreas (Furukawa et al., 2011).
[0162] EIF3C encodes eukaryotic translation initiation factor 3,
subunit C and is located on chromosome 16p11.2 (RefSeq, 2002).
EIF3C is over-expressed and promotes cell proliferation in human
U-87 MG cells (Hao et al., 2015). EIF3C is highly expressed in
colon cancer (Song et al., 2013). EIF3C mRNA is over-expressed in
testicular seminomas (Rothe et al., 2000).
[0163] EIF3CL encodes eukaryotic translation initiation factor 3,
subunit C-like. It is located on chromosome 16p11.2 (RefSeq,
2002).
[0164] EIF3E encodes eukaryotic translation initiation factor 3,
subunit E and is located on chromosome 8q22-q23 (RefSeq, 2002).
EIF3E might play a role in the carcinogenesis of oral squamous cell
carcinoma (Yong et al., 2014). EIF3E is essential for proliferation
and survival of glioblastoma cells (Sesen et al., 2014). EIF3E has
an oncogenic role in breast cancer progression. Decreased EIF3E
expression causes epithelial to mesenchymal transition in breast
epithelial cells (Gillis and Lewis, 2013; Grzmil et al., 2010).
EIF3E expression level is significantly increased in bladder cancer
(Chen et al., 2011b). EIF3E is involved in non-small lung carcinoma
(Marchetti et al., 2001).
[0165] EXT2 encodes exostosin glycosyltransferase 2, one of two
glycosyltransferases involved in the chain elongation step of
heparin sulfate biosynthesis. Mutations in his gene cause the type
II form of multiple exostoses (RefSeq, 2002). EXT2 mutation plays a
role in chondrosarcoma (Samuel et al., 2014). EXT2 mutation induces
multiple osteochondroma syndrome (Jochmann et al., 2014). EXT2
mutation causes hereditary multiple exostoses, leading to heparan
sulfate deficiency (Huegel et al., 2013).
[0166] F2R (also known as PAR1) encodes coagulation factor II
thrombin receptor, a transmembrane receptor involved in the
regulation of thrombotic response (RefSeq, 2002). F2R binds to the
pleckstrin homology (PH) domain of Etk/Bmx. A F2R mutant, which is
unable to bind the PH domain, reduces mammary tumors and
extravillous trophoblast invasion (Kancharla et al., 2015). F2R is
thought to promote cancer invasion and metastasis by facilitating
tumor cell migration, angiogenesis, and interactions with host
vascular cells (Wojtukiewicz et al., 2015). Down-regulation of F2R
leads to cancer cell death (Burns and Thevenin, 2015).
Polymorphisms in F2R are associated with acute injury in rectal
cancer patients (Zhang et al., 2015a). F2R is correlated with poor
prognosis specifically in ER-negative breast cancer patients
(Lidfeldt et al., 2015). F2R-deficient mice show reduced colonic
adenocarcinoma growth (Adams et al., 2015). Matrix
metalloproteinase (MMP)-1 activates F2R to induce angiogenesis (Fan
et al., 2015). F2R is involved in PTEN down-regulation in lung
cancer (Xu et al., 2015). F2R activation induces the Hippo-YAP
pathway which is correlated with epithelial mesenchymal transition
(Jia et al., 2015; Owens et al., 2015; Yang et al., 2015a; Fujimoto
et al., 2015). Inhibition of F2R activation reduces cancer cell
migration and invasion in HER-2 negative breast cancer,
hepatocellular carcinoma and gastric cancer (Mussbach et al., 2015;
Wang et al., 2015g; Gonda et al., 2015).
[0167] FADS2 encodes fatty acid desaturase 2, a member of the fatty
acid desaturase gene family. Desaturase enzymes regulate
unsaturation of fatty acids through the introduction of double
bonds between defined carbons of the fatty acyl chain (RefSeq,
2002). FADS2 is up-regulated in hepatocellular carcinoma (Muir et
al., 2013). FADS2 activity is increased in breast cancer tissue
(Pender-Cudlip et al., 2013). FADS2 expression is associated with
aggressiveness of breast cancer (Lane et al., 2003). FADS2
inhibition impedes intestinal tumorigenesis (Hansen-Petrik et al.,
2002).
[0168] FADS3 encodes fatty acid desaturase 3. Desaturase enzymes
regulate unsaturation of fatty acids through the introduction of
double bonds between defined carbons of the fatty acyl chain
(RefSeq, 2002).
[0169] FAM83D encodes family with sequence similarity 83, member D
and is located on chromosome 20q11.23 (RefSeq, 2002). Up-regulation
of FAM83D affects the proliferation and invasion of hepatocellular
carcinoma cells (Wang et al., 2015a; Liao et al., 2015). FAM83D is
significantly elevated in breast cancer cell lines and in primary
human breast cancers (Wang et al., 2013e).
[0170] FN1 encodes fibronectin 1, a glycoprotein present in a
soluble dimeric form in plasma, and in a dimeric or a multimeric
form at the cell surface and in extracellular matrix. It is
involved in cell adhesion and migration processes including
embryogenesis, wound healing, blood coagulation, host defense, and
metastasis (RefSeq, 2002). FN1 is an important tumor-associated
angiogenesis targeting agent (Sollini et al., 2015). FN1 is one of
several biomarkers for pancreatic cancer (Ansari et al., 2014). FN1
is one of many factors responsible for endocrine resistance in
breast cancer. FN1 is significantly deregulated and promotes tumor
progression and metastatic spread in breast cancer (Oskarsson,
2013; Zheng et al., 2014). It is a biomarker of
epithelial-mesenchymal transition in squamous cell carcinoma
(Scanlon et al., 2013). FN1 plays an important role in multiple
myeloma (Neri and Bahlis, 2012).
[0171] FUCA2, secreted human a-l-fucosidase 2, was identified to be
the key enzyme responsible for the transfer of l-fucose. The
hydrolytic enzyme was found to be essential for H. pylori adhesion
to human gastric cancer cells and shows great potential as a
diagnostic marker and a target for therapeutic treatment H. pylori
associated gastric cancer (Liu et al., 2009).
[0172] GCG encodes glucagon. It is a pancreatic hormone that
counteracts the glucose lowering action of insulin by stimulating
glycogenolysis and gluconeogenesis. It is a ligand for a specific
G-protein linked receptor whose signaling pathway controls cell
proliferation (RefSeq, 2002). GCG receptor imaging seems to be a
potential tool to evaluate pancreatic beta cell mass. It might also
become a target for imaging other tumors such as gastrinoma,
pheochromocytoma and medullary thyroid cancer (Hubalewska-Dydejczyk
et al., 2015). GCG plays a key role in colon carcinogenesis (Kannen
et al., 2013). GCG is an emerging tracer for neuroendocrine tumors
(Reubi and Maecke, 2008).
[0173] GFPT2 encodes glutamine fructose 6 phosphate transaminase 2
and is located on chromosome 5q34-q35 (RefSeq, 2002). GFPT2 plays
an important role in breast cancer and lymphocytic leukemia (Kuang
et al., 2008; Simpson et al., 2012).
[0174] GPN1 encodes GPN loop GTPase 1 and is located on chromosome
2p23.3 (RefSeq, 2002). GPN1 is a cytoplasmic GTPase involved in
nuclear localization of the DNA repair gene XPA, a critical factor
controlling nucleotide excision repair signaling pathways (Nitta et
al., 2000).
[0175] GRIK2 encodes glutamate receptor, ionotropic, kainite 2.
Mutations in this gene have been associated with autosomal
recessive mental retardation (RefSeq, 2002). TRMT11-GRIK2 is one of
several fusion transcripts found in prostate cancer and is
associated with tumor aggressiveness (Yu et al., 2014). GRIK2 SNPs
are associated with increased risk or susceptibility to oral cancer
(Bhatnagar et al., 2012). GRIK2 is a potential biomarker for lung
cancer (Rauch et al., 2012). GRIK2 inactivation by chromosomal
deletion may contribute to the onset of T-cell lymphomas. GRIK2
inactivation plays a role in gastric carcinogenesis (Resende et
al., 2011; Lopez-Nieva et al., 2012).
[0176] GRIK3 encodes glutamate receptor, ionotropic, kainite 3. It
belongs to a family of glutamate receptors, which are the
predominant excitatory neurotransmitter receptors in the mammalian
brain and are activated in a variety of normal neurophysiologic
processes (RefSeq, 2002). GRIK3 is associated with lung
adenocarcinoma (methylation, functional modifications), pediatric
central nervous system tumors, lymphocytic leukemia, and
neuroblastoma (Pradhan et al., 2013). GRIK3 is differentially
expressed in several pediatric tumors of the central nervous system
(Brocke et al., 2010).
[0177] GSK3B encodes glycogen synthase kinase 3 beta. It is
involved in energy metabolism, neuronal cell development, and body
pattern formation (RefSeq, 2002). Aberrant regulation of GSK3B has
been shown to promote cell growth in some cancers, while
suppressing it in others, and may play an important role in
esophageal cancer (Gao et al., 2014b). GSK3B is dysregulated in
glioblastoma multiforme (Atkins et al., 2013). Deregulated GSK3B
promotes gastrointestinal, pancreatic, and liver cancers (Miyashita
et al., 2009).
[0178] HLA-A encodes the major histocompatibility complex class 1 A
that plays a central role in the immune system by presenting
peptides derived from the endoplasmic reticulum lumen (RefSeq,
2002). The loss of HLA-A antigens is a common feature in human
tumors. Decrease in the percentage of HLA-A, HLA-B, and
HLA-C-positive cells, selective loss of particular antigens and
total loss of class 1 molecule expression is documented in
melanomas, carcinomas, lymphomas, neuroblastomas and acute
leukemias (Garrido and Ruiz-Cabello, 1991; Salerno et al., 1990).
HLA-A expression is predominantly regulated by the MAPK pathway in
gastric and esophageal cancer and in part influenced by the Akt
pathway with a strong inverse correlation between p-Erk expression
and HLA class 1 expression in clinical tumor samples (Mimura et
al., 2013).
[0179] HNRNPU (also called SAF-A) encodes the heterogeneous nuclear
ribonucleoprotein U that belongs to the RNA binding subfamily of
heterogeneous nuclear riboproteins (hnRNPs), which is associated
with pre-mRNA processing and other aspects of mRNA metabolism and
transport in the nucleus. HNRNPU is thought to be involved in the
packaging of hnRNA into large ribonucleoprotein complexes (RefSeq,
2002). Up-regulation of the miR-193a-3p that inhibits the
metastasis of lung cancer cells down-regulates the expression of
HNRNPU (Deng et al., 2015b). The long non-coding RNA H19 can--via
association with the HNRNPU/PCAF/RNAPol II protein
complex--activate the miR-200 pathway, thus contributing to
mesenchymal-to-epithelial cell transition and to the suppression of
tumor metastasis in hepatocellular carcinoma (Zhang et al., 2013d).
HNRNPU interacts with SOX2, a key gene for maintaining the stemness
of embryonic and adult stem cells that appears to be re-activated
in several human cancers (Fang et al., 2011).
[0180] HSPA2 encodes the testis specific heat-shock protein 70-2,
essential for the growth of spermatocytes and cancer cells.
Different studies suggest an important role of HSPA2 in disease
progression of cervical cancer, renal cell carcinoma and bladder
cancer. Polymorphisms within the gene are associated with the
development of gastric cancer (Ferrer-Ferrer et al., 2013; Garg et
al., 2010a; Garg et al., 2010b; Singh and Suri, 2014).
[0181] HSPA8 was shown to be over-expressed in esophageal squamous
cell carcinoma. High expression levels of HSPA8 in esophageal
cancer cells counter-acted oxidative stress-induced apoptosis of
these cells in vitro. Furthermore, HSPA8 is over-expressed in
multiple myeloma and colonic carcinoma and BCR-ABL1-induced
expression of HSPA8 promotes cell survival in chronic myeloid
leukemia (Chatterjee et al., 2013; Dadkhah et al., 2013;
Jose-Eneriz et al., 2008; Kubota et al., 2010; Wang et al.,
2013a).
[0182] HSPA8P8 is a pseudogene (RefSeq, 2002).
[0183] HSPA9 encodes heat shock 70 kDa protein 9. This protein
plays a role in cell proliferation, stress response and maintenance
of the mitochondria (RefSeq, 2002). HSPA9 regulates cellular
processes ranging from viral infection to neurodegeneration, which
also includes carcinogenesis (Flachbartova and Kovacech, 2013).
HSPA9 is up-regulated in hepatocellular carcinoma and colorectal
cancer (Rozenberg et al., 2013; Chen et al., 2014a; Kuramitsu and
Nakamura, 2005). HSPA9 plays a role in the development of gastric
cancer (Ando et al., 2014). HSPA9 is a potential therapeutic target
for improved treatment of drug-resistant ovarian cancer (Yang et
al., 2013).
[0184] IGDCC4 encodes immunoglobulin superfamily, DCC subclass,
member 4 and is located on chromosome 15q22.31 (RefSeq, 2002).
GDCC4 is expressed in hepatocellular carcinoma (Joy and Burns,
1988; Marquardt et al., 2011). GDCC4 plays a role in acute
lymphoblastic leukemia (Taylor et al., 2007).
[0185] IGF2BP3 encodes insulin-like growth factor II mRNA binding
protein 3, an oncofetal protein, which represses translation of
insulin-like growth factor II (RefSeq, 2002). Several studies have
shown that IGF2BP3 acts in various important aspects of cell
function, such as cell polarization, migration, morphology,
metabolism, proliferation and differentiation. In vitro studies
have shown that IGF2BP3 promotes tumor cell proliferation,
adhesion, and invasion. Furthermore, IGF2BP3 has been shown to be
associated with aggressive and advanced cancers (Bell et al., 2013;
Gong et al., 2014). IGF2BP3 over-expression has been described in
numerous tumor types and correlated with poor prognosis, advanced
tumor stage and metastasis, as for example in neuroblastoma,
colorectal carcinoma, intrahepatic cholangiocarcinoma,
hepatocellular carcinoma, prostate cancer, and renal cell carcinoma
(Bell et al., 2013; Findeis-Hosey and Xu, 2012; Hu et al., 2014a;
Szarvas et al., 2014; Jeng et al., 2009; Chen et al., 2011c; Chen
et al., 2013; Hoffmann et al., 2008; Lin et al., 2013b; Yuan et
al., 2009).
[0186] IPO5 encodes importin 5, a member of the importin beta
family. Importins are essential in the translocation of proteins
through the nuclear pore complex (RefSeq, 2002). IPO7 encodes
importin 7. The importin alpha/beta complex and the GTPase Ran
mediate nuclear import of proteins with a classical nuclear
localization signal (RefSeq, 2002). IPO7 is frequently
over-expressed in cancers (Golomb et al., 2012). IPO7 is
dysregulated in glioblastoma, Hodgkin lymphoma and breast cancer
(Jung et al., 2013; Ju et al., 2013; Nagel et al., 2014; Xue et
al., 2015). IPO7 is a microRNA target that is down-regulated in
prostate carcinoma (Szczyrba et al., 2013). Elevated levels of IPO7
mRNA in colorectal carcinoma are associated with increased
proliferation (Li et al., 2000).
[0187] IQGAP3 encodes a member of the IQ-motif-containing GAP
family which acts at the interface between cellular signaling and
the cytoskeleton. IQGAP3 regulates the Rac1/Cdc42-promoted neurite
outgrowth and interacts directly with calmodulin and the myosin
light chain (Wang et al., 2007; Atcheson et al., 2011). IQGAP3 is
over-expressed in lung cancer and is associated with tumor cell
growth, migration and invasion. Furthermore, it is up-regulated by
chromosomal amplification in hepatocellular carcinoma and the
expression of IQGAP3 is increased in p53-mutated colorectal cancer
patients with poor survival (Katkoori et al., 2012; Yang et al.,
2014b; Skawran et al., 2008). IQGAP3 is modulating the EGFR/Ras/ERK
signaling cascade and interacts with Rac/Cdc42 (Yang et al., 2014b;
Kunimoto et al., 2009).
[0188] KDELR1 encodes KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum
protein retention receptor 1. KDELR1 is structurally and
functionally similar to the yeast ERD2 gene product (RefSeq, 2002).
KDELR1 has a role in tumorigenesis (Yi et al., 2009). Decreased
KDELR1 levels are found in hepatoma cells (Hou et al., 2015).
Down-regulation of KDELR1 is seen in acute myeloid leukemia
(Caldarelli et al., 2013).
[0189] KPNA2 encodes karyopherin alpha 2. KPNA2 may be involved in
the nuclear transport of proteins (RefSeq, 2002). KPNA2 expression
is dysregulated in epithelial ovarian cancer (Lin et al., 2015).
KPNA2 is down-regulated in large oral squamous cell carcinoma
tumors in comparison to small tumors (Diniz et al., 2015). KPNA2
contributes to the aberrant localization of key proteins and to
poor prognosis of breast cancer (Alshareeda et al., 2015). The
expression of KPNA2 is significantly up-regulated in the upper
tract urothelial carcinoma and in endometrial cancers (Ikenberg et
al., 2014; Shi et al., 2015). KPNA2 promotes tumor growth in
hepatocellular carcinoma (Hu et al., 2014b).
[0190] KRT19 encodes a member of the keratin family. Keratins are
intermediate filament proteins responsible for the structural
integrity of epithelial cells and are subdivided into cytokeratins
and hair keratins. KRT19 is specifically expressed in the periderm,
the transiently superficial layer that envelopes the developing
epidermis (RefSeq, 2002). KRT19 expression in tumor cells is a
prognostic marker for several tumor entities such as breast, lung,
ovarian and hepatocellular cancer (Skondra et al., 2014; Gao et
al., 2014a; Liu et al., 2013a; Lee et al., 2013). KRT19 has been
shown to be an independent prognostic factor for pancreatic
neuroendocrine tumors, especially the insulin-negative tumors.
KRT19 positive tumors are associated with poor outcome irrespective
of the established pathologic parameters such as size, mitoses,
lymphovascular invasion, and necrosis (Jain et al., 2010).
[0191] KRT8 (also called CK8) encodes a member of type II keratin
family that dimerizes with keratin 18 to form an intermediate
filament in single-layered epithelial cells. KRT8 plays a role in
maintaining cellular structural integrity and also has a function
in signal transduction and cellular differentiation (RefSeq, 2002).
KRT8 is up-regulated and secreted from different cancer cells
including lung, prostate and breast cancer. High levels of KRT8
correlate with increased migration and invasion (Gonias et al.,
2001; Kuchma et al., 2012; Fukunaga et al., 2002; Takei et al.,
1995). The MEK/ERK pathway regulates
sphingosylphosphorycholine-induced KRT8 phosphorylation at Ser431.
This leads to keratin cytoskeleton re-organization and consequently
enhances the migration of tumor cells (Busch et al., 2012). The
tumor suppressor SMAR down-regulates KRT8 expression and this leads
to a decreased migration and invasiveness of cells (Pavithra et
al., 2009; Mukhopadhyay and Roth, 1996).
[0192] KRT8P44 encodes keratin 8 pseudogene 44, which is located on
chromosome 6q26 (RefSeq, 2002).
[0193] MACC1 encodes a key regulator of the hepatocyte growth
factor (HGF) receptor pathway which is involved in cellular growth,
epithelial-mesenchymal transition, angiogenesis, cell motility,
invasiveness and metastasis (RefSeq, 2002). MACC1 is over-expressed
in many cancer entities including gastric, colorectal, lung and
breast cancer and is associated with cancer progression, metastasis
and poor survival of patients (Huang et al., 2013b; Ma et al.,
2013; Stein, 2013; Wang et al., 2015b; Wang et al., 2015m; Ilm et
al., 2015). MACC1 promotes carcinogenesis through targeting
beta-catenin and PI3K/AKT signaling pathways, which leads to an
increase of c-Met and beta-catenin and their downstream target
genes including c-Myc, cyclin D1, caspase9, BAD and MMP9 (Zhen et
al., 2014; Yao et al., 2015).
[0194] MAGED2 encodes melanoma antigen family D, 2, a member of a
new defined MAGE-D cluster in Xp11.2, a hot spot for X-linked
mental retardation. MAGED2 is expressed ubiquitously with high
expression levels in specific brain regions and in the interstitium
of testes. MAGED2 is a potential negative regulator of wildtype p53
activity (Langnaese et al., 2001; Papageorgio et al., 2007). MAGED2
over-expression is associated with melanoma, breast cancer and
colon cancer (Li et al., 2004; Strekalova et al., 2015).
[0195] MAN2A1 encodes mannosidase alpha class 2A, member 1, which
is localized in the Golgi and catalyzes the final hydrolytic step
in the asparagine-linked oligosaccharide maturation pathway
(RefSeq, 2002). Swainsonine inhibits MAN2A1, resulting in the
inhibition of the production of beta 1,6-branched N-linked glycans,
which are related to the malignant phenotype of tumor cells (Yagel
et al., 1990; Gerber-Lemaire and Juillerat-Jeanneret, 2010; Santos
et al., 2011; Przybylo et al., 2005; Dennis and Laferte, 1987;
Baptista et al., 1994; Goss et al., 1994; Fujieda et al., 1994;
Korczak and Dennis, 1993; Roberts et al., 1998; Goss et al., 1997;
Goss et al., 1995; Seftor et al., 1991). A SNP in MAN2A1 is
strongly associated with childhood acute lymphoblastic leukemia
(Han et al., 2010).
[0196] MAP1A encodes microtubule associated protein 1A which is
involved in microtubule assembly, an essential step in neurogenesis
(RefSeq, 2002). MAP1A accumulates in retinoic acid-induced P19
embryonal carcinoma cells (Vaillant and Brown, 1995). MAP1A is
down-regulated in the tumor-adjacent stroma of prostate cancer (Zhu
et al., 2015b). MAP1A may play a role in cell proliferation
(Matsuno et al., 2004). Danusertib significantly increases the
expression level of membrane-bound MAP1A in breast cancer (Li et
al., 2015c). Baicalein up-regulates MAP1A in the hepatocellular
carcinoma cell line HepG2 (Wang et al., 2015l). MAP1A is inversely
correlated to p62 in cutaneous squamous cell carcinoma (Yoshihara
et al., 2014). Gamma-tocotrienol induces an increased conversion of
MAP1A from its cytosolic to its lipidated isoform (Tiwari et al.,
2014).
[0197] MAT2A encodes methionine adenosyltransferase 2A which
catalyzes the production of S-adenosylmethionine from methionine
and ATP (RefSeq, 2002). MAT2A is up-regulated in
tamoxifen-resistant MCF-7 breast cancer cells (Phuong et al.,
2015). There are higher levels of sumoylated and total MAT2A in
colon cancer. Interaction between Ubc9, Bc12, and MAT2A enhance
growth and survival of cancer cells (Tomasi et al., 2015). MAT2A
expression is down-regulated in renal cell carcinoma and in the
S-adenosylmethionine-treated hepatocellular carcinoma cell line
WCH17 (Kuang et al., 2014; Wang et al., 2014b). The MAT1A:MAT2A
switch is associated with global DNA hypomethylation, decreased DNA
repair, genomic instability, and signaling deregulation in
hepatocellular carcinoma (Woodburn et al., 2013; Frau et al.,
2013). MAT2A is up-regulated in hepatocellular cell carcinoma,
gastric cancer, and colon cancer (Frau et al., 2012; Zhang et al.,
2013e; Tomasi et al., 2013; Frau et al., 2013; Lo et al., 2013).
MAT2A is correlated with tumor classification, lymph node
metastasis, and poor tumor differentiation in gastric cancer
patients (Liu et al., 2011b; Zhang et al., 2013e). MAT2A is a
transcriptional co-repressor of the oncoprotein MafK (Katoh et al.,
2011). MAT2A is linked to tumor growth and progression in liver
cancer (Vazquez-Chantada et al., 2010; Liu et al., 2011a; Lu and
Mato, 2008).
[0198] MBTPS2 is a membrane-embedded zinc metalloprotease that
activates signaling of proteins involved in sterol control of
transcription and plays a role in ER stress response (Oeffner et
al., 2009).
[0199] MCM4 encodes the minichromosome maintenance complex
component 4 which is essential for the initiation of eukaryotic
genome replication (RefSeq, 2002). MCM4 expression is associated
with up-regulated carbonic anhydrase IX, a transmembrane
glycoprotein which is correlated with decreased survival and cancer
progression in several entities including esophageal cancer (Huber
et al., 2015). Has-miR-615-3p may be involved in nasopharyngeal
carcinoma by regulating MCM4 (Chen et al., 2015b). MCM4 might play
a role in the development of bladder cancer (Zekri et al., 2015). A
gain-of-function mutant of p53 increases the expression of MCM4 in
breast cancer (Polotskaia et al., 2015). There is a mutation of
MCM4 in human skin cancer which shows reduced DNA helicase activity
(Ishimi and Irie, 2015). MCM4 over-expression alone is only weakly
associated with shorter survival in breast cancer. Over-expression
of all six parts of the MCM complex is strongly associated with
shorter survival (Kwok et al., 2015). MCM4 is differentially
expressed in lung adenocarcinoma and laryngeal squamous cell
carcinoma (Lian et al., 2013; Zhang et al., 2014c). MCM4 is
significantly over-expressed in cervical cancer (Das et al., 2013;
Das et al., 2015). MCM4 may be used as a biomarker for colorectal
cancer (Fijneman et al., 2012).
[0200] MIER1 (also called MI-ER1) encodes a transcriptional
regulator that was first identified in Xenopus leavis (RefSeq,
2002). MIER1 is up-regulated in chronic myeloid leukemia (CML) and
breast cancer, where loss of the nuclear transcript variant alpha
is associated with cancer progression and proliferation (McCarthy
et al., 2008; Ding et al., 2003; Mascarenhas et al., 2014). The
transcriptional repressor MIER1 functions due to interaction with
HDAC1 (Ding et al., 2003).
[0201] MIR2861 is a short non-coding RNAs that is involved in
post-transcriptional regulation of gene expression by affecting
both the stability and translation of mRNAs (RefSeq, 2002). MIR2861
expression is up-regulated in papillary thyroid carcinoma (PTC)
with lymph node metastasis in comparison to PTC without lymph node
metastasis (Wang et al., 2013f).
[0202] MLEC encodes malectin, which is a type I membrane-anchored
ER protein. MLEC has an affinity for Glc2Man9GlcNAc2
(G2M9)N-glycans and is involved in regulating glycosylation in the
ER. MLEC has also been shown to interact with ribophorin I and may
be involved in directing the degradation of misfolded proteins
(RefSeq, 2002; Pierce and Taniguchi, 2009). MLEC is de-regulated in
colorectal cancer and enhanced in glioblastoma (Sethi et al., 2015;
Demeure et al., 2016). MLEC might be a biomarker for thyroid
papillary carcinoma (Ban et al., 2012).
[0203] MVP encodes the major compartment of the vault complex, a
protein which may play a role in multiple cellular processes by
regulating MAPK, JAK/STAT and PI3K/Akt signaling pathways. It also
plays a role in multidrug resistance, innate immunity, cell
survival and differentiation, and expression of this gene may be a
prognostic marker for several types of cancer (RefSeq, 2002; Tucci
et al., 2009; Lara et al., 2011; Scagliotti et al., 1999; van den
Heuvel-Eibrink M M et al., 2000; Perez-Tomas, 2006; Scheffer et
al., 2000; Ramachandran, 2007; Sekine et al., 2007; Lu and
Shervington, 2008). MVP is highly expressed in several central
nervous system tumors (Yang et al., 2012a). MVP is highly expressed
in cancer, and in several chemoresistant cancer cell lines
(Szaflarski et al., 2011; Mossink et al., 2003). MVP expression
level increases with age and facilitates apoptosis resistance (Ryu
and Park, 2009).
[0204] MYBBP1A (also called p160) encodes a nucleolar
transcriptional regulator that was first identified by its ability
to bind to the Myb proto-oncogene protein. MYBBP1A might play a
role in many cellular processes, including response to nucleolar
stress, tumor suppression and synthesis of ribosomal DNA (RefSeq,
2002). MYBBP1A is de-regulated in different cancer entities,
including lung, breast and head and neck cancer. It is associated
with cell proliferation and metastasis (Bidkhori et al., 2013;
George et al., 2015; Acuna Sanhueza et al., 2012; Akaogi et al.,
2013). MYBBP1A promotes transcriptional activity via p53 activation
as well as Myb binding and regulates cell cycle and mitosis leading
to G2/M arrest or anomalous mitosis by affecting the control of
chromosomal segregation (Tavner et al., 1998; Tsuchiya et al.,
2011; Mori et al., 2012; Ono et al., 2013).
[0205] NCAPD2 (also called CNAP1) encodes non-SMC condensin I
complex subunit D2 that is involved in chromosome condensation and
associated with Alzheimer's disease (Ball, Jr. et al., 2002; Zhang
et al., 2014b). NCAPD2 over-expression was found in the development
of ovarian cancer together with its amplification and mutation
during tumor progression (Emmanuel et al., 2011).
[0206] NCAPG encodes the non-SMC condensing I complex subunit G
which is responsible for the condensation and stabilization of
chromosomes during mitosis and meiosis (RefSeq, 2002). NCAPG is
down-regulated in patients with multiple myeloma, acute myeloid
leukemia, and leukemic cells from blood or myeloma cells (Cohen et
al., 2014). NCAPG may be a multi-drug resistant gene in colorectal
cancer (Li et al., 2012). NCAPG is highly up-regulated in the
chromophobe subtype of human cell carcinoma but not in conventional
human renal cell carcinoma (Kim et al., 2010a). Up-regulation of
NCAPG is associated with melanoma progression (Ryu et al., 2007).
NCAPG is associated with uveal melanoma (Van Ginkel et al., 1998).
NCAPG shows variable expression in different tumor cells (Jager et
al., 2000).
[0207] NLE1 encodes a notchless homolog and member of the
WD40-repeat protein family that is involved in embryonic
development through different signal pathways and seems to play a
role in ribosome maturation (Beck-Cormier et al., 2014; Romes et
al., 2016; Lossie et al., 2012).
[0208] NOMO1 (also called PM5) encodes Nodal modulator 1, a protein
that might be part of a protein complex that participates in the
Nodal signaling pathway during vertebrate development (RefSeq,
2002). NOMO1 is de-regulated in prostate cancer and in T-cell
lymphoma cells (Stubbs et al., 1999; Lange et al., 2009).
[0209] NOMO2 encodes Nodal modulator 2, a protein that might be
part of a protein complex that participates in the Nodal signaling
pathway during vertebrate development (RefSeq, 2002). NOMO2 is
up-regulated at the epithelium/stroma cell interface in the
transition to cervical intraepithelial neoplasia (CIN) 3 and
cervical cancer as part of a pro-invasive genomic signature that
may be a response to epithelial tumor cell over-crowding (Gius et
al., 2007).
[0210] NOMO3 encodes Nodal modulator 3, a protein that might be
part of a protein complex that participates in the Nodal signaling
pathway during vertebrate development (RefSeq, 2002). NOMO3 is
de-regulated by DNA methylation in non-small cell lung cancer
(Mullapudi et al., 2015). NOMO3 is an enriched membrane protein
associated with glycosylation in ovarian cancer tissues (Allam et
al., 2015).
[0211] NONO (also known as p54nrb) encodes non-POU domain
containing, octamer-binding. NONO is an RNA-binding protein which
plays various roles in the nucleus, including transcriptional
regulation and RNA splicing. A rearrangement between this gene and
the transcription factor E3 has been observed in papillary renal
cell carcinoma (RefSeq, 2002; Macher-Goeppinger et al., 2012). NONO
expression strongly correlates with vascular invasion and decreased
survival (Barboro et al., 2008). Furospinosulin selectively
inhibits the growth of hypoxia-adapted cancer cells, maybe through
direct binding to NONO (Arai et al., 2016). NONO mediates
MIA/CD-RAP action to promote chondrogenesis and progression of
malignant melanoma (Schmid et al., 2013). NONO expression
correlates with the expression of c-Myc, cyclin D1, and CDK4
(Nelson et al., 2012). Knock-out of NONO in YB-1 over-expressing
colorectal cancers can sensitize them to oxaliplatin (Tsofack et
al., 2011). Simvastatin strongly down-regulates NONO and reduces
melanoma progression (Schiffner et al., 2011; Zanfardino et al.,
2013). NONO is over-expressed in breast cancer and melanoma
(Schiffner et al., 2011; Zhu et al., 2015d).
[0212] NPC1 encodes Niemann-Pick disease, type C1, a large protein
that resides in the limiting membrane of endosomes and lysosomes
and mediates intracellular cholesterol trafficking via binding of
cholesterol to its N-terminal domain (RefSeq, 2002). The promotor
of NPC1 is hypo-methylated and NPC1 expression is up-regulated in
esophageal cancer (Singh et al., 2015). NPC1 is differentially
expressed in isogenic metastatic cancer cell lines, human embryonic
stem cells, and human embryonal carcinoma cells (Lund et al., 2015;
Dormeyer et al., 2008). NPC1 degradation is regulated by Akt. Thus
NPC1 is linked to cell proliferation and migration in cervical
cancer (Du et al., 2015). Treatment with sildenafil reduces NPC1
expression and kills brain cancer stem cells (Booth et al., 2015).
Inhibitors of cholesterol metabolism, including NPC1 for
cholesterol uptake, are thought to be beneficial for cancer
treatment (Ali-Rahmani et al., 2014). NPC1 is up-regulated in
TNF-alpha-resistant MCF-7 breast adenocarcinoma cells (Vincent et
al., 2010; Moussay et al., 2011).
[0213] NPC2 encodes a protein with a lipid recognition domain that
may function in regulating the transport of cholesterol through the
late endosomal/lysosomal system. Mutations in this gene are
associated with Niemann-Pick disease and frontal lobe atrophy
(RefSeq, 2002). NPC2 is de-regulated in different cancer entities,
including breast, colon, lung, kidney and liver cancer (McDonald et
al., 2004; Garcia-Lorenzo et al., 2012; Liao et al., 2013).
NPC-related cholesterol perturbation induces abnormal signaling
pathways leading to p38 MAPK activation, Mdm2-mediated p53
degradation, ROCK activation and increased RhoA synthesis (Qin et
al., 2010).
[0214] NUP160 encodes a nucleoporin of 160 kDa that is part of the
nuclear pore complex that mediates the nucleoplasmic transport
(RefSeq, 2002). NUP160-SLC43A3 is a recurrent fusion oncogene in
angiosarcoma and associated with tumor progression (Shimozono et
al., 2015).
[0215] NUP205 encodes nucleoporin 205 kDa (RefSeq, 2002). NUP205 is
stabilized by TMEM209. This interaction is a critical driver for
lung cancer proliferation (Fujitomo et al., 2012).
[0216] NUP98 encodes nucleoporin 98 kDa which participates in many
cellular processes, including nuclear import, nuclear export,
mitotic progression, and regulation of gene expression.
Translocations between this gene and many other partner genes have
been observed in different leukemias. Rearrangements typically
result in chimeras with the N-terminal GLGF domain of this gene to
the C-terminus of the partner gene (RefSeq, 2002). NUP98
rearrangement induces leukemia in mice. It enhances proliferation
and disrupts differentiation in primary human hematopoietic
precursors (Takeda and Yaseen, 2014). Dys-regulation of homeobox
genes, which cause NUP98 rearrangement, result in leukemic
transformation (Gough et al., 2011; De et al., 2014; Slape and
Aplan, 2004; Grier et al., 2005; Abramovich et al., 2005; Nakamura,
2005; Shimada et al., 2000; Argiropoulos and Humphries, 2007).
NUP98 rearranges with several partners in hematopoietic
malignancies, including acute myeloid leukemia, chronic myeloid
leukemia in blast crisis, myelodysplastic syndrome, acute
lymphoblastic leukemia, and bilineage/biphenotypic leukemia (Tosic
et al., 2009; Haznedaroglu and Beyazit, 2010; Shi et al., 2011;
Gough et al., 2011; Panagopoulos et al., 2003; Morerio et al.,
2006; Moore et al., 2007; Ahuja et al., 2001; McCormack et al.,
2008; Lam and Aplan, 2001). NUP98 is linked to tumorigenesis (Xu
and Powers, 2009; Simon and Rout, 2014). NUP98 is a modulator of
genomic stability and a suppressor of tumor development (Rao et
al., 2009).
[0217] OXSR1 encodes a the Ser/Thr protein kinase that regulates
down-stream kinases in response to oxidative stress and may play a
role in regulating the actin cytoskeleton (RefSeq, 2002). OXSR1 is
up-regulated in the tumor stroma from human breast cancer patients
and associated with recurrence (Pavlides et al., 2010).
[0218] PCSK9 encodes a member of the subtilisin-like proprotein
convertase family, which includes proteases that process protein
and peptide precursors trafficking through regulated or
constitutive branches of the secretory pathway. It plays a role in
cholesterol and fatty acid metabolism (RefSeq, 2002). PCSK9 is
de-regulated in different cancer entities including liver, lung and
gastric cancer (Bhat et al., 2015; Marimuthu et al., 2013; Demidyuk
et al., 2013). PCSK9 deficiency reduces liver metastasis by its
ability to lower cholesterol levels and by enhancing
TNFalpha-mediated apoptosis. Other studies show in contrast no
effect of cholesterol levels on cancer risk (Folsom et al., 2007;
Sun et al., 2012).
[0219] PDAP1 encodes a phosphoprotein that may up-regulate the
PDGFA-stimulated growth of fibroblasts and also down-regulate the
mitogenicity of PDGFB (RefSeq, 2002). PDAP1 is over-expressed in
different cancer types, including gastric and rectal cancer, and
could thereby play a role as a biomarker (Choi et al., 2011;
Marimuthu et al., 2013).
[0220] PDIA3 (also known as ERp57) encodes the protein disulfide
isomerase family A member 3, a protein of the endoplasmic reticulum
that interacts with lectin chaperons, calreticulin, and calnexin to
modulate folding of newly synthesized glycoproteins (RefSeq, 2002;
Coe and Michalak, 2010). PDIA3 may be used as a biomarker and in
the diagnosis of tumors (Shishkin et al., 2013). PDIA3 is
differentially expressed in gliomas (Deighton et al., 2010). PDIA3
is implicated in human pathology including cancer and Alzheimer's
disease (Coe and Michalak, 2010). PDIA3 is an auxiliary factor of
TAP which loads viral and self-peptides on MHC class I (Coe and
Michalak, 2010; Abele and Tampe, 2011).
[0221] PFDN1 encodes prefoldin subunit 1, one of six subunits of
prefoldin, a molecular chaperone complex that binds and stabilizes
newly synthesized polypeptides, thereby allowing them to fold
correctly (RefSeq, 2002). PFDN1 is involved in colorectal cancer
progression, and is positively correlated with tumor size and
invasion (Wang et al., 2015e). PFDN1 is up-regulated in several
cancers including colorectal cancer (Wang et al., 2015e). PFDN1 can
be used as a reference gene in nasopharyngeal carcinoma (Guo et
al., 2010).
[0222] PHB encodes prohibitin which is proposed to play a role in
human cellular senescence and tumor suppression (RefSeq, 2002;
Mishra et al., 2010; Theiss and Sitaraman, 2011; Zhou and Qin,
2013; Mishra et al., 2005; McClung et al., 1995; Rajalingam and
Rudel, 2005). PHB activates the Raf/MEK/ERK pathway which is
involved in cell growth and malignant transformation (Rajalingam
and Rudel, 2005). PHB is a potential biomarker in nasopharyngeal
carcinoma that predicts the treatment response to radiotherapy
(Chen et al., 2015e). PHB was identified in the proteomic analysis
of drug-resistant cancer cells, drug action, and disease state
tissues (Guo et al., 2013). PHB is over-expressed in many cancer
entities (Zhou and Qin, 2013). The core protein of hepatitis C
virus, which is a major risk factor for hepatocellular carcinoma,
induces over-production of oxidative stress by impairing prohibitin
(Theiss and Sitaraman, 2011; Schrier and Falk, 2011; Koike, 2014).
PHB is differentially expressed in gliomas (Deighton et al.,
2010).
[0223] PKM2 encodes pyruvate kinase, muscle, a protein involved in
glycolysis. PKM2 interacts with thyroid hormone and thus may
mediate cellular metabolic effects induced by thyroid hormones. It
is also thought to be involved in bacterial pathogenesis (RefSeq,
2002; Israelsen and Vander Heiden, 2015). PKM2 was shown to be
crucial for cancer cell proliferation and tumor growth (Chen et
al., 2014b; Li et al., 2014c; DeLaBarre et al., 2014). N-myc acts
as a transcriptional regulator for PKM2 in medulloblastoma (Tech et
al., 2015). PKM2 seems to play a role in hepatocarcinogenesis,
epithelial mesenchymal transition, and angiogenesis (Nakao et al.,
2014). PKM2 is one of the two key factors of the Warburg effect in
oncology (Tamada et al., 2012; Warner et al., 2014; Ng et al.,
2015). Expression of PKM2 is up-regulated in cancer cells (Chaneton
and Gottlieb, 2012; Luo and Semenza, 2012; Wu and Le, 2013). In
malignant cells PKM2 functions in glycolysis, as a transcriptional
coactivator and as a protein kinase. In the latter function it
translocates to the nucleus and phosphorylates histone 3 which
finally causes the progress of the cell cycle in glioblastomas
(Semenza, 2011; Luo and Semenza, 2012; Tamada et al., 2012; Venneti
and Thompson, 2013; Yang and Lu, 2013; Gupta et al., 2014; Iqbal et
al., 2014; Chen et al., 2014b; Warner et al., 2014). The
low-activity-dimeric PKM2 might play a role in cancer instead of
the active tetrameric form (Mazurek, 2011; Wong et al., 2015; Iqbal
et al., 2014; Mazurek, 2007).
[0224] PKP3 encodes plakophilin, 3 a member of the arm-repeat and
plakophilin family, which is localized to desmosomes and nuclei and
participates in linking cadherins to intermediate filaments in the
cytoskeleton. PKP3 may act in cellular desmosome-dependent adhesion
and signaling pathways (RefSeq, 2002). Increased PKP3 mRNA in the
blood of gastrointestinal cancer patients can be used as a
biomarker and predictor for disease outcome (Valladares-Ayerbes et
al., 2010). Over-expression of PKP3 was correlated with a poor
outcome in breast, lung and prostate cancer, whereas
down-regulation in bladder cancer is linked to invasive behavior
(Furukawa et al., 2005; Breuninger et al., 2010; Demirag et al.,
2012; Takahashi et al., 2012). Loss of PKP3 leads to increased
protein levels of MMP7 and PRL3, which are required for cell
migration and tumor formation (Khapare et al., 2012; Basu et al.,
2015b).
[0225] PLEC encodes the plakin family member plectin, a protein
involved in the cross-linking and organization of the cytoskeleton
and adhesion complexes (Bouameur et al., 2014). PLEC is
over-expressed in colorectal adenocarcinoma, head and neck squamous
cell carcinoma and pancreatic cancer (Lee et al., 2004; Katada et
al., 2012; Bausch et al., 2011).
[0226] PLXNA2 encodes plexin A2 which is a semaphorin co-receptor.
PLXNA2 is thought to transduce signals from semaphorin 3A and 3C
(RefSeq, 2002). KIAA1199 binds to PLXNA2, resulting in the
inhibition of semaphorin 3A mediated cell death via EGFR
stabilization and signaling (Shostak et al., 2014). PLXNA2 is
up-regulated in TMPRSS2-ERG-positive prostate cancer and metastatic
prostate cancer, resulting in enhanced cell migration and invasion
(Tian et al., 2014). PLXNA2 has higher expression levels in more
aggressive breast cancer and is associated with tumorigenesis
(Gabrovska et al., 2011).
[0227] POLA2 encodes an accessory subunit of DNA polymerase alpha
(also called 70/68 kDa or B subunit) that plays an important role
in the initiation of DNA replication by tethering the catalytic
subunit A and the primase complex (Collins et al., 1993; Pollok et
al., 2003). POLA2 is de-regulated in different cancer types
including gastrointestinal stromal tumors and non-small cell lung
cancer (Mah et al., 2014; Kang et al., 2015). During S-phase, POLA2
is attached to telomeres. It is associated with telomerase activity
and is important for proper telomeric overhang processing through
fill-in synthesis (Diotti et al., 2015).
[0228] PPM1 G encodes protein phosphatase, Mg2+/Mn2+ dependent, 1
G. This protein is found to be responsible for the
dephosphorylation of pre-mRNA splicing factors, which is important
for the formation of functional spliceosomes (RefSeq, 2002). PPM1 G
regulates the E3 ligase VVWP2 which differentially regulates
cellular p73 and DeltaNp73 (Chaudhary and Maddika, 2014). PPM1G is
able to bind apoptosis-stimulating proteins of p53 which are
uniquely over-expressed in various entities (Skene-Arnold et al.,
2013). PPM1 G down-regulates USP7S by dephosphorylation, resulting
in p53 accumulation (Khoronenkova et al., 2012).
[0229] PPP1R15B encodes a protein phosphatase-1 (PP1) interacting
protein. PPP1R15B promotes de-phosphorylation of the transcription
initiation factor EIF2-alpha through recruitment of PP1 catalytic
subunits (RefSeq, 2002). Down-regulation of PPP1R15B results in
impaired proliferation due to unsuccessful transition from G1 to S
phase of the cell cycle, induction of apoptosis by increased
activity of caspase 3/7, and regulation of ERalpha activity
(Shahmoradgoli et al., 2013).
[0230] PPY encodes a protein that is synthesized as a 95 amino acid
polypeptide precursor in the pancreatic islets of Langerhans. It is
cleaved into two peptide products; the active hormone of 36 amino
acids and an icosapeptide of unknown function. The hormone acts as
a regulator of pancreatic and gastrointestinal functions and may be
important in the regulation of food intake (RefSeq, 2002). Patients
with diabetes melitus secondary to pancreatic cancer have a blunted
PPY response to a mixed meal compared to patients with type 2
diabetes melitus. However, the blunted PPY response is only
observed in those pancreas carcinoma patients with a tumor located
in the head of the pancreas (Hart et al., 2015).
[0231] PRKDC encodes the catalytic subunit of the DNA-dependent
protein kinase (DNA-PK) (RefSeq, 2002). PRKDC is a frequently
mutated gene in endometriosis-associated ovarian cancer and breast
cancer (Er et al., 2016; Wheler et al., 2015). PRKDC is
up-regulated in cancerous tissues compared with normal tissues in
colorectal carcinoma. Patients with high PRKDC expression show
poorer overall survival (Sun et al., 2016b).
[0232] PSEN1 encodes presenilin 1 which is linked to Alzheimer's
disease. It is part of the gamma-secretase complex which is
required for Notch activation (RefSeq, 2002; Ponnurangam et al.,
2015). Over-expression of PSEN1 by a small interfering RNA
sensitizes chemoresistant bladder cancer cells to drug-triggered
cell death (Deng et al., 2015a). PSEN1 plays a key role in
epithelial-mesenchymal transition and chemoresistance by
down-regulating E-cadherin (Sehrawat et al., 2014; Dinicola et al.,
2016). TRAF6-mediated PSEN1 activation results in promotion of
tumor invasiveness (Gudey et al., 2014; Sundar et al., 2015).
Down-regulated expression of the gamma-secretase complex is thought
to be a risk factor for breast cancer specific mortality (Peltonen
et al., 2013). PSEN1 is differentially expressed in T-cell acute
lymphoblastic leukemia caused by dys-regulated Notch1 (Paryan et
al., 2013). PSEN1 is over-expressed in oral squamous cell carcinoma
cell lines and primary oral keratinocytes isolated from oral
squamous cell carcinoma tissue. PSEN1 over-expression results in
reduced cell adhesion in oral squamous cell carcinoma by affecting
P-cadherin (Bauer et al., 2013). The endocannabinoid anandamide
increases the expression and recruitment of PSEN1 in
cholangiocarcinoma (Frampton et al., 2010). p53 is able to regulate
PSEN1 expression (Checler et al., 2010). PSEN1 is involved in tumor
reversion (Telerman and Amson, 2009).
[0233] PSEN2 encodes presenilin 2 which is linked to Alzheimer's
disease. It is part of the gamma-secretase complex which is
required for Notch activation (RefSeq, 2002). Oxidative stress and
p53 expression level is increased in PC12 cells carrying a mutated
PSEN2 gene (Nguyen et al., 2007). PSEN2 is a useful prognostic
factor in breast cancer. The novel PSEN2 alleles R62H and R71W
affect PSEN2 function and may potentially confer a moderate risk of
susceptibility to breast cancer (To et al., 2006; Xu et al., 2006).
PSEN2 is part of a 10-gene signature set which is associated with
recurrence-free survival time but not overall survival time in
ovarian carcinoma (Chen and Liu, 2015). Loss of PSEN2 may cause
lung tumor development by up-regulating iPLA2 (Yun et al., 2014).
Down-regulated expression of the gamma-secretase complex is thought
to be a risk factor for breast cancer specific mortality (Peltonen
et al., 2013). PSEN2 is differentially expressed in megakaryocytic
leukemia and gastric cancer. PSEN2 expression correlates with tumor
type, UICC tumor stage, tumor grade, and patient survival (Warneke
et al., 2013; Hao et al., 2006). The promotor of PSEN2 is
de-methylated in glioma tissues, causing PSEN2 over-expression (Liu
et al., 2012). 2-arachidonylglycerol increases the expression and
recruitment of PSEN2 in cholangiocarcinoma (Frampton et al., 2010).
PSEN2 causes tumor cell proliferation in rat pancreatic cancer by
cleaving EpC (Maetzel et al., 2009; Thuma and Zoller, 2013).
[0234] PTGS1 (also known as Cox1) encodes the
prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase
and cyclooxygenase). PTGS1 is constitutively expressed and
catalyzes the conversion of arachinodate to prostaglandin. The
encoded protein regulates angiogenesis in endothelial cells, and is
inhibited by non-steroidal anti-inflammatory drugs, such as
aspirin. Based on its ability to function as both a cyclooxygenase
and as a peroxidase, PTGS1 has been identified as a moonlighting
protein. The protein may promote cell proliferation during tumor
progression (RefSeq, 2002; Tietz et al., 2013). PTGS1 may be
involved in tumorigenesis (Rouzer and Marnett, 2009). Enhanced
tumor growth is supported by up-regulation of PTGS1 which plays a
role in prostaglandin and VEGF production (Campione et al., 2015).
PTGS1 is associated with decreased survival for recurrent minor
salivary gland carcinoma (Haymerle et al., 2015). PTGS1 is
associated with breast carcinogenesis (Basu et al., 2015a; Serra et
al., 2016). PTGS1 is frequently de-regulated in the progression of
cancer (Karnezis et al., 2012). Deletion of PTGS1 results in robust
decrease of basal cell carcinoma (Arbiser, 2010). Aspirin inhibits
PTGS1-induced platelet activation which is thought to be involved
in the development of inflammation and cancer, including colorectal
carcinoma, head and neck cancer, gastrointestinal cancer, and
pancreatic cancer (Pereira et al., 2009; Perrone et al., 2010;
Schror, 2011; Garcia Rodriguez et al., 2013; Bruno et al., 2012;
Yue et al., 2014; Sostres et al., 2014; Schror and Rauch, 2013;
Guillem-Llobat et al., 2014; Patrignani and Patrono, 2015; Patrono,
2015; Dovizio et al., 2015; Jimenez et al., 2007; Klass and Shin,
2007).
[0235] PTGS2 (also called COX-2) encodes prostaglandin-endoperoxide
synthase 2 (cyclooxygenase), the key enzyme in prostaglandin
biosynthesis that acts as a dioxygenase and as a peroxidase
(RefSeq, 2002). Expression of PTGS2 and prostaglandins is
associated with various cancer types including breast, lung,
gastric, pancreatic, colorectal and prostate tumors. The expression
level is also directly proportional to tumor aggressiveness
including metastasis (Shao et al., 2012; Kunzmann et al., 2013;
Misra and Sharma, 2014; Aziz and Qiu, 2014; Thill et al., 2014;
Knab et al., 2014; Huang and Huang, 2014; Wang et al., 2014c).
Anti-inflammatory agents with activity against PTGS2 have a strong
potential for the chemoprevention of cancer (Harris, 2009; Ghosh et
al., 2010).
[0236] PTPN14 encodes protein tyrosine phosphatase, non-receptor
type 14, which appears to regulate lymphatic development in
mammals. A loss-of-function mutation has been found in kindred with
a lymphedema-choanal atresia (RefSeq, 2002). PTPN14 induces
TGF-beta signaling, regulates endothelial-mesenchymal transition,
and organogenesis (Wyatt and Khew-Goodall, 2008). PTPN14 is
down-regulated in cholangiocarcinoma and is inversely correlated
with clinical-pathological features and survival (Wang et al.,
2015d; Wang et al., 2015c). PTPN14 inhibits trafficking of soluble
and membrane-bound proteins, resulting in prevention of metastasis
(Belle et al., 2015). PTPN14 negatively regulates the oncoprotein
Yes-associated protein (YAP), a key protein in the Hippo pathway,
which is responsible for organ size and tumorigenesis (Liu et al.,
2013b; Huang et al., 2013a; Lin et al., 2013a). Loss-of-function
mutations in PTPN14 are involved in neuroblastoma relapse, breast
cancer, and colorectal cancer (Laczmanska and Sasiadek, 2011; Wang
et al., 2004; Schramm et al., 2015; Wyatt and Khew-Goodall,
2008).
[0237] RABGGTB is the beta subunit of Rab geranylgeranyltransferase
that catalyzes the posttranslational geranylgeranylation of Rab
GTPases (Pylypenko et al., 2003). RABGGTB is over-expressed in
chemotherapy-refractory diffuse large B-cell lymphoma (Linderoth et
al., 2008).
[0238] RAC1 encodes the ras-related C3 botulinum toxin substrate 1
(rho family, small GTP binding protein Rac1), a GTPase which
belongs to the RAS superfamily of small GTP-binding proteins.
Members of this superfamily appear to regulate a diverse array of
cellular events, including the control of cell growth, cytoskeletal
reorganization, and the activation of protein kinases (RefSeq,
2002). RAC1 is important for neural crest development and can
prevent melanoma formation (Shakhova, 2014). RAC1 can be activated
by the hepatocyte growth factor and the Met tyrosine kinase
receptor, resulting in proliferation and migration of endothelial
cells (Barrow-McGee and Kermorgant, 2014; Gallo et al., 2015). RAC1
induces ROS in the viral oncogenesis of Kaposi's sarcoma (Mesri et
al., 2013). RAC1 is involved in melanoma initiation and
progression, in breast cancer, and in head and neck cancer (Alan
and Lundquist, 2013; Imianitov, 2013; Meierjohann, 2014). Tiam1 is
able to regulate RAC1, which in turn regulates signaling pathways
involved in cytoskeletal activity, cell polarity, endocytosis and
membrane trafficking, cell migration, adhesion and invasion, cell
growth and survival, metastasis, angiogenesis, and carcinogenesis
(Bid et al., 2013; Boissier and Huynh-Do, 2014). RAC1 is thought to
be an oncogene (Kunz, 2013; Kunz, 2014). Mutations in RAC1 can
cause a variety of disorders, including malignant transformation
(Read, 2013; Chi et al., 2013). Activation of Rac1 results in
formation of actin stress fibers, membrane ruffles, lamellipodia,
and filopodia (Klopocka et al., 2013; van and van Buul, 2012; Lane
et al., 2014). RAC1 is down-regulated in astrocytic tumors, but is
over-expressed in medulloblastoma tumors (Khalil and EI-Sibai,
2012).
[0239] RAS3 encodes ras-related C3 botulinum toxin substrate 3 (rho
family, small GTP binding protein Rac3), a GTPase which belongs to
the RAS superfamily of small GTP-binding proteins. Members of this
superfamily appear to regulate a diverse array of cellular events,
including the control of cell growth, cytoskeletal reorganization,
and the activation of protein kinases (RefSeq, 2002).
Over-expression of RAC3 is associated with poor prognosis in
endometrial carcinoma (Balmer et al., 2006). RAC3 is a target of
ARHGAP6 which acts as a tumor suppressor in cervical cancer (Li et
al., 2015b). RAC3 is involved in the organization of the
cytoskeleton, cell migration, and invasion (Liu et al., 2015c).
RAC3 is differentially expressed in leukemia and non-small cell
lung cancer, and is involved in tumor growth (Tan and Chen, 2014;
Liu et al., 2015c; Koldehoff et al., 2008). RAC3 is involved in the
TGF-beta-induced down-regulation of E-cadherin in esophageal cancer
(Dong et al., 2014; Xu et al., 2007). Rac3 induces the
Rac3/ERK-2/NF-kappaB signaling pathway that triggers breast cancer
cell aggressiveness. Endogenous Rac activity correlates with high
metastatic potential in breast cancer cells (Gest et al., 2013;
Baugher et al., 2005). RAC3 is up-regulated in several cancers,
including leukemia, prostate cancer, and breast cancer (Fernandez
Larrosa et al., 2012; Liu et al., 2015c; Culig and Bartsch, 2006;
Calaf and Roy, 2007; Engers et al., 2007; Colo et al., 2007a; Colo
et al., 2007b). RAC3 is an NF-kappaB coactivator which regulates
cyclin D1 expression (Rubio et al., 2012; Colo et al., 2007b).
Over-expression of RAC3 in ERalpha-positive breast cancer results
in enhanced cell migration (Walker et al., 2011; Rubio et al.,
2006).
[0240] RAD54 encodes a protein belonging to the DEAD-like helicase
superfamily. It shares similarity with Saccharomyces cerevisiae
RAD54 and RDH54, both of which are involved in homologous
recombination and repair of DNA. This protein binds to
double-stranded DNA, and displays ATPase activity in the presence
of DNA. This gene is highly expressed in testis and spleen, which
suggests active roles in meiotic and mitotic recombination (RefSeq,
2002). Homozygous mutations of RAD54B were observed in primary
lymphoma and colon cancer (Hiramoto et al., 1999). RAD54B
counteracts genome-destabilizing effects of direct binding of RAD51
to dsDNA in human tumor cells (Mason et al., 2015).
[0241] RAI14 (also called NORPEG) encodes retinoic acid induced 14.
The gene is detected in retinal pigment epithelial cells where it
is inducible by all-trans-retinoic acid that is ubiquitously
expressed in human tissues and may have a role in human testis
development and spermatogenesis (Kutty et al., 2001; Yuan et al.,
2005). RAI14 is de-regulated in gastric cancer and connected with
cell proliferation. It is a prognostic marker for relapse-free
survival for lung and breast cancer patients (Zhou et al., 2015a;
Hsu et al., 2013).
[0242] RBM19 encodes a nucleolar protein that contains six
RNA-binding motifs and may be involved in ribosome biogenesis
(RefSeq, 2002). RBM19 is widely expressed in human colorectal
carcinoma (Lorenzen et al., 2005). Mutational inactivation of RBM19
results in elevated p53 activity and increased apoptosis in mice
(Zhang et al., 2008; Deisenroth and Zhang, 2010).
[0243] RPF1 (also called BXDC5) encodes a nucleolar RNA binding
protein that contains a sigma(70)-like motif and is required for
ribosome biogenesis (Wehner and Baserga, 2002).
[0244] RPL13A encodes a member of the L13P family of ribosomal
proteins that is a component of the 60S ribosomal subunit. The
encoded protein also plays a role in the repression of inflammatory
genes as a component of the IFN-gamma-activated inhibitor of
translation (GAIT) complex (RefSeq, 2002). RPL13A is de-regulated
in different cancer types including prostate, liver and colorectal
cancer (Kasai et al., 2003; Ohl et al., 2005; Yoon et al., 2006).
Depletion of RPL13A causes significant reduction of methylation of
ribosomal RNA and of cap-independent translation mediated by IRES
elements derived from p27, p53 and SNAT2 mRNAs (Chaudhuri et al.,
2007).
[0245] RPL13AP20 encodes ribosomal protein L13a pseudogene that is
located on chromosome 12p13.1 (Balasubramanian et al., 2009).
[0246] RPL13AP5 encodes a ribosomal protein L13a pseudogene that is
located on chromosome 10q24.1 (Balasubramanian et al., 2009).
[0247] RPL34 encodes the ribosomal protein L34 which is a component
of the 60S subunit. Over-expression of this gene has been observed
in some cancer cells (RefSeq, 2002). Over-expression of RPL34
results in the promotion of malignant proliferation in non-small
cell lung cancer (Yang et al., 2016). RPL34 plays a critical role
in cell proliferation, cell cycle distribution and apoptosis of
human malignant gastric cells (Liu et al., 2015a).
[0248] RPTOR (also known as RAPTOR) encodes the regulatory
associated protein of mTOR, complex 1. The protein is a compartment
of a signaling pathway that regulates cell growth in response to
nutrient and insulin levels. The protein positively regulates the
down-stream effector ribosomal protein S6 kinase, and negatively
regulates the mTOR kinase (RefSeq, 2002). In the absence of either
tuberous sclerosis complex 1 or 2, mTOR-RPTOR signaling gets
constitutively activated, resulting in enhanced and de-regulated
protein synthesis and cell growth (Avruch et al., 2005; Kwiatkowski
and Manning, 2005). mTOR positively regulates cell growth and
survival primarily through direct interaction with RPTOR (Sun,
2013). In complex with mTOR, RPTOR controls cap-dependent
translation, and this function is essential for PI3K-initiated
oncogenesis (Vogt et al., 2010). Rapalogs are agents that primarily
inhibit the mTOR-RPTOR complex 1 (mTORC1, rapamycin-sensitive) and
are used in breast cancer therapy (Wysocki, 2009; De et al., 2013;
Vinayak and Carlson, 2013; Le et al., 2008).
[0249] SEC24D encodes SEC24 homolog D, COPII coat complex
component. SEC24D has similarity to yeast Sec24p component of
COPII. COPII is the coat protein complex responsible for vesicle
budding from the ER. This gene product is implicated in the shaping
of the vesicle, and also in cargo selection and concentration.
Mutations in this gene have been associated with Cole-Carpenter
syndrome, a disorder affecting bone formation, resulting in
craniofacial malformations and bones that break easily (RefSeq,
2002). The induction ratio of SEC24D is enhanced in the human
prostate cancer cell line LNCaP (DePrimo et al., 2002; Zhao et al.,
2004). SEC24D can be phosphorylated by Akt (Sharpe et al.,
2011).
[0250] SEPT10 encodes a member of the septin family of
filament-forming cytoskeletal GTPases. It is localized to the
cytoplasm and nucleus and displays GTP-binding and GTPase activity
(RefSeq, 2002). SEPT10 is down-regulated in different cancer types
including bladder, breast, liver, lung, pancreas and prostate
cancer as well as melanoma and leukemia. It is associated with poor
prognosis for survival (Kienle et al., 2010; Liu et al.,
2010b).
[0251] SEPT11 encodes a member of the conserved septin family of
filament-forming cytoskeletal GTPases that are involved in a
variety of cellular functions including cytokinesis and vesicle
trafficking (RefSeq, 2002). SEPT11 is over-expressed in different
cancer entities including brain, cervix, pancreas and prostate
cancer, melanoma and leukemia (Liu et al., 2010b). Loss of
heterozygosity (LOH) of SEPT11 is associated with poor prognosis in
hepatocellular carcinomas. A fusion transcript with MLL has been
identified in myeloid neoplasia (Huang et al., 2010; Cerveira et
al., 2011).
[0252] SEPT8 encodes a member of the septin family of nucleotide
binding proteins which is highly conserved and plays a role in the
regulation of cytoskeletal organization and cytokinesis (RefSeq,
2002). SEPT8 is up-regulated in different cancer types including
bladder, liver, pancreas and lung cancer as well as leukemia (Liu
et al., 2010b).
[0253] SERPINB2 (also known as PAI2) encodes serpin peptidase
inhibitor, Glade B (ovalbumin), member 2 and is located on
chromosome 18q21.3. It is a non-conventional serine protease
inhibitor (SERPIN) which influences gene expression, cell
proliferation and differentiation, and apoptosis (RefSeq, 2002;
Medcalf and Stasinopoulos, 2005). SERPINB2 encodes serpin peptidase
inhibitor, Glade B (ovalbumin), member 2, an inhibitor of
extracellular protease urokinase plasminogen activator and tissue
plasminogen activator (Schroder et al., 2014). SERPINB2 is
expressed in a number of different tumors. SERPINB2 expression is
associated with favorable prognosis in breast and pancreatic
cancers, but poor prognosis in endometrial, ovarian, and colorectal
cancers (Schroder et al., 2014). SERPINB2 is an invasion- and
metastasis-related gene (Pucci et al., 2016). SERPINB2 regulates
urokinase-type plasminogen activator (uPA) which triggers the
conversion of plasminogen to plasm in. Plasm in is able to degrade
the extracellular matrix (ECM), an important process of tumor
progression (Gershtein and Kushlinskii, 1999; Ulisse et al., 2009;
Berger, 2002; Baldini et al., 2012; Mekkawy et al., 2014; Andreasen
et al., 2000). Degradation of the ECM results in tumor progression,
tumor mass expansion, tumor growth factor release, cytokine
activation, tumor cell proliferation, migration, and invasion
(Hildenbrand et al., 2010; Magdolen et al., 2003; Halamkova et al.,
2012; Duffy, 2004; Mekkawy et al., 2014; Dass et al., 2008). Many
tumors show a correlation between uPA system components and tumor
aggressiveness and survival (Mekkawy et al., 2014; Duffy and
Duggan, 2004; Han et al., 2005). High levels of SERPINB2 decrease
tumor growth and metastasis (Croucher et al., 2008). SH3BP4 encodes
the SH3-domain binding protein 4 which is involved in
cargo-specific control of clathrin-mediated endocytosis,
specifically controlling the internalization of a specific protein
receptor (RefSeq, 2002). SH3BP4 expression is 7-fold increased in
the retinoblastoma cell line Y79 (Khanobdee et al., 2004).
Fibroblast growth factor receptor 10 stimulation in SH3BP4-depleted
cells causes a decreased cell migration in breast cancer cells and
the inhibition of epithelial branching in mouse lung explants
(Francavilla et al., 2013).
[0254] SHCBP1 encodes a protein that associates with human
centralspindlin and is one of the crucial factors involved in
midbody organization and cytokinesis completion (Asano et al.,
2014). SHCBP1 is up-regulated in human hepatocellular carcinoma.
Targeting SHCBP1 inhibits cell proliferation in human
hepatocellular carcinoma cell lines (Tao et al., 2013). Among 16
genes with concomitant genomic alterations, SHCBP1 may be involved
in tumorigenesis and in the processes of invasion and progression
from pre-invasive ductal carcinoma in situ to invasive ductal
carcinoma (Colak et al., 2013).
[0255] SIGMAR1 (also called OPRS1 or SIG-1R) encodes a sigma
non-opioid intracellular receptor that interacts with a variety of
psychotomimetic drugs, including cocaine and amphetamines.
Mutations in this gene are associated with a juvenile amyotrophic
lateral sclerosis (RefSeq, 2002). SIGMAR1 is over-expressed in
tumor cell lines and tumors of various cancer tissues, including
lung, colon, skin, and breast cancer. SIGMAR1 over-expression is
associated with cell proliferation (Vilner et al., 1995; Aydar et
al., 2004; Aydar et al., 2006; Bem et al., 1991; Skrzycki and
Czeczot, 2013). SIGMAR1 promotes hERG/betl-integrin signaling,
triggers the activation of the PI3K/Akt pathway, and induces the
phosphorylation of translational regulator proteins like p70S6K, S6
and 4E-BP1. SIGMAR1 increases motility and VEGF secretion, thus
enhancing the aggressiveness of tumor cells (Crottes et al., 2016;
Kim et al., 2012a).
[0256] SLC16A3 encodes solute carrier family 16 member 3, a
proton-linked monocarboxylate transporter (RefSeq, 2002). Most
solid tumors are known to rely on glycolysis for energy production.
High rates of glycolysis result in an increased production of
lactate which has been associated with poor clinical outcome and
direct contribution to tumor growth and progression. SLC16A3 is one
of few monocarboxylate transporters which facilitate the lactate
export in cancer cells (Dhup et al., 2012; Draoui and Feron, 2011).
The SLC16A3 expression has been associated with poor prognosis in
hepatocellular cancer patients and increased cell proliferation,
migration and invasion in cell line experiments (Gao et al., 2015).
The functional involvement of SLC16A3 in the tumorigenesis was
shown in a subset of pancreatic cancer (Baek et al., 2014).
[0257] SLC1A4 (also known as ASCT1) encodes solute carrier family
(glutamate/neutral amino acid transporter), member 4 which is
located on chromosome 2p15-p13 (RefSeq, 2002). The hepatocellular
carcinoma cell line C3A enhances SLC1A4 expression after cysteine
deprivation (Lee et al., 2008b). SLC1A4 acts as a recruiter of
amino acids in esophageal adenocarcinoma (Younes et al., 2000).
Knock-down of ASCT2 enhances SLC1A4 mRNA levels in human hepatoma
cells (Fuchs et al., 2004). Activation of the v-myc
myelocytomatosis viral oncogene homologue gene leads to an
up-regulation of SLC1A4 in the human glioma cell line Hs683 (Jiang
et al., 2012). Glutamine deprivation does not lead to an
up-regulation of SLC1A4 in neuroblastoma (Wasa et al., 2002).
[0258] SLC1A5 (also known as ASCT2) encodes solute carrier family
(glutamate/neutral amino acid transporter), member 5, which is a
sodium-dependent neutral amino acid transporter that can act as a
receptor for RD114/type D retrovirus (RefSeq, 2002). c-Myc
activation increases SLC1A5 expression (Perez-Escuredo et al.,
2016). Over-expression of SLC1A5 is associated with poor prognosis
in clear-cell renal cell carcinoma (Liu et al., 2015d). A high
expression of CD147 is significantly associated with SLC1A5 in
patients with pancreatic cancer (Kaira et al., 2015). SLC1A5 might
be a biomarker for non-small cell lung cancer (Hassanein et al.,
2015; Hassanein et al., 2016). The ubiquitin ligase RNF5 regulates
SLC1A5 in breast cancer (Jeon et al., 2015). SLC1A5 is
over-expressed in several cancer entities, including advanced
laryngeal cancer, prostate cancer, and adenoid cystic carcinoma
(Koo and Yoon, 2015; Wang et al., 2015f; Bhutia et al., 2015;
Nikkuni et al., 2015; Ganapathy et al., 2009). Inhibition of SLC1A5
in breast cancer leads to reduced glutamine uptake and
proliferation (Chen et al., 2015d; van et al., 2015).
[0259] SLC1A5 may stimulate tumor growth by regulating mTOR
(Nakanishi and Tamai, 2011; Fuchs and Bode, 2005; Corbet et al.,
2016; McGivan and Bungard, 2007).
[0260] SLC26A6 encodes a member of the solute carrier family 26
which consists of anion transport proteins. SLC26A6 is involved in
transporting chloride, oxalate, sulfate and bicarbonate ions
(RefSeq, 2002). Mutations of SLC26A6 have been identified in
different colorectal cancer cell lines (Donnard et al., 2014).
SLC26A6 gene expression and promoter activity are inhibited by
IFN-gamma (Saksena et al., 2010).
[0261] SLC52A3 (also called RFT2 or C20orf54) encodes a member of
the solute carrier family 52. It is a riboflavin transporter
protein that likely plays a role in intestinal absorption of
riboflavin (RefSeq, 2002). SLC52A3 is de-regulated in different
cancer entities including gastric cancer, esophageal squamous cell
carcinoma and cervical cancer. Single nucleotide polymorphisms of
SLC52A3 correlate with cancer risks in esophageal squamous cell
carcinoma and gastric cardia adenocarcinomas (Jiang et al., 2014b;
Duan et al., 2015; Matnuri et al., 2015; Eli et al., 2012; Aili et
al., 2013). Knock-down of SLC52A3 increases p21 and p27 protein
levels and decreases their down-stream targets cyclin E1 and Cdk2,
leading to cell cycle arrest at G1-G1/S. Knock-down of SLC52A3 also
leads to the activation of caspase-3 and apoptosis (Jiang et al.,
2014b).
[0262] SLC6A15 encodes a member of the solute carrier family 6
which transports neutral amino acids. SLC6A15 might play a role in
neuronal amino acid transport and might be associated with major
depression (RefSeq, 2002). SLC6A15 is hyper-methylated and thereby
down-regulated in colorectal cancer and may be a candidate
biomarker for a stool-based assay (Kim et al., 2011b; Mitchell et
al., 2014).
[0263] SMIM10 (also called CXorf69 or LOC644538) encodes a small
integral membrane protein located on chromosome Xq26.3 (RefSeq,
2002).
[0264] SNX14 encodes a member of the sorting nexin family and
contains a regulator of G protein signaling (RGS) domain (RefSeq,
2002). SNX14 is down-regulated upon rasV12/E1A transformation of
mouse embryonic fibroblasts and may be associated with tumor
development (Vasseur et al., 2005).
[0265] SSH1 (also called SSH1 L) encodes a member of the slingshot
homolog (SSH) family of phosphatases. The SSH family appears to
play a role in actin dynamics by reactivating cofilin proteins
(RefSeq, 2002). SSH1 is over-expressed in pancreatic cancer and
associated with tumor cell migration (Wang et al., 2015k).
Inhibition of PKD1 by neuregulin leads to the localization of SSH1
to F-actin, increased cofilin activity and increased reorganization
of the actin cytoskeleton and cell migration. The SSH1-dependent
activation of cofilin is induced by the PI3K/Akt signaling pathway
(Wang et al., 2010; Doppler et al., 2013).
[0266] STAT2 operates as a positive regulator in the
transcriptional activation response elicited by IFNs (Steen and
Gamero, 2012). STAT2 may regulate tumor cell response to
interferons (Shodeinde et al., 2013). A link between STAT2 and
tumorigenesis was observed in transgenic mice lacking STAT2 (Yue et
al., 2015). or expressing constitutively IFN-alpha in the brain
(Wang et al., 2003).
[0267] SUPT16H encodes a subunit of FACT (facilitates chromatin
transcription), an accessory factor which is needed for the
transcription of DNA packaged into chromatin (RefSeq, 2002).
SUPT16H is de-regulated in endothelial and stromal components of
juvenile nasopharyngeal angiofibroma (JNA) and could thereby play a
role as a potential molecular marker (Silveira et al., 2012).
SUPT16H is involved in DNA double-strand break repair by remodeling
of chromatin. SUPT16H activates p53 by forming a complex with CK2
(Keller et al., 2001; Kari et al., 2011).
[0268] SUSD1 encodes a sushi domain containing protein and is
associated with an increased risk of venous thromboembolism (Tang
et al., 2013). The heterozygous SUSD1-ROD1/PTBP3 fusion transcript
is expressed in a human breast cancer cell line (Newman et al.,
2013).
[0269] TAF6L encodes a protein with structurally similarity to the
histone like TATA-box binding protein associated factor 6 (TAF6).
It is a component of the PCAF histone acetylase complex which is
required for myogenic transcription and differentiation (RefSeq,
2002). The expression of miR-145 and miR-196a negatively correlates
with the expression of TAF6L (Havelange et al., 2011). TAF6L is
inactivated in the small cell lung cancer cell line H187 by forming
the fusion transcript TAF6L-GNG3 (Fernandez-Cuesta et al.,
2015).
[0270] TEP1 encodes telomerase associated protein 1, a component of
the ribonucleoprotein complex responsible for telomerase activity,
which catalyzes the addition of new telomeres on the chromosome
ends (RefSeq, 2002; Szaflarski et al., 2011). TEP1 is a main part
of vaults to which also major vault protein (MVP) belongs (Lara et
al., 2011; Mossink et al., 2003). TEP1 is expressed in thyroid
carcinoma (Hoang-Vu et al., 2002).
[0271] TFPI encodes tissue factor pathway inhibitor, a protease
inhibitor that regulates the tissue factor (TF)-dependent pathway
of blood coagulation (RefSeq, 2002). TFPI is expressed in breast
cancer, colorectal cancer, and pancreatic cancer cell lines (Kurer,
2007). TFPI induces HIF1alpha, c-Myc, c-SRC, and HDAC2 in breast
cancer (Davies et al., 2014). TFPI expression level is decreased in
sarcomas compared to non-malignant lesions (Savitskaya et al.,
2012). TFPI inhibits the protease activity of the TF-VIIa complex
which is involved in metastasis (Fischer et al., 1999; Sandset and
Abildgaard, 1991; Lindahl et al., 1991).
[0272] TFPI2 encodes tissue factor pathway inhibitor 2 which can
inhibit a variety of serine proteases including factor Vila/tissue
factor, factor Xa, plasmin, trypsin, chymotrypsin, and plasma
kallikrein. This gene has been identified as a tumor suppressor
gene in several types of cancer (RefSeq, 2002; Sierko et al.,
2007). TFPI2 may be used as a biomarker for relapse prediction in
pancreatic carcinoma (Zhai et al., 2015c). DNA methylation of TFPI2
can be used as a biomarker for colorectal cancer in a fecal occult
blood test (Koga et al., 2015). TFPI2 induces apoptosis and
inhibits invasiveness, growth of neoplasms, metastasis, and
angiogenesis (Ghilardi et al., 2015; Amirkhosravi et al., 2007;
Sierko et al., 2007). TFPI2 is hyper-methylated and down-regulated
in cancer, and expression is correlated with the degree of cancer,
early tumor recurrence, and poor prognosis (Sun et al., 2016a;
Sierko et al., 2007). TFPI2 is down-regulated in pancreatic cancer
and cholangiocarcinoma (Chu et al., 2015; Zhai et al., 2015a; Zhai
et al., 2015b). TFPI2 is methylated in gastric cancer, canine
diffuse large B-cell lymphoma, acute myeloid leukemia, non-small
cell lung cancer, cervical cancer, oral squamous cell carcinoma,
inflammation-associated colon cancer, and hepatocellular carcinoma
(Qu et al., 2013; Ferraresso et al., 2014; Liu et al., 2014b; Shao
et al., 2014; Lai et al., 2014; Hamamoto et al., 2015; Li et al.,
2015d; Gerecke et al., 2015; Dong et al., 2015; Sun et al., 2016a).
TFPI2 is a well-validated DNA methylation biomarker in cancer
(Fukushige and Horii, 2013; Huisman et al., 2015).
[0273] TGFBI encodes an RGD-containing protein that binds to type
I, II and IV collagens, is induced by transforming growth
factor-beta which plays a role in cell-collagen interactions and
acts to inhibit cell adhesion (RefSeq, 2002). TGFBI expression was
shown to be elevated in cholangiocarcinoma, hepatic carcinoma,
gastric carcinoma, esophageal squamous cell carcinoma and clear
cell renal cell carcinoma. Furthermore, TGFBI was shown to be
associated with colorectal cancer (Lebdai et al., 2015; Ozawa et
al., 2014; Zhu et al., 2015a; Han et al., 2015).
[0274] TGIF2-C20orf24 encodes a fusion protein that shares sequence
identity with TGIF2 and C20orf24 (RefSeq, 2002).
[0275] TMEM154 encodes a transmembrane protein that is associated
with an increased risk for type 2 diabetes and that seems to play a
role in beta cell function (Harder et al., 2015).
[0276] TRAM2 encodes translocation associated membrane protein 2.
It is a component of the translocon, a gated macromolecular channel
that controls the posttranslational processing of nascent secretory
and membrane proteins at the endoplasmic reticulum (ER) membrane
(RefSeq, 2002). Runx2 may regulate TRAM2 expression (Pregizer et
al., 2007). SNPs in TRAM2 can increase the risk of bone fracture in
ER-positive breast cancer patients (Liu et al., 2014a).
[0277] TRPV2 encodes an ion channel that is activated by
temperatures above 52 degrees Celsius. It may be involved in
transduction of high-temperature heat response s in sensory ganglia
(RefSeq, 2002). TRPV2 is de-regulated in different cancer types
including esophageal, prostate, liver and bladder cancer and
leukemia. Loss or alterations of TRPV2 lead to uncontrolled
proliferation and resistance to apoptotic stimuli (Liberati et al.,
2014a; Zhou et al., 2014; Liberati et al., 2014b; Liu et al.,
2010a; Morelli et al., 2013). Silencing of TRPV2 in glioma cells
leads to down-regulation of Fas and pro-caspase 8 as well as
up-regulation of Cyclin E1, CDK2 E2F1 and Bcl-2-associated X
protein. TRPV2 over-expression in bladder cancer cells leads to an
enhanced cell migration and invasion (Nabissi et al., 2010; Liu and
Wang, 2013).
[0278] TSEN15 encodes tRNA splicing endonuclease subunit 15. This
endonuclease catalyzes the removal of introns from tRNA precursors
(RefSeq, 2002; Trotta et al., 2006). TSEN15 is a target of
miRNA-449a, which functions as a tumor suppressor in neuroblastoma.
TSEN15 plays an important role in mediating the
differentiation-inducing function of miRNA-449a (Zhao et al.,
2015). TSEN15 is associated with cell differentiation potential in
human fetal femur-derived cells (Mirmalek-Sani et al., 2009).
[0279] UBE2C (also called UBCH10) encodes a member of the E2
ubiquitin-conjugating enzyme family. It is required for the
destruction of mitotic cyclins and cell cycle progression (RefSeq,
2002). UBE2C is often up-regulated by gene amplification, as
observed in patients with breast, lung and colorectal cancer. UBE2C
up-regulation correlates with poor prognosis and tumor progression
(Okamoto et al., 2003; Wagner et al., 2004; Fujita et al., 2009;
Chen et al., 2010; Hao et al., 2012). UBE2C is up-regulated in U251
glioma cells and in tissues from colorectal carcinoma (CRC)
patients. UBE2C knock-down induces apoptosis through the induction
of Bax and p53, down-regulation of Bcl-2 and G2/M arrest of the
cell cycle. UBE2C suppression de-regulates cyclin B and ERK1 in CRC
(Cacciola et al., 2015; Jiang et al., 2010).
[0280] UBIAD1 (also called TERE1) encodes a protein containing an
UbiA prenyltransferase domain that might be involved in cholesterol
and phospholipid metabolism (RefSeq, 2002). The tumor suppressor
UBIAD1 is down-regulated in different cancer entities, including
bladder, prostate and renal cancer, and is associated with growth
regulation (McGarvey et al., 2001; Fredericks et al., 2011;
McGarvey et al., 2003; Fredericks et al., 2013). UBIAD1 regulates
the phosphorylation of the growth factor-related p42/44 MAP kinase.
The proper Golgi localization of UBIAD1 influences its tumor
suppressor activities including apoptosis (McGarvey et al., 2005;
Wang et al., 2013d).
[0281] UBR1 encodes ubiquitin protein ligase E3 component
N-recognin 1. It binds to a destabilizing N-terminal residue of a
substrate protein and participates in the formation of a
substrate-linked multi-ubiquitin chain, addressing the protein for
the proteolytic pathway of the ubiquitin system (RefSeq, 2002).
Loss or reduction of UBR1 expression is associated with spontaneous
B-cell lymphomas and T-cell acute lymphoblastic leukemia (Chen et
al., 2006). UBR1 regulates the homeostasis of MGMT, a DNA repair
enzyme that protects cells from carcinogenic effects of alkylating
agents (Leng et al., 2015).
[0282] UBR2 encodes an E3 ubiquitin ligase of the N-end rule
proteolytic pathway that targets proteins with destabilizing
N-terminal residues for polyubiquitylation and proteasome-mediated
degradation (RefSeq, 2002). Autoantibodies against UBR2 are
detected in serum of patients with autoimmune pancreatitis and
pancreatic cancer (Frulloni et al., 2009). UBR2 is up-regulated by
tumor cell-induced cachectic stimuli via activation of
p38beta/MAPK, C/EBPbeta phosphorylation and binding to the UBR2
promotor (Zhang et al., 2013b).
[0283] URB1 is required for ribosome biogenesis during early
maturation of 60S ribosomal subunits (Rosado and de la Cruz,
2004).
[0284] USP11 encodes ubiquitin specific peptidase 11. Protein
ubiquitination controls many intracellular processes, including
cell cycle progression, transcriptional activation, and signal
transduction (RefSeq, 2002). USP11 is a novel regulator of p53,
which is required for p53 activation in response to DNA damage (Ke
et al., 2014a). USP11 plays a major role in promyelocytic leukemia
and pancreatic cancer (Burkhart et al., 2013; Wu et al., 2014).
[0285] USP22 encodes ubiquitin specific peptidase 22 and is located
on chromosome 17p11.2 (RefSeq, 2002). High expression of USP22 was
observed in hepatocellular carcinoma, colon carcinoma, gastric
carcinoma, epithelial ovarian cancer, pancreatic cancer, glioma,
salivary adenoid cystic carcinoma, and papillary thyroid carcinoma
(Wang et al., 2013b; Dai et al., 2014; Liang et al., 2014a; Liang
et al., 2014b; Ji et al., 2015; He et al., 2015; Wang et al.,
2015n; Tang et al., 2015). USP22 promotes tumor progression and
induces epithelial mesenchymal transition in lung adenocarcinoma
(Hu et al., 2015a). USP22 acts as an oncogene by regulating the
stability of cyclooxygenase 2 in non-small cell lung cancer (Xiao
et al., 2015). USP22 plays a critical regulatory role in the
pathologic processes of nasopharyngeal carcinoma, and it may be a
potential treatment target (Zhuang et al., 2015). Over-expression
of USP22 may contribute to the progression of breast cancer (Zhang
et al., 2011).
[0286] UTP20 is a component of the U3 small nucleolar RNA protein
complex and is involved in 18s rRNA processing (RefSeq, 2002).
UTP20 expression is decreased in metastatic human breast tumor cell
lines (Schwirzke et al., 1998; Goodison et al., 2003). UTP20 is
expressed at high levels in gastric cancer tissues and premalignant
lesions implicating the involvement of UTP20 in cell transformation
(Xing et al., 2005).
[0287] WLS (also called EVI or GPR177) encodes Wntless Wnt ligand
secretion mediator. WLS represents an ancient partner for Wnts
dedicated to promoting their secretion into the extracellular
milieu (Banziger et al., 2006). WLS is over-expressed in different
cancer entities including breast, gastric, ovarian and colorectal
cancer as well as leukemia and is associated with poor outcome
(Chiou et al., 2014; Stewart et al., 2015; Lu et al., 2015;
Voloshanenko et al., 2013). WLS is important for the secretion of
all Wnt proteins. It regulates the expression of beta-catenin and
cyclin-D1, thereby influencing cell proliferation (Yang et al.,
2015b; Banziger et al., 2006).
[0288] YIF1A encodes Yip1 interacting factor homolog A and is
located on chromosome 11q13 (RefSeq, 2002). Several mutations
(amplifications and deletions) have been detected in the YIF1A gene
in hepatocellular carcinoma (Nalesnik et al., 2012). YIF1A
expression shows a significant difference between normal and
squamous cell carcinoma samples (Sugimoto et al., 2009).
[0289] ZRANB3 encodes zinc finger, RAN-binding domain containing 3
and is located on chromosome 2q21.3 (RefSeq, 2002). ZRANB3 encodes
a zinc finger protein that is a structure-specific ATP-dependent
endonuclease. It is involved in replication stress response to
maintain genomic integrity (Ciccia et al., 2012; Weston et al.,
2012). Single nucleotide polymorphism rs4954256, located in ZRANB3
on chromosome 2q21.3, was associated with a 3.93-fold increase in
pathologic complete response to concurrent chemoradiation therapy
in the treatment of esophageal cancer (Chen et al., 2012). ZRANB3
is frequently mutated in endometrial cancer (Lawrence et al.,
2014).
DETAILED DESCRIPTION OF THE INVENTION
[0290] Stimulation of an immune response is dependent upon the
presence of antigens recognized as foreign by the host immune
system. The discovery of the existence of tumor associated antigens
has raised the possibility of using a host's immune system to
intervene in tumor growth. Various mechanisms of harnessing both
the humoral and cellular arms of the immune system are currently
being explored for cancer immunotherapy.
[0291] Specific elements of the cellular immune response are
capable of specifically recognizing and destroying tumor cells. The
isolation of T-cells from tumor-infiltrating cell populations or
from peripheral blood suggests that such cells play an important
role in natural immune defense against cancer. CD8-positive T-cells
in particular, which recognize class I molecules of the major
histocompatibility complex (MHC)-bearing peptides of usually 8 to
10 amino acid residues derived from proteins or defect ribosomal
products (DRIPS) located in the cytosol, play an important role in
this response. The MHC-molecules of the human are also designated
as human leukocyte-antigens (HLA).
[0292] The term "T-cell response" means the specific proliferation
and activation of effector functions induced by a peptide in vitro
or in vivo. For MHC class I restricted cytotoxic T cells, effector
functions may be lysis of peptide-pulsed, peptide-precursor pulsed
or naturally peptide-presenting target cells, secretion of
cytokines, preferably Interferon-gamma, TNF-alpha, or IL-2 induced
by peptide, secretion of effector molecules, preferably granzymes
or perforins induced by peptide, or degranulation.
[0293] The term "peptide" is used herein to designate a series of
amino acid residues, connected one to the other typically by
peptide bonds between the alpha-amino and carbonyl groups of the
adjacent amino acids. The peptides are preferably 9 amino acids in
length, but can be as short as 8 amino acids in length, and as long
as 10, 11, 12, or 13 amino acids or longer, and in case of MHC
class II peptides (elongated variants of the peptides of the
invention) they can be as long as 14, 15, 16, 17, 18, 19 or 20 or
more amino acids in length.
[0294] Furthermore, the term "peptide" shall include salts of a
series of amino acid residues, connected one to the other typically
by peptide bonds between the alpha-amino and carbonyl groups of the
adjacent amino acids. Preferably, the salts are pharmaceutical
acceptable salts of the peptides, such as, for example, the
chloride or acetate (trifluoroacetate) salts. It has to be noted
that the salts of the peptides according to the present invention
differ substantially from the peptides in their state(s) in vivo,
as the peptides are not salts in vivo.
[0295] The term "peptide" shall also include "oligopeptide". The
term "oligopeptide" is used herein to designate a series of amino
acid residues, connected one to the other typically by peptide
bonds between the alpha-amino and carbonyl groups of the adjacent
amino acids. The length of the oligopeptide is not critical to the
invention, as long as the correct epitope or epitopes are
maintained therein. The oligopeptides are typically less than about
30 amino acid residues in length, and greater than about 15 amino
acids in length.
[0296] The term "polypeptide" designates a series of amino acid
residues, connected one to the other typically by peptide bonds
between the alpha-amino and carbonyl groups of the adjacent amino
acids. The length of the polypeptide is not critical to the
invention as long as the correct epitopes are maintained. In
contrast to the terms peptide or oligopeptide, the term polypeptide
is meant to refer to molecules containing more than about 30 amino
acid residues.
[0297] A peptide, oligopeptide, protein or polynucleotide coding
for such a molecule is "immunogenic" (and thus is an "immunogen"
within the present invention), if it is capable of inducing an
immune response. In the case of the present invention,
immunogenicity is more specifically defined as the ability to
induce a T-cell response. Thus, an "immunogen" would be a molecule
that is capable of inducing an immune response, and in the case of
the present invention, a molecule capable of inducing a T-cell
response. In another aspect, the immunogen can be the peptide, the
complex of the peptide with MHC, oligopeptide, and/or protein that
is used to raise specific antibodies or TCRs against it.
[0298] A class I T cell "epitope" requires a short peptide that is
bound to a class I MHC receptor, forming a ternary complex (MHC
class I alpha chain, beta-2-microglobulin, and peptide) that can be
recognized by a T cell bearing a matching T-cell receptor binding
to the MHC/peptide complex with appropriate affinity. Peptides
binding to MHC class I molecules are typically 8-14 amino acids in
length, and most typically 9 amino acids in length.
[0299] In humans there are three different genetic loci that encode
MHC class I molecules (the MHC-molecules of the human are also
designated human leukocyte antigens (HLA)): HLA-A, HLA-B, and
HLA-C. HLA-A*01, HLA-A*02, and HLA-B*07 are examples of different
MHC class I alleles that can be expressed from these loci.
TABLE-US-00006 TABLE 5 Expression frequencies F of HLA-A*02 and
HLA-A*24 and the most frequent HLA-DR serotypes. Frequencies are
deduced from haplotype frequencies Gf within the American
population adapted from Mori et al. (Mori et al., 1997) employing
the Hardy-Weinberg formula F = 1 - (1 - Gf).sup.2. Combinations of
A*02 or A*24 with certain HLA-DR alleles might be enriched or less
frequent than expected from their single frequencies due to linkage
disequilibrium. For details refer to Chanock et al. (Chanock et
al., 2004). Calculated phenotype from Allele Population allele
frequency A*02 Caucasian (North America) 49.1% A*02 African
American (North America) 34.1% A*02 Asian American (North America)
43.2% A*02 Latin American (North American) 48.3% DR1 Caucasian
(North America) 19.4% DR2 Caucasian (North America) 28.2% DR3
Caucasian (North America) 20.6% DR4 Caucasian (North America) 30.7%
DR5 Caucasian (North America) 23.3% DR6 Caucasian (North America)
26.7% DR7 Caucasian (North America) 24.8% DR8 Caucasian (North
America) 5.7% DR9 Caucasian (North America) 2.1% DR1 African
(North) American 13.20% DR2 African (North) American 29.80% DR3
African (North) American 24.80% DR4 African (North) American 11.10%
DR5 African (North) American 31.10% DR6 African (North) American
33.70% DR7 African (North) American 19.20% DR8 African (North)
American 12.10% DR9 African (North) American 5.80% DR1 Asian
(North) American 6.80% DR2 Asian (North) American 33.80% DR3 Asian
(North) American 9.20% DR4 Asian (North) American 28.60% DR5 Asian
(North) American 30.00% DR6 Asian (North) American 25.10% DR7 Asian
(North) American 13.40% DR8 Asian (North) American 12.70% DR9 Asian
(North) American 18.60% DR1 Latin (North) American 15.30% DR2 Latin
(North) American 21.20% DR3 Latin (North) American 15.20% DR4 Latin
(North) American 36.80% DR5 Latin (North) American 20.00% DR6 Latin
(North) American 31.10% DR7 Latin (North) American 20.20% DR8 Latin
(North) American 18.60% DR9 Latin (North) American 2.10% A*24
Philippines 65% A*24 Russia Nenets 61% A*24:02 Japan 59% A*24
Malaysia 58% A*24:02 Philippines 54% A*24 India 47% A*24 South
Korea 40% A*24 Sri Lanka 37% A*24 China 32% A*24:02 India 29% A*24
Australia West 22% A*24 USA 22% A*24 Russia Samara 20% A*24 South
America 20% A*24 Europe 18%
[0300] The peptides of the invention, preferably when included into
a vaccine of the invention as described herein bind to A*02. A
vaccine may also include pan-binding MHC class II peptides.
Therefore, the vaccine of the invention can be used to treat cancer
in patients that are A*02 positive, whereas no selection for MHC
class II allotypes is necessary due to the pan-binding nature of
these peptides.
[0301] If A*02 peptides of the invention are combined with peptides
binding to another allele, for example A*24, a higher percentage of
any patient population can be treated compared with addressing
either MHC class I allele alone. While in most populations less
than 50% of patients could be addressed by either allele alone, a
vaccine comprising HLA-A*24 and HLA-A*02 epitopes can treat at
least 60% of patients in any relevant population. Specifically, the
following percentages of patients will be positive for at least one
of these alleles in various regions: USA 61%, Western Europe 62%,
China 75%, South Korea 77%, Japan 86% (calculated from
www.allelefrequencies.net).
[0302] In a preferred embodiment, the term "nucleotide sequence"
refers to a heteropolymer of deoxyribonucleotides.
[0303] The nucleotide sequence coding for a particular peptide,
oligopeptide, or polypeptide may be naturally occurring or they may
be synthetically constructed. Generally, DNA segments encoding the
peptides, polypeptides, and proteins of this invention are
assembled from cDNA fragments and short oligonucleotide linkers, or
from a series of oligonucleotides, to provide a synthetic gene that
is capable of being expressed in a recombinant transcriptional unit
comprising regulatory elements derived from a microbial or viral
operon.
[0304] As used herein the term "a nucleotide coding for (or
encoding) a peptide" refers to a nucleotide sequence coding for the
peptide including artificial (man-made) start and stop codons
compatible for the biological system the sequence is to be
expressed by, for example, a dendritic cell or another cell system
useful for the production of TCRs.
[0305] As used herein, reference to a nucleic acid sequence
includes both single stranded and double stranded nucleic acid.
Thus, for example for DNA, the specific sequence, unless the
context indicates otherwise, refers to the single strand DNA of
such sequence, the duplex of such sequence with its complement
(double stranded DNA) and the complement of such sequence.
[0306] The term "coding region" refers to that portion of a gene
which either naturally or normally codes for the expression product
of that gene in its natural genomic environment, i.e., the region
coding in vivo for the native expression product of the gene.
[0307] The coding region can be derived from a non-mutated
("normal"), mutated or altered gene, or can even be derived from a
DNA sequence, or gene, wholly synthesized in the laboratory using
methods well known to those of skill in the art of DNA
synthesis.
[0308] The term "expression product" means the polypeptide or
protein that is the natural translation product of the gene and any
nucleic acid sequence coding equivalents resulting from genetic
code degeneracy and thus coding for the same amino acid(s).
[0309] The term "fragment", when referring to a coding sequence,
means a portion of DNA comprising less than the complete coding
region, whose expression product retains essentially the same
biological function or activity as the expression product of the
complete coding region.
[0310] The term "DNA segment" refers to a DNA polymer, in the form
of a separate fragment or as a component of a larger DNA construct,
which has been derived from DNA isolated at least once in
substantially pure form, i.e., free of contaminating endogenous
materials and in a quantity or concentration enabling
identification, manipulation, and recovery of the segment and its
component nucleotide sequences by standard biochemical methods, for
example, by using a cloning vector. Such segments are provided in
the form of an open reading frame uninterrupted by internal
non-translated sequences, or introns, which are typically present
in eukaryotic genes. Sequences of non-translated DNA may be present
downstream from the open reading frame, where the same do not
interfere with manipulation or expression of the coding
regions.
[0311] The term "primer" means a short nucleic acid sequence that
can be paired with one strand of DNA and provides a free 3'-OH end
at which a DNA polymerase starts synthesis of a deoxyribonucleotide
chain.
[0312] The term "promoter" means a region of DNA involved in
binding of RNA polymerase to initiate transcription.
[0313] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment, if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or polypeptide, separated
from some or all of the coexisting materials in the natural system,
is isolated. Such polynucleotides could be part of a vector and/or
such polynucleotides or polypeptides could be part of a
composition, and still be isolated in that such vector or
composition is not part of its natural environment.
[0314] The polynucleotides, and recombinant or immunogenic
polypeptides, disclosed in accordance with the present invention
may also be in "purified" form. The term "purified" does not
require absolute purity; rather, it is intended as a relative
definition, and can include preparations that are highly purified
or preparations that are only partially purified, as those terms
are understood by those of skill in the relevant art. For example,
individual clones isolated from a cDNA library have been
conventionally purified to electrophoretic homogeneity.
Purification of starting material or natural material to at least
one order of magnitude, preferably two or three orders, and more
preferably four or five orders of magnitude is expressly
contemplated. Furthermore, a claimed polypeptide which has a purity
of preferably 99.999%, or at least 99.99% or 99.9%; and even
desirably 99% by weight or greater is expressly encompassed.
[0315] The nucleic acids and polypeptide expression products
disclosed according to the present invention, as well as expression
vectors containing such nucleic acids and/or such polypeptides, may
be in "enriched form". As used herein, the term "enriched" means
that the concentration of the material is at least about 2, 5, 10,
100, or 1000 times its natural concentration (for example),
advantageously 0.01%, by weight, preferably at least about 0.1% by
weight. Enriched preparations of about 0.5%, 1%, 5%, 10%, and 20%
by weight are also contemplated. The sequences, constructs,
vectors, clones, and other materials comprising the present
invention can advantageously be in enriched or isolated form. The
term "active fragment" means a fragment, usually of a peptide,
polypeptide or nucleic acid sequence, that generates an immune
response (i.e., has immunogenic activity) when administered, alone
or optionally with a suitable adjuvant or in a vector, to an
animal, such as a mammal, for example, a rabbit or a mouse, and
also including a human, such immune response taking the form of
stimulating a T-cell response within the recipient animal, such as
a human. Alternatively, the "active fragment" may also be used to
induce a T-cell response in vitro.
[0316] As used herein, the terms "portion", "segment" and
"fragment", when used in relation to polypeptides, refer to a
continuous sequence of residues, such as amino acid residues, which
sequence forms a subset of a larger sequence. For example, if a
polypeptide were subjected to treatment with any of the common
endopeptidases, such as trypsin or chymotrypsin, the oligopeptides
resulting from such treatment would represent portions, segments or
fragments of the starting polypeptide. When used in relation to
polynucleotides, these terms refer to the products produced by
treatment of said polynucleotides with any of the
endonucleases.
[0317] In accordance with the present invention, the term "percent
identity" or "percent identical", when referring to a sequence,
means that a sequence is compared to a claimed or described
sequence after alignment of the sequence to be compared (the
"Compared Sequence") with the described or claimed sequence (the
"Reference Sequence"). The percent identity is then determined
according to the following formula:
percent identity=100[1-(C/R)]
wherein C is the number of differences between the Reference
Sequence and the Compared Sequence over the length of alignment
between the Reference Sequence and the Compared Sequence, wherein
(i) each base or amino acid in the Reference Sequence that does not
have a corresponding aligned base or amino acid in the Compared
Sequence and (ii) each gap in the Reference Sequence and (iii) each
aligned base or amino acid in the Reference Sequence that is
different from an aligned base or amino acid in the Compared
Sequence, constitutes a difference and (iiii) the alignment has to
start at position 1 of the aligned sequences; And R is the number
of bases or amino acids in the Reference Sequence over the length
of the alignment with the Compared Sequence with any gap created in
the Reference Sequence also being counted as a base or amino
acid.
[0318] If an alignment exists between the Compared Sequence and the
Reference Sequence for which the percent identity as calculated
above is about equal to or greater than a specified minimum Percent
Identity, then the Compared Sequence has the specified minimum
percent identity to the Reference Sequence even though alignments
may exist in which the herein above calculated percent identity is
less than the specified percent identity.
[0319] As mentioned above, the present invention thus provides a
peptide comprising a sequence that is selected from the group of
consisting of SEQ ID NO: 1 to SEQ ID NO: 161 or a variant thereof
which is 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 161, or a
variant thereof that will induce T cells cross-reacting with said
peptide. The peptides of the invention have the ability to bind to
a molecule of the human major histocompatibility complex (MHC)
Class-I or elongated versions of said peptides to class II.
[0320] In the present invention, the term "homologous" refers to
the degree of identity (see percent identity above) between
sequences of two amino acid sequences, i.e. peptide or polypeptide
sequences. The aforementioned "homology" is determined by comparing
two sequences aligned under optimal conditions over the sequences
to be compared. Such a sequence homology can be calculated by
creating an alignment using, for example, the ClustalW algorithm.
Commonly available sequence analysis software, more specifically,
Vector NTI, GENETYX or other tools are provided by public
databases.
[0321] A person skilled in the art will be able to assess, whether
T cells induced by a variant of a specific peptide will be able to
cross-react with the peptide itself (Appay et al., 2006; Colombetti
et al., 2006; Fong et al., 2001; Zaremba et al., 1997).
[0322] By a "variant" of the given amino acid sequence the
inventors mean that the side chains of, for example, one or two of
the amino acid residues are altered (for example by replacing them
with the side chain of another naturally occurring amino acid
residue or some other side chain) such that the peptide is still
able to bind to an HLA molecule in substantially the same way as a
peptide consisting of the given amino acid sequence in consisting
of SEQ ID NO: 1 to SEQ ID NO: 161. For example, a peptide may be
modified so that it at least maintains, if not improves, the
ability to interact with and bind to the binding groove of a
suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it
at least maintains, if not improves, the ability to bind to the TCR
of activated T cells.
[0323] These T cells can subsequently cross-react with cells and
kill cells that express a polypeptide that contains the natural
amino acid sequence of the cognate peptide as defined in the
aspects of the invention. As can be derived from the scientific
literature and databases (Rammensee et al., 1999; Godkin et al.,
1997), certain positions of HLA binding peptides are typically
anchor residues forming a core sequence fitting to the binding
motif of the HLA receptor, which is defined by polar,
electrophysical, hydrophobic and spatial properties of the
polypeptide chains constituting the binding groove. Thus, one
skilled in the art would be able to modify the amino acid sequences
set forth in SEQ ID NO: 1 to SEQ ID NO 161, by maintaining the
known anchor residues, and would be able to determine whether such
variants maintain the ability to bind MHC class I or II molecules.
The variants of the present invention retain the ability to bind to
the TCR of activated T cells, which can subsequently cross-react
with and kill cells that express a polypeptide containing the
natural amino acid sequence of the cognate peptide as defined in
the aspects of the invention.
[0324] The original (unmodified) peptides as disclosed herein can
be modified by the substitution of one or more residues at
different, possibly selective, sites within the peptide chain, if
not otherwise stated. Preferably those substitutions are located at
the end of the amino acid chain. Such substitutions may be of a
conservative nature, for example, where one amino acid is replaced
by an amino acid of similar structure and characteristics, such as
where a hydrophobic amino acid is replaced by another hydrophobic
amino acid. Even more conservative would be replacement of amino
acids of the same or similar size and chemical nature, such as
where leucine is replaced by isoleucine. In studies of sequence
variations in families of naturally occurring homologous proteins,
certain amino acid substitutions are more often tolerated than
others, and these are often show correlation with similarities in
size, charge, polarity, and hydrophobicity between the original
amino acid and its replacement, and such is the basis for defining
"conservative substitutions."
[0325] Conservative substitutions are herein defined as exchanges
within one of the following five groups: Group 1-small aliphatic,
nonpolar or slightly polar residues (Ala, Ser, Thr, Pro, Gly);
Group 2-polar, negatively charged residues and their amides (Asp,
Asn, Glu, Gln); Group 3-polar, positively charged residues (His,
Arg, Lys); Group 4-large, aliphatic, nonpolar residues (Met, Leu,
Ile, Val, Cys); and Group 5-large, aromatic residues (Phe, Tyr,
Trp).
[0326] Less conservative substitutions might involve the
replacement of one amino acid by another that has similar
characteristics but is somewhat different in size, such as
replacement of an alanine by an isoleucine residue. Highly
non-conservative replacements might involve substituting an acidic
amino acid for one that is polar, or even for one that is basic in
character. Such "radical" substitutions cannot, however, be
dismissed as potentially ineffective since chemical effects are not
totally predictable and radical substitutions might well give rise
to serendipitous effects not otherwise predictable from simple
chemical principles.
[0327] Of course, such substitutions may involve structures other
than the common L-amino acids. Thus, D-amino acids might be
substituted for the L-amino acids commonly found in the antigenic
peptides of the invention and yet still be encompassed by the
disclosure herein. In addition, non-standard amino acids (i.e.,
other than the common naturally occurring proteinogenic amino
acids) may also be used for substitution purposes to produce
immunogens and immunogenic polypeptides according to the present
invention. If substitutions at more than one position are found to
result in a peptide with substantially equivalent or greater
antigenic activity as defined below, then combinations of those
substitutions will be tested to determine if the combined
substitutions result in additive or synergistic effects on the
antigenicity of the peptide. At most, no more than 4 positions
within the peptide would be simultaneously substituted.
[0328] A peptide consisting essentially of the amino acid sequence
as indicated herein can have one or two non-anchor amino acids (see
below regarding the anchor motif) exchanged without that the
ability to bind to a molecule of the human major histocompatibility
complex (MHC) Class-I or -II is substantially changed or is
negatively affected, when compared to the non-modified peptide. In
another embodiment, in a peptide consisting essentially of the
amino acid sequence as indicated herein, one or two amino acids can
be exchanged with their conservative exchange partners (see herein
below) without that the ability to bind to a molecule of the human
major histocompatibility complex (MHC) Class-I or -II is
substantially changed, or is negatively affected, when compared to
the non-modified peptide.
[0329] The amino acid residues that do not substantially contribute
to interactions with the T-cell receptor can be modified by
replacement with other amino acids whose incorporation do not
substantially affect T-cell reactivity and does not eliminate
binding to the relevant MHC. Thus, apart from the proviso given,
the peptide of the invention may be any peptide (by which term the
inventors include oligopeptide or polypeptide), which includes the
amino acid sequences or a portion or variant thereof as given.
TABLE-US-00007 TABLE 6 Preferred variants and motif of the peptides
according to SEQ ID NO: 7, 32, 46, and 76. Position 1 2 3 4 5 6 7 8
9 SEQ ID NO. 7 A L V D I V R S L Variants V I A M V M I M M A A V A
I A A A V V V I V V A T V T I T T A Q V Q I Q Q A Position 1 2 3 4
5 6 7 8 9 SEQ ID NO. 32 Y V D D G L I S L Variants I V I I I I A M
V M I M M A A V A I A A A L V L I L L A T V T I T T A Q V Q I Q Q A
Position 1 2 3 4 5 6 7 8 9 SEQ ID NO. 46 T M V E H N Y Y V Variants
L I A A L A I A A A Position 1 3 4 5 6 7 8 10 11 SEQ ID NO. 76 L S
F S S D V P K Variants V A I L L I A V A I L L I A V A I L L I A Q
V Q A I L A T V T I T L T A Q V Q I indicates data missing or
illegible when filed
[0330] Longer (elongated) peptides may also be suitable. It is
possible that MHC class I epitopes, although usually between 8 and
11 amino acids long, are generated by peptide processing from
longer peptides or proteins that include the actual epitope. It is
preferred that the residues that flank the actual epitope are
residues that do not substantially affect proteolytic cleavage
necessary to expose the actual epitope during processing.
[0331] The peptides of the invention can be elongated by up to four
amino acids, that is 1, 2, 3 or 4 amino acids can be added to
either end in any combination between 4:0 and 0:4. Combinations of
the elongations according to the invention can be found in Table
7.
TABLE-US-00008 TABLE 7 Combinations of the elongations of peptides
of the invention C-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1
0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0
3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or
4
[0332] The amino acids for the elongation/extension can be the
peptides of the original sequence of the protein or any other amino
acid(s). The elongation can be used to enhance the stability or
solubility of the peptides.
[0333] Thus, the epitopes of the present invention may be identical
to naturally occurring tumor-associated or tumor-specific epitopes
or may include epitopes that differ by no more than four residues
from the reference peptide, as long as they have substantially
identical antigenic activity.
[0334] In an alternative embodiment, the peptide is elongated on
either or both sides by more than 4 amino acids, preferably to a
total length of up to 30 amino acids. This may lead to MHC class II
binding peptides. Binding to MHC class II can be tested by methods
known in the art.
[0335] Accordingly, the present invention provides peptides and
variants of MHC class I epitopes, wherein the peptide or variant
has an overall length of between 8 and 100, preferably between 8
and 30, and most preferred between 8 and 14, namely 8, 9, 10, 11,
12, 13, 14 amino acids, in case of the elongated class II binding
peptides the length can also be 15, 16, 17, 18, 19, 20, 21 or 22
amino acids.
[0336] Of course, the peptide or variant according to the present
invention will have the ability to bind to a molecule of the human
major histocompatibility complex (MHC) class I or II. Binding of a
peptide or a variant to a MHC complex may be tested by methods
known in the art.
[0337] Preferably, when the T cells specific for a peptide
according to the present invention are tested against the
substituted peptides, the peptide concentration at which the
substituted peptides achieve half the maximal increase in lysis
relative to background is no more than about 1 mM, preferably no
more than about 1 .mu.M, more preferably no more than about 1 nM,
and still more preferably no more than about 100 .mu.M, and most
preferably no more than about 10 .mu.M. It is also preferred that
the substituted peptide be recognized by T cells from more than one
individual, at least two, and more preferably three
individuals.
[0338] In a particularly preferred embodiment of the invention the
peptide consists or consists essentially of an amino acid sequence
according to SEQ ID NO: 1 to SEQ ID NO: 161.
[0339] "Consisting essentially of" shall mean that a peptide
according to the present invention, in addition to the sequence
according to any of SEQ ID NO: 1 to SEQ ID NO 161 or a variant
thereof contains additional N- and/or C-terminally located
stretches of amino acids that are not necessarily forming part of
the peptide that functions as an epitope for MHC molecules
epitope.
[0340] Nevertheless, these stretches can be important to provide an
efficient introduction of the peptide according to the present
invention into the cells. In one embodiment of the present
invention, the peptide is part of a fusion protein which comprises,
for example, the 80 N-terminal amino acids of the HLA-DR
antigen-associated invariant chain (p33, in the following "Ii") as
derived from the NCBI, GenBank Accession number X00497. In other
fusions, the peptides of the present invention can be fused to an
antibody as described herein, or a functional part thereof, in
particular into a sequence of an antibody, so as to be specifically
targeted by said antibody, or, for example, to or into an antibody
that is specific for dendritic cells as described herein.
[0341] In addition, the peptide or variant may be modified further
to improve stability and/or binding to MHC molecules in order to
elicit a stronger immune response. Methods for such an optimization
of a peptide sequence are well known in the art and include, for
example, the introduction of reverse peptide bonds or non-peptide
bonds.
[0342] In a reverse peptide bond amino acid residues are not joined
by peptide (--CO--NH--) linkages but the peptide bond is reversed.
Such retro-inverso peptidomimetics may be made using methods known
in the art, for example such as those described in Meziere et al
(1997) (Meziere et al., 1997), incorporated herein by reference.
This approach involves making pseudopeptides containing changes
involving the backbone, and not the orientation of side chains.
Meziere et al. (Meziere et al., 1997) show that for MHC binding and
T helper cell responses, these pseudopeptides are useful.
Retro-inverse peptides, which contain NH--CO bonds instead of
CO--NH peptide bonds, are much more resistant to proteolysis.
[0343] A non-peptide bond is, for example, --CH.sub.2--NH,
--CH.sub.2S--, --CH.sub.2CH.sub.2--, --CH.dbd.CH--, --COCH.sub.2--,
--CH(OH)CH.sub.2--, and --CH.sub.2SO--. U.S. Pat. No. 4,897,445
provides a method for the solid phase synthesis of non-peptide
bonds (--CH.sub.2--NH) in polypeptide chains which involves
polypeptides synthesized by standard procedures and the non-peptide
bond synthesized by reacting an amino aldehyde and an amino acid in
the presence of NaCNBH.sub.3.
[0344] Peptides comprising the sequences described above may be
synthesized with additional chemical groups present at their amino
and/or carboxy termini, to enhance the stability, bioavailability,
and/or affinity of the peptides. For example, hydrophobic groups
such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl groups may be
added to the peptides' amino termini. Likewise, an acetyl group or
a 9-fluorenylmethoxy-carbonyl group may be placed at the peptides'
amino termini. Additionally, the hydrophobic group,
t-butyloxycarbonyl, or an am ido group may be added to the
peptides' carboxy termini.
[0345] Further, the peptides of the invention may be synthesized to
alter their steric configuration. For example, the D-isomer of one
or more of the amino acid residues of the peptide may be used,
rather than the usual L-isomer. Still further, at least one of the
amino acid residues of the peptides of the invention may be
substituted by one of the well-known non-naturally occurring amino
acid residues. Alterations such as these may serve to increase the
stability, bioavailability and/or binding action of the peptides of
the invention.
[0346] Similarly, a peptide or variant of the invention may be
modified chemically by reacting specific amino acids either before
or after synthesis of the peptide. Examples for such modifications
are well known in the art and are summarized e.g. in R. Lundblad,
Chemical Reagents for Protein Modification, 3rd ed. CRC Press, 2004
(Lundblad, 2004), which is incorporated herein by reference.
Chemical modification of amino acids includes but is not limited
to, modification by acylation, amidination, pyridoxylation of
lysine, reductive alkylation, trinitrobenzylation of amino groups
with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amide
modification of carboxyl groups and sulphydryl modification by
performic acid oxidation of cysteine to cysteic acid, formation of
mercurial derivatives, formation of mixed disulphides with other
thiol compounds, reaction with maleimide, carboxymethylation with
iodoacetic acid or iodoacetamide and carbamoylation with cyanate at
alkaline pH, although without limitation thereto. In this regard,
the skilled person is referred to Chapter 15 of Current Protocols
In Protein Science, Eds. Coligan et al. (John Wiley and Sons NY
1995-2000) (Coligan et al., 1995) for more extensive methodology
relating to chemical modification of proteins.
[0347] Briefly, modification of e.g. arginyl residues in proteins
is often based on the reaction of vicinal dicarbonyl compounds such
as phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form
an adduct. Another example is the reaction of methylglyoxal with
arginine residues. Cysteine can be modified without concomitant
modification of other nucleophilic sites such as lysine and
histidine. As a result, a large number of reagents are available
for the modification of cysteine. The websites of companies such as
Sigma-Aldrich (http://www.sigma-aldrich.com) provide information on
specific reagents.
[0348] Selective reduction of disulfide bonds in proteins is also
common. Disulfide bonds can be formed and oxidized during the heat
treatment of biopharmaceuticals. Woodward's Reagent K may be used
to modify specific glutamic acid residues. N-(3-(dimethyl
amino)propyl)-N'-ethylcarbodiimide can be used to form
intra-molecular crosslinks between a lysine residue and a glutamic
acid residue. For example, diethylpyrocarbonate is a reagent for
the modification of histidyl residues in proteins. Histidine can
also be modified using 4-hydroxy-2-nonenal. The reaction of lysine
residues and other a-amino groups is, for example, useful in
binding of peptides to surfaces or the cross-linking of
proteins/peptides. Lysine is the site of attachment of
poly(ethylene)glycol and the major site of modification in the
glycosylation of proteins. Methionine residues in proteins can be
modified with e.g. iodoacetamide, bromoethylamine, and chloramine
T.
[0349] Tetranitromethane and N-acetylimidazole can be used for the
modification of tyrosyl residues. Cross-linking via the formation
of dityrosine can be accomplished with hydrogen peroxide/copper
ions.
[0350] Recent studies on the modification of tryptophan have used
N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or
3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole
(BPNS-skatole).
[0351] Successful modification of therapeutic proteins and peptides
with PEG is often associated with an extension of circulatory
half-life while cross-linking of proteins with glutaraldehyde,
polyethylene glycol diacrylate and formaldehyde is used for the
preparation of hydrogels. Chemical modification of allergens for
immunotherapy is often achieved by carbamylation with potassium
cyanate.
[0352] A peptide or variant, wherein the peptide is modified or
includes non-peptide bonds is a preferred embodiment of the
invention. Generally, peptides and variants (at least those
containing peptide linkages between amino acid residues) may be
synthesized by the Fmoc-polyamide mode of solid-phase peptide
synthesis as disclosed by Lukas et al. (Lukas et al., 1981) and by
references as cited therein. Temporary N-amino group protection is
afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group.
Repetitive cleavage of this highly base-labile protecting group is
done using 20% piperidine in N, N-dimethylformamide. Side-chain
functionalities may be protected as their butyl ethers (in the case
of serine threonine and tyrosine), butyl esters (in the case of
glutamic acid and aspartic acid), butyloxycarbonyl derivative (in
the case of lysine and histidine), trityl derivative (in the case
of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl
derivative (in the case of arginine). Where glutamine or asparagine
are C-terminal residues, use is made of the
4,4'-dimethoxybenzhydryl group for protection of the side chain
amido functionalities. The solid-phase support is based on a
polydimethyl-acrylamide polymer constituted from the three monomers
dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine
(cross linker) and acryloylsarcosine methyl ester (functionalizing
agent). The peptide-to-resin cleavable linked agent used is the
acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All
amino acid derivatives are added as their preformed symmetrical
anhydride derivatives with the exception of asparagine and
glutamine, which are added using a reversed N,
N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling
procedure. All coupling and deprotection reactions are monitored
using ninhydrin, trinitrobenzene sulphonic acid or isotin test
procedures. Upon completion of synthesis, peptides are cleaved from
the resin support with concomitant removal of side-chain protecting
groups by treatment with 95% trifluoroacetic acid containing a 50%
scavenger mix. Scavengers commonly used include ethanedithiol,
phenol, anisole and water, the exact choice depending on the
constituent amino acids of the peptide being synthesized. Also a
combination of solid phase and solution phase methodologies for the
synthesis of peptides is possible (see, for example, (Bruckdorfer
et al., 2004), and the references as cited therein).
[0353] Trifluoroacetic acid is removed by evaporation in vacuo,
with subsequent trituration with diethyl ether affording the crude
peptide. Any scavengers present are removed by a simple extraction
procedure which on lyophilization of the aqueous phase affords the
crude peptide free of scavengers. Reagents for peptide synthesis
are generally available from e.g. Calbiochem-Novabiochem
(Nottingham, UK).
[0354] Purification may be performed by any one, or a combination
of, techniques such as re-crystallization, size exclusion
chromatography, ion-exchange chromatography, hydrophobic
interaction chromatography and (usually) reverse-phase high
performance liquid chromatography using e.g. acetonitrile/water
gradient separation.
[0355] Analysis of peptides may be carried out using thin layer
chromatography, electrophoresis, in particular capillary
electrophoresis, solid phase extraction (CSPE), reverse-phase high
performance liquid chromatography, amino-acid analysis after acid
hydrolysis and by fast atom bombardment (FAB) mass spectrometric
analysis, as well as MALDI and ESI-Q-TOF mass spectrometric
analysis.
[0356] In order to select over-presented peptides, a presentation
profile is calculated showing the median sample presentation as
well as replicate variation. The profile juxtaposes samples of the
tumor entity of interest to a baseline of normal tissue samples.
Each of these profiles can then be consolidated into an
over-presentation score by calculating the p-value of a Linear
Mixed-Effects Model (Pinheiro et al., 2015) adjusting for multiple
testing by False Discovery Rate (Benjamini and Hochberg, 1995).
[0357] For the identification and relative quantitation of HLA
ligands by mass spectrometry, HLA molecules from shock-frozen
tissue samples were purified and HLA-associated peptides were
isolated. The isolated peptides were separated and sequences were
identified by online nano-electrospray-ionization (nanoESI) liquid
chromatography-mass spectrometry (LC-MS) experiments. The resulting
peptide sequences were verified by comparison of the fragmentation
pattern of TUMAPs recorded from pancreatic cancer samples (N=20
A*02-positive samples) with the fragmentation patterns of
corresponding synthetic reference peptides of identical sequences.
Since the peptides were directly identified as ligands of HLA
molecules of tumor cells, these results provide direct evidence for
the processing and presentation of the identified peptides on
pancreatic cancer.
[0358] The discovery pipeline XPRESIDENT.RTM. v2.1 (see, for
example, US 2013-0096016, which is hereby incorporated by reference
in its entirety) allows the identification and selection of
relevant over-presented peptide vaccine candidates based on direct
relative quantitation of HLA-restricted peptide levels on cancer
tissues in comparison to several different non-cancerous tissues
and organs. This was achieved by the development of label-free
differential quantitation using the acquired LC-MS data processed
by a proprietary data analysis pipeline, combining algorithms for
sequence identification, spectral clustering, ion counting,
retention time alignment, charge state deconvolution and
normalization.
[0359] Presentation levels including error estimates for each
peptide and sample were established. Peptides exclusively presented
on tumor tissue and peptides over-presented in tumor versus
non-cancerous tissues and organs have been identified.
[0360] HLA-peptide complexes from pancreatic cancer samples were
purified and HLA-associated peptides were isolated and analyzed by
LC-MS (see examples). All TUMAPs contained in the present
application were identified with this approach on pancreatic cancer
samples confirming their presentation on pancreatic cancer.
[0361] TUMAPs identified on multiple pancreatic cancer and normal
tissues were quantified using ion-counting of label-free LC-MS
data. The method assumes that LC-MS signal areas of a peptide
correlate with its abundance in the sample. All quantitative
signals of a peptide in various LC-MS experiments were normalized
based on central tendency, averaged per sample and merged into a
bar plot, called presentation profile. The presentation profile
consolidates different analysis methods like protein database
search, spectral clustering, charge state deconvolution
(decharging) and retention time alignment and normalization.
[0362] The present invention provides peptides that are useful in
treating cancers/tumors, preferably pancreatic cancer, that over-
or exclusively present the peptides of the invention. These
peptides were shown by mass spectrometry to be naturally presented
by HLA molecules on human pancreatic cancer samples.
[0363] Many of the source gene/proteins (also designated
"full-length proteins" or "underlying proteins") from which the
peptides are derived were shown to be highly over-expressed in
cancer compared with normal tissues--"normal tissues" in relation
to this invention shall mean either healthy pancreatic cells or
other normal tissue cells, demonstrating a high degree of tumor
association of the source genes (see Example 2). Moreover, the
peptides themselves are strongly over-presented on tumor
tissue--"tumor tissue" in relation to this invention shall mean a
pancreatic cancer sample, but not on normal tissues (see Example
1).
[0364] HLA-bound peptides can be recognized by the immune system,
specifically T lymphocytes. T cells can destroy the cells
presenting the recognized HLA/peptide complex, e.g. pancreatic
cancer cells presenting the derived peptides.
[0365] The peptides of the present invention have been shown to be
capable of stimulating T cell responses and/or are over-presented
and thus can be used for the production of antibodies and/or TCRs,
such as soluble TCRs, according to the present invention (see
Example 3, Example 4). Furthermore, the peptides when complexed
with the respective MHC can be used for the production of
antibodies and/or TCRs, in particular sTCRs, according to the
present invention, as well. Respective methods are well known to
the person of skill, and can be found in the respective literature
as well. Thus, the peptides of the present invention are useful for
generating an immune response in a patient by which tumor cells can
be destroyed. An immune response in a patient can be induced by
direct administration of the described peptides or suitable
precursor substances (e.g. elongated peptides, proteins, or nucleic
acids encoding these peptides) to the patient, ideally in
combination with an agent enhancing the immunogenicity (i.e. an
adjuvant). The immune response originating from such a therapeutic
vaccination can be expected to be highly specific against tumor
cells because the target peptides of the present invention are not
presented on normal tissues in comparable copy numbers, preventing
the risk of undesired autoimmune reactions against normal cells in
the patient.
[0366] The present description further relates to T-cell receptors
(TCRs) comprising an alpha chain and a beta chain ("alpha/beta
TCRs"). Also provided are HAVCR1-001 peptides capable of binding to
TCRs and antibodies when presented by an MHC molecule. The present
description also relates to nucleic acids, vectors and host cells
for expressing TCRs and peptides of the present description; and
methods of using the same.
[0367] The term "T-cell receptor" (abbreviated TCR) refers to a
heterodimeric molecule comprising an alpha polypeptide chain (alpha
chain) and a beta polypeptide chain (beta chain), wherein the
heterodimeric receptor is capable of binding to a peptide antigen
presented by an HLA molecule. The term also includes so-called
gamma/delta TCRs.
[0368] In one embodiment the description provides a method of
producing a TCR as described herein, the method comprising
culturing a host cell capable of expressing the TCR under
conditions suitable to promote expression of the TCR.
[0369] The description in another aspect relates to methods
according to the description, wherein the antigen is loaded onto
class I or II MHC molecules expressed on the surface of a suitable
antigen-presenting cell or artificial antigen-presenting cell by
contacting a sufficient amount of the antigen with an
antigen-presenting cell or the antigen is loaded onto class I or II
MHC tetramers by tetramerizing the antigen/class I or II MHC
complex monomers.
[0370] The alpha and beta chains of alpha/beta TCR's, and the gamma
and delta chains of gamma/delta TCRs, are generally regarded as
each having two "domains", namely variable and constant domains.
The variable domain consists of a concatenation of variable region
(V), and joining region (J). The variable domain may also include a
leader region (L). Beta and delta chains may also include a
diversity region (D). The alpha and beta constant domains may also
include C-terminal transmembrane (TM) domains that anchor the alpha
and beta chains to the cell membrane.
[0371] With respect to gamma/delta TCRs, the term "TCR gamma
variable domain" as used herein refers to the concatenation of the
TCR gamma V (TRGV) region without leader region (L), and the TCR
gamma J (TRGJ) region, and the term TCR gamma constant domain
refers to the extracellular TRGC region, or to a C-terminal
truncated TRGC sequence. Likewise the term "TCR delta variable
domain" refers to the concatenation of the TCR delta V (TRDV)
region without leader region (L) and the TCR delta D/J (TRDD/TRDJ)
region, and the term "TCR delta constant domain" refers to the
extracellular TRDC region, or to a C-terminal truncated TRDC
sequence.
[0372] TCRs of the present description preferably bind to an
HAVCR1-001 peptide-HLA molecule complex with a binding affinity
(KD) of about 100 .mu.M or less, about 50 .mu.M or less, about 25
.mu.M or less, or about 10 .mu.M or less. More preferred are high
affinity TCRs having binding affinities of about 1 .mu.M or less,
about 100 nM or less, about 50 nM or less, about 25 nM or less.
Non-limiting examples of preferred binding affinity ranges for TCRs
of the present invention include about 1 nM to about 10 nM; about
10 nM to about 20 nM; about 20 nM to about 30 nM; about 30 nM to
about 40 nM; about 40 nM to about 50 nM; about 50 nM to about 60
nM; about 60 nM to about 70 nM; about 70 nM to about 80 nM; about
80 nM to about 90 nM; and about 90 nM to about 100 nM.
[0373] As used herein in connect with TCRs of the present
description, "specific binding" and grammatical variants thereof
are used to mean a TCR having a binding affinity (KD) for an
HAVCR1-001 peptide-HLA molecule complex of 100 .mu.M or less.
[0374] Alpha/beta heterodimeric TCRs of the present description may
have an introduced disulfide bond between their constant domains.
Preferred TCRs of this type include those which have a TRAC
constant domain sequence and a TRBC1 or TRBC2 constant domain
sequence except that Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2
are replaced by cysteine residues, the said cysteines forming a
disulfide bond between the TRAC constant domain sequence and the
TRBC1 or TRBC2 constant domain sequence of the TCR.
[0375] With or without the introduced inter-chain bond mentioned
above, alpha/beta hetero-dimeric TCRs of the present description
may have a TRAC constant domain sequence and a TRBC1 or TRBC2
constant domain sequence, and the TRAC constant domain sequence and
the TRBC1 or TRBC2 constant domain sequence of the TCR may be
linked by the native disulfide bond between Cys4 of exon 2 of TRAC
and Cys2 of exon 2 of TRBC1 or TRBC2.
[0376] TCRs of the present description may comprise a detectable
label selected from the group consisting of a radionuclide, a
fluorophore and biotin. TCRs of the present description may be
conjugated to a therapeutically active agent, such as a
radionuclide, a chemotherapeutic agent, or a toxin.
[0377] In an embodiment, a TCR of the present description having at
least one mutation in the alpha chain and/or having at least one
mutation in the beta chain has modified glycosylation compared to
the unmutated TCR.
[0378] In an embodiment, a TCR comprising at least one mutation in
the TCR alpha chain and/or TCR beta chain has a binding affinity
for, and/or a binding half-life for, a HAVCR1-001 peptide-HLA
molecule complex, which is at least double that of a TCR comprising
the unmutated TCR alpha chain and/or unmutated TCR beta chain.
Affinity-enhancement of tumor-specific TCRs, and its exploitation,
relies on the existence of a window for optimal TCR affinities. The
existence of such a window is based on observations that TCRs
specific for HLA-A2-restricted pathogens have KD values that are
generally about 10-fold lower when compared to TCRs specific for
HLA-A2-restricted tumor-associated self-antigens. It is now known,
although tumor antigens have the potential to be immunogenic,
because tumors arise from the individual's own cells only mutated
proteins or proteins with altered translational processing will be
seen as foreign by the immune system. Antigens that are upregulated
or overexpressed (so called self-antigens) will not necessarily
induce a functional immune response against the tumor: T-cells
expressing TCRs that are highly reactive to these antigens will
have been negatively selected within the thymus in a process known
as central tolerance, meaning that only T-cells with low-affinity
TCRs for self-antigens remain. Therefore, affinity of TCRs or
variants of the present description to HAVCR1-001 can be enhanced
by methods well known in the art.
[0379] The present description further relates to a method of
identifying and isolating a TCR according to the present
description, said method comprising incubating PBMCs from
HLA-A*02-negative healthy donors with A2/HAVCR1-001 monomers,
incubating the PBMCs with tetramer-phycoerythrin (PE) and isolating
the high avidity T-cells by fluorescence activated cell sorting
(FACS)-Calibur analysis.
[0380] The present description further relates to a method of
identifying and isolating a TCR according to the present
description, said method comprising obtaining a transgenic mouse
with the entire human TCR.alpha..beta. gene loci (1.1 and 0.7 Mb),
whose T-cells express a diverse human TCR repertoire that
compensates for mouse TCR deficiency, immunizing the mouse with
HAVCR1-001, incubating PBMCs obtained from the transgenic mice with
tetramer-phycoerythrin (PE), and isolating the high avidity T-cells
by fluorescence activated cell sorting (FACS)-Calibur analysis.
[0381] In one aspect, to obtain T-cells expressing TCRs of the
present description, nucleic acids encoding TCR-alpha and/or
TCR-beta chains of the present description are cloned into
expression vectors, such as gamma retrovirus or lentivirus. The
recombinant viruses are generated and then tested for
functionality, such as antigen specificity and functional avidity.
An aliquot of the final product is then used to transduce the
target T-cell population (generally purified from patient PBMCs),
which is expanded before infusion into the patient.
[0382] In another aspect, to obtain T-cells expressing TCRs of the
present description, TCR RNAs are synthesized by techniques known
in the art, e.g., in vitro transcription systems. The in
vitro-synthesized TCR RNAs are then introduced into primary CD8+
T-cells obtained from healthy donors by electroporation to
re-express tumor specific TCR-alpha and/or TCR-beta chains.
[0383] To increase the expression, nucleic acids encoding TCRs of
the present description may be operably linked to strong promoters,
such as retroviral long terminal repeats (LTRs), cytomegalovirus
(CMV), murine stem cell virus (MSCV) U3, phosphoglycerate kinase
(PGK), .beta.-actin, ubiquitin, and a simian virus 40 (SV40)/CD43
composite promoter, elongation factor (EF)-1 a and the spleen
focus-forming virus (SFFV) promoter. In a preferred embodiment, the
promoter is heterologous to the nucleic acid being expressed.
[0384] In addition to strong promoters, TCR expression cassettes of
the present description may contain additional elements that can
enhance transgene expression, including a central polypurine tract
(cPPT), which promotes the nuclear translocation of lentiviral
constructs (Follenzi et al., 2000), and the woodchuck hepatitis
virus posttranscriptional regulatory element (wPRE), which
increases the level of transgene expression by increasing RNA
stability (Zufferey et al., 1999).
[0385] The alpha and beta chains of a TCR of the present invention
may be encoded by nucleic acids located in separate vectors, or may
be encoded by polynucleotides located in the same vector.
[0386] Achieving high-level TCR surface expression requires that
both the TCR-alpha and TCR-beta chains of the introduced TCR be
transcribed at high levels. To do so, the TCR-alpha and TCR-beta
chains of the present description may be cloned into bi-cistronic
constructs in a single vector, which has been shown to be capable
of over-coming this obstacle. The use of a viral intraribosomal
entry site (IRES) between the TCR-alpha and TCR-beta chains results
in the coordinated expression of both chains, because the TCR-alpha
and TCR-beta chains are generated from a single transcript that is
broken into two proteins during translation, ensuring that an equal
molar ratio of TCR-alpha and TCR-beta chains are produced. (Schmitt
et al. 2009).
[0387] Nucleic acids encoding TCRs of the present description may
be codon optimized to increase expression from a host cell.
Redundancy in the genetic code allows some amino acids to be
encoded by more than one codon, but certain codons are less
"op-timal" than others because of the relative availability of
matching tRNAs as well as other factors (Gustafsson et al., 2004).
Modifying the TCR-alpha and TCR-beta gene sequences such that each
amino acid is encoded by the optimal codon for mammalian gene
expression, as well as eliminating mRNA instability motifs or
cryptic splice sites, has been shown to significantly enhance
TCR-alpha and TCR-beta gene expression (Scholten et al., 2006).
[0388] Furthermore, mispairing between the introduced and
endogenous TCR chains may result in the acquisition of
specificities that pose a significant risk for autoimmunity. For
example, the formation of mixed TCR dimers may reduce the number of
CD3 molecules available to form properly paired TCR complexes, and
therefore can significantly decrease the functional avidity of the
cells expressing the introduced TCR (Kuball et al., 2007).
[0389] To reduce mispairing, the C-terminus domain of the
introduced TCR chains of the present description may be modified in
order to promote interchain affinity, while de-creasing the ability
of the introduced chains to pair with the endogenous TCR. These
strategies may include replacing the human TCR-alpha and TCR-beta
C-terminus domains with their murine counterparts (murinized
C-terminus domain); generating a second interchain disulfide bond
in the C-terminus domain by introducing a second cysteine residue
into both the TCR-alpha and TCR-beta chains of the introduced TCR
(cysteine modification); swapping interacting residues in the
TCR-alpha and TCR-beta chain C-terminus domains ("knob-in-hole");
and fusing the variable domains of the TCR-alpha and TCR-beta
chains directly to CD3 (CD3 fusion). (Schmitt et al. 2009).
[0390] In an embodiment, a host cell is engineered to express a TCR
of the present description. In preferred embodiments, the host cell
is a human T-cell or T-cell progenitor. In some embodiments the
T-cell or T-cell progenitor is obtained from a cancer patient. In
other embodiments the T-cell or T-cell progenitor is obtained from
a healthy donor. Host cells of the present description can be
allogeneic or autologous with respect to a patient to be treated.
In one embodiment, the host is a gamma/delta T-cell transformed to
express an alpha/beta TCR.
[0391] A "pharmaceutical composition" is a composition suitable for
administration to a human being in a medical setting. Preferably, a
pharmaceutical composition is sterile and produced according to GMP
guidelines.
[0392] The pharmaceutical compositions comprise the peptides either
in the free form or in the form of a pharmaceutically acceptable
salt (see also above). As used herein, "a pharmaceutically
acceptable salt" refers to a derivative of the disclosed peptides
wherein the peptide is modified by making acid or base salts of the
agent. For example, acid salts are prepared from the free base
(typically wherein the neutral form of the drug has a neutral--NH2
group) involving reaction with a suitable acid. Suitable acids for
preparing acid salts include both organic acids, e.g., acetic acid,
propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic
acid, malonic acid, succinic acid, maleic acid, fumaric acid,
tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic
acid, methane sulfonic acid, ethane sulfonic acid,
p-toluenesulfonic acid, salicylic acid, and the like, as well as
inorganic acids, e.g., hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid phosphoric acid and the like.
Conversely, preparation of basic salts of acid moieties which may
be present on a peptide are prepared using a pharmaceutically
acceptable base such as sodium hydroxide, potassium hydroxide,
ammonium hydroxide, calcium hydroxide, trimethylamine or the
like.
[0393] In an especially preferred embodiment, the pharmaceutical
compositions comprise the peptides as salts of acetic acid
(acetates), trifluoro acetates or hydrochloric acid
(chlorides).
[0394] Preferably, the medicament of the present invention is an
immunotherapeutic such as a vaccine. It may be administered
directly into the patient, into the affected organ or systemically
i.d., i.m., s.c., i.p. and i.v., or applied ex vivo to cells
derived from the patient or a human cell line which are
subsequently administered to the patient, or used in vitro to
select a subpopulation of immune cells derived from the patient,
which are then re-administered to the patient. If the nucleic acid
is administered to cells in vitro, it may be useful for the cells
to be transfected so as to co-express immune-stimulating cytokines,
such as interleukin-2. The peptide may be substantially pure, or
combined with an immune-stimulating adjuvant (see below) or used in
combination with immune-stimulatory cytokines, or be administered
with a suitable delivery system, for example liposomes. The peptide
may also be conjugated to a suitable carrier such as keyhole limpet
haemocyanin (KLH) or mannan (see WO 95/18145 and (Longenecker et
al., 1993)). The peptide may also be tagged, may be a fusion
protein, or may be a hybrid molecule. The peptides whose sequence
is given in the present invention are expected to stimulate CD4 or
CD8 T cells. However, stimulation of CD8 T cells is more efficient
in the presence of help provided by CD4 T-helper cells. Thus, for
MHC Class I epitopes that stimulate CD8 T cells the fusion partner
or sections of a hybrid molecule suitably provide epitopes which
stimulate CD4-positive T cells. CD4- and CD8-stimulating epitopes
are well known in the art and include those identified in the
present invention.
[0395] In one aspect, the vaccine comprises at least one peptide
having the amino acid sequence set forth SEQ ID No. 1 to SEQ ID No.
161, and at least one additional peptide, preferably two to 50,
more preferably two to 25, even more preferably two to 20 and most
preferably two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or
eighteen peptides. The peptide(s) may be derived from one or more
specific TAAs and may bind to MHC class I molecules.
[0396] A further aspect of the invention provides a nucleic acid
(for example a polynucleotide) encoding a peptide or peptide
variant of the invention. The polynucleotide may be, for example,
DNA, cDNA, PNA, RNA or combinations thereof, either single- and/or
double-stranded, or native or stabilized forms of polynucleotides,
such as, for example, polynucleotides with a phosphorothioate
backbone and it may or may not contain introns so long as it codes
for the peptide. Of course, only peptides that contain naturally
occurring amino acid residues joined by naturally occurring peptide
bonds are encodable by a polynucleotide. A still further aspect of
the invention provides an expression vector capable of expressing a
polypeptide according to the invention.
[0397] A variety of methods have been developed to link
polynucleotides, especially DNA, to vectors for example via
complementary cohesive termini. For instance, complementary
homopolymer tracts can be added to the DNA segment to be inserted
to the vector DNA. The vector and DNA segment are then joined by
hydrogen bonding between the complementary homopolymeric tails to
form recombinant DNA molecules.
[0398] Synthetic linkers containing one or more restriction sites
provide an alternative method of joining the DNA segment to
vectors. Synthetic linkers containing a variety of restriction
endonuclease sites are commercially available from a number of
sources including International Biotechnologies Inc. New Haven,
Conn., USA.
[0399] A desirable method of modifying the DNA encoding the
polypeptide of the invention employs the polymerase chain reaction
as disclosed by Saiki R K, et al. (Saiki et al., 1988). This method
may be used for introducing the DNA into a suitable vector, for
example by engineering in suitable restriction sites, or it may be
used to modify the DNA in other useful ways as is known in the art.
If viral vectors are used, pox- or adenovirus vectors are
preferred.
[0400] The DNA (or in the case of retroviral vectors, RNA) may then
be expressed in a suitable host to produce a polypeptide comprising
the peptide or variant of the invention. Thus, the DNA encoding the
peptide or variant of the invention may be used in accordance with
known techniques, appropriately modified in view of the teachings
contained herein, to construct an expression vector, which is then
used to transform an appropriate host cell for the expression and
production of the polypeptide of the invention. Such techniques
include those disclosed, for example, in U.S. Pat. Nos. 4,440,859,
4,530,901, 4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463,
4,757,006, 4,766,075, and 4,810,648.
[0401] The DNA (or in the case of retroviral vectors, RNA) encoding
the polypeptide constituting the compound of the invention may be
joined to a wide variety of other DNA sequences for introduction
into an appropriate host. The companion DNA will depend upon the
nature of the host, the manner of the introduction of the DNA into
the host, and whether episomal maintenance or integration is
desired.
[0402] Generally, the DNA is inserted into an expression vector,
such as a plasmid, in proper orientation and correct reading frame
for expression. If necessary, the DNA may be linked to the
appropriate transcriptional and translational regulatory control
nucleotide sequences recognized by the desired host, although such
controls are generally available in the expression vector. The
vector is then introduced into the host through standard
techniques. Generally, not all of the hosts will be transformed by
the vector. Therefore, it will be necessary to select for
transformed host cells. One selection technique involves
incorporating into the expression vector a DNA sequence, with any
necessary control elements, that codes for a selectable trait in
the transformed cell, such as antibiotic resistance.
[0403] Alternatively, the gene for such selectable trait can be on
another vector, which is used to co-transform the desired host
cell.
[0404] Host cells that have been transformed by the recombinant DNA
of the invention are then cultured for a sufficient time and under
appropriate conditions known to those skilled in the art in view of
the teachings disclosed herein to permit the expression of the
polypeptide, which can then be recovered.
[0405] Many expression systems are known, including bacteria (for
example E. coli and Bacillus subtilis), yeasts (for example
Saccharomyces cerevisiae), filamentous fungi (for example
Aspergillus spec.), plant cells, animal cells and insect cells.
Preferably, the system can be mammalian cells such as CHO cells
available from the ATCC Cell Biology Collection.
[0406] A typical mammalian cell vector plasmid for constitutive
expression comprises the CMV or SV40 promoter with a suitable poly
A tail and a resistance marker, such as neomycin. One example is
pSVL available from Pharmacia, Piscataway, N.J., USA. An example of
an inducible mammalian expression vector is pMSG, also available
from Pharmacia. Useful yeast plasmid vectors are pRS403-406 and
pRS413-416 and are generally available from Stratagene Cloning
Systems, La Jolla, Calif. 92037, USA. Plasmids pRS403, pRS404,
pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and
incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3.
Plasm ids pRS413-416 are Yeast Centromere plasmids (Ycps). CMV
promoter-based vectors (for example from Sigma-Aldrich) provide
transient or stable expression, cytoplasmic expression or
secretion, and N-terminal or C-terminal tagging in various
combinations of FLAG, 3.times.FLAG, c-myc or MAT. These fusion
proteins allow for detection, purification and analysis of
recombinant protein. Dual-tagged fusions provide flexibility in
detection.
[0407] The strong human cytomegalovirus (CMV) promoter regulatory
region drives constitutive protein expression levels as high as 1
mg/L in COS cells. For less potent cell lines, protein levels are
typically-0.1 mg/L. The presence of the SV40 replication origin
will result in high levels of DNA replication in SV40 replication
permissive COS cells. CMV vectors, for example, can contain the
pMB1 (derivative of pBR322) origin for replication in bacterial
cells, the b-lactamase gene for ampicillin resistance selection in
bacteria, hGH polyA, and the f1 origin. Vectors containing the
pre-pro-trypsin leader (PPT) sequence can direct the secretion of
FLAG fusion proteins into the culture medium for purification using
ANTI-FLAG antibodies, resins, and plates. Other vectors and
expression systems are well known in the art for use with a variety
of host cells.
[0408] In another embodiment two or more peptides or peptide
variants of the invention are encoded and thus expressed in a
successive order (similar to "beads on a string" constructs). In
doing so, the peptides or peptide variants may be linked or fused
together by stretches of linker amino acids, such as for example
LLLLLL, or may be linked without any additional peptide(s) between
them. These constructs can also be used for cancer therapy, and may
induce immune responses both involving MHC I and MHC II.
[0409] The present invention also relates to a host cell
transformed with a polynucleotide vector construct of the present
invention. The host cell can be either prokaryotic or eukaryotic.
Bacterial cells may be preferred prokaryotic host cells in some
circumstances and typically are a strain of E. coli such as, for
example, the E. coli strains DH5 available from Bethesda Research
Laboratories Inc., Bethesda, Md., USA, and RR1 available from the
American Type Culture Collection (ATCC) of Rockville, Md., USA (No
ATCC 31343). Preferred eukaryotic host cells include yeast, insect
and mammalian cells, preferably vertebrate cells such as those from
a mouse, rat, monkey or human fibroblastic and colon cell lines.
Yeast host cells include YPH499, YPHSO0 and YPHSO1, which are
generally available from Stratagene Cloning Systems, La Jolla,
Calif. 92037, USA. Preferred mammalian host cells include Chinese
hamster ovary (CHO) cells available from the ATCC as CCL61, NIH
Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL
1658, monkey kidney-derived COS-1 cells available from the ATCC as
CRL 1650 and 293 cells which are human embryonic kidney cells.
Preferred insect cells are Sf9 cells which can be transfected with
baculovirus expression vectors. An overview regarding the choice of
suitable host cells for expression can be found in, for example,
the textbook of Paulina Balbas and Argelia Lorence "Methods in
Molecular Biology Recombinant Gene Expression, Reviews and
Protocols," Part One, Second Edition, ISBN 978-1-58829-262-9, and
other literature known to the person of skill.
[0410] Transformation of appropriate cell hosts with a DNA
construct of the present invention is accomplished by well-known
methods that typically depend on the type of vector used. With
regard to transformation of prokaryotic host cells, see, for
example, Cohen et al. (Cohen et al., 1972) and (Green and Sambrook,
2012). Transformation of yeast cells is described in Sherman et al.
(Sherman et al., 1986). The method of Beggs (Beggs, 1978) is also
useful. With regard to vertebrate cells, reagents useful in
transfecting such cells, for example calcium phosphate and
DEAE-dextran or liposome formulations, are available from
Stratagene Cloning Systems, or Life Technologies Inc.,
Gaithersburg, Md. 20877, USA. Electroporation is also useful for
transforming and/or transfecting cells and is well known in the art
for transforming yeast cell, bacterial cells, insect cells and
vertebrate cells.
[0411] Successfully transformed cells, i.e. cells that contain a
DNA construct of the present invention, can be identified by
well-known techniques such as PCR. Alternatively, the presence of
the protein in the supernatant can be detected using
antibodies.
[0412] It will be appreciated that certain host cells of the
invention are useful in the preparation of the peptides of the
invention, for example bacterial, yeast and insect cells. However,
other host cells may be useful in certain therapeutic methods. For
example, antigen-presenting cells, such as dendritic cells, may
usefully be used to express the peptides of the invention such that
they may be loaded into appropriate MHC molecules. Thus, the
current invention provides a host cell comprising a nucleic acid or
an expression vector according to the invention.
[0413] In a preferred embodiment the host cell is an antigen
presenting cell, in particular a dendritic cell or antigen
presenting cell. APCs loaded with a recombinant fusion protein
containing prostatic acid phosphatase (PAP) were approved by the
U.S. Food and Drug Administration (FDA) on Apr. 29, 2010, to treat
asymptomatic or minimally symptomatic metastatic HRPC
(Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).
[0414] A further aspect of the invention provides a method of
producing a peptide or its variant, the method comprising culturing
a host cell and isolating the peptide from the host cell or its
culture medium.
[0415] In another embodiment, the peptide, the nucleic acid or the
expression vector of the invention are used in medicine. For
example, the peptide or its variant may be prepared for intravenous
(i.v.) injection, sub-cutaneous (s.c.) injection, intradermal
(i.d.) injection, intraperitoneal (i.p.) injection, intramuscular
(i.m.) injection. Preferred methods of peptide injection include
s.c., i.d., i.p., i.m., and i.v. Preferred methods of DNA injection
include i.d., i.m., s.c., i.p. and i.v. Doses of e.g. between 50
.mu.g and 1.5 mg, preferably 125 .mu.g to 500 .mu.g, of peptide or
DNA may be given and will depend on the respective peptide or DNA.
Dosages of this range were successfully used in previous trials
(Walter et al., 2012).
[0416] The polynucleotide used for active vaccination may be
substantially pure, or contained in a suitable vector or delivery
system. The nucleic acid may be DNA, cDNA, PNA, RNA or a
combination thereof. Methods for designing and introducing such a
nucleic acid are well known in the art. An overview is provided by
e.g. Teufel et al. (Teufel et al., 2005). Polynucleotide vaccines
are easy to prepare, but the mode of action of these vectors in
inducing an immune response is not fully understood. Suitable
vectors and delivery systems include viral DNA and/or RNA, such as
systems based on adenovirus, vaccinia virus, retroviruses, herpes
virus, adeno-associated virus or hybrids containing elements of
more than one virus. Non-viral delivery systems include cationic
lipids and cationic polymers and are well known in the art of DNA
delivery. Physical delivery, such as via a "gene-gun" may also be
used. The peptide or peptides encoded by the nucleic acid may be a
fusion protein, for example with an epitope that stimulates T cells
for the respective opposite CDR as noted above.
[0417] The medicament of the invention may also include one or more
adjuvants. Adjuvants are substances that non-specifically enhance
or potentiate the immune response (e.g., immune responses mediated
by CD8-positive T cells and Helper-T (TH) cells to an antigen, and
would thus be considered useful in the medicament of the present
invention. Suitable adjuvants include, but are not limited to, 1018
ISS, aluminum salts, AMPLIVAX.RTM., AS15, BCG, CP-870,893, CpG7909,
CyaA, dSLIM, flagellin or TLR5 ligands derived from flagellin, FLT3
ligand, GM-CSF, IC30, IC31, Imiquimod (ALDARA.RTM.), resiquimod,
ImuFact IMP321, Interleukins as IL-2, IL-13, IL-21,
Interferon-alpha or -beta, or pegylated derivatives thereof, IS
Patch, ISS, ISCOMATRIX, ISCOMs, JuvImmune.RTM., LipoVac, MALP2,
MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA
206, Montanide ISA 50V, Montanide ISA-51, water-in-oil and
oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA,
PepTel.RTM. vector system, poly(lactid co-glycolid) [PLG]-based and
dextran microparticles, talactoferrin SRL172, Virosomes and other
Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan,
Pam3Cys, Aquila's QS21 stimulon, which is derived from saponin,
mycobacterial extracts and synthetic bacterial cell wall mimics,
and other proprietary adjuvants such as Ribi's Detox, Quil, or
Superfos. Adjuvants such as Freund's or GM-CSF are preferred.
Several immunological adjuvants (e.g., MF59) specific for dendritic
cells and their preparation have been described previously (Allison
and Krummel, 1995). Also cytokines may be used. Several cytokines
have been directly linked to influencing dendritic cell migration
to lymphoid tissues (e.g., TNF-), accelerating the maturation of
dendritic cells into efficient antigen-presenting cells for
T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No.
5,849,589, specifically incorporated herein by reference in its
entirety) and acting as immunoadjuvants (e.g., IL-12, IL-15, IL-23,
IL-7, IFN-alpha. IFN-beta) (Gabrilovich et al., 1996).
[0418] CpG immunostimulatory oligonucleotides have also been
reported to enhance the effects of adjuvants in a vaccine setting.
Without being bound by theory, CpG oligonucleotides act by
activating the innate (non-adaptive) immune system via Toll-like
receptors (TLR), mainly TLR9. CpG triggered TLR9 activation
enhances antigen-specific humoral and cellular responses to a wide
variety of antigens, including peptide or protein antigens, live or
killed viruses, dendritic cell vaccines, autologous cellular
vaccines and polysaccharide conjugates in both prophylactic and
therapeutic vaccines. More importantly it enhances dendritic cell
maturation and differentiation, resulting in enhanced activation of
TH1 cells and strong cytotoxic T-lymphocyte (CTL) generation, even
in the absence of CD4 T cell help. The TH1 bias induced by TLR9
stimulation is maintained even in the presence of vaccine adjuvants
such as alum or incomplete Freund's adjuvant (IFA) that normally
promote a TH2 bias. CpG oligonucleotides show even greater adjuvant
activity when formulated or co-administered with other adjuvants or
in formulations such as microparticles, nanoparticles, lipid
emulsions or similar formulations, which are especially necessary
for inducing a strong response when the antigen is relatively weak.
They also accelerate the immune response and enable the antigen
doses to be reduced by approximately two orders of magnitude, with
comparable antibody responses to the full-dose vaccine without CpG
in some experiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1
describes the combined use of CpG oligonucleotides, non-nucleic
acid adjuvants and an antigen to induce an antigen-specific immune
response. A CpG TLR9 antagonist is dSLIM (double Stem Loop
Immunomodulator) by Mologen (Berlin, Germany) which is a preferred
component of the pharmaceutical composition of the present
invention. Other TLR binding molecules such as RNA binding TLR 7,
TLR 8 and/or TLR 9 may also be used.
[0419] Other examples for useful adjuvants include, but are not
limited to chemically modified CpGs (e.g. CpR, Idera), dsRNA
analogues such as Poly(I:C) and derivates thereof (e.g.
AmpliGen.RTM., Hiltonol.RTM., poly-(ICLC), poly(IC-R),
poly(I:C12U), non-CpG bacterial DNA or RNA as well as immunoactive
small molecules and antibodies such as cyclophosphamide, sunitinib,
Bevacizumab.RTM., celebrex, NCX-4016, sildenafil, tadalafil,
vardenafil, sorafenib, temozolomide, temsirolimus, XL-999,
CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other
antibodies targeting key structures of the immune system (e.g.
anti-CD40, anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which
may act therapeutically and/or as an adjuvant. The amounts and
concentrations of adjuvants and additives useful in the context of
the present invention can readily be determined by the skilled
artisan without undue experimentation. Preferred adjuvants are
anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide,
sunitinib, Bevacizumab, interferon-alpha, CpG oligonucleotides and
derivates, poly-(I:C) and derivates, RNA, sildenafil, and
particulate formulations with PLG or virosomes.
[0420] In a preferred embodiment, the pharmaceutical composition
according to the invention the adjuvant is selected from the group
consisting of colony-stimulating factors, such as Granulocyte
Macrophage Colony Stimulating Factor (GM-CSF, sargramostim),
cyclophosphamide, imiquimod, resiquimod, and interferon-alpha.
[0421] In a preferred embodiment, the pharmaceutical composition
according to the invention the adjuvant is selected from the group
consisting of colony-stimulating factors, such as Granulocyte
Macrophage Colony Stimulating Factor (GM-CSF, sargramostim),
cyclophosphamide, imiquimod and resiquimod. In a preferred
embodiment of the pharmaceutical composition according to the
invention, the adjuvant is cyclophosphamide, imiquimod or
resiquimod. Even more preferred adjuvants are Montanide IMS 1312,
Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, poly-ICLC
(Hiltonol.RTM.) and anti-CD40 mAB, or combinations thereof.
[0422] This composition is used for parenteral administration, such
as subcutaneous, intradermal, intramuscular or oral administration.
For this, the peptides and optionally other molecules are dissolved
or suspended in a pharmaceutically acceptable, preferably aqueous
carrier. In addition, the composition can contain excipients, such
as buffers, binding agents, blasting agents, diluents, flavors,
lubricants, etc. The peptides can also be administered together
with immune stimulating substances, such as cytokines. An extensive
listing of excipients that can be used in such a composition, can
be, for example, taken from A. Kibbe, Handbook of Pharmaceutical
Excipients (Kibbe, 2000). The composition can be used for a
prevention, prophylaxis and/or therapy of adenomatous or cancerous
diseases. Exemplary formulations can be found in, for example,
EP2112253.
[0423] It is important to realize that the immune response
triggered by the vaccine according to the invention attacks the
cancer in different cell-stages and different stages of
development. Furthermore, different cancer associated signaling
pathways are attacked. This is an advantage over vaccines that
address only one or few targets, which may cause the tumor to
easily adapt to the attack (tumor escape). Furthermore, not all
individual tumors express the same pattern of antigens. Therefore,
a combination of several tumor-associated peptides ensures that
every single tumor bears at least some of the targets. The
composition is designed in such a way that each tumor is expected
to express several of the antigens and cover several independent
pathways necessary for tumor growth and maintenance. Thus, the
vaccine can easily be used "off-the-shelf" for a larger patient
population. This means that a pre-selection of patients to be
treated with the vaccine can be restricted to HLA typing, does not
require any additional biomarker assessments for antigen
expression, but it is still ensured that several targets are
simultaneously attacked by the induced immune response, which is
important for efficacy (Banchereau et al., 2001; Walter et al.,
2012).
[0424] As used herein, the term "scaffold" refers to a molecule
that specifically binds to an (e.g. antigenic) determinant. In one
embodiment, a scaffold is able to direct the entity to which it is
attached (e.g. a (second) antigen binding moiety) to a target site,
for example to a specific type of tumor cell or tumor stroma
bearing the antigenic determinant (e.g. the complex of a peptide
with MHC, according to the application at hand). In another
embodiment a scaffold is able to activate signaling through its
target antigen, for example a T cell receptor complex antigen.
Scaffolds include but are not limited to antibodies and fragments
thereof, antigen binding domains of an antibody, comprising an
antibody heavy chain variable region and an antibody light chain
variable region, binding proteins comprising at least one Ankyrin
repeat motif and single domain antigen binding (SDAB) molecules,
aptamers, (soluble) TCRs and (modified) cells such as allogenic or
autologous T cells. To assess whether a molecule is a scaffold
binding to a target, binding assays can be performed.
[0425] "Specific" binding means that the scaffold binds the
peptide-MHC-complex of interest better than other naturally
occurring peptide-MHC-complexes, to an extent that a scaffold armed
with an active molecule that is able to kill a cell bearing the
specific target is not able to kill another cell without the
specific target but presenting other peptide-MHC complex(es).
Binding to other peptide-MHC complexes is irrelevant if the peptide
of the cross-reactive peptide-MHC is not naturally occurring, i.e.
not derived from the human HLA-peptidome. Tests to assess target
cell killing are well known in the art. They should be performed
using target cells (primary cells or cell lines) with unaltered
peptide-MHC presentation, or cells loaded with peptides such that
naturally occurring peptide-MHC levels are reached.
[0426] Each scaffold can comprise a labelling which provides that
the bound scaffold can be detected by determining the presence or
absence of a signal provided by the label. For example, the
scaffold can be labelled with a fluorescent dye or any other
applicable cellular marker molecule. Such marker molecules are well
known in the art. For example, a fluorescence-labelling, for
example provided by a fluorescence dye, can provide a visualization
of the bound aptamer by fluorescence or laser scanning microscopy
or flow cytometry.
[0427] Each scaffold can be conjugated with a second active
molecule such as for example IL-21, anti-CD3, and anti-CD28.
[0428] For further information on polypeptide scaffolds see for
example the background section of WO 2014/071978A1 and the
references cited therein.
[0429] The present invention further relates to aptamers. Aptamers
(see for example WO 2014/191359 and the literature as cited
therein) are short single-stranded nucleic acid molecules, which
can fold into defined three-dimensional structures and recognize
specific target structures. They have appeared to be suitable
alternatives for developing targeted therapies. Aptamers have been
shown to selectively bind to a variety of complex targets with high
affinity and specificity.
[0430] Aptamers recognizing cell surface located molecules have
been identified within the past decade and provide means for
developing diagnostic and therapeutic approaches. Since aptamers
have been shown to possess almost no toxicity and immunogenicity
they are promising candidates for biomedical applications. Indeed,
aptamers, for example prostate-specific membrane-antigen
recognizing aptamers, have been successfully employed for targeted
therapies and shown to be functional in xenograft in vivo models.
Furthermore, aptamers recognizing specific tumor cell lines have
been identified.
[0431] DNA aptamers can be selected to reveal broad-spectrum
recognition properties for various cancer cells, and particularly
those derived from solid tumors, while non-tumorigenic and primary
healthy cells are not recognized. If the identified aptamers
recognize not only a specific tumor sub-type but rather interact
with a series of tumors, this renders the aptamers applicable as
so-called broad-spectrum diagnostics and therapeutics.
[0432] Further, investigation of cell-binding behavior with flow
cytometry showed that the aptamers revealed very good apparent
affinities that are within the nanomolar range.
[0433] Aptamers are useful for diagnostic and therapeutic purposes.
Further, it could be shown that some of the aptamers are taken up
by tumor cells and thus can function as molecular vehicles for the
targeted delivery of anti-cancer agents such as siRNA into tumor
cells.
[0434] Aptamers can be selected against complex targets such as
cells and tissues and complexes of the peptides comprising,
preferably consisting of, a sequence according to any of SEQ ID NO
1 to SEQ ID NO 161, according to the invention at hand with the MHC
molecule, using the cell-SELEX (Systematic Evolution of Ligands by
Exponential enrichment) technique.
[0435] The peptides of the present invention can be used to
generate and develop specific antibodies against MHC/peptide
complexes. These can be used for therapy, targeting toxins or
radioactive substances to the diseased tissue. Another use of these
antibodies can be targeting radionuclides to the diseased tissue
for imaging purposes such as PET. This use can help to detect small
metastases or to determine the size and precise localization of
diseased tissues.
[0436] Therefore, it is a further aspect of the invention to
provide a method for producing a recombinant antibody specifically
binding to a human major histocompatibility complex (MHC) class I
or II being complexed with a HLA-restricted antigen, the method
comprising: immunizing a genetically engineered non-human mammal
comprising cells expressing said human major histocompatibility
complex (MHC) class I or II with a soluble form of a MHC class I or
II molecule being complexed with said HLA-restricted antigen;
isolating mRNA molecules from antibody producing cells of said
non-human mammal; producing a phage display library displaying
protein molecules encoded by said mRNA molecules; and isolating at
least one phage from said phage display library, said at least one
phage displaying said antibody specifically binding to said human
major histocompatibility complex (MHC) class I or II being
complexed with said HLA-restricted antigen.
[0437] It is a further aspect of the invention to provide an
antibody that specifically binds to a human major
histocompatibility complex (MHC) class I or II being complexed with
a HLA-restricted antigen, wherein the antibody preferably is a
polyclonal antibody, monoclonal antibody, bi-specific antibody
and/or a chimeric antibody.
[0438] Respective methods for producing such antibodies and single
chain class I major histocompatibility complexes, as well as other
tools for the production of these antibodies are disclosed in WO
03/068201, WO 2004/084798, WO 01/72768, WO 03/070752, and in
publications (Cohen et al., 2003a; Cohen et al., 2003b; Denkberg et
al., 2003), which for the purposes of the present invention are all
explicitly incorporated by reference in their entireties.
[0439] Preferably, the antibody is binding with a binding affinity
of below 20 nanomolar, preferably of below 10 nanomolar, to the
complex, which is also regarded as "specific" in the context of the
present invention.
[0440] The present invention relates to a peptide comprising a
sequence that is selected from the group consisting of SEQ ID NO: 1
to SEQ ID NO: 161, or a variant thereof which is at least 88%
homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 161
or a variant thereof that induces T cells cross-reacting with said
peptide, wherein said peptide is not the underlying full-length
polypeptide.
[0441] The present invention further relates to a peptide
comprising a sequence that is selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 161 or a variant thereof which is at
least 88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ
ID NO: 161, wherein said peptide or variant has an overall length
of between 8 and 100, preferably between 8 and 30, and most
preferred between 8 and 14 amino acids.
[0442] The present invention further relates to the peptides
according to the invention that have the ability to bind to a
molecule of the human major histocompatibility complex (MHC)
Class-I or -II.
[0443] The present invention further relates to the peptides
according to the invention wherein the peptide consists or consists
essentially of an amino acid sequence according to SEQ ID NO: 1 to
SEQ ID NO: 161.
[0444] The present invention further relates to the peptides
according to the invention, wherein the peptide is (chemically)
modified and/or includes non-peptide bonds.
[0445] The present invention further relates to the peptides
according to the invention, wherein the peptide is part of a fusion
protein, in particular comprising N-terminal amino acids of the
HLA-DR antigen-associated invariant chain (Ii), or wherein the
peptide is fused to (or into) an antibody, such as, for example, an
antibody that is specific for dendritic cells.
[0446] The present invention further relates to a nucleic acid,
encoding the peptides according to the invention, provided that the
peptide is not the complete (full) human protein.
[0447] The present invention further relates to the nucleic acid
according to the invention that is DNA, cDNA, PNA, RNA or
combinations thereof.
[0448] The present invention further relates to an expression
vector capable of expressing a nucleic acid according to the
present invention.
[0449] The present invention further relates to a peptide according
to the present invention, a nucleic acid according to the present
invention or an expression vector according to the present
invention for use in medicine, in particular in the treatment of
pancreatic cancer.
[0450] The present invention further relates to a host cell
comprising a nucleic acid according to the invention or an
expression vector according to the invention.
[0451] The present invention further relates to the host cell
according to the present invention that is an antigen presenting
cell, and preferably a dendritic cell.
[0452] The present invention further relates to a method of
producing a peptide according to the present invention, said method
comprising culturing the host cell according to the present
invention, and isolating the peptide from said host cell or its
culture medium.
[0453] The present invention further relates to the method
according to the present invention, where-in the antigen is loaded
onto class I or II MHC molecules expressed on the surface of a
suitable antigen-presenting cell by contacting a sufficient amount
of the antigen with an antigen-presenting cell.
[0454] The present invention further relates to the method
according to the invention, wherein the antigen-presenting cell
comprises an expression vector capable of expressing said peptide
containing SEQ ID NO: 1 to SEQ ID NO: 161 or said variant amino
acid sequence.
[0455] The present invention further relates to activated T cells,
produced by the method according to the present invention, wherein
said T cells selectively recognizes a cell which aberrantly
expresses a polypeptide comprising an amino acid sequence according
to the present invention.
[0456] The present invention further relates to a method of killing
target cells in a patient which target cells aberrantly express a
polypeptide comprising any amino acid sequence according to the
present invention, the method comprising administering to the
patient an effective number of T cells as according to the present
invention.
[0457] The present invention further relates to the use of any
peptide described, a nucleic acid according to the present
invention, an expression vector according to the present invention,
a cell according to the present invention, or an activated
cytotoxic T lymphocyte according to the present invention as a
medicament or in the manufacture of a medicament. The present
invention further relates to a use according to the present
invention, wherein the medicament is active against cancer.
[0458] The present invention further relates to a use according to
the invention, wherein the medicament is a vaccine. The present
invention further relates to a use according to the invention,
wherein the medicament is active against cancer.
[0459] The present invention further relates to a use according to
the invention, wherein said cancer cells are pancreatic cancer
cells or other solid or hematological tumor cells such as lung
cancer, kidney cancer, brain cancer, stomach cancer, colon or
rectal cancer, liver cancer, prostate cancer, leukemia, breast
cancer, Merkel cell carcinoma (MCC), melanoma, ovarian cancer,
esophageal cancer, urinary bladder cancer, endometrial cancer, gall
bladder cancer, and bile duct cancer.
[0460] The present invention further relates to particular marker
proteins and biomarkers based on the peptides according to the
present invention, herein called "targets" that can be used in the
diagnosis and/or prognosis of pancreatic cancer. The present
invention also relates to the use of these novel targets for cancer
treatment.
[0461] The term "antibody" or "antibodies" is used herein in a
broad sense and includes both polyclonal and monoclonal antibodies.
In addition to intact or "full" immunoglobulin molecules, also
included in the term "antibodies" are fragments (e.g. CDRs, Fv, Fab
and Fc fragments) or polymers of those immunoglobulin molecules and
humanized versions of immunoglobulin molecules, as long as they
exhibit any of the desired properties (e.g., specific binding of a
pancreatic cancer marker (poly)peptide, delivery of a toxin to a
pancreatic cancer cell expressing a cancer marker gene at an
increased level, and/or inhibiting the activity of a pancreatic
cancer marker polypeptide) according to the invention.
[0462] Whenever possible, the antibodies of the invention may be
purchased from commercial sources. The antibodies of the invention
may also be generated using well-known methods. The skilled artisan
will understand that either full length pancreatic cancer marker
polypeptides or fragments thereof may be used to generate the
antibodies of the invention. A polypeptide to be used for
generating an antibody of the invention may be partially or fully
purified from a natural source, or may be produced using
recombinant DNA techniques.
[0463] For example, a cDNA encoding a peptide according to the
present invention, such as a peptide according to SEQ ID NO: 1 to
SEQ ID NO: 161 polypeptide, or a variant or fragment thereof, can
be expressed in prokaryotic cells (e.g., bacteria) or eukaryotic
cells (e.g., yeast, insect, or mammalian cells), after which the
recombinant protein can be purified and used to generate a
monoclonal or polyclonal antibody preparation that specifically
bind the pancreatic cancer marker polypeptide used to generate the
antibody according to the invention.
[0464] One of skill in the art will realize that the generation of
two or more different sets of monoclonal or polyclonal antibodies
maximizes the likelihood of obtaining an antibody with the
specificity and affinity required for its intended use (e.g.,
ELISA, immunohistochemistry, in vivo imaging, immunotoxin therapy).
The antibodies are tested for their desired activity by known
methods, in accordance with the purpose for which the antibodies
are to be used (e.g., ELISA, immunohistochemistry, immunotherapy,
etc.; for further guidance on the generation and testing of
antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). For
example, the antibodies may be tested in ELISA assays or, Western
blots, immunohistochemical staining of formalin-fixed cancers or
frozen tissue sections. After their initial in vitro
characterization, antibodies intended for therapeutic or in vivo
diagnostic use are tested according to known clinical testing
methods.
[0465] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a substantially homogeneous population of
antibodies, i.e.; the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. The monoclonal
antibodies herein specifically include "chimeric" antibodies in
which a portion of the heavy and/or light chain is identical with
or homologous to corresponding sequences in antibodies derived from
a particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is identical with or
homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, so long as they exhibit
the desired antagonistic activity (U.S. Pat. No. 4,816,567, which
is hereby incorporated in its entirety).
[0466] Monoclonal antibodies of the invention may be prepared using
hybridoma methods. In a hybridoma method, a mouse or other
appropriate host animal is typically immunized with an immunizing
agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the immunizing
agent. Alternatively, the lymphocytes may be immunized in
vitro.
[0467] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies).
[0468] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly Fab fragments, can be accomplished using routine
techniques known in the art. For instance, digestion can be
performed using papain. Examples of papain digestion are described
in WO 94/29348 and U.S. Pat. No. 4,342,566. Papain digestion of
antibodies typically produces two identical antigen binding
fragments, called Fab fragments, each with a single antigen binding
site, and a residual Fc fragment. Pepsin treatment yields a F(ab')2
fragment and a pFc' fragment.
[0469] The antibody fragments, whether attached to other sequences
or not, can also include insertions, deletions, substitutions, or
other selected modifications of particular regions or specific
amino acids residues, provided the activity of the fragment is not
significantly altered or impaired compared to the non-modified
antibody or antibody fragment. These modifications can provide for
some additional property, such as to remove/add amino acids capable
of disulfide bonding, to increase its bio-longevity, to alter its
secretory characteristics, etc. In any case, the antibody fragment
must possess a bioactive property, such as binding activity,
regulation of binding at the binding domain, etc. Functional or
active regions of the antibody may be identified by mutagenesis of
a specific region of the protein, followed by expression and
testing of the expressed polypeptide. Such methods are readily
apparent to a skilled practitioner in the art and can include
site-specific mutagenesis of the nucleic acid encoding the antibody
fragment.
[0470] The antibodies of the invention may further comprise
humanized antibodies or human antibodies. Humanized forms of
non-human (e.g., murine) antibodies are chimeric immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab'
or other antigen-binding subsequences of antibodies) which contain
minimal sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues from a complementary determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework (FR) residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin.
[0471] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0472] Transgenic animals (e.g., mice) that are capable, upon
immunization, of producing a full repertoire of human antibodies in
the absence of endogenous immunoglobulin production can be
employed. For example, it has been described that the homozygous
deletion of the antibody heavy chain joining region gene in
chimeric and germ-line mutant mice results in complete inhibition
of endogenous antibody production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge. Human
antibodies can also be produced in phage display libraries.
[0473] Antibodies of the invention are preferably administered to a
subject in a pharmaceutically acceptable carrier. Typically, an
appropriate amount of a pharmaceutically-acceptable salt is used in
the formulation to render the formulation isotonic. Examples of the
pharmaceutically-acceptable carrier include saline, Ringer's
solution and dextrose solution. The pH of the solution is
preferably from about 5 to about 8, and more preferably from about
7 to about 7.5. Further carriers include sustained release
preparations such as semipermeable matrices of solid hydrophobic
polymers containing the antibody, which matrices are in the form of
shaped articles, e.g., films, liposomes or microparticles. It will
be apparent to those persons skilled in the art that certain
carriers may be more preferable depending upon, for instance, the
route of administration and concentration of antibody being
administered.
[0474] The antibodies can be administered to the subject, patient,
or cell by injection (e.g., intravenous, intraperitoneal,
subcutaneous, intramuscular), or by other methods such as infusion
that ensure its delivery to the bloodstream in an effective form.
The antibodies may also be administered by intratumoral or
peritumoral routes, to exert local as well as systemic therapeutic
effects. Local or intravenous injection is preferred.
[0475] Effective dosages and schedules for administering the
antibodies may be determined empirically, and making such
determinations is within the skill in the art. Those skilled in the
art will understand that the dosage of antibodies that must be
administered will vary depending on, for example, the subject that
will receive the antibody, the route of administration, the
particular type of antibody used and other drugs being
administered. A typical daily dosage of the antibody used alone
might range from about 1 (.mu.g/kg to up to 100 mg/kg of body
weight or more per day, depending on the factors mentioned above.
Following administration of an antibody, preferably for treating
pancreatic cancer, the efficacy of the therapeutic antibody can be
assessed in various ways well known to the skilled practitioner.
For instance, the size, number, and/or distribution of cancer in a
subject receiving treatment may be monitored using standard tumor
imaging techniques. A therapeutically-administered antibody that
arrests tumor growth, results in tumor shrinkage, and/or prevents
the development of new tumors, compared to the disease course that
would occur in the absence of antibody administration, is an
efficacious antibody for treatment of cancer.
[0476] It is a further aspect of the invention to provide a method
for producing a soluble T-cell receptor (sTCR) recognizing a
specific peptide-MHC complex. Such soluble T-cell receptors can be
generated from specific T-cell clones, and their affinity can be
increased by mutagenesis targeting the complementarity-determining
regions. For the purpose of T-cell receptor selection, phage
display can be used (US 2010/0113300, (Liddy et al., 2012)). For
the purpose of stabilization of T-cell receptors during phage
display and in case of practical use as drug, alpha and beta chain
can be linked e.g. by non-native disulfide bonds, other covalent
bonds (single-chain T-cell receptor), or by dimerization domains
(Boulter et al., 2003; Card et al., 2004; Willcox et al., 1999).
The T-cell receptor can be linked to toxins, drugs, cytokines (see,
for example, US 2013/0115191), and domains recruiting effector
cells such as an anti-CD3 domain, etc., in order to execute
particular functions on target cells. Moreover, it could be
expressed in T cells used for adoptive transfer. Further
information can be found in WO 2004/033685A1 and WO 2004/074322A1.
A combination of sTCRs is described in WO 2012/056407A1. Further
methods for the production are disclosed in WO 2013/057586A1.
[0477] In addition, the peptides and/or the TCRs or antibodies or
other binding molecules of the present invention can be used to
verify a pathologist's diagnosis of a cancer based on a biopsied
sample.
[0478] The antibodies or TCRs may also be used for in vivo
diagnostic assays. Generally, the antibody is labeled with a
radionucleotide (such as .sup.111In, .sup.99Tc, .sup.14C,
.sup.131I, .sup.3H, .sup.32P or .sup.35S) so that the tumor can be
localized using immunoscintiography. In one embodiment, antibodies
or fragments thereof bind to the extracellular domains of two or
more targets of a protein selected from the group consisting of the
above-mentioned proteins, and the affinity value (Kd) is less than
1.times. 10 .mu.M.
[0479] Antibodies for diagnostic use may be labeled with probes
suitable for detection by various imaging methods. Methods for
detection of probes include, but are not limited to, fluorescence,
light, confocal and electron microscopy; magnetic resonance imaging
and spectroscopy; fluoroscopy, computed tomography and positron
emission tomography. Suitable probes include, but are not limited
to, fluorescein, rhodamine, eosin and other fluorophores,
radioisotopes, gold, gadolinium and other lanthanides, paramagnetic
iron, fluorine-18 and other positron-emitting radionuclides.
Additionally, probes may be bi- or multi-functional and be
detectable by more than one of the methods listed. These antibodies
may be directly or indirectly labeled with said probes. Attachment
of probes to the antibodies includes covalent attachment of the
probe, incorporation of the probe into the antibody, and the
covalent attachment of a chelating compound for binding of probe,
amongst others well recognized in the art. For
immunohistochemistry, the disease tissue sample may be fresh or
frozen or may be embedded in paraffin and fixed with a preservative
such as formalin. The fixed or embedded section contains the sample
are contacted with a labeled primary antibody and secondary
antibody, wherein the antibody is used to detect the expression of
the proteins in situ.
[0480] Another aspect of the present invention includes an in vitro
method for producing activated T cells, the method comprising
contacting in vitro T cells with antigen loaded human MHC molecules
expressed on the surface of a suitable antigen-presenting cell for
a period of time sufficient to activate the T cell in an antigen
specific manner, wherein the antigen is a peptide according to the
invention. Preferably a sufficient amount of the antigen is used
with an antigen-presenting cell.
[0481] Preferably the mammalian cell lacks or has a reduced level
or function of the TAP peptide transporter. Suitable cells that
lack the TAP peptide transporter include T2, RMA-S and Drosophila
cells. TAP is the transporter associated with antigen
processing.
[0482] The human peptide loading deficient cell line T2 is
available from the American Type Culture Collection, 12301 Parklawn
Drive, Rockville, Md. 20852, USA under Catalogue No CRL 1992; the
Drosophila cell line Schneider line 2 is available from the ATCC
under Catalogue No CRL 19863; the mouse RMA-S cell line is
described in Ljunggren et al. (Ljunggren and Karre, 1985).
[0483] Preferably, before transfection the host cell expresses
substantially no MHC class I molecules. It is also preferred that
the stimulator cell expresses a molecule important for providing a
co-stimulatory signal for T-cells such as any of B7.1, B7.2, ICAM-1
and LFA 3. The nucleic acid sequences of numerous MHC class I
molecules and of the co-stimulator molecules are publicly available
from the GenBank and EMBL databases.
[0484] In case of a MHC class I epitope being used as an antigen,
the T cells are CD8-positive T cells.
[0485] If an antigen-presenting cell is transfected to express such
an epitope, preferably the cell comprises an expression vector
capable of expressing a peptide containing SEQ ID NO: 1 to SEQ ID
NO: 161, or a variant amino acid sequence thereof.
[0486] A number of other methods may be used for generating T cells
in vitro. For example, autologous tumor-infiltrating lymphocytes
can be used in the generation of CTL. Plebanski et al. (Plebanski
et al., 1995) made use of autologous peripheral blood lymphocytes
(PLBs) in the preparation of T cells. Furthermore, the production
of autologous T cells by pulsing dendritic cells with peptide or
polypeptide, or via infection with recombinant virus is possible.
Also, B cells can be used in the production of autologous T cells.
In addition, macrophages pulsed with peptide or polypeptide, or
infected with recombinant virus, may be used in the preparation of
autologous T cells. S. Walter et al. (Walter et al., 2003) describe
the in vitro priming of T cells by using artificial antigen
presenting cells (aAPCs), which is also a suitable way for
generating T cells against the peptide of choice. In the present
invention, aAPCs were generated by the coupling of preformed
MHC:peptide complexes to the surface of polystyrene particles
(microbeads) by biotin:streptavidin biochemistry. This system
permits the exact control of the MHC density on aAPCs, which allows
to selectively elicit high- or low-avidity antigen-specific T cell
responses with high efficiency from blood samples. Apart from
MHC:peptide complexes, aAPCs should carry other proteins with
co-stimulatory activity like anti-CD28 antibodies coupled to their
surface. Furthermore, such aAPCs-based systems often require the
addition of appropriate soluble factors, e. g. cytokines, like
interleukin-12.
[0487] Allogeneic cells may also be used in the preparation of T
cells and a method is described in detail in WO 97/26328,
incorporated herein by reference. For example, in addition to
Drosophila cells and T2 cells, other cells may be used to present
antigens such as CHO cells, baculovirus-infected insect cells,
bacteria, yeast, and vaccinia-infected target cells. In addition,
plant viruses may be used (see, for example, Porta et al. (Porta et
al., 1994) which describes the development of cowpea mosaic virus
as a high-yielding system for the presentation of foreign
peptides.
[0488] The activated T cells that are directed against the peptides
of the invention are useful in therapy. Thus, a further aspect of
the invention provides activated T cells obtainable by the
foregoing methods of the invention.
[0489] Activated T cells, which are produced by the above method,
will selectively recognize a cell that aberrantly expresses a
polypeptide that comprises an amino acid sequence of SEQ ID NO: 1
to SEQ ID NO 161.
[0490] Preferably, the T cell recognizes the cell by interacting
through its TCR with the HLA/peptide-complex (for example,
binding). The T cells are useful in a method of killing target
cells in a patient whose target cells aberrantly express a
polypeptide comprising an amino acid sequence of the invention
wherein the patient is administered an effective number of the
activated T cells. The T cells that are administered to the patient
may be derived from the patient and activated as described above
(i.e. they are autologous T cells). Alternatively, the T cells are
not from the patient but are from another individual. Of course, it
is preferred if the individual is a healthy individual. By "healthy
individual" the inventors mean that the individual is generally in
good health, preferably has a competent immune system and, more
preferably, is not suffering from any disease that can be readily
tested for, and detected.
[0491] In vivo, the target cells for the CD8-positive T cells
according to the present invention can be cells of the tumor (which
sometimes express MHC class II) and/or stromal cells surrounding
the tumor (tumor cells) (which sometimes also express MHC class II;
(Dengjel et al., 2006)).
[0492] The T cells of the present invention may be used as active
ingredients of a therapeutic composition. Thus, the invention also
provides a method of killing target cells in a patient whose target
cells aberrantly express a polypeptide comprising an amino acid
sequence of the invention, the method comprising administering to
the patient an effective number of T cells as defined above.
[0493] By "aberrantly expressed" the inventors also mean that the
polypeptide is over-expressed compared to normal levels of
expression or that the gene is silent in the tissue from which the
tumor is derived but in the tumor it is expressed. By
"over-expressed" the inventors mean that the polypeptide is present
at a level at least 1.2-fold of that present in normal tissue;
preferably at least 2-fold, and more preferably at least 5-fold or
10-fold the level present in normal tissue.
[0494] T cells may be obtained by methods known in the art, e.g.
those described above.
[0495] Protocols for this so-called adoptive transfer of T cells
are well known in the art. Reviews can be found in: Gattioni et al.
and Morgan et al. (Gattinoni et al., 2006; Morgan et al.,
2006).
[0496] Another aspect of the present invention includes the use of
the peptides complexed with MHC to generate a T-cell receptor whose
nucleic acid is cloned and is introduced into a host cell,
preferably a T cell. This engineered T cell can then be transferred
to a patient for therapy of cancer.
[0497] Any molecule of the invention, i.e. the peptide, nucleic
acid, antibody, expression vector, cell, activated T cell, T-cell
receptor or the nucleic acid encoding it, is useful for the
treatment of disorders, characterized by cells escaping an immune
response. Therefore, any molecule of the present invention may be
used as medicament or in the manufacture of a medicament. The
molecule may be used by itself or combined with other molecule(s)
of the invention or (a) known molecule(s).
[0498] The present invention is further directed at a kit
comprising:
[0499] (a) A container containing a pharmaceutical composition as
described above, in solution or in lyophilized form;
[0500] (b) Optionally a second container containing a diluent or
reconstituting solution for the lyophilized formulation; and
[0501] (c) Optionally, instructions for (i) use of the solution or
(ii) reconstitution and/or use of the lyophilized formulation.
[0502] The kit may further comprise one or more of (iii) a buffer,
(iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe. The
container is preferably a bottle, a vial, a syringe or test tube;
and it may be a multi-use container. The pharmaceutical composition
is preferably lyophilized.
[0503] Kits of the present invention preferably comprise a
lyophilized formulation of the present invention in a suitable
container and instructions for its reconstitution and/or use.
Suitable containers include, for example, bottles, vials (e.g. dual
chamber vials), syringes (such as dual chamber syringes) and test
tubes. The container may be formed from a variety of materials such
as glass or plastic. Preferably the kit and/or container contain/s
instructions on or associated with the container that indicates
directions for reconstitution and/or use. For example, the label
may indicate that the lyophilized formulation is to be
reconstituted to peptide concentrations as described above. The
label may further indicate that the formulation is useful or
intended for subcutaneous administration.
[0504] The container holding the formulation may be a multi-use
vial, which allows for repeat administrations (e.g., from 2-6
administrations) of the reconstituted formulation. The kit may
further comprise a second container comprising a suitable diluent
(e.g., sodium bicarbonate solution).
[0505] Upon mixing of the diluent and the lyophilized formulation,
the final peptide concentration in the reconstituted formulation is
preferably at least 0.15 mg/mL/peptide (=75 .mu.g) and preferably
not more than 3 mg/mL/peptide (=1500 .mu.g). The kit may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use.
[0506] Kits of the present invention may have a single container
that contains the formulation of the pharmaceutical compositions
according to the present invention with or without other components
(e.g., other compounds or pharmaceutical compositions of these
other compounds) or may have distinct container for each
component.
[0507] Preferably, kits of the invention include a formulation of
the invention packaged for use in combination with the
co-administration of a second compound (such as adjuvants (e.g.
GM-CSF), a chemotherapeutic agent, a natural product, a hormone or
antagonist, an anti-angiogenesis agent or inhibitor, an
apoptosis-inducing agent or a chelator) or a pharmaceutical
composition thereof. The components of the kit may be pre-complexed
or each component may be in a separate distinct container prior to
administration to a patient. The components of the kit may be
provided in one or more liquid solutions, preferably, an aqueous
solution, more preferably, a sterile aqueous solution. The
components of the kit may also be provided as solids, which may be
converted into liquids by addition of suitable solvents, which are
preferably provided in another distinct container.
[0508] The container of a therapeutic kit may be a vial, test tube,
flask, bottle, syringe, or any other means of enclosing a solid or
liquid. Usually, when there is more than one component, the kit
will contain a second vial or other container, which allows for
separate dosing. The kit may also contain another container for a
pharmaceutically acceptable liquid. Preferably, a therapeutic kit
will contain an apparatus (e.g., one or more needles, syringes, eye
droppers, pipette, etc.), which enables administration of the
agents of the invention that are components of the present kit.
[0509] The present formulation is one that is suitable for
administration of the peptides by any acceptable route such as oral
(enteral), nasal, ophthal, subcutaneous, intradermal,
intramuscular, intravenous or transdermal. Preferably, the
administration is s.c., and most preferably i.d. administration may
be by infusion pump.
[0510] Since the peptides of the invention were isolated from
pancreatic cancer, the medicament of the invention is preferably
used to treat pancreatic cancer.
[0511] The present invention further relates to a method for
producing a personalized pharmaceutical for an individual patient
comprising manufacturing a pharmaceutical composition comprising at
least one peptide selected from a warehouse of pre-screened TUMAPs,
wherein the at least one peptide used in the pharmaceutical
composition is selected for suitability in the individual patient.
In one embodiment, the pharmaceutical composition is a vaccine. The
method could also be adapted to produce T cell clones for
down-stream applications, such as TCR isolations, or soluble
antibodies, and other treatment options.
[0512] A "personalized pharmaceutical" shall mean specifically
tailored therapies for one individual patient that will only be
used for therapy in such individual patient, including actively
personalized cancer vaccines and adoptive cellular therapies using
autologous patient tissue.
[0513] As used herein, the term "warehouse" shall refer to a group
or set of peptides that have been pre-screened for immunogenicity
and/or over-presentation in a particular tumor type. The term
"warehouse" is not intended to imply that the particular peptides
included in the vaccine have been pre-manufactured and stored in a
physical facility, although that possibility is contemplated. It is
expressly contemplated that the peptides may be manufactured de
novo for each individualized vaccine produced, or may be
pre-manufactured and stored. The warehouse (e.g. in the form of a
database) is composed of tumor-associated peptides which were
highly overexpressed in the tumor tissue of pancreatic cancer
patients with various HLA-A HLA-B and HLA-C alleles. It may contain
MHC class I and MHC class II peptides or elongated MHC class I
peptides. In addition to the tumor associated peptides collected
from several pancreatic cancer samples, the warehouse may contain
HLA-A*02 and HLA-A*24 marker peptides. These peptides allow
comparison of the magnitude of T-cell immunity induced by TUMAPS in
a quantitative manner and hence allow important conclusion to be
drawn on the capacity of the vaccine to elicit anti-tumor
responses. Secondly, they function as important positive control
peptides derived from a "non-self" antigen in the case that any
vaccine-induced T-cell responses to TUMAPs derived from "self"
antigens in a patient are not observed. And thirdly, it may allow
conclusions to be drawn, regarding the status of immunocompetence
of the patient.
[0514] TUMAPs for the warehouse are identified by using an
integrated functional genomics approach combining gene expression
analysis, mass spectrometry, and T-cell immunology (XPresident C)).
The approach assures that only TUMAPs truly present on a high
percentage of tumors but not or only minimally expressed on normal
tissue, are chosen for further analysis. For initial peptide
selection, pancreatic cancer samples and blood from healthy donors
were analyzed in a stepwise approach:
[0515] 1. HLA ligands from the malignant material were identified
by mass spectrometry
[0516] 2. Genome-wide messenger ribonucleic acid (mRNA) expression
analysis was used to identify genes over-expressed in the malignant
tissue (pancreatic cancer) compared with a range of normal organs
and tissues
[0517] 3. Identified HLA ligands were compared to gene expression
data. Peptides over-presented or selectively presented on tumor
tissue, preferably encoded by selectively expressed or
over-expressed genes as detected in step 2 were considered suitable
TUMAP candidates for a multi-peptide vaccine.
[0518] 4. Literature research was performed in order to identify
additional evidence supporting the relevance of the identified
peptides as TUMAPs
[0519] 5. The relevance of over-expression at the mRNA level was
confirmed by redetection of selected TUMAPs from step 3 on tumor
tissue and lack of (or infrequent) detection on healthy
tissues.
[0520] 6. In order to assess, whether an induction of in vivo
T-cell responses by the selected peptides may be feasible, in vitro
immunogenicity assays were performed using human T cells from
healthy donors as well as from pancreatic cancer patients.
[0521] In an aspect, the peptides are pre-screened for
immunogenicity before being included in the warehouse. By way of
example, and not limitation, the immunogenicity of the peptides
included in the warehouse is determined by a method comprising in
vitro T-cell priming through repeated stimulations of CD8+ T cells
from healthy donors with artificial antigen presenting cells loaded
with peptide/MHC complexes and anti-CD28 antibody.
[0522] This method is preferred for rare cancers and patients with
a rare expression profile. In contrast to multi-peptide cocktails
with a fixed composition as currently developed, the warehouse
allows a significantly higher matching of the actual expression of
antigens in the tumor with the vaccine. Selected single or
combinations of several "off-the-shelf" peptides will be used for
each patient in a multitarget approach. In theory an approach based
on selection of e.g. 5 different antigenic peptides from a library
of 50 would already lead to approximately 17 million possible drug
product (DP) compositions.
[0523] In an aspect, the peptides are selected for inclusion in the
vaccine based on their suitability for the individual patient based
on the method according to the present invention as described
herein, or as below.
[0524] The HLA phenotype, transcriptomic and peptidomic data is
gathered from the patient's tumor material, and blood samples to
identify the most suitable peptides for each patient containing
"warehouse" and patient-unique (i.e. mutated) TUMAPs. Those
peptides will be chosen, which are selectively or over-expressed in
the patients' tumor and, where possible, show strong in vitro
immunogenicity if tested with the patients' individual PBMCs.
[0525] Preferably, the peptides included in the vaccine are
identified by a method comprising: (a) identifying tumor-associated
peptides (TUMAPs) presented by a tumor sample from the individual
patient; (b) comparing the peptides identified in (a) with a
warehouse (database) of peptides as described above; and (c)
selecting at least one peptide from the warehouse (database) that
correlates with a tumor-associated peptide identified in the
patient. For example, the TUMAPs presented by the tumor sample are
identified by: (a1)) comparing expression data from the tumor
sample to expression data from a sample of normal tissue
corresponding to the tissue type of the tumor sample to identify
proteins that are over-expressed or aberrantly expressed in the
tumor sample; and (a2) correlating the expression data with
sequences of MHC ligands bound to MHC class I and/or class II
molecules in the tumor sample to identify MHC ligands derived from
proteins over-expressed or aberrantly expressed by the tumor.
Preferably, the sequences of MHC ligands are identified by eluting
bound peptides from MHC molecules isolated from the tumor sample,
and sequencing the eluted ligands. Preferably, the tumor sample and
the normal tissue are obtained from the same patient.
[0526] In addition to, or as an alternative to, selecting peptides
using a warehousing (database) model, TUMAPs may be identified in
the patient de novo, and then included in the vaccine. As one
example, candidate TUMAPs may be identified in the patient by (a1)
comparing expression data from the tumor sample to expression data
from a sample of normal tissue corresponding to the tissue type of
the tumor sample to identify proteins that are over-expressed or
aberrantly expressed in the tumor sample; and (a2) correlating the
expression data with sequences of MHC ligands bound to MHC class I
and/or class II molecules in the tumor sample to identify MHC
ligands derived from proteins over-expressed or aberrantly
expressed by the tumor. As another example, proteins may be
identified containing mutations that are unique to the tumor sample
relative to normal corresponding tissue from the individual
patient, and TUMAPs can be identified that specifically target the
mutation. For example, the genome of the tumor and of corresponding
normal tissue can be sequenced by whole genome sequencing: For
discovery of non-synonymous mutations in the protein-coding regions
of genes, genomic DNA and RNA are extracted from tumor tissues and
normal non-mutated genomic germline DNA is extracted from
peripheral blood mononuclear cells (PBMCs). The applied NGS
approach is confined to the re-sequencing of protein coding regions
(exome re-sequencing). For this purpose, exonic DNA from human
samples is captured using vendor-supplied target enrichment kits,
followed by sequencing with e.g. a HiSeq2000 (Illumina).
Additionally, tumor mRNA is sequenced for direct quantification of
gene expression and validation that mutated genes are expressed in
the patients' tumors. The resultant millions of sequence reads are
processed through software algorithms. The output list contains
mutations and gene expression. Tumor-specific somatic mutations are
determined by comparison with the PBMC-derived germline variations
and prioritized. The de novo identified peptides can then be tested
for immunogenicity as described above for the warehouse, and
candidate TUMAPs possessing suitable immunogenicity are selected
for inclusion in the vaccine.
[0527] In one exemplary embodiment, the peptides included in the
vaccine are identified by: (a) identifying tumor-associated
peptides (TUMAPs) presented by a tumor sample from the individual
patient by the method as described above; (b) comparing the
peptides identified in a) with a warehouse of peptides that have
been prescreened for immunogenicity and overpresentation in tumors
as compared to corresponding normal tissue; (c) selecting at least
one peptide from the warehouse that correlates with a
tumor-associated peptide identified in the patient; and (d)
optionally, selecting at least one peptide identified de novo in
(a) confirming its immunogenicity.
[0528] In one exemplary embodiment, the peptides included in the
vaccine are identified by: (a) identifying tumor-associated
peptides (TUMAPs) presented by a tumor sample from the individual
patient; and (b) selecting at least one peptide identified de novo
in (a) and confirming its immunogenicity.
[0529] Once the peptides for a personalized peptide based vaccine
are selected, the vaccine is produced. The vaccine preferably is a
liquid formulation consisting of the individual peptides dissolved
in between 20-40% DMSO, preferably about 30-35% DMSO, such as about
33% DMSO.
[0530] Each peptide to be included into a product is dissolved in
DMSO. The concentration of the single peptide solutions has to be
chosen depending on the number of peptides to be included into the
product. The single peptide-DMSO solutions are mixed in equal parts
to achieve a solution containing all peptides to be included in the
product with a concentration of-2.5 mg/ml per peptide. The mixed
solution is then diluted 1:3 with water for injection to achieve a
concentration of 0.826 mg/ml per peptide in 33% DMSO. The diluted
solution is filtered through a 0.22 .mu.m sterile filter. The final
bulk solution is obtained.
[0531] Final bulk solution is filled into vials and stored at
-20.degree. C. until use. One vial contains 700 .mu.L solution,
containing 0.578 mg of each peptide. Of this, 500 .mu.L (approx.
400 .mu.g per peptide) will be applied for intradermal
injection.
[0532] In addition to being useful for treating cancer, the
peptides of the present invention are also useful as diagnostics.
Since the peptides were generated from pancreatic cancer samples
and since it was determined that these peptides are not or at lower
levels present in normal tissues, these peptides can be used to
diagnose the presence of a cancer.
[0533] The presence of claimed peptides on tissue biopsies in blood
samples can assist a pathologist in diagnosis of cancer. Detection
of certain peptides by means of antibodies, mass spectrometry or
other methods known in the art can tell the pathologist that the
tissue sample is malignant or inflamed or generally diseased, or
can be used as a biomarker for pancreatic cancer. Presence of
groups of peptides can enable classification or sub-classification
of diseased tissues.
[0534] The detection of peptides on diseased tissue specimen can
enable the decision about the benefit of therapies involving the
immune system, especially if T-lymphocytes are known or expected to
be involved in the mechanism of action. Loss of MHC expression is a
well described mechanism by which infected of malignant cells
escape immuno-surveillance. Thus, presence of peptides shows that
this mechanism is not exploited by the analyzed cells.
[0535] The peptides of the present invention might be used to
analyze lymphocyte responses against those peptides such as T cell
responses or antibody responses against the peptide or the peptide
complexed to MHC molecules. These lymphocyte responses can be used
as prognostic markers for decision on further therapy steps. These
responses can also be used as surrogate response markers in
immunotherapy approaches aiming to induce lymphocyte responses by
different means, e.g. vaccination of protein, nucleic acids,
autologous materials, adoptive transfer of lymphocytes. In gene
therapy settings, lymphocyte responses against peptides can be
considered in the assessment of side effects. Monitoring of
lymphocyte responses might also be a valuable tool for follow-up
examinations of transplantation therapies, e.g. for the detection
of graft versus host and host versus graft diseases.
[0536] The present invention will now be described in the following
examples which describe preferred embodiments thereof, and with
reference to the accompanying figures, nevertheless, without being
limited thereto. For the purposes of the present invention, all
references as cited herein are incorporated by reference in their
entireties.
FIGURES
[0537] FIGS. 1A to AF show the over-presentation of various
peptides in normal tissues (white bars) and pancreatic cancer
(black bars). FIG. 1A) Gene symbol(s): PTGS1, PTGS2, Peptide:
ILIGETIKI (SEQ ID NO.: 3), Tissues from left to right: 1 adipose
tissue, 3 adrenal glands, 6 arteries, 5 bone marrows, 7 brains, 3
breasts, 1 nerve, 13 colons, 1 ovary, 8 esophagi, 2 gallbladders, 5
hearts, 16 kidneys, 21 livers, 46 lungs, 3 lymph nodes, 4 leukocyte
samples, 3 ovaries, 4 peripheral nerves, 1 peritoneum, 3 pituitary
glands, 2 placentas, 3 pleuras, 3 prostates, 6 recti, 7 salivary
glands, 3 skeletal muscles, 5 skins, 2 small intestines, 4 spleens,
7 stomachs, 4 testes, 3 thymi, 4 thyroid glands, 7 tracheas, 3
ureters, 6 urinary bladders, 2 uteri, 2 veins, 7 pancreas, 20
pancreatic cancer cell line and xenograft samples. The peptide has
additionally been detected on 4/91 lung cancers, 1/20 ovarian
cancers, 1/24 colorectal cancers, 1/18 kidney cancers, and 1/4
urinary bladder cancers (not shown). FIG. 1B) Gene symbol(s):
COL1A2, Peptide: FVDTRTLL (SEQ ID NO.: 1), Tissues from left to
right: 1 adipose tissue, 3 adrenal glands, 6 arteries, 5 bone
marrows, 7 brains, 3 breasts, 1 nerve, 13 colons, 1 ovary, 8
esophagi, 2 gallbladders, 5 hearts, 16 kidneys, 21 livers, 46
lungs, 3 lymph nodes, 4 leukocyte samples, 3 ovaries, 4 peripheral
nerves, 1 peritoneum, 3 pituitary glands, 2 placentas, 3 pleuras, 3
prostates, 6 recti, 7 salivary glands, 3 skeletal muscles, 5 skins,
2 small intestines, 4 spleens, 7 stomachs, 4 testes, 3 thymi, 4
thyroid glands, 7 tracheas, 3 ureters, 6 urinary bladders, 2 uteri,
2 veins, 7 pancreas, 20 pancreatic cancer cell line and xenograft
samples. The peptide has additionally been detected on 3/91 lung
cancers and 1/17 esophageal cancers. FIG. 1C) Gene symbol(s):
PTPN14, Peptide: AQYKFVYQV (SEQ ID NO.: 12), Tissues from left to
right: 1 adipose tissue, 3 adrenal glands, 6 arteries, 5 bone
marrows, 7 brains, 3 breasts, 1 nerve, 13 colons, 1 ovary, 8
esophagi, 2 gallbladders, 5 hearts, 16 kidneys, 21 livers, 46
lungs, 3 lymph nodes, 4 leukocyte samples, 3 ovaries, 4 peripheral
nerves, 1 peritoneum, 3 pituitary glands, 2 placentas, 3 pleuras, 3
prostates, 6 recti, 7 salivary glands, 3 skeletal muscles, 5 skins,
2 small intestines, 4 spleens, 7 stomachs, 4 testes, 3 thymi, 4
thyroid glands, 7 tracheas, 3 ureters, 6 urinary bladders, 2 uteri,
2 veins, 7 pancreas, 20 pancreatic cancer cell line and xenograft
samples. The peptide has additionally been detected on 1/20 ovarian
cancers, 2/17 esophageal cancers, 1/46 stomach cancers, 1/91 lung
cancers, and 1/18 kidney cancers. FIG. 1D) Gene symbol(s): UBR1,
Peptide: SLMDPNKFLLL (SEQ ID NO.: 115), Tissues from left to right:
13 pancreatic cell lines, 2 PBMC cultures, 1 prostate cell culture,
3 skin cell lines, 7 normal tissues (1 liver, 2 lungs, 2 spleens, 1
stomach, 1 trachea), 62 cancer tissues (8 brain cancers, 2 breast
cancers, 2 colon cancers, 1 esophageal cancer, 1 gallbladder
cancer, 5 kidney cancers, 3 leukemias, 6 liver cancers, 19 lung
cancers, 5 ovarian cancers, 1 pancreatic cancer, 3 prostate
cancers, 3 rectal cancers, 1 skin cancer, 2 urinary bladder
cancers). The normal tissue panel (no disease) and the cancer cell
lines and xenografts tested were the same as in FIG. 1A-C,
consisting of 1 adipose tissue, 3 adrenal glands, 6 arteries, 5
bone marrows, 7 brains, 3 breasts, 1 nerve, 13 colons, 1 ovary, 8
esophagi, 2 gallbladders, 5 hearts, 16 kidneys, 21 livers, 46
lungs, 3 lymph nodes, 4 leukocyte samples, 3 ovaries, 4 peripheral
nerves, 1 peritoneum, 3 pituitary glands, 2 placentas, 3 pleuras, 3
prostates, 6 recti, 7 salivary glands, 3 skeletal muscles, 5 skins,
2 small intestines, 4 spleens, 7 stomachs, 4 testes, 3 thymi, 4
thyroid glands, 7 tracheas, 3 ureters, 6 urinary bladders, 2 uteri,
2 veins, 7 pancreas, 20 pancreatic cancer cell line and xenograft
samples. The peptide has additionally been detected on 1/6 breast
cancers, 5/24 colorectal cancers, 1/2 gallbladder/bile duct
cancers, 6/16 liver cancers, 1/2 melanomas, 5/20 ovarian cancers,
1/17 esophageal cancers, 3/12 leukemias, 7/29 brain cancers, 16/91
non-small cell lung carcinomas, 3/33 prostate cancers, 3/18 kidney
cancers, 3/14 small cell lung carcinomas, and 1/4 urinary bladder
cancers. Discrepancies regarding the list of tumor types between
FIG. 1D and table 4 may be due to the more stringent selection
criteria applied in table 4 (for details please refer to table 4).
FIG. 1D shows all samples with detectable presentation of the
peptide Y, regardless of over-presentation parameters and technical
sample quality test. FIG. 1E) Gene symbol(s): NUP205, Peptide:
ALLTGIISKA (SEQ ID NO.: 5), Tissues from left to right: 6 adipose
tissues, 8 adrenal glands, 24 blood cells, 15 blood vessels, 10
bone marrows, 14 brains, 7 breasts, 9 esophagi, 2 eyes, 3
gallbladders, 16 hearts, 17 kidneys, 23 large intestines, 23
livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 6
parathyroid glands, 1 peritoneum, 6 pituitary glands, 7 placentas,
1 pleura, 3 prostates, 7 salivary glands, 10 skeletal muscles, 11
skins, 8 small intestines, 12 spleens, 7 stomachs, 5 testes, 3
thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8 urinary
bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell line
and xenograft samples. The peptide has additionally been found on
2/34 brain cancers, 1/18 breast cancers, 2/29 colon or rectum
cancers, 1/18 esophageal cancers, 1/8 head and neck cancers, 1/21
liver cancers, 8/107 lung cancers, 1/20 lymph node cancers, 1/20
ovarian cancers, 1/18 skin cancers, 2/15 urinary bladder cancers,
1/16 uterus cancers. FIG. 1F) Gene symbol(s): NUP160, Peptide:
ALWHDAENQTVV (SEQ ID NO.: 19), Tissues from left to right: 6
adipose tissues, 8 adrenal glands, 24 blood cells, 15 blood
vessels, 10 bone marrows, 14 brains, 7 breasts, 9 esophagi, 2 eyes,
3 gallbladders, 16 hearts, 17 kidneys, 23 large intestines, 23
livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 6
parathyroid glands, 1 peritoneum, 6 pituitary glands, 7 placentas,
1 pleura, 3 prostates, 7 salivary glands, 10 skeletal muscles, 11
skins, 8 small intestines, 12 spleens, 7 stomachs, 5 testes, 3
thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8 urinary
bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell line
and xenograft samples. The peptide has additionally been found on
2/17 gallbladder or bile duct cancers, 2/34 brain cancers, 1/18
breast cancers, 1/18 esophageal cancers, 1/21 liver cancers, 8/107
lung cancers, 2/18 skin cancers, 2/15 urinary bladder cancers, 1/16
uterus cancers. FIG. 1G) Gene symbol(s): C11orf80, Peptide:
ILSTEIFGV (SEQ ID NO.: 22), Tissues from left to right: 6 adipose
tissues, 8 adrenal glands, 24 blood cells, 15 blood vessels, 10
bone marrows, 14 brains, 7 breasts, 9 esophagi, 2 eyes, 3
gallbladders, 16 hearts, 17 kidneys, 23 large intestines, 23
livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 6
parathyroid glands, 1 peritoneum, 6 pituitary glands, 7 placentas,
1 pleura, 3 prostates, 7 salivary glands, 10 skeletal muscles, 11
skins, 8 small intestines, 12 spleens, 7 stomachs, 5 testes, 3
thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8 urinary
bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell line
and xenograft samples. The peptide has additionally been found on
3/18 breast cancers, 1/17 gallbladder cancers, 1/8 head and neck
cancers, 5/17 leukocytic leukemia cancers, 6/107 lung cancers, 4/20
lymph node cancers, 1/20 ovarian cancers, 1/19 pancreas cancers,
1/18 skin cancers, 1/21 stomach cancers. FIG. 1H) Gene symbol(s):
FAM83D, Peptide: FLNPDEVHAI (SEQ ID NO.: 37), Tissues from left to
right: 6 adipose tissues, 8 adrenal glands, 24 blood cells, 15
blood vessels, 10 bone marrows, 14 brains, 7 breasts, 9 esophagi, 2
eyes, 3 gallbladders, 16 hearts, 17 kidneys, 23 large intestines,
23 livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 6
parathyroid glands, 1 peritoneum, 6 pituitary glands, 7 placentas,
1 pleura, 3 prostates, 7 salivary glands, 10 skeletal muscles, 11
skins, 8 small intestines, 12 spleens, 7 stomachs, 5 testes, 3
thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8 urinary
bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell line
and xenograft samples. The peptide has additionally been found on
2/17 gallbladder or bile duct cancers, 2/34 brain cancers, 3/18
breast cancers, 6/29 colon or rectum cancers, 2/18 esophageal
cancers, 2/8 head and neck cancers, 1/23 kidney cancers, 5/21 liver
cancers, 25/107 lung cancers, 4/20 lymph node cancers, 7/20 ovarian
cancers, 1/87 prostate cancers, 2/18 skin cancers, 2/45 stomach
cancers, 6/15 urinary bladder cancers, 3/16 uterus cancers. FIG.
11) Gene symbol(s): DCBLD2, Peptide: TMVEHNYYV (SEQ ID NO.: 46),
Tissues from left to right: 6 adipose tissues, 8 adrenal glands, 24
blood cells, 15 blood vessels, 10 bone marrows, 14 brains, 7
breasts, 9 esophagi, 2 eyes, 3 gallbladders, 16 hearts, 17 kidneys,
23 large intestines, 23 livers, 49 lungs, 7 lymph nodes, 12 nerves,
2 ovaries, 6 parathyroid glands, 1 peritoneum, 6 pituitary glands,
7 placentas, 1 pleura, 3 prostates, 7 salivary glands, 10 skeletal
muscles, 11 skins, 8 small intestines, 12 spleens, 7 stomachs, 5
testes, 3 thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8
urinary bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell
line and xenograft samples. The peptide has additionally been found
on 1/18 esophageal cancer, 1/17 gallbladder cancers, 1/8 head and
neck cancers, 3/23 kidney cancers, 9/107 lung cancers, 7/20 ovarian
cancers, 1/19 pancreas cancers, 1/18 skin cancers, 1/45 stomach
cancers, 2/15 urinary bladder cancers, 1/16 uterus cancers. FIG.
1J) Gene symbol(s): SHCBP1, Peptide: RLSELGITQA (SEQ ID NO.: 57),
Tissues from left to right: 6 adipose tissues, 8 adrenal glands, 24
blood cells, 15 blood vessels, 10 bone marrows, 14 brains, 7
breasts, 9 esophagi, 2 eyes, 3 gallbladders, 16 hearts, 17 kidneys,
23 large intestines, 23 livers, 49 lungs, 7 lymph nodes, 12 nerves,
2 ovaries, 6 parathyroid glands, 1 peritoneum, 6 pituitary glands,
7 placentas, 1 pleura, 3 prostates, 7 salivary glands, 10 skeletal
muscles, 11 skins, 8 small intestines, 12 spleens, 7 stomachs, 5
testes, 3 thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8
urinary bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell
line and xenograft samples. The peptide has additionally been found
on 1/34 brain cancers, 1/18 breast cancers, 2/18 esophageal
cancers, 2/8 head and neck cancers, 1/21 liver cancers, 8/107 lung
cancers, 4/20 lymph node cancers, 1/18 myeloid cell cancers, 4/20
ovarian cancers, 4/18 skin cancers, 2/15 urinary bladder cancers,
1/16 uterus cancers. FIG. 1K) Gene symbol(s): CTHRC1, Peptide:
VLFSGSLRL (SEQ ID NO.: 69), Tissues from left to right: 6 adipose
tissues, 8 adrenal glands, 24 blood cells, 15 blood vessels, 10
bone marrows, 14 brains, 7 breasts, 9 esophagi, 2 eyes, 3
gallbladders, 16 hearts, 17 kidneys, 23 large intestines, 23
livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 6
parathyroid glands, 1 peritoneum, 6 pituitary glands, 7 placentas,
1 pleura, 3 prostates, 7 salivary glands, 10 skeletal muscles, 11
skins, 8 small intestines, 12 spleens, 7 stomachs, 5 testes, 3
thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8 urinary
bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell line
and xenograft samples. The peptide has additionally been found on
2/18 breast cancers, 1/18 esophageal cancers, 1/17 gallbladder
cancers, 9/107 lung cancers, 1/20 ovarian cancers. FIG. 1L) Gene
symbol(s): CDC27, Peptide: KISTITPQI (SEQ ID NO.: 123), Tissues
from left to right: 6 adipose tissues, 8 adrenal glands, 24 blood
cells, 15 blood vessels, 10 bone marrows, 14 brains, 7 breasts, 9
esophagi, 2 eyes, 3 gallbladders, 16 hearts, 17 kidneys, 23 large
intestines, 23 livers, 49 lungs, 7 lymph nodes, 12 nerves, 2
ovaries, 6 parathyroid glands, 1 peritoneum, 6 pituitary glands, 7
placentas, 1 pleura, 3 prostates, 7 salivary glands, 10 skeletal
muscles, 11 skins, 8 small intestines, 12 spleens, 7 stomachs, 5
testes, 3 thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8
urinary bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell
line and xenograft samples. The peptide has additionally been found
on 2/34 brain cancers, 2/8 head and neck cancers, 1/23 kidney
cancers, 1/17 leukocytic leukemia cancers, 2/21 liver cancers,
7/107 lung cancers, 2/20 lymph node cancers, 1/18 myeloid cell
cancers, 1/18 skin cancers, 1/45 stomach cancers, 2/15 urinary
bladder cancers, 3/16 uterus cancers. FIG. 1M) Gene symbol(s):
UBE2C, Peptide: ALYDVRTILL (SEQ ID NO.: 128), Tissues from left to
right: 6 adipose tissues, 8 adrenal glands, 24 blood cells, 15
blood vessels, 10 bone marrows, 14 brains, 7 breasts, 9 esophagi, 2
eyes, 3 gallbladders, 16 hearts, 17 kidneys, 23 large intestines,
23 livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 6
parathyroid glands, 1 peritoneum, 6 pituitary glands, 7 placentas,
1 pleura, 3 prostates, 7 salivary glands, 10 skeletal muscles, 11
skins, 8 small intestines, 12 spleens, 7 stomachs, 5 testes, 3
thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8 urinary
bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell line
and xenograft samples. The peptide has additionally been found on
2/18 breast cancers, 3/29 colon or rectum cancers, 1/17 leukocytic
leukemia cancers, 8/107 lung cancers, 1/20 lymph node cancers, 1/20
ovarian cancers, 1/15 urinary bladder cancers. FIG. 1N) Gene
symbol(s): MBTPS2, Peptide: VLISGVVHEI (SEQ ID NO.: 146), Tissues
from left to right: 6 adipose tissues, 8 adrenal glands, 24 blood
cells, 15 blood vessels, 10 bone marrows, 14 brains, 7 breasts, 9
esophagi, 2 eyes, 3 gallbladders, 16 hearts, 17 kidneys, 23 large
intestines, 23 livers, 49 lungs, 7 lymph nodes, 12 nerves, 2
ovaries, 6 parathyroid glands, 1 peritoneum, 6 pituitary glands, 7
placentas, 1 pleura, 3 prostates, 7 salivary glands, 10 skeletal
muscles, 11 skins, 8 small intestines, 12 spleens, 7 stomachs, 5
testes, 3 thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8
urinary bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell
line and xenograft samples. The peptide has additionally been found
on 7/34 brain cancers, 1/18 breast cancers, 2/29 colon or rectum
cancers, 1/18 esophageal cancers, 1/23 kidney cancers, 3/21 liver
cancers, 5/107 lung cancers, 1/20 lymph node cancers, 2/20 ovarian
cancers, 1/87 prostate cancers, 3/18 skin cancers, 1/16 uterus
cancers. FIG. 1O) Gene symbol(s): PFDN1, Peptide: KLADIQIEQL (SEQ
ID NO.: 89), Tissues from left to right: 6 adipose tissues, 8
adrenal glands, 24 blood cells, 15 blood vessels, 10 bone marrows,
14 brains, 7 breasts, 9 esophagi, 2 eyes, 3 gallbladders, 16
hearts, 17 kidneys, 23 large intestines, 23 livers, 49 lungs, 7
lymph nodes, 12 nerves, 2 ovaries, 6 parathyroid glands, 1
peritoneum, 6 pituitary glands, 7 placentas, 1 pleura, 3 prostates,
7 salivary glands, 10 skeletal muscles, 11 skins, 8 small
intestines, 12 spleens, 7 stomachs, 5 testes, 3 thymi, 3 thyroid
glands, 15 tracheas, 7 ureters, 8 urinary bladders, 6 uteri, 10
pancreases, 20 pancreatic cancer cell line and xenograft samples.
The peptide has additionally been found on 2/29 colon or rectum
cancers, 1/17 leukocytic leukemia cancers, 4/107 lung cancers, 4/20
ovarian cancers, 4/16 urinary bladder cancers.
FIG. 1P) Gene symbol(s): PKP3, Peptide: ALVEENGIFEL (SEQ ID NO.:
101), Tissues from left to right: 6 adipose tissues, 8 adrenal
glands, 24 blood cells, 15 blood vessels, 10 bone marrows, 14
brains, 7 breasts, 9 esophagi, 2 eyes, 3 gallbladders, 16 hearts,
17 kidneys, 23 large intestines, 23 livers, 49 lungs, 7 lymph
nodes, 12 nerves, 2 ovaries, 6 parathyroid glands, 1 peritoneum, 6
pituitary glands, 7 placentas, 1 pleura, 3 prostates, 7 salivary
glands, 10 skeletal muscles, 11 skins, 8 small intestines, 12
spleens, 7 stomachs, 5 testes, 3 thymi, 3 thyroid glands, 15
tracheas, 7 ureters, 8 urinary bladders, 6 uteri, 10 pancreases, 20
pancreatic cancer cell line and xenograft samples. The peptide has
additionally been found on 1/17 bile duct cancers, 2/18 breast
cancers, 2/29 colon or rectum cancers, 2/18 esophageal cancers, 2/8
head and neck cancers, 1/21 liver cancers, 7/107 lung cancers, 6/20
ovarian cancers, 3/87 prostate cancers, 4/15 urinary bladder
cancers, 1/16 uterus cancers. FIG. 1Q) Gene symbol(s): GFPT2,
Peptide: LMMSEDRISL (SEQ ID NO.: 113), Tissues from left to right:
6 adipose tissues, 8 adrenal glands, 24 blood cells, 15 blood
vessels, 10 bone marrows, 14 brains, 7 breasts, 9 esophagi, 2 eyes,
3 gallbladders, 16 hearts, 17 kidneys, 23 large intestines, 23
livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 6
parathyroid glands, 1 peritoneum, 6 pituitary glands, 7 placentas,
1 pleura, 3 prostates, 7 salivary glands, 10 skeletal muscles, 11
skins, 8 small intestines, 12 spleens, 7 stomachs, 5 testes, 3
thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8 urinary
bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell line
and xenograft samples. The peptide has additionally been found on
3/17 gallbladder or bile duct cancers, 5/34 brain cancers, 3/18
breast cancers, 2/29 colon or rectum cancers, 2/18 esophageal
cancers, 1/8 head and neck cancers, 1/21 liver cancers, 18/107 lung
cancers, 3/20 lymph node cancers, 1/19 pancreas cancers, 1/87
prostate cancers, 2/18 skin cancers, 2/15 urinary bladder cancers,
1/16 uterus cancers. FIG. 1R) Gene symbol(s): CCT4, Peptide:
ALSDLALHFL (SEQ ID NO.: 127), Tissues from left to right: 6 adipose
tissues, 8 adrenal glands, 24 blood cells, 15 blood vessels, 10
bone marrows, 14 brains, 7 breasts, 9 esophagi, 2 eyes, 3
gallbladders, 16 hearts, 17 kidneys, 23 large intestines, 23
livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 6
parathyroid glands, 1 peritoneum, 6 pituitary glands, 7 placentas,
1 pleura, 3 prostates, 7 salivary glands, 10 skeletal muscles, 11
skins, 8 small intestines, 12 spleens, 7 stomachs, 5 testes, 3
thymi, 3 thyroid glands, 15 tracheas, 7 ureters, 8 urinary
bladders, 6 uteri, 10 pancreases, 20 pancreatic cancer cell line
and xenograft samples. The peptide has additionally been found on
1/34 brain cancers, 2/18 breast cancers, 2/8 head and neck cancers,
3/17 leukocytic leukemia cancers, 1/21 liver cancers, 3/107 lung
cancers, 4/20 lymph node cancers, 2/18 myeloid cell cancers, 1/20
ovarian cancers, 3/18 skin cancers, 4/15 urinary bladder cancers.
FIG. 1S) Gene symbol(s): NUP205, Peptide: ALLTGIISKA (SEQ ID NO.:
5), Tissues from left to right: 12 cancer cell lines, 1 normal
tissue (1 spleen), 22 cancer tissues (2 brain cancers, 1 breast
cancer, 1 colon cancer, 1 esophageal cancer, 1 head and neck
cancer, 1 liver cancer, 8 lung cancers, 1 lymph node cancer, 1
ovarian cancer, 1 rectum cancer, 1 skin cancer, 2 urinary bladder
cancers, 1 uterus cancer). The normal tissue panel tested was the
same as in FIG. 1E-R. FIG. 1T) Gene symbol(s): NUP160, Peptide:
ALWHDAENQTVV (SEQ ID NO.: 19), Tissues from left to right: 13
cancer cell lines, 1 primary culture, 1 normal tissue (1 spleen),
20 cancer tissues (1 bile duct cancer, 2 brain cancers, 1 breast
cancer, 1 esophageal cancer, 1 gallbladder cancer, 1 liver cancer,
8 lung cancers, 2 skin cancers, 2 urinary bladder cancers, 1 uterus
cancer). The normal tissue panel tested was the same as in FIG.
1E-R. FIG. 1U) Gene symbol(s): C11orf80, Peptide: ILSTEIFGV (SEQ ID
NO.: 22), Tissues from left to right: 1 cancer cell line, 3 primary
cultures, 1 normal tissue (1 lymph node), 24 cancer tissues (3
breast cancers, 1 gallbladder cancer, 1 head and neck cancer, 5
leukocytic leukemia cancers, 6 lung cancers, 4 lymph node cancers,
1 ovarian cancer, 1 pancreas cancer, 1 skin cancer, 1 stomach
cancer). The normal tissue panel tested was the same as in FIG.
1E-R. FIG. 1V) Gene symbol(s): FAM83D, Peptide: FLNPDEVHAI (SEQ ID
NO.: 37), Tissues from left to right: 16 cancer cell lines, 3
primary cultures, 1 normal tissue (1 trachea), 73 cancer tissues (1
bile duct cancer, 2 brain cancers, 3 breast cancers, 4 colon
cancers, 2 esophageal cancers, 1 gallbladder cancer, 2 head and
neck cancers, 1 kidney cancer, 5 liver cancers, 25 lung cancers, 4
lymph node cancers, 7 ovarian cancers, 1 prostate cancer, 2 rectum
cancers, 2 skin cancers, 2 stomach cancers, 6 urinary bladder
cancers, 3 uterus cancers). The normal tissue panel tested was the
same as in FIG. 1E-R. FIG. 1W) Gene symbol(s): DCBLD2, Peptide:
TMVEHNYYV (SEQ ID NO.: 46), Tissues from left to right: 4 cancer
cell lines, 1 primary culture, 28 cancer tissues (1 esophageal
cancer, 1 gallbladder cancer, 1 head and neck cancer, 3 kidney
cancers, 9 lung cancers, 7 ovarian cancers, 1 pancreas cancer, 1
skin cancer, 1 stomach cancer, 2 urinary bladder cancers, 1 uterus
cancer). The normal tissue panel tested was the same as in FIG.
1E-R. FIG. 1X) Gene symbol(s): SHCBP1, Peptide: RLSELGITQA (SEQ ID
NO.: 57), Tissues from left to right: 20 cancer cell lines, 2
primary cultures, 2 normal tissues (1 bone marrow, 1 placenta), 31
cancer tissues (1 brain cancer, 1 breast cancer, 2 esophageal
cancers, 2 head and neck cancers, 1 liver cancer, 8 lung cancers, 4
lymph node cancers, 1 myeloid cell cancer, 4 ovarian cancers, 4
skin cancers, 2 urinary bladder cancers, 1 uterus cancer). The
normal tissue panel tested was the same as in FIG. 1E-R. FIG. 1Y)
Gene symbol(s): CTHRC1, Peptide: VLFSGSLRL (SEQ ID NO.: 69),
Tissues from left to right: 5 cancer cell lines, 14 cancer tissues
(2 breast cancers, 1 esophageal cancer, 1 gallbladder cancer, 9
lung cancers, 1 ovarian cancer). The normal tissue panel tested was
the same as in FIG. 1E-R. FIG. 1Z) Gene symbol(s): CDC27, Peptide:
KISTITPQI (SEQ ID NO.: 123), Tissues from left to right: 19 cancer
cell lines, 2 primary cultures, 3 normal tissues (1 adrenal gland,
1 liver, 1 placenta), 25 cancer tissues (2 brain cancers, 2 head
and neck cancers, 1 kidney cancer, 1 leukocytic leukemia cancer, 2
liver cancers, 7 lung cancers, 2 lymph node cancers, 1 myeloid cell
cancer, 1 skin cancer, 1 stomach cancer, 2 urinary bladder cancers,
3 uterus cancers). The normal tissue panel tested was the same as
in FIG. 1E-R. FIG. 1AA) Gene symbol(s): UBE2C, Peptide: ALYDVRTILL
(SEQ ID NO.: 128), Tissues from left to right: 10 cancer cell
lines, 17 cancer tissues (2 breast cancers, 1 cecum cancer, 2 colon
cancers, 1 leukocytic leukemia cancer, 8 lung cancers, 1 lymph node
cancer, 1 ovarian cancer, 1 urinary bladder cancer). The normal
tissue panel tested was the same as in FIG. 1E-R. FIG. 1AB) Gene
symbol(s): MBTPS2, Peptide: VLISGVVHEI (SEQ ID NO.: 146), Tissues
from left to right: 16 cancer cell lines, 2 primary cultures, 2
normal tissues (1 spleen, 1 uterus), 28 cancer tissues (7 brain
cancers, 1 breast cancer, 2 colon cancers, 1 esophageal cancer, 1
kidney cancer, 3 liver cancers, 5 lung cancers, 1 lymph node
cancer, 2 ovarian cancers, 1 prostate cancer, 3 skin cancers, 1
uterus cancer). The normal tissue panel tested was the same as in
FIG. 1E-R. FIG. 1AC) Gene symbol(s): PFDN1, Peptide: KLADIQIEQL
(SEQ ID NO.: 89), Tissues from left to right: 11 cancer cell lines,
2 normal tissues (2 adrenal glands), 15 cancer tissues (2 colon
cancers, 1 leukocytic leukemia cancer, 4 lung cancers, 4 ovarian
cancers, 4 urinary bladder cancers). The normal tissue panel tested
was the same as in FIG. 1E-R. FIG. 1AD) Gene symbol(s): PKP3,
Peptide: ALVEENGIFEL (SEQ ID NO.: 101), Tissues from left to right:
3 cancer cell lines, 3 primary cultures, 2 normal tissues (2
colons), 31 cancer tissues (1 bile duct cancer, 2 breast cancers, 1
cecum cancer, 1 colon cancer, 2 esophageal cancers, 2 head and neck
cancers, 1 liver cancer, 7 lung cancers, 6 ovarian cancers, 3
prostate cancers, 4 urinary bladder cancers, 1 uterus cancer). The
normal tissue panel tested was the same as in FIG. 1E-R. FIG. 1AE)
Gene symbol(s): GFPT2, Peptide: LMMSEDRISL (SEQ ID NO.: 113),
Tissues from left to right: 8 cancer cell lines, 1 normal tissue (1
eye), 45 cancer tissues (1 bile duct cancer, 5 brain cancers, 3
breast cancers, 1 colon cancer, 2 esophageal cancers, 2 gallbladder
cancers, 1 head and neck cancer, 1 liver cancer, 18 lung cancers, 3
lymph node cancers, 1 pancreas cancer, 1 prostate cancer, 1 rectum
cancer, 2 skin cancers, 2 urinary bladder cancers, 1 uterus
cancer). The normal tissue panel tested was the same as in FIG.
1E-R. FIG. 1AF) Gene symbol(s): CCT4, Peptide: ALSDLALHFL (SEQ ID
NO.: 127), Tissues from left to right: 9 cancer cell lines, 26
cancer tissues (1 bone marrow cancer, 1 brain cancer, 2 breast
cancers, 2 head and neck cancers, 3 leukocytic leukemia cancers, 1
liver cancer, 3 lung cancers, 4 lymph node cancers, 1 myeloid cell
cancer, 1 ovarian cancer, 3 skin cancers, 4 urinary bladder
cancers). The normal tissue panel tested was the same as in FIG.
1E-R.
[0538] FIGS. 2A to C show exemplary expression profiles (relative
expression compared to normal pancreas) of source genes of the
present invention that are highly over-expressed or exclusively
expressed in pancreatic cancer in a panel of normal tissues (white
bars) and 9 pancreatic cancer samples (black bars). Tissues from
left to right: adrenal gland, artery, bone marrow, brain (whole),
breast, colon, esophagus, heart, kidney (triplicate), leukocytes,
liver, lung, lymph node, ovary, pancreas, placenta, prostate,
salivary gland, skeletal muscle, skin, small intestine, spleen,
stomach, testis, thymus, thyroid gland, urinary bladder, uterine
cervix, uterus, vein, 9 pancreatic cancer samples. FIG. 2A) SHCBP1;
FIG. 2B) FN1; and FIG. 2C) PLEC.
[0539] FIG. 3A to D show exemplary immunogenicity data: flow
cytometry results after peptide-specific multimer staining. CD8+ T
cells were primed using artificial APCs coated with anti-CD28 mAb
and HLA-A*02 in complex with SeqID No 125 peptide (A, left panel),
SeqID No 148 peptide (B, left panel), SeqID No 156 peptide (C, left
panel), SeqID No 178 peptide (D, left panel, top), and SeqID No 177
peptide (D, left panel, bottom), respectively. After three cycles
of stimulation, the detection of peptide-reactive cells was
performed by 2D multimer staining with A*02/SeqID No 125 (A),
A*02/SeqID No 148 (B) or A*02/SeqID No 156 (C). Right panels (A, B,
C, and D) show control staining of cells stimulated with irrelevant
A*02/peptide complexes. Viable singlet cells were gated for CD8+
lymphocytes. Boolean gates helped excluding false-positive events
detected with multimers specific for different peptides.
Frequencies of specific multimer+ cells among CD8+ lymphocytes are
indicated.
EXAMPLES
Example 1
Identification and Quantitation of Tumor Associated Peptides
Presented on the Cell Surface Tissue Samples
[0540] Patients' tumor tissues and cell lines were obtained from
University Hospital of Tubingen, Germany, University Hospital of
Heidelberg, Germany, NMI Reutlingen, Germany, Md. Anderson Cancer
Center, Houston, Tex., USA. Normal tissues were obtained from
Asterand, Detroit, USA and Royston, Herts, UK; Bio-Options Inc.,
CA, USA; BioServe, Beltsville, Md., USA; Capital BioScience Inc.,
Rockville, Md., USA; Geneticist Inc., Glendale, Calif., USA; Tissue
Solutions Ltd, Glasgow, Scotland, UK; University Hospital of
Geneva; University Hospital of Heidelberg; Kyoto Prefectural
University of Medicine (KPUM); University Hospital Munich;
ProteoGenex Inc., Culver City, Calif., USA; University Hospital of
Tubingen, Germany. Written informed consents of all donors had been
given before surgery or autopsy. Tissues were shock-frozen
immediately after excision and stored until isolation of TUMAPs at
-70.degree. C. or below.
Isolation of HLA Peptides from Tissue Samples
[0541] HLA peptide pools from frozen tissue samples were obtained
by immune precipitation according to a slightly modified protocol
(Falk et al., 1991; Seeger et al., 1999) using the
HLA-A*02-specific antibody BB7.2, the HLA-A, --B, C-specific
antibody W6/32, CNBr-activated sepharose, acid treatment, and
ultrafiltration.
Mass Spectrometry Analyses
[0542] The HLA peptide pools as obtained were separated according
to their hydrophobicity by reversed-phase chromatography
(nanoAcquity UPLC system, Waters) and the eluting peptides were
analyzed in LTQ-velos and fusion hybrid mass spectrometers
(ThermoElectron) equipped with an ESI source. Peptide pools were
loaded directly onto the analytical fused-silica micro-capillary
column (75 .mu.m i.d..times.250 mm) packed with 1.7 .mu.m C18
reversed-phase material (Waters) applying a flow rate of 400 nL per
minute. Subsequently, the peptides were separated using a two-step
180 minute-binary gradient from 10% to 33% B at a flow rate of 300
nL per minute. The gradient was composed of Solvent A (0.1% formic
acid in water) and solvent B (0.1% formic acid in acetonitrile). A
gold coated glass capillary (PicoTip, New Objective) was used for
introduction into the nanoESI source. The LTQ-Orbitrap mass
spectrometers were operated in the data-dependent mode using a TOP5
strategy. In brief, a scan cycle was initiated with a full scan of
high mass accuracy in the Orbitrap (R=30 000), which was followed
by MS/MS scans also in the Orbitrap (R=7500) on the 5 most abundant
precursor ions with dynamic exclusion of previously selected ions.
Tandem mass spectra were interpreted by SEQUEST and additional
manual control. The identified peptide sequence was assured by
comparison of the generated natural peptide fragmentation pattern
with the fragmentation pattern of a synthetic sequence-identical
reference peptide.
[0543] Label-free relative LC-MS quantitation was performed by ion
counting i.e. by extraction and analysis of LC-MS features (Mueller
et al., 2007). The method assumes that the peptide's LC-MS signal
area correlates with its abundance in the sample. Extracted
features were further processed by charge state deconvolution and
retention time alignment (Mueller et al., 2008; Sturm et al.,
2008). Finally, all LC-MS features were cross-referenced with the
sequence identification results to combine quantitative data of
different samples and tissues to peptide presentation profiles. The
quantitative data were normalized in a two-tier fashion according
to central tendency to account for variation within technical and
biological replicates. Thus each identified peptide can be
associated with quantitative data allowing relative quantification
between samples and tissues. In addition, all quantitative data
acquired for peptide candidates was inspected manually to assure
data consistency and to verify the accuracy of the automated
analysis. For each peptide a presentation profile was calculated
showing the mean sample presentation as well as replicate
variations. The profiles juxtapose pancreatic cancer samples to a
baseline of normal tissue samples. Presentation profiles of
exemplary over-presented peptides are shown in FIG. 1. Presentation
scores for exemplary peptides are shown in Table 8.
TABLE-US-00009 TABLE 8 Presentation scores. The table lists
peptides that are very highly over-presented on tumors compared to
a panel of normal tissues (+++), highly over- presented on tumors
compared to a panel of normal tissues (++) or over-presented on
tumors compared to a panel of normal tissues (+). SEQ ID Peptide
No. Sequence Presentation 1 FVDTRTLL +++ 3 ILIGETIKI +++ 4
ALDPAAQAFLL +++ 5 ALLTGIISKA +++ 6 ALTGIPLPLI +++ 7 ALVDIVRSL +++ 8
ALYTGSALDFV +++ 10 VLLDKIKNL + 11 ALYYNPHLL +++ 12 AQYKFVYQV +++ 14
FIIDNPQDLKV +++ 15 FILANEHNV +++ 16 GLIDYDTGI +++ 17 GLIDYDTGIRL ++
18 ALFVRLLAL +++ 19 ALWHDAENQTVV +++ 21 GLVDGRDLVIV +++ 22
ILSTEIFGV +++ 23 KLDSSGGAVQL ++ 24 KLSENAGIQSL +++ 25 LINPNIATV +++
27 TLLAHPVTL + 29 YILPFSEVL +++ 30 YIYKDTIQV +++ 31 YLDSMYIML ++ 34
FLEDDDIAAV +++ 35 FLFPSQYVDV +++ 37 FLNPDEVHAI + 39 FLTPSIFII +++
40 GLAPQIHDL +++ 41 GLLAGNEKLTM ++ 42 ILSDMRSQYEV +++ 43 HLGVKVFSV
+++ 44 ILAQVGFSV +++ 45 ILYSDDGQKWTV +++ 46 TMVEHNYYV +++ 47
LIYKDLVSV + 48 LLDENGVLKL +++ 49 LLDGFPRTV +++ 50 LLFGSDGYYV +++ 51
LLGPAGARA +++ 52 LLSDPIPEV ++ 53 LLWDPSTGKQV +++ 54 LTQPGPIASA +++
55 NLAPAPLNA +++ 56 NLIGVTAEL +++ 57 RLSELGITQA ++ 58 RQYPWGVVQV
+++ 59 SLSESFFMV + 60 SLWEDYPHV ++ 61 SMYDGLLQA ++ 62 SVFPGARLL +++
63 SVTGIIVGV +++ 64 TLFSEPKFAQV ++ 67 VIWGTDVNV ++ 68 VLFDVTGQV +++
69 VLFSGSLRL +++ 70 VLGVIWGV +++ 71 VLLPEGGITAI +++ 72 VMASPGGLSAV
+++ 73 VMVDGKPVNL + 74 YIDKDLEYV +++ 75 FSFVDLRLL +++ 77 RLFPGSSFL
+++ 79 VVYEGQLISI + 80 LLPGTEYVVSV + 81 VVYDDSTGLIRL +++ 82
ALIAEGIAL ++ 83 ALSKEIYVI +++ 85 FLSDGTIISV ++ 86 GLGDFIFYSV + 88
IIDDTIFNL ++ 90 KLLTPITTL + 91 LLFNDVQTL + 92 YLTNEGIAHL + 93
SIDSEPALV +++ 94 VMMEEFVQL + 95 ALADDDFLTV ++ 96 ALAPATGGGSLLL + 98
ALDQKVRSV + 99 ALESFLKQV + 100 ALFGAGPASI +++ 102 ALYPGTDYTV + 104
FLQPDLDSL +++ 105 FLSEVFHQA + 106 FVWSGTAEA +++ 107 FVYGGPQVQL +
108 IADGGFTEL +++ 109 ILASVILNV ++ 111 LLLAAARLAAA + 114 SLFPHNPQFI
+++ 115 SLMDPNKFLLL ++ 116 SMMDPNHFL ++ 118 TLWYRPPEL ++ 119
VLGDDPQLMKV + 120 VLVNDFFLV ++ 122 MQAPRAALVFA + 123 KISTITPQI +++
124 ALFEESGLIRI +++ 125 ALLGKLDAINV +++ 126 ALLSLDPAAV +++ 128
ALYDVRTILL +++ 130 FLFGEEPSKL + 131 FLIEEQKIVV +++ 132 FLWAGGRASYGV
+++ 133 ILDDVSLTHL +++ 134 ILLAEGRLVNL +++ 135 KLDDTYIKA +++ 136
KLFPGFEIETV +++ 137 KLGPEGELL +++ 138 NIFPNPEATFV ++ 140
SLLNPPETLNL +++ 142 SLYGYLRGA +++ 143 TADPLDYRL ++ 144 TAVALLRLL
+++ 145 TTFPRPVTV +++ 146 VLISGVVHEI +++ 147 YAFPKAVSV +++ 148
YLHNQGIGV + 149 ILGTEDLIVEV +
150 ALFQPHLINV ++ 151 ALLDIIRSL +++ 153 ALPKEDPTAV + 154 KVADLVLML
+ 155 LLLDPDTAVLKL ++ 156 LLLPPPPCPA + 157 MLLEIPYMAA ++ 158
SLIEKYFSV + 159 SLLDLHTKV + 160 VLLPDERTISL +++ 162 NADPQAVTM +++
163 VMAPRTLVL ++ 164 YLGRLAHEV ++ 165 YLLSYIQSI ++ 166 SLFPGQVVI
+++ 167 MLFGHPLLVSV +++ 169 FMLPDPQNI +++ 171 LLLDVTPLSL ++ 172
TMMSRPPVL ++ 173 SLAGDVALQQL +++ 174 TLDPRSFLL ++ 175 ALLESSLRQA ++
176 YLMPGFIHL +++
Example 2
Expression Profiling of Genes Encoding the Peptides of the
Invention
[0544] Over-presentation or specific presentation of a peptide on
tumor cells compared to normal cells is sufficient for its
usefulness in immunotherapy, and some peptides are tumor-specific
despite their source protein occurring also in normal tissues.
Still, mRNA expression profiling adds an additional level of safety
in selection of peptide targets for immunotherapies. Especially for
therapeutic options with high safety risks, such as
affinity-matured TCRs, the ideal target peptide will be derived
from a protein that is unique to the tumor and not found on normal
tissues.
RNA Sources and Preparation
[0545] Surgically removed tissue specimens were provided as
indicated above (see Example 1) after written informed consent had
been obtained from each patient. Tumor tissue specimens were
snap-frozen immediately after surgery and later homogenized with
mortar and pestle under liquid nitrogen. Total RNA was prepared
from these samples using TRI Reagent (Ambion, Darmstadt, Germany)
followed by a cleanup with RNeasy (QIAGEN, Hilden, Germany); both
methods were performed according to the manufacturer's
protocol.
[0546] Total RNA from healthy human tissues was obtained
commercially (Ambion, Huntingdon, UK; Clontech, Heidelberg,
Germany; Stratagene, Amsterdam, Netherlands; BioChain, Hayward,
Calif., USA). The RNA from several individuals (between 2 and 123
individuals) was mixed such that RNA from each individual was
equally weighted.
[0547] Quality and quantity of all RNA samples were assessed on an
Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) using the
RNA 6000 Pico LabChip Kit (Agilent).
Microarray Experiments
[0548] Gene expression analysis of all tumor and normal tissue RNA
samples was performed by Affymetrix Human Genome (HG) U133A or
HG-U133 Plus 2.0 oligonucleotide microarrays (Affymetrix, Santa
Clara, Calif., USA). All steps were carried out according to the
Affymetrix manual. Briefly, double-stranded cDNA was synthesized
from 5-8 .mu.g of total RNA, using SuperScript RTII (Invitrogen)
and the oligo-dT-T7 primer (MWG Biotech, Ebersberg, Germany) as
described in the manual. In vitro transcription was performed with
the BioArray High Yield RNA Transcript Labelling Kit (ENZO
Diagnostics, Inc., Farmingdale, N.Y., USA) for the U133A arrays or
with the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus
2.0 arrays, followed by cRNA fragmentation, hybridization, and
staining with streptavidin-phycoerythrin and biotinylated
anti-streptavidin antibody (Molecular Probes, Leiden, Netherlands).
Images were scanned with the Agilent 2500A GeneArray Scanner
(U133A) or the Affymetrix Gene-Chip Scanner 3000 (U133 Plus 2.0),
and data were analyzed with the GCOS software (Affymetrix), using
default settings for all parameters. For normalization, 100
housekeeping genes provided by Affymetrix were used. Relative
expression values were calculated from the signal log ratios given
by the software and the normal kidney sample was arbitrarily set to
1.0. Exemplary expression profiles of source genes of the present
invention that are highly over-expressed or exclusively expressed
in pancreatic cancer are shown in FIG. 2. Expression scores for
further exemplary genes are shown in Table 9.
TABLE-US-00010 TABLE 9 Expression scores. The table lists peptides
from genes that are very highly over-expressed in tumors compared
to a panel of normal tissues (+++), highly over- expressed in
tumors compared to a panel of normal tissues (++) or over-expressed
in tumors compared to a panel of normal tissues (+). SEQ Gene ID No
Gene name Sequence Expression 1 COL1A2 FVDTRTLL ++ 2 COL1A2
FGYDGDFYRA ++ 3 PTGS1, PTGS2 ILIGETIKI +++ 6 CDK2 ALTGIPLPLI + 7
FADS3 ALVDIVRSL ++ 8 COL6A3 ALYTGSALDFV + 9 COL6A3 QIIDAINKV + 10
COL6A3 VLLDKIKNL + 11 IPO7 ALYYNPHLL + 12 PTPN14 AQYKFVYQV + 18
TGFBI ALFVRLLAL +++ 24 RAI14 KLSENAGIQSL + 26 MAN2A1 SLYTALTEA + 31
ADAM9 YLDSMYIML + 34 GFPT2 FLEDDDIAAV ++ 38 TFPI2 FLTEAALGDA + 43
COL6A1 HLGVKVFSV +++ 44 SLC6A15 ILAQVGFSV ++ 45 DCBLD2 ILYSDDGQKWTV
++ 46 DCBLD2 TMVEHNYYV ++ 53 NLE1 LLWDPSTGKQV + 54 CXCL5 LTQPGPIASA
+ 56 ARMC9 NLIGVTAEL ++ 57 SHCBP1 RLSELGITQA +++ 58 SEPT10, SEPT8,
RQYPWGVVQV ++ SEPT11 60 TRAM2 SLWEDYPHV ++ 61 TRPV2 SMYDGLLQA ++ 67
MCM4 VIWGTDVNV +++ 75 COL1A1 FSFVDLRLL +++ 77 CREB3L1 RLFPGSSFL ++
79 FN1 VVYEGQLISI +++ 80 FN1 LLPGTEYVVSV +++ 84 SLC1A4, SLC1A5
FILPIGATV + 90 COL6A3 KLLTPITTL + 91 PLEC LLFNDVQTL +++ 92 PLEC
YLTNEGIAHL +++ 95 MCM4 ALADDDFLTV +++ 99 PRKDC ALESFLKQV + 105
SERPINB2 FLSEVFHQA + 113 GFPT2 LMMSEDRISL ++ 119 TAF6L VLGDDPQLMKV
+ 123 CDC27 KISTITPQI + 124 CELSR3, SLC26A6 ALFEESGLIRI + 126 PRKDC
ALLSLDPAAV + 128 UBE2C ALYDVRTILL + 132 HNRNPU FLWAGGRASYGV + 136
ASNS KLFPGFEIETV +++ 137 SLC1A5 KLGPEGELL + 139 STAT2 SIDRNPPQL +
140 CCNA2 SLLNPPETLNL ++ 145 NONO TTFPRPVTV + 146 MBTPS2 VLISGVVHEI
+ 151 FADS2 ALLDIIRSL ++ 153 COPG1 ALPKEDPTAV + 165 NCAPG YLLSYIQSI
+++ 166 POLA2 SLFPGQVVI + 173 NCAPD2 SLAGDVALQQL + 175 CCND1
ALLESSLRQA +
Example 3
In Vitro Immunogenicity for MHC Class I Presented Peptides
[0549] In order to obtain information regarding the immunogenicity
of the TUMAPs of the present invention, the inventors performed
investigations using an in vitro T-cell priming assay based on
repeated stimulations of CD8+ T cells with artificial antigen
presenting cells (aAPCs) loaded with peptide/MHC complexes and
anti-CD28 antibody. This way the inventors could show
immunogenicity for 22 HLA-A*0201 restricted TUMAPs of the invention
so far, demonstrating that these peptides are T-cell epitopes
against which CD8+ precursor T cells exist in humans (Table
10).
In Vitro Priming of CD8+ T Cells
[0550] In order to perform in vitro stimulations by artificial
antigen presenting cells loaded with peptide-MHC complex (pMHC) and
anti-CD28 antibody, the inventors first isolated CD8+ T cells from
fresh HLA-A*02 leukapheresis products via positive selection using
CD8 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) of
healthy donors obtained from the University clinics Mannheim,
Germany, after informed consent.
[0551] PBMCs and isolated CD8+ lymphocytes were incubated in T-cell
medium (TCM) until use consisting of RPMI-Glutamax (Invitrogen,
Karlsruhe, Germany) supplemented with 10% heat inactivated human AB
serum (PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100
.mu.g/ml Streptomycin (Cambrex, Cologne, Germany), 1 mM sodium
pyruvate (CC Pro, Oberdorla, Germany), 20 .mu.g/ml Gentamycin
(Cambrex). 2.5 ng/ml IL-7 (PromoCell, Heidelberg, Germany) and 10
U/ml IL-2 (Novartis Pharma, Nurnberg, Germany) were also added to
the TCM at this step.
[0552] Generation of pMHC/anti-CD28 coated beads, T-cell
stimulations and readout was performed in a highly defined in vitro
system using four different pMHC molecules per stimulation
condition and 8 different pMHC molecules per readout condition.
[0553] The purified co-stimulatory mouse IgG2a anti human CD28 Ab
9.3 (Jung et al., 1987) was chemically biotinylated using
Sulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer
(Perbio, Bonn, Germany). Beads used were 5.6 .mu.m diameter
streptavidin coated polystyrene particles (Bangs Laboratories,
Illinois, USA).
[0554] pMHC used for positive and negative control stimulations
were A*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 179) from
modified Melan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5,
SEQ ID NO. 180), respectively.
[0555] 800.000 beads/200 .mu.l were coated in 96-well plates in the
presence of 4.times.12.5 ng different biotin-pMHC, washed and 600
ng biotin anti-CD28 were added subsequently in a volume of 200
.mu.l. Stimulations were initiated in 96-well plates by
co-incubating 1.times.10.sup.6 CD8+ T cells with 2.times.10.sup.5
washed coated beads in 200 .mu.l TCM supplemented with 5 ng/ml
IL-12 (PromoCell) for 3 days at 37.degree. C. Half of the medium
was then exchanged by fresh TCM supplemented with 80 U/ml IL-2 and
incubating was continued for 4 days at 37.degree. C. This
stimulation cycle was performed for a total of three times. For the
pMHC multimer readout using 8 different pMHC molecules per
condition, a two-dimensional combinatorial coding approach was used
as previously described (Andersen et al., 2012) with minor
modifications encompassing coupling to 5 different fluorochromes.
Finally, multimeric analyses were performed by staining the cells
with Live/dead near IR dye (Invitrogen, Karlsruhe, Germany),
CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany) and
fluorescent pMHC multimers. For analysis, a BD LSRII SORP cytometer
equipped with appropriate lasers and filters was used. Peptide
specific cells were calculated as percentage of total CD8+ cells.
Evaluation of multimeric analysis was done using the FlowJo
software (Tree Star, Oregon, USA). In vitro priming of specific
multimer+CD8+ lymphocytes was detected by comparing to negative
control stimulations. Immunogenicity for a given antigen was
detected if at least one evaluable in vitro stimulated well of one
healthy donor was found to contain a specific CD8+ T-cell line
after in vitro stimulation (i.e. this well contained at least 1% of
specific multimer+ among CD8+ T-cells and the percentage of
specific multimer+ cells was at least 10.times. the median of the
negative control stimulations).
In Vitro Immunogenicity for Pancreatic Cancer Peptides
[0556] For tested HLA class I peptides, in vitro immunogenicity
could be demonstrated by generation of peptide specific T-cell
lines. Exemplary flow cytometry results after TUMAP-specific
multimer staining for 2 peptides of the invention are shown in FIG.
3 together with corresponding negative controls. Results for 4
peptides from the invention are summarized in Table 10.
TABLE-US-00011 TABLE 10 In vitro immunogenicity of HLA class I
peptides of the invention Exemplary results of in vitro
immunogenicity experiments conducted by the applicant for HLA-A*02
restricted peptides of the invention. Results of in vitro
immunogenicity experiments are indicated. Percentage of positive
wells and donors (among evaluable) are summarized as indicated
<20% = +; 20% - 49% = ++; 50% - 69% = +++; > = 70% = ++++ SEQ
ID No Sequence Wells positive [%] 17 GLIDYDTGIRL ''+'' 81
VVYDDSTGLIRL ''+'' 122 MQAPRAALVFA ''++'' 165 YLLSYIQSI ''++'' 167
MLFGHPLLVSV ''++'' 172 TMMSRPPVL ''+'' 173 SLAGDVALQQL ''+'' 174
TLDPRSFLL ''++'' 119 VLGDDPQLMKV ''+'' 125 ALLGKLDAINV ''+'' 135
KLDDTYIKA ''+++'' 137 KLGPEGELL ''+'' 147 YAFPKAVSV ''+'' 148
YLHNQGIGV ''+++'' 149 ILGTEDLIVEV ''++'' 156 LLLPPPPCPA ''++''
Example 4
Synthesis of Peptides
[0557] All peptides were synthesized using standard and
well-established solid phase peptide synthesis using the
Fmoc-strategy. Identity and purity of each individual peptide have
been determined by mass spectrometry and analytical RP-HPLC. The
peptides were obtained as white to off-white lyophilizates
(trifluoro acetate salt) in purities of >50%. All TUMAPs are
preferably administered as trifluoro-acetate salts or acetate
salts, other salt-forms are also possible.
Example 5
MHC Binding Assays
[0558] Candidate peptides for T cell based therapies according to
the present invention were further tested for their MHC binding
capacity (affinity). The individual peptide-MHC complexes were
produced by UV-ligand exchange, where a UV-sensitive peptide is
cleaved upon UV-irradiation, and exchanged with the peptide of
interest as analyzed. Only peptide candidates that can effectively
bind and stabilize the peptide-receptive MHC molecules prevent
dissociation of the MHC complexes. To determine the yield of the
exchange reaction, an ELISA was performed based on the detection of
the light chain (.beta.2m) of stabilized MHC complexes. The assay
was performed as generally described in Rodenko et al. (Rodenko et
al., 2006).
[0559] 96 well MAXISorp plates (NUNC) were coated over night with 2
ug/ml streptavidin in PBS at room temperature, washed 4.times. and
blocked for 1h at 37.degree. C. in 2% BSA containing blocking
buffer. Refolded HLA-A*02:01/MLA-001 monomers served as standards,
covering the range of 15-500 ng/ml. Peptide-MHC monomers of the
UV-exchange reaction were diluted 100 fold in blocking buffer.
Samples were incubated for 1 h at 37.degree. C., washed four times,
incubated with 2 ug/ml HRP conjugated anti-.beta.2m for 1h at
37.degree. C., washed again and detected with TMB solution that is
stopped with NH2SO4. Absorption was measured at 450 nm. Candidate
peptides that show a high exchange yield (preferably higher than
50%, most preferred higher than 75%) are generally preferred for a
generation and production of antibodies or fragments thereof,
and/or T cell receptors or fragments thereof, as they show
sufficient avidity to the MHC molecules and prevent dissociation of
the MHC complexes.
[0560] MHC class I binding scores. Binding of HLA-class I
restricted peptides to HLA-A*02:01 was ranged by peptide exchange
yield: .gtoreq.10%=+; .gtoreq.20%=++; .gtoreq.50=+++;
.gtoreq.75%=++++
TABLE-US-00012 SEQ ID No Sequence Peptide exchange 1 FVDTRTLL
''++'' 2 FGYDGDFYRA ''+++'' 3 ILIGETIKI ''+++'' 4 ALDPAAQAFLL
''+++'' 5 ALLTGIISKA ''+++'' 6 ALTGIPLPLI ''+++'' 7 ALVDIVRSL
''+++'' 8 ALYTGSALDFV ''+++'' 9 QIIDAINKV ''++'' 10 VLLDKIKNL
''++'' 11 ALYYNPHLL ''++'' 12 AQYKFVYQV ''+++'' 13 FIDSSNPGL ''++''
14 FIIDNPQDLKV ''+++'' 15 FILANEHNV ''+++'' 16 GLIDYDTGI ''+++'' 17
GLIDYDTGIRL ''+++'' 18 ALFVRLLAL ''++'' 19 ALWHDAENQTVV ''+++'' 20
GLIDIENPNRV ''+++'' 21 GLVDGRDLVIV ''+++'' 22 ILSTEIFGV ''+++'' 23
KLDSSGGAVQL ''++'' 24 KLSENAGIQSL ''++'' 25 LINPNIATV ''++'' 26
SLYTALTEA ''+++'' 27 TLLAHPVTL ''+++'' 28 VLDEFYSSL ''+++'' 29
YILPFSEVL ''++++'' 30 YIYKDTIQV ''++'' 31 YLDSMYIML ''+++'' 32
YVDDGLISL ''++'' 34 FLEDDDIAAV ''++'' 35 FLFPSQYVDV ''+++'' 36
FLGDLSHLL ''++'' 37 FLNPDEVHAI ''++'' 38 FLTEAALGDA ''+++'' 39
FLTPSIFII ''++'' 40 GLAPQIHDL ''++'' 41 GLLAGNEKLTM ''++'' 42
ILSDMRSQYEV ''+++'' 43 HLGVKVFSV ''++'' 44 ILAQVGFSV ''+++'' 45
ILYSDDGQKWTV ''+++'' 46 TMVEHNYYV ''+++'' 47 LIYKDLVSV ''+++'' 48
LLDENGVLKL ''+++'' 49 LLDGFPRTV ''++'' 50 LLFGSDGYYV ''+++'' 51
LLGPAGARA ''++'' 52 LLSDPIPEV ''+++'' 53 LLWDPSTGKQV ''+++'' 54
LTQPGPIASA ''+++'' 55 NLAPAPLNA ''++'' 56 NLIGVTAEL ''++'' 57
RLSELGITQA ''+++'' 58 RQYPWGVVQV ''++'' 59 SLSESFFMV ''+++'' 60
SLWEDYPHV ''++++'' 61 SMYDGLLQA ''++'' 62 SVFPGARLL ''+'' 63
SVTGIIVGV ''++++'' 64 TLFSEPKFAQV ''++++'' 65 TLNEKLTAL ''+++'' 67
VIWGTDVNV ''++++'' 68 VLFDVTGQV ''+++'' 69 VLFSGSLRL ''+++'' 70
VLGVIWGV ''++++'' 71 VLLPEGGITAI ''+++'' 72 VMASPGGLSAV ''+++'' 73
VMVDGKPVNL ''++++'' 74 YIDKDLEYV ''+++'' 77 RLFPGSSFL ''++++'' 78
SLQDTEEKSRS ''+++'' 79 VVYEGQLISI ''+++'' 80 LLPGTEYVVSV ''+++'' 81
VVYDDSTGLIRL ''+++'' 82 ALIAEGIAL ''+++'' 83 ALSKEIYVI ''+++'' 84
FILPIGATV ''++++'' 85 FLSDGTIISV ''++++'' 86 GLGDFIFYSV ''++++'' 87
GLLPALVAL ''+++'' 88 IIDDTIFNL ''+++'' 89 KLADIQIEQL ''+++'' 90
KLLTPITTL ''+++'' 91 LLFNDVQTL ''+++'' 92 YLTNEGIAHL ''++++'' 93
SIDSEPALV ''+++'' 94 VMMEEFVQL ''+++'' 95 ALADDDFLTV ''+++'' 96
ALAPATGGGSLLL ''+++'' 97 ALDDMISTL ''+++'' 98 ALDQKVRSV ''++'' 99
ALESFLKQV ''+++'' 100 ALFGAGPASI ''+++'' 101 ALVEENGIFEL ''+++''
102 ALYPGTDYTV ''+++'' 103 AVAAVLTQV ''+++'' 104 FLQPDLDSL ''+++''
105 FLSEVFHQA ''+++'' 106 FVWSGTAEA ''+++'' 107 FVYGGPQVQL ''+++''
109 ILASVILNV ''++++'' 110 ILLTGTPAL ''+++'' 111 LLLAAARLAAA
''+++'' 112 LLSDVRFVL ''+++'' 113 LMMSEDRISL ''+++'' 114 SLFPHNPQFI
''+++'' 115 SLMDPNKFLLL ''+++'' 116 SMMDPNHFL ''++++'' 117
SVDGVIKEV ''+++'' 118 TLWYRPPEL ''+++'' 119 VLGDDPQLMKV ''+++'' 121
YLDEDTIYHL ''++'' 122 MQAPRAALVFA ''++++'' 123 KISTITPQI ''++'' 124
ALFEESGLIRI ''+++'' 125 ALLGKLDAINV ''+++'' 126 ALLSLDPAAV ''++++''
127 ALSDLALHFL ''++++'' 128 ALYDVRTILL ''+++'' 129 ALYEKDNTYL
''+++'' 130 FLFGEEPSKL ''+++''
131 FLIEEQKIVV ''+++'' 132 FLWAGGRASYGV ''+++'' 133 ILDDVSLTHL
''++'' 134 ILLAEGRLVNL ''+++'' 135 KLDDTYIKA ''+++'' 136
KLFPGFEIETV ''++++'' 137 KLGPEGELL ''+++'' 138 NIFPNPEATFV ''+++''
139 SIDRNPPQL ''+++'' 140 SLLNPPETLNL ''+++'' 141 SLTEQVHSL ''+++''
142 SLYGYLRGA ''+++'' 144 TAVALLRLL ''++'' 145 TTFPRPVTV ''+++''
146 VLISGVVHEI ''+++'' 147 YAFPKAVSV ''++'' 148 YLHNQGIGV ''++''
149 ILGTEDLIVEV ''+++'' 150 ALFQPHLINV ''++++'' 151 ALLDIIRSL
''++++'' 152 ALLEPEFILKA ''++++'' 153 ALPKEDPTAV ''+++'' 154
KVADLVLML ''+++'' 155 LLLDPDTAVLKL ''+++'' 156 LLLPPPPCPA ''+++''
157 MLLEIPYMAA ''+++'' 158 SLIEKYFSV ''++++'' 159 SLLDLHTKV ''+++''
160 VLLPDERTISL ''++++'' 161 YLPDIIKDQKA ''+++''
REFERENCE LIST
[0561] Abele, R. et al., Essays Biochem. 50 (2011): 249-264 [0562]
Abramovich, C. et al., Ann. N.Y. Acad. Sci. 1044 (2005): 109-116
[0563] Acuna Sanhueza, G. A. et al., BMC. Cancer 12 (2012): 72
[0564] Adams, G. N. et al., Cancer Res 75 (2015): 4235-4243 [0565]
Agarwal, R., Biochem. Pharmacol. 60 (2000): 1051-1059 [0566] Ahn,
J. W. et al., Genome Med. 6 (2014): 18 [0567] Aili, A. et al.,
PLoS. One. 8 (2013): e79937 [0568] Aisa, Y. et al., Int. J Hematol.
82 (2005): 266-269 [0569] Akaogi, K. et al., BMC. Cancer 13 (2013):
65 [0570] Akiyama, Y. et al., Oncol. Rep. 31 (2014): 1683-1690
[0571] Alagaratnam, S. et al., Mol. Cell Endocrinol. 306 (2009):
75-80 [0572] Alan, J. K. et al., Small GTPases. 4 (2013): 159-163
[0573] Ali-Rahmani, F. et al., PLoS. One. 9 (2014): e88724 [0574]
Allam, H. et al., J Proteome. Res 14 (2015): 434-446 [0575]
Allison, J. P. et al., Science 270 (1995): 932-933 [0576]
Alshareeda, A. T. et al., Br. J Cancer 112 (2015): 1929-1937 [0577]
Ambrosini, G. et al., Mol. Cancer Ther. 13 (2014): 2073-2080 [0578]
Amirkhosravi, A. et al., Semin. Thromb. Hemost. 33 (2007): 643-652
[0579] Andersen, R. S. et al., Nat. Protoc. 7 (2012): 891-902
[0580] Andersen, V. et al., Aliment. Pharmacol. Ther. 37 (2013):
383-391 [0581] Ando, K. et al., Gastric. Cancer 17 (2014): 255-262
[0582] Ansari, D. et al., J Cancer Res Clin Oncol 141 (2015):
369-380 [0583] Ansari, D. et al., J Transl. Med. 12 (2014): 87
[0584] Aoki, T. et al., Science 212 (1981): 463-465 [0585] Appay,
V. et al., Eur. J Immunol. 36 (2006): 1805-1814 [0586] Arai, M. et
al., Chembiochem. 17 (2016): 181-189 [0587] Argiropoulos, B. et
al., Oncogene 26 (2007): 6766-6776 [0588] Asad, M. et al., Cell
Death. Dis. 5 (2014): e1346 [0589] Asano, E. et al., Cell Cycle 13
(2014): 2744-2751 [0590] Atcheson, E. et al., Biosci. Rep. 31
(2011): 371-379 [0591] Atkins, R. J. et al., J Clin Neurosci. 20
(2013): 1185-1192 [0592] Avery-Kiejda, K. A. et al., BMC. Cancer 14
(2014): 253 [0593] Avruch, J. et al., Curr. Opin. Clin Nutr. Metab
Care 8 (2005): 67-72 [0594] Aydar, E. et al., Cancer Lett. 242
(2006): 245-257 [0595] Aydar, E. et al., Cancer Res 64 (2004):
5029-5035 [0596] Aylon, Y. et al., Mol. Oncol 5 (2011): 315-323
[0597] Aziz, F. et al., Curr. Drug Targets. 15 (2014): 469-476
[0598] Baek, G. et al., Cell Rep. 9 (2014): 2233-2249 [0599]
Balasubramanian, S. et al., Genome Biol 10 (2009): R2 [0600]
Baldwin, R. M. et al., World J Biol Chem 5 (2014): 115-129 [0601]
Ball, A. R., Jr. et al., Mol. Cell Biol 22 (2002): 5769-5781 [0602]
Balmer, N. N. et al., Mod. Pathol. 19 (2006): 1593-1605 [0603] Ban,
Y. et al., J Thyroid Res 2012 (2012): 815079 [0604] Banchereau, J.
et al., Cell 106 (2001): 271-274 [0605] Banziger, C. et al., Cell
125 (2006): 509-522 [0606] Baptista, J. A. et al., Clin Chem 40
(1994): 426-430 [0607] Barboro, P. et al., Cell Oncol 30 (2008):
13-26 [0608] Barrow-McGee, R. et al., Int. J Biochem. Cell Biol 49
(2014): 69-74 [0609] Basu, S. et al., PLoS. One. 10 (2015a):
e0138443 [0610] Basu, S. et al., PLoS. One. 10 (2015b): e0123979
[0611] Bausch, D. et al., Clin Cancer Res 17 (2011): 302-309 [0612]
Beatty, G. et al., J Immunol 166 (2001): 2276-2282 [0613]
Beck-Cormier, S. et al., PLoS. One. 9 (2014): e98507 [0614] Beggs,
J. D., Nature 275 (1978): 104-109 [0615] Bell, J. L. et al., Cell
Mol Life Sci. 70 (2013): 2657-2675 [0616] Belle, L. et al., Sci.
Signal. 8 (2015): ra18 [0617] Bern, W. T. et al., Cancer Res 51
(1991): 6558-6562 [0618] Benjamini, Y. et al., Journal of the Royal
Statistical Society. Series B (Methodological), [0619] Vol. 57
(1995): 289-300 [0620] Bhat, M. et al., BMC. Gastroenterol. 15
(2015): 176 [0621] Bhatnagar, R. et al., Oral Oncol 48 (2012):
831-835 [0622] Bhutia, Y. D. et al., Cancer Res 75 (2015):
1782-1788 [0623] Bidkhori, G. et al., PLoS. One. 8 (2013): e67552
[0624] Blanco, I. et al., PLoS. One. 10 (2015): e0120020 [0625]
Blanco, M. A. et al., Cell Res 22 (2012): 1339-1355 [0626] Boige,
V. et al., JAMA Oncol (2016) [0627] Booth, L. et al., J Cell
Physiol 230 (2015): 1661-1676 [0628] Bouameur, J. E. et al., J
Invest Dermatol. 134 (2014): 885-894 [0629] Boulter, J. M. et al.,
Protein Eng 16 (2003): 707-711 [0630] Braumuller, H. et al., Nature
(2013) [0631] Breuninger, S. et al., Am. J Pathol. 176 (2010):
2509-2519 [0632] Brocke, K. S. et al., Cancer Biol Ther. 9 (2010):
455-468 [0633] Brossart, P. et al., Blood 90 (1997): 1594-1599
[0634] Bruckdorfer, T. et al., Curr. Pharm. Biotechnol. 5 (2004):
29-43 [0635] Burkhart, R. A. et al., Mol. Cancer Res 11 (2013):
901-911 [0636] Burns, K. E. et al., Biochem. J 472 (2015): 287-295
[0637] Busch, T. et al., J Cell Sci. 125 (2012): 2148-2159 [0638]
Cacciola, N. A. et al., Mol. Carcinog (2015) [0639] Cai, X. et al.,
Lung Cancer 65 (2009): 299-305 [0640] Cai, Y. et al., Oncogene 33
(2014): 2157-2168 [0641] Caldarelli, A. et al., Leukemia 27 (2013):
2301-2310 [0642] Camos, M. et al., Cancer Res 66 (2006): 6947-6954
[0643] Campione, E. et al., Drug Des Devel. Ther. 9 (2015):
5843-5850 [0644] Cance, W. G. et al., Breast Cancer Res Treat. 35
(1995): 105-114 [0645] Capurso, G. et al., J Mol. Endocrinol. 49
(2012): R37-R50 [0646] Card, K. F. et al., Cancer Immunol
Immunother. 53 (2004): 345-357 [0647] Carvalho, B. et al., Gut 58
(2009): 79-89 [0648] Cascon, A. et al., Hum. Mutat. 28 (2007):
613-621 [0649] Cavalcante, G. C. et al., Anticancer Res 35 (2015):
6971-6977 [0650] Cerveira, N. et al., Biol Chem 392 (2011): 713-724
[0651] Chandramouli, A. et al., Carcinogenesis 28 (2007): 2028-2035
[0652] Chaneton, B. et al., Trends Biochem. Sci. 37 (2012): 309-316
[0653] Chanock, S. J. et al., Hum. Immunol. 65 (2004): 1211-1223
[0654] Chatterjee, M. et al., Haematologica 98 (2013): 1132-1141
[0655] Chaudhary, N. et al., Mol. Cell Biol 34 (2014): 3754-3764
[0656] Chaudhuri, S. et al., RNA. 13 (2007): 2224-2237 [0657] Chen,
C. et al., PLoS. One. 10 (2015a): e0135074 [0658] Chen, E. et al.,
Oncogene 25 (2006): 5752-5763 [0659] Chen, F. et al., Oncol Lett.
10 (2015): 1704-1708 [0660] Chen, J. et al., Int. J Oncol 44
(2014a): 247-255 [0661] Chen, J. et al., Int. J Clin Exp. Pathol. 8
(2015b): 2026-2032 [0662] Chen, L. et al., Int. J Mol. Sci. 15
(2014b): 11435-11445 [0663] Chen, P. C. et al., Int. J Radiat.
Oncol Biol Phys. 82 (2012): 1996-2003 [0664] Chen, Q. et al.,
Zhongguo Shi Yan. Xue. Ye. Xue. Za Zhi. 19 (2011a): 1171-1175
[0665] Chen, R. et al., J Int. Med. Res 39 (2011b): 533-540 [0666]
Chen, S. et al., J Cancer Res Clin Oncol 136 (2010): 419-426 [0667]
Chen, S. T. et al., Cancer Sci. 102 (2011c): 2191-2198 [0668] Chen,
Y. et al., J Cell Biochem. 100 (2007): 1337-1345 [0669] Chen, Y. L.
et al., Int J Surg. 11 (2013): 85-91 [0670] Chen, Z. T. et al.,
Int. J Mol. Sci. 16 (2015c): 15497-15530 [0671] Chiou, S. S. et
al., Carcinogenesis 35 (2014): 2357-2364 [0672] Chohan, T. A. et
al., Curr. Med. Chem 22 (2015): 237-263 [0673] Choi, S. Y. et al.,
Clin Exp. Med. 11 (2011): 219-226 [0674] Chu, X. et al., Int. J
Clin Exp. Pathol. 8 (2015): 328-336 [0675] Chudnovsky, Y. et al.,
Cell Rep. 6 (2014): 313-324 [0676] Ciccia, A. et al., Mol. Cell 47
(2012): 396-409 [0677] Coe, H. et al., Int. J Biochem. Cell Biol 42
(2010): 796-799 [0678] Cohen, C. J. et al., J Mol Recognit. 16
(2003a): 324-332 [0679] Cohen, C. J. et al., J Immunol 170 (2003b):
4349-4361 [0680] Cohen, S. N. et al., Proc. Natl. Acad. Sci. U.S.A
69 (1972): 2110-2114 [0681] Cohen, Y. et al., Hematology. 19
(2014): 286-292 [0682] Colak, D. et al., PLoS. One. 8 (2013):
e63204 [0683] Coligan, J. E. et al., Current Protocols in Protein
Science (1995) [0684] Collins, K. L. et al., EMBO J 12 (1993):
4555-4566 [0685] Colombetti, S. et al., J Immunol. 176 (2006):
2730-2738 [0686] Coppola, D. et al., J Geriatr. Oncol 5 (2014):
389-399 [0687] Crottes, D. et al., Cancer Res 76 (2016): 607-618
[0688] Cui, H. et al., Cancer Res 67 (2007): 3345-3355 [0689]
Dadkhah, E. et al., Arch. Iran Med. 16 (2013): 463-470 [0690] Dai,
W. et al., PLoS. One. 9 (2014): e87148 [0691] Davies, G. F. et al.,
PLoS. One. 9 (2014): e84611 [0692] De, Braekeleer E. et al.,
Future. Oncol 10 (2014): 475-495 [0693] De, Falco G. et al., Cancer
Biol Ther. 1 (2002): 342-347 [0694] De, P. et al., Cancer Treat.
Rev 39 (2013): 403-412 [0695] Deighton, R. F. et al., Brain Pathol.
20 (2010): 691-703 [0696] Deisenroth, C. et al., Oncogene 29
(2010): 4253-4260 [0697] DeLaBarre, B. et al., Chem Biol 21 (2014):
1143-1161 [0698] Delas, A. et al., Pathol. Res Pract. 209 (2013):
115-119 [0699] Demeure, K. et al., Mol. Cell Proteomics. 15 (2016):
481-492 [0700] Demidyuk, I. V. et al., PLoS. One. 8 (2013): e55752
[0701] Demirag, G. G. et al., Med. Oncol 29 (2012): 1518-1522
[0702] Deng, H. et al., Biochim. Biophys. Acta 1852 (2015a):
520-528 [0703] Deng, W. et al., Cell Physiol Biochem. 35 (2015b):
1677-1688 [0704] Dengjel, J. et al., Clin Cancer Res 12 (2006):
4163-4170 [0705] Denkberg, G. et al., J Immunol 171 (2003):
2197-2207 [0706] Dennis, J. W. et al., Cancer Metastasis Rev 5
(1987): 185-204 [0707] DePrimo, S. E. et al., Genome Biol 3 (2002):
RESEARCH0032 [0708] Derivery, E. et al., PLoS. One. 3 (2008): e2462
[0709] Dhup, S. et al., Curr. Pharm. Des 18 (2012): 1319-1330
[0710] Ding, Z. et al., Mol. Cell Biol 23 (2003): 250-258 [0711]
Dinicola, S. et al., Life Sci. 145 (2016): 174-183 [0712] Diniz, M.
G. et al., Tumour. Biol (2015) [0713] Diotti, R. et al., Mol.
Cancer Res 13 (2015): 402-410 [0714] Dong, S. et al., Mol. Cancer
13 (2014): 76 [0715] Donnard, E. et al., Oncotarget. 5 (2014):
9199-9213 [0716] Doppler, H. et al., J Biol Chem 288 (2013):
455-465 [0717] Dormeyer, W. et al., J Proteome. Res 7 (2008):
2936-2951 [0718] Draoui, N. et al., Dis. Model. Mech. 4 (2011):
727-732 [0719] Du, X. et al., Biochem. J 471 (2015): 243-253 [0720]
Duan, F. et al., Sci. Rep. 5 (2015): 11961 [0721] Duensing, S. et
al., Oncogene 21 (2002): 6241-6248 [0722] Dworakowska, D.,
Pneumonol. Alergol. Pol. 73 (2005): 297-300 [0723] Ebrahimi, F. et
al., Exp. Mol. Pathol. 96 (2014): 98-107 [0724] Efthimiou, E. et
al., Pancreatology. 1 (2001): 571-575 [0725] Elakoum, R. et al.,
Biochimie 97 (2014): 210-218 [0726] Eli, M. et al., World J
Gastroenterol. 18 (2012): 3112-3118 [0727] Emmanuel, C. et al.,
PLoS. One. 6 (2011): e17617 [0728] Er, T. K. et al., J Mol. Med.
(Berl) (2016) [0729] Erkan, M. et al., Mol. Cancer 9 (2010): 88
[0730] Escobar, B. et al., Cancer Res 70 (2010): 9349-9359 [0731]
Fagin, J. A., Mol. Endocrinol. 16 (2002): 903-911 [0732] Falk, K.
et al., Nature 351 (1991): 290-296 [0733] Fan, C. W. et al., Scand.
J Gastroenterol. 39 (2004): 464-469 [0734] Fan, H. X. et al., Onco.
Targets. Ther. 8 (2015): 1619-1626 [0735] Fan, J. et al., Clin
Cancer Res 17 (2011): 2908-2918 [0736] Fang, W. Y. et al., Acta
Biochim. Biophys. Sin. (Shanghai) 37 (2005): 541-546 [0737] Fang,
X. et al., Proteomics. 11 (2011): 921-934 [0738] Feldner, J. C. et
al., Exp. Cell Res 272 (2002): 93-108 [0739] Feng, H. et al., J
Clin Invest 124 (2014): 3741-3756 [0740] Fernandez-Cuesta, L. et
al., Genome Biol 16 (2015): 7 [0741] Ferrer-Ferrer, M. et al.,
Arch. Med. Res 44 (2013): 467-474 [0742] Findeis-Hosey, J. J. et
al., Biotech. Histochem. 87 (2012): 24-29 [0743] Fischer, E. G. et
al., J Clin Invest 104 (1999): 1213-1221 [0744] Flachbartova, Z. et
al., Acta Virol. 57 (2013): 3-15 [0745] Folsom, A. R. et al.,
Cancer Epidemiol. Biomarkers Prev. 16 (2007): 2455-2458 [0746]
Fong, L. et al., Proc. Natl. Acad. Sci. U.S.A 98 (2001): 8809-8814
[0747] Foster, J. S. et al., Trends Endocrinol. Metab 12 (2001):
320-327 [0748] Francavilla, C. et al., Mol. Cell 51 (2013): 707-722
[0749] Frau, M. et al., J Hepatol. 59 (2013): 830-841 [0750] Frau,
M. et al., Hepatology 56 (2012): 165-175 [0751] Fredericks, W. J.
et al., DNA Cell Biol 30 (2011): 851-864 [0752] Fredericks, W. J.
et al., Int. J Oncol 43 (2013): 638-652 [0753] Frugtniet, B. et
al., Breast Cancer (Dove. Med. Press) 7 (2015): 99-109 [0754]
Frulloni, L. et al., N. Engl. J Med. 361 (2009): 2135-2142 [0755]
Fuchs, B. C. et al., Am. J Physiol Gastrointest. Liver Physiol 286
(2004): G467-G478 [0756] Fujieda, S. et al., Arch. Otolaryngol.
Head Neck Surg. 120 (1994): 389-394 [0757] Fujita, A. et al.,
Genet. Mol. Res 7 (2008): 371-378 [0758] Fujita, T. et al., Cancer
Sci. 100 (2009): 238-248 [0759] Fujitomo, T. et al., Cancer Res 72
(2012): 4110-4118 [0760] Fujiwara, K. et al., PLoS. One. 9 (2014):
e107247 [0761] Fukunaga, Y. et al., Lung Cancer 38 (2002): 31-38
[0762] Furukawa, C. et al., Cancer Res 65 (2005): 7102-7110 [0763]
Gabrilovich, D. I. et al., Nat Med. 2 (1996): 1096-1103 [0764]
Gabrovska, P. N. et al., Gene 489 (2011): 63-69 [0765] Gallo, S. et
al., Clin Sci. (Lond) 129 (2015): 1173-1193 [0766] Ganapathy, V. et
al., Pharmacol. Ther. 121 (2009): 29-40 [0767] Gao, H. J. et al., J
Cancer Res. Clin Oncol 141 (2015): 1151-1162 [0768] Gao, J. et al.,
PLoS. One. 9 (2014a): e101979 [0769] Gao, S. et al., Arch. Immunol.
Ther. Exp. (Warsz.) 62 (2014b): 131-144 [0770] Garcia-Lorenzo, A.
et al., Int. J Mol. Sci. 13 (2012): 14401-14420 [0771] Garg, M. et
al., Cancer 116 (2010a): 3785-3796 [0772] Garg, M. et al., Eur. J
Cancer 46 (2010b): 207-215 [0773] Garrido, F. et al., Semin. Cancer
Biol 2 (1991): 3-10 [0774] Gattinoni, L. et al., Nat Rev. Immunol 6
(2006): 383-393 [0775] Gautschi, O. et al., Lung Cancer 55 (2007):
1-14 [0776] George, B. et al., Cancer Lett. 358 (2015): 191-199
[0777] Georgitsi, M., Best. Pract. Res Clin Endocrinol. Metab 24
(2010): 425-437 [0778] Gerber-Lemaire, S. et al., Chimia (Aarau.)
64 (2010): 634-639 [0779] Ghilardi, C. et al., Oncotarget. 6
(2015): 28389-28400 [0780] Ghosh, N. et al., Pharmacol. Rep. 62
(2010): 233-244 [0781] Gillis, L. D. et al., Oncogene 32 (2013):
3598-3605 [0782] Gius, D. et al., Cancer Res 67 (2007): 7113-7123
[0783] Gnjatic, S. et al., Proc Natl. Acad. Sci. U.S.A 100 (2003):
8862-8867 [0784] Godkin, A. et al., Int. Immunol 9 (1997): 905-911
[0785] Golomb, L. et al., Mol. Cell 45 (2012): 222-232 [0786] Gong,
Y. et al., Adv. Anat. Pathol. 21 (2014): 191-200 [0787] Gonias, S.
L. et al., Front Biosci. 6 (2001): D1403-D1411 [0788]
Gonzalez-Exposito, R. et al., Clin Transl. Oncol (2015) [0789]
Goodison, S. et al., BMC. Genomics 4 (2003): 39 [0790] Goss, P. E.
et al., Clin Cancer Res 1 (1995): 935-944 [0791] Goss, P. E. et
al., Cancer Res 54 (1994): 1450-1457 [0792] Goss, P. E. et al.,
Clin Cancer Res 3 (1997): 1077-1086 [0793] Gough, S. M. et al.,
Blood 118 (2011): 6247-6257 [0794] Grady, W. M., Cancer Metastasis
Rev 23 (2004): 11-27 [0795] Graves, L. M. et al., Nature 403
(2000): 328-332 [0796] Green, M. R. et al., Molecular Cloning, A
Laboratory Manual 4th (2012) [0797] Greenfield, E. A., Antibodies:
A Laboratory Manual 2nd (2014) [0798] Grier, D. G. et al., J
Pathol. 205 (2005): 154-171 [0799] Grzmil, M. et al., Oncogene 29
(2010): 4080-4089 [0800] Gudey, S. K. et al., Sci. Signal. 7
(2014): ra2 [0801] Guillemette, S. et al., Genes Dev. 29 (2015):
489-494 [0802] Guo, H. et al., Tumour. Biol 36 (2015): 5299-5304
[0803] Guo, S. et al., Drug Des Devel. Ther. 7 (2013):
1259-1271
[0804] Guo, Y. et al., Acta Pharmacol. Sin. 31 (2010): 1487-1494
[0805] Han, B. et al., Mol. Cancer 14 (2015): 64 [0806] Han, S. et
al., Leuk. Res 34 (2010): 1271-1274 [0807] Hansen-Petrik, M. B. et
al., Cancer Lett. 175 (2002): 157-163 [0808] Hao, J. et al., Oncol
Lett. 9 (2015): 2525-2533 [0809] Hao, Z. et al., Tumour. Biol 33
(2012): 723-730 [0810] Harder, M. N. et al., PLoS. One. 10 (2015):
e0120890 [0811] Harris, R. E., Inflammopharmacology. 17 (2009):
55-67 [0812] Hart, P. A. et al., Pancreatology. 15 (2015): 162-166
[0813] Hassanein, M. et al., Mol. Imaging Biol 18 (2016): 18-23
[0814] Hassanein, M. et al., Int. J Cancer 137 (2015): 1587-1597
[0815] Havelange, V. et al., Cancer 117 (2011): 4696-4706 [0816]
Havens, M. A. et al., PLoS. Genet. 10 (2014): e1004312 [0817]
Haymerle, G. et al., Eur. Arch. Otorhinolaryngol. (2015) [0818] He,
L. et al., Toxicology 312 (2013): 36-47 [0819] He, Y. et al.,
Transl. Res 165 (2015): 407-416 [0820] Hiramoto, T. et al.,
Oncogene 18 (1999): 3422-3426 [0821] Hoang-Vu, C. et al., Int. J
Oncol 21 (2002): 265-272 [0822] Hoffmann, N. E. et al., Cancer 112
(2008): 1471-1479 [0823] Hou, J. et al., Mol. Oncol 9 (2015):
1312-1323 [0824] Hsu, Y. C. et al., BMC. Med. 11 (2013): 106 [0825]
Hu, J. et al., Lung Cancer 88 (2015): 239-245 [0826] Hu, S. et al.,
J Cancer Res Clin Oncol 140 (2014a): 883-893 [0827] Hu, X. T. et
al., Cell Prolif. 47 (2014): 200-210 [0828] Hu, Z. Y. et al., J
Exp. Clin Cancer Res 33 (2014b): 61 [0829] Huang, G. L. et al.,
World J Gastroenterol. 16 (2010): 2046-2054 [0830] Huang, J. M. et
al., Oncogene 32 (2013a): 2220-2229 [0831] Huang, Q. C. et al.,
Cancer Lett. 354 (2014): 28-32 [0832] Huang, Y. et al., Cell
Biosci. 3 (2013b): 16 [0833] Hubalewska-Dydejczyk, A. et al., Q. J
Nucl. Med. Mol. Imaging 59 (2015): 152-160 [0834] Huber, A. R. et
al., BMC. Gastroenterol. 15 (2015): 80 [0835] Huegel, J. et al.,
Dev. Dyn. 242 (2013): 1021-1032 [0836] Hwang, M. L. et al., J
Immunol. 179 (2007): 5829-5838 [0837] Ikenberg, K. et al., J
Pathol. 234 (2014): 239-252 [0838] Ilm, K. et al., Mol. Cancer 14
(2015): 38 [0839] Imianitov, E. N., Arkh. Patol. 75 (2013): 63-72
[0840] Ip, W. et al., J Cutan. Med. Surg. 15 (2011): 103-110 [0841]
Ishimi, Y. et al., J Biochem. 157 (2015): 561-569 [0842] Israelsen,
W. J. et al., Semin. Cell Dev. Biol 43 (2015): 43-51 [0843] Jager,
D. et al., Cancer Res 60 (2000): 3584-3591 [0844] Jain, R. et al.,
Appl. Immunohistochem. Mol Morphol. 18 (2010): 9-15 [0845] Jeng, Y.
M. et al., Br. J Surg. 96 (2009): 66-73 [0846] Jeon, Y. J. et al.,
Cancer Cell 27 (2015): 354-369 [0847] Ji, M. et al., Oncol Rep. 33
(2015): 133-140 [0848] Jia, A. Y. et al., Br. J Cancer 110 (2014):
2945-2954 [0849] Jiang, L. et al., J Cancer Res Clin Oncol 136
(2010): 211-217 [0850] Jiang, L. et al., Tumour. Biol 35 (2014a):
12645-12654 [0851] Jiang, X. R. et al., Cancer Lett. 353 (2014b):
78-86 [0852] Jiang, Y. X. et al., J Int. Med. Res 40 (2012):
887-898 [0853] Jochmann, K. et al., Matrix Biol 34 (2014): 55-63
[0854] Jose-Eneriz, E. S. et al., Br. J Haematol. 142 (2008):
571-582 [0855] Joy, R. M. et al., Neurotoxicology 9 (1988): 637-643
[0856] Ju, J. H. et al., Clin Cancer Res 19 (2013): 4335-4346
[0857] Jung, G. et al., Proc Natl Acad Sci USA 84 (1987): 4611-4615
[0858] Jung, J. H. et al., Evid. Based. Complement Alternat. Med.
2013 (2013): 879746 [0859] Kaira, K. et al., Am. J Transl. Res 7
(2015): 356-363 [0860] Kancharla, A. et al., Nat Commun. 6 (2015):
8853 [0861] Kang, C. Y. et al., J Gastrointest. Surg. 18 (2014):
7-15 [0862] Kang, G. et al., Oncotarget. (2015) [0863] Kannen, V.
et al., Pharmacol. Ther. 139 (2013): 87-94 [0864] Karess, R. E. et
al., Int. Rev Cell Mol. Biol 306 (2013): 223-273 [0865] Kari, V. et
al., Cell Cycle 10 (2011): 3495-3504 [0866] Kasai, H. et al., J
Histochem. Cytochem. 51 (2003): 567-574 [0867] Katada, K. et al., J
Proteomics. 75 (2012): 1803-1815 [0868] Katkoori, V. R. et al.,
PLoS. One. 7 (2012): e30020 [0869] Katoh, M. et al., Int. J Mol.
Med. 20 (2007): 405-409 [0870] Ke, J. Y. et al., J Zhejiang. Univ
Sci. B 15 (2014a): 1032-1038 [0871] Ke, Z. et al., Oncotarget. 5
(2014b): 9410-9424 [0872] Keller, D. M. et al., Mol. Cell 7 (2001):
283-292 [0873] Khanobdee, K. et al., Mol. Vis. 10 (2004): 933-942
[0874] Khapare, N. et al., PLoS. One. 7 (2012): e38561 [0875] Khor,
G. H. et al., Asian Pac. J Cancer Prev. 15 (2014): 8957-8961 [0876]
Khoronenkova, S. V. et al., Mol. Cell 45 (2012): 801-813 [0877]
Kibbe, A. H., Handbook of Pharmaceutical Excipients rd (2000)
[0878] Kienle, D. et al., Haematologica 95 (2010): 102-109 [0879]
Kim, D. S. et al., J Proteome. Res 9 (2010a): 3710-3719 [0880] Kim,
F. J. et al., Biochem. Biophys. Res Commun. 426 (2012a): 177-182
[0881] Kim, H. et al., PLoS. One. 8 (2013a): e63468 [0882] Kim, H.
E. et al., PLoS. One. 7 (2012b): e43223 [0883] Kim, H. J. et al., J
Proteome. Res 8 (2009): 1368-1379 [0884] Kim, J. H. et al., Pathol.
Oncol Res 19 (2013b): 731-737 [0885] Kim, M. et al., Mol Cancer Res
6 (2008): 222-230 [0886] Kim, M. S. et al., Histopathology 58
(2011a): 660-668 [0887] Kim, Y. et al., Oncotarget. (2016) [0888]
Kim, Y. et al., J Biol Chem 288 (2013c): 36502-36518 [0889] Kim, Y.
et al., J Biol Chem 285 (2010b): 25957-25968 [0890] Kim, Y. H. et
al., Ann. Surg. Oncol 18 (2011b): 2338-2347 [0891] Kiniwa, Y. et
al., Cancer Res 61 (2001): 7900-7907 [0892] Kirkbride, K. C. et
al., Cell Adh. Migr. 5 (2011): 187-198 [0893] Kittang, A. O. et
al., Curr. Top. Microbiol. Immunol. 341 (2010): 149-172 [0894]
Kleist, B. et al., J Clin Pathol. (2015) [0895] Knab, L. M. et al.,
World J Gastroenterol. 20 (2014): 10729-10739 [0896] Koga, Y. et
al., Rinsho Byori 63 (2015): 361-368 [0897] Koike, K., Recent
Results Cancer Res 193 (2014): 97-111 [0898] Koldehoff, M. et al.,
Int. J Hematol. 87 (2008): 39-47 [0899] Kolla, V. et al., Cancer
Res 74 (2014): 652-658 [0900] Koo, J. S. et al., Am. J Clin Pathol.
143 (2015): 584-592 [0901] Korczak, B. et al., Int. J Cancer 53
(1993): 634-639 [0902] Koshikawa, K. et al., Oncogene 21 (2002):
2822-2828 [0903] Krieg, A. M., Nat Rev. Drug Discov. 5 (2006):
471-484 [0904] Kuang, S. Q. et al., Leukemia 22 (2008): 1529-1538
[0905] Kuang, Y. et al., Mol. Imaging Biol 16 (2014): 459-468
[0906] Kubo, N. et al., Biochem. Biophys. Res Commun. 430 (2013):
1034-1039 [0907] Kubota, H. et al., Cell Stress. Chaperones. 15
(2010): 1003-1011 [0908] Kuchma, M. H. et al., Protein J 31 (2012):
195-205 [0909] Kunimoto, K. et al., J Cell Physiol 220 (2009):
621-631 [0910] Kunzmann, A. T. et al., Cancer Epidemiol. Biomarkers
Prev. 22 (2013): 1490-1497 [0911] Kuramitsu, Y. et al., Expert. Rev
Proteomics. 2 (2005): 589-601 [0912] Kurer, M. A., Mol. Biol Rep.
34 (2007): 221-224 [0913] Kurisu, S. et al., Cancer Sci. 101
(2010): 2093-2104 [0914] Kutty, R. K. et al., J Biol Chem 276
(2001): 2831-2840 [0915] Kwiatkowski, D. J. et al., Hum. Mol.
Genet. 14 Spec No. 2 (2005): R251-R258 [0916] Kwok, H. F. et al.,
Am. J Cancer Res 5 (2015): 52-71 [0917] Laczmanska, I. et al., Acta
Biochim. Pol. 58 (2011): 467-470 [0918] Lambros, M. B. et al., Hum.
Pathol. 38 (2007): 1105-1122 [0919] Lane, J. et al., Int. J Mol.
Med. 12 (2003): 253-257 [0920] Lange, A. et al., Exp. Dermatol. 18
(2009): 527-535 [0921] Langnaese, K. et al., Cytogenet. Cell Genet.
94 (2001): 233-240 [0922] Lara, P. C. et al., Radiat. Oncol 6
(2011): 148 [0923] Lau, L. F., Cell Mol. Life Sci. 68 (2011):
3149-3163 [0924] Lawrence, M. S. et al., Nature 505 (2014): 495-501
[0925] Le, Tourneau C. et al., Br. J Cancer 99 (2008): 1197-1203
[0926] Leal, J. F. et al., Carcinogenesis 29 (2008): 2089-2095
[0927] Lebdai, S. et al., Urol. Oncol 33 (2015): 69-8 [0928] Lee,
A. M. et al., Pharmacogenet. Genomics 26 (2016): 133-137 [0929]
Lee, C. F. et al., World J Gastroenterol. 14 (2008a): 6072-6077
[0930] Lee, C. W. et al., World J Surg. Oncol 11 (2013): 136 [0931]
Lee, H. J. et al., Nat Cell Biol 9 (2007): 1303-1310 [0932] Lee, J.
I. et al., Physiol Genomics 33 (2008b): 218-229 [0933] Lee, K. Y.
et al., J Med. 35 (2004): 141-149 [0934] Lei, Y. et al., Oncogene
34 (2015): 485-495 [0935] Leng, S. et al., Cancer Res 75 (2015):
3108-3117 [0936] Li, H. et al., Biotechnol. Appl. Biochem. (2015a)
[0937] Li, J. et al., J Invest Surg. (2013) [0938] Li, J. et al.,
Tumour. Biol (2015b) [0939] Li, J. F. et al., Zhonghua Wei Chang
Wai Ke. Za Zhi. 15 (2012): 388-391 [0940] Li, J. P. et al., Drug
Des Devel. Ther. 9 (2015c): 1027-1062 [0941] Li, M. et al., Int. J
Oncol. 24 (2004): 305-312 [0942] Li, M. et al., Gene 542 (2014a):
134-140 [0943] Li, Q. et al., Mol. Biol Rep. 41 (2014b): 2409-2417
[0944] Li, S. R. et al., Biochem. Biophys. Res Commun. 271 (2000):
537-543 [0945] Li, Y. et al., J Cell Physiol 212 (2007): 675-681
[0946] Li, Z. et al., Biochim. Biophys. Acta 1846 (2014c): 285-296
[0947] Liang, J. et al., Med. Oncol 31 (2014a): 899 [0948] Liang,
J. X. et al., Oncol Rep. 32 (2014b): 2726-2734 [0949] Liao, W. et
al., Oncotarget. 6 (2015): 24132-24147 [0950] Liao, Y. J. et al.,
PLoS. One. 8 (2013): e77586 [0951] Liberati, S. et al., Cells 3
(2014a): 112-128 [0952] Liberati, S. et al., Curr. Protein Pept.
Sci. 15 (2014b): 732-737 [0953] Liddy, N. et al., Nat Med. 18
(2012): 980-987 [0954] Lidfeldt, J. et al., PLoS. One. 10 (2015):
e0134932 [0955] Liggins, A. P. et al., Cancer Immun. 10 (2010): 8
[0956] Limm, K. et al., Eur. J Cancer 49 (2013): 1305-1313 [0957]
Lin, C. Y. et al., Cancer Res 74 (2014a): 5229-5243 [0958] Lin, H.
S. et al., Arch. Otolaryngol. Head Neck Surg. 130 (2004): 311-316
[0959] Lin, J. et al., Leuk. Res 38 (2014b): 601-607 [0960] Lin, J.
et al., Oncotarget. 6 (2015): 23793-23806 [0961] Lin, J. I. et al.,
Sci. Signal. 6 (2013a): e4 [0962] Lin, L. et al., Oncol Lett. 6
(2013b): 740-744 [0963] Lin, S. T. et al., J Proteomics. 75 (2012):
5822-5847 [0964] Lindahl, A. K. et al., Thromb. Res 64 (1991):
155-168 [0965] Lindberg, J. et al., Eur. Urol. 63 (2013): 702-708
[0966] Linderoth, J. et al., Br. J Haematol. 141 (2008): 423-432
[0967] Liu, G. et al., Cancer Genet. Cytogenet. 197 (2010a): 54-59
[0968] Liu, H. et al., Oncol Rep. 34 (2015a): 2267-2272 [0969] Liu,
M. et al., Mol. Endocrinol. 28 (2014): 1740-1751 [0970] Liu, M. et
al., Reprod. Sci. 20 (2013a): 605-615 [0971] Liu, M. et al., Mol.
Biol Rep. 37 (2010b): 3601-3608 [0972] Liu, Q. et al., Exp. Ther.
Med. 6 (2013): 1277-1282 [0973] Liu, R. et al., Clin Cancer Res 21
(2015b): 854-863 [0974] Liu, T. Q. et al., Asian Pac. J Cancer
Prev. 16 (2015c): 3061-3065 [0975] Liu, T. W. et al., Proc. Natl.
Acad. Sci. U.S.A 106 (2009): 14581-14586 [0976] Liu, X. et al.,
Oncogene 32 (2013b): 1266-1273 [0977] Liu, Y. et al., Sci. Rep. 5
(2015d): 16954 [0978] Liu, Z. et al., Mol. Cancer Res 3 (2005):
21-31 [0979] Ljunggren, H. G. et al., J Exp. Med. 162 (1985):
1745-1759 [0980] Lo, T. F. et al., PLoS. One. 8 (2013): e75628
[0981] Longenecker, B. M. et al., Ann N.Y. Acad. Sci. 690 (1993):
276-291 [0982] Lopez-Nieva, P. et al., Carcinogenesis 33 (2012):
452-458 [0983] Lorenzen, J. A. et al., Gene Expr. Patterns. 6
(2005): 45-56 [0984] Lorenzi, P. L. et al., Mol. Cancer Ther. 7
(2008): 3123-3128 [0985] Lorenzi, P. L. et al., Mol. Cancer Ther. 5
(2006): 2613-2623 [0986] Lorenzi, P. L. et al., Drug News Perspect.
22 (2009): 61-64 [0987] Lossie, A. C. et al., BMC. Genet. 13
(2012): 106 [0988] Lu, C. et al., Mol. Cell Biochem. 312 (2008):
71-80 [0989] Lu, D. et al., Med. Oncol 32 (2015): 140 [0990] Lukas,
T. J. et al., Proc. Natl. Acad. Sci. U.S.A 78 (1981): 2791-2795
[0991] Lund, R. R. et al., Mol. Cell Proteomics. 14 (2015):
2988-2999 [0992] Lundblad, R. L., Chemical Reagents for Protein
Modification 3rd (2004) [0993] Luo, W. et al., Trends Endocrinol.
Metab 23 (2012): 560-566 [0994] Ma, J. et al., Pathol. Oncol Res 19
(2013): 821-832 [0995] Mac, S. M. et al., Mol. Carcinog. 27 (2000):
84-96 [0996] Macher-Goeppinger, S. et al., Mod. Pathol. 25 (2012):
308-315 [0997] Maekawa, R. et al., J Reprod. Dev. 57 (2011):
604-612 [0998] Mah, T. L. et al., BMC. Genomics 15 Suppl 9 (2014):
S20 [0999] Mak, G. W. et al., Cancer Res 71 (2011): 2949-2958
[1000] Mak, G. W. et al., PLoS. One. 7 (2012): e42210 [1001]
Malta-Vacas, J. et al., Clin Chem Lab Med. 47 (2009): 427-431
[1002] Malumbres, M. et al., Curr. Opin. Genet. Dev. 17 (2007):
60-65 [1003] Marchetti, A. et al., Int. J Oncol 18 (2001): 175-179
[1004] Marimuthu, A. et al., Proteomics. Clin Appl. 7 (2013):
355-366 [1005] Marquardt, J. U. et al., Int. J Cancer 128 (2011):
2353-2363 [1006] Mascarenhas, Cdo C. et al., Leuk. Lymphoma 55
(2014): 1861-1869 [1007] Mason, J. M. et al., Nucleic Acids Res. 43
(2015): 3180-3196 [1008] Matnuri, M. et al., Int. J Clin Exp.
Pathol. 8 (2015): 13339-13345 [1009] Matsuno, A. et al., Br. J
Neurosurg. 18 (2004): 343-346 [1010] McCarthy, P. L. et al., Br. J
Cancer 99 (2008): 639-646 [1011] McClung, J. K. et al., Exp.
Gerontol. 30 (1995): 99-124 [1012] McDonald, S. L. et al., Cancer
Biol Ther. 3 (2004): 110-120 [1013] McGarvey, T. W. et al.,
Prostate 54 (2003): 144-155 [1014] McGarvey, T. W. et al., Oncogene
20 (2001): 1042-1051 [1015] McGarvey, T. W. et al., J Cell Biochem.
95 (2005): 419-428 [1016] Medcalf, R. L. et al., FEBS J 272 (2005):
4858-4867 [1017] Meierjohann, S., Eur. J Cell Biol 93 (2014): 36-41
[1018] Mellor, P. et al., Mol. Cell Biol 33 (2013): 4985-4995
[1019] Mesri, E. A. et al., Immunol. Res 57 (2013): 159-165 [1020]
Meziere, C. et al., J Immunol 159 (1997): 3230-3237 [1021]
Midorikawa, Y. et al., Jpn. J Cancer Res 93 (2002): 636-643 [1022]
Mimura, K. et al., J Immunol. 191 (2013): 6261-6272 [1023]
Mirmalek-Sani, S. H. et al., J Cell Mol. Med. 13 (2009): 3541-3555
[1024] Mishra, S. et al., FEBS J 277 (2010): 3937-3946 [1025]
Mishra, S. et al., Trends Mol. Med. 11 (2005): 192-197 [1026]
Misra, S. et al., Curr. Drug Targets. 15 (2014): 347-359 [1027]
Mitchell, S. M. et al., BMC. Cancer 14 (2014): 54 [1028] Miyashita,
K. et al., Anticancer Agents Med. Chem 9 (2009): 1114-1122 [1029]
Mizukoshi, E. et al., Hepatology 53 (2011): 1206-1216 [1030]
Morelli, M. B. et al., Curr. Mol. Pharmacol. 6 (2013): 137-148
[1031] Morgan, R. A. et al., Science 314 (2006): 126-129 [1032]
Mori, M. et al., Transplantation 64 (1997): 1017-1027 [1033] Mori,
S. et al., PLoS. One. 7 (2012): e39723 [1034] Morin, A. et al.,
FASEB J 26 (2012): 460-467 [1035] Mortara, L. et al., Clin Cancer
Res. 12 (2006): 3435-3443 [1036] Mossink, M. H. et al., Oncogene 22
(2003): 7458-7467 [1037] Moussay, E. et al., Autophagy. 7 (2011):
760-770 [1038] Mueller, L. N. et al., J Proteome. Res 7 (2008):
51-61 [1039] Mueller, L. N. et al., Proteomics. 7 (2007): 3470-3480
[1040] Muir, K. et al., Cancer Res 73 (2013): 4722-4731 [1041]
Mukhopadhyay, T. et al., Anticancer Res 16 (1996): 105-112 [1042]
Mullapudi, N. et al., PLoS. One. 10 (2015): e0143826 [1043]
Mumberg, D. et al., Proc. Natl. Acad. Sci. U.S.A 96 (1999):
8633-8638 [1044] Nabissi, M. et al., Carcinogenesis 31 (2010):
794-803 [1045] Nagel, S. et al., Genes Chromosomes. Cancer 53
(2014): 917-933 [1046] Nakamura, T., Int. J Hematol. 82 (2005):
21-27
[1047] Nakao, K. et al., J Gastroenterol. 49 (2014): 589-593 [1048]
Nalesnik, M. A. et al., Am. J Pathol. 180 (2012): 1495-1508 [1049]
Navarro, A. et al., Semin. Hematol. 48 (2011): 155-165 [1050]
Nelson, L. D. et al., Mol. Cancer 11 (2012): 38 [1051] Nelson, M.
A. et al., Cancer Genet. Cytogenet. 108 (1999): 91-99 [1052] Neri,
P. et al., Curr. Cancer Drug Targets. 12 (2012): 776-796 [1053]
Newman, S. et al., PLoS. One. 8 (2013): e64991 [1054] Ng, S. K. et
al., Clin Experiment. Ophthalmol. 43 (2015): 367-376 [1055] Nguyen,
H. N. et al., Biochem. Biophys. Res Commun. 357 (2007): 174-180
[1056] Nikkuni, O. et al., Pathol. Oncol Res 21 (2015): 1175-1181
[1057] Nio, K. et al., J Hepatol. 63 (2015): 1164-1172 [1058]
Nitta, M. et al., Nucleic Acids Res 28 (2000): 4212-4218 [1059]
Noorlag, R. et al., Virchows Arch. 466 (2015): 363-373 [1060]
Nurnberg, A. et al., Nat Rev Cancer 11 (2011): 177-187 [1061]
O'Shea, C. et al., Int. J Cancer 105 (2003): 754-761 [1062]
Oeffner, F. et al., Am J Hum. Genet. 84 (2009): 459-467 [1063] Ohl,
F. et al., J Mol. Med. (Berl) 83 (2005): 1014-1024 [1064] Okamoto,
Y. et al., Cancer Res 63 (2003): 4167-4173 [1065] Olakowski, M. et
al., Folia Histochem. Cytobiol. 47 (2009): 249-255 [1066] Ono, W.
et al., Biochem. Biophys. Res Commun. 434 (2013): 659-663 [1067]
Osada, S. et al., Oncol Rep. 30 (2013): 1669-1674 [1068] Oskarsson,
T., Breast 22 Suppl 2 (2013): S66-S72 [1069] Otani, S. et al., Br.
J Ophthalmol. 90 (2006): 773-777 [1070] Ozawa, D. et al., Ann.
Surg. Oncol (2014) [1071] Pandi, N. S. et al., Gene 545 (2014):
23-29 [1072] Panosyan, E. H. et al., Mol. Cancer Res 12 (2014):
694-702 [1073] Papageorgio, C. et al., Int. J Oncol. 31 (2007):
1205-1211 [1074] Parihar, J. S. et al., Rev Urol. 16 (2014):
118-121 [1075] Park, H. J. et al., J Proteome. Res 7 (2008):
1138-1150 [1076] Park, J. H. et al., Cancer Res 65 (2005):
2804-2814 [1077] Parker, L. P. et al., Cancer Genomics Proteomics.
6 (2009): 189-194 [1078] Paryan, M. et al., Mol. Biol Rep. 40
(2013): 5531-5540 [1079] Pavithra, L. et al., Int. J Biochem. Cell
Biol 41 (2009): 862-871 [1080] Pavlides, S. et al., Cell Cycle 9
(2010): 3485-3505 [1081] Pawar, S. A. et al., Proc. Natl. Acad.
Sci. U.S.A 107 (2010): 9210-9215 [1082] Peltonen, H. M. et al.,
PLoS. One. 8 (2013): e79249 [1083] Pender-Cudlip, M. C. et al.,
Cancer Sci. 104 (2013): 760-764 [1084] Perez-Escuredo, J. et al.,
Cell Cycle 15 (2016): 72-83 [1085] Perez-Fernandez, J. et al.,
Nucleic Acids Res 39 (2011): 8105-8121 [1086] Perez-Tomas, R.,
Curr. Med. Chem 13 (2006): 1859-1876 [1087] Peters, D. G. et al.,
Cancer Epidemiol. Biomarkers Prev. 14 (2005): 1717-1723 [1088]
Phuong, N. T. et al., Oncotarget. (2015) [1089] Pierce, J. M. et
al., Proteomics. 9 (2009): 1738-1741 [1090] Pinheiro, J. et al.,
nlme: Linear and Nonlinear Mixed Effects Models (2015) [1091]
Plebanski, M. et al., Eur. J Immunol 25 (1995): 1783-1787 [1092]
Pohler, E. et al., Nat Genet. 44 (2012): 1272-1276 [1093] Pollok,
S. et al., Biochem. Soc. Trans. 31 (2003): 266-269 [1094]
Polotskaia, A. et al., Proc. Natl. Acad. Sci. U.S.A 112 (2015):
E1220-E1229 [1095] Ponnurangam, S. et al., Oncotarget. (2015)
[1096] Porta, C. et al., Virology 202 (1994): 949-955 [1097]
Pradhan, M. P. et al., BMC. Syst. Biol 7 (2013): 141 [1098]
Pregizer, S. et al., J Cell Biochem. 102 (2007): 1458-1471 [1099]
Prieto-Granada, C. et al., Genes Chromosomes. Cancer 54 (2015):
28-38 [1100] Przybylo, M. et al., Biochimie 87 (2005): 133-142
[1101] Pucci, S. et al., Oncotarget. (2016) [1102] Pylypenko, O. et
al., Mol Cell 11 (2003): 483-494 [1103] Qin, Q. et al., PLoS. One.
5 (2010): e9999 [1104] Rajalingam, K. et al., Cell Cycle 4 (2005):
1503-1505 [1105] Rajkumar, T. et al., Indian J Biochem. Biophys. 42
(2005): 271-278 [1106] Ramachandran, C., Curr. Pharm. Biotechnol. 8
(2007): 99-104 [1107] Rammensee, H. G. et al., Immunogenetics 50
(1999): 213-219 [1108] Rana, S. et al., Expert. Rev Anticancer
Ther. 8 (2008): 1461-1470 [1109] Rao, C. V. et al., Carcinogenesis
30 (2009): 1469-1474 [1110] Rappa, G. et al., Mol. Cancer Res 12
(2014): 1840-1850 [1111] Raso, E. et al., Magy. Onkol. 57 (2013):
79-83 [1112] Rauch, T. A. et al., Tumour. Biol 33 (2012): 287-296
[1113] RefSeq, The NCBI handbook [Internet], Chapter 18, (2002),
[1114] http://www. ncbi. nlm. nih. gov/books/NBK21091/Ren, [1115]
Y. Q. et al., Med. Sci. Monit. 21 (2015): 1297-1303 [1116] Resende,
C. et al., Helicobacter. 15 Suppl 1 (2010): 34-39 [1117] Resende,
C. et al., Helicobacter. 16 Suppl 1 (2011): 38-44 [1118] Reubi, J.
C. et al., J Nucl. Med. 49 (2008): 1735-1738 [1119] Rini, B. I. et
al., Cancer 107 (2006): 67-74 [1120] Roberts, J. D. et al., Cancer
Detect. Prev. 22 (1998): 455-462 [1121] Rock, K. L. et al., Science
249 (1990): 918-921 [1122] Roignot, J. et al., Cell Adh. Migr. 3
(2009): 167-170 [1123] Romes, E. M. et al., J Biol Chem 291 (2016):
882-893 [1124] Ronkainen, H. et al., Oncol Rep. 25 (2011): 129-133
[1125] Rosado, I. V. et al., RNA. 10 (2004): 1073-1083 [1126] Rose,
M. et al., Epigenetics. 9 (2014): 1626-1640 [1127] Rothe, M. et
al., Am. J Pathol. 157 (2000): 1597-1604 [1128] Rouzer, C. A. et
al., J Lipid Res 50 Suppl (2009): S29-S34 [1129] Roy, D. et al.,
Oncol Rep. 23 (2010): 1383-1391 [1130] Rozenberg, P. et al., Int. J
Cancer 133 (2013): 514-518 [1131] Rucki, A. A. et al., World J
Gastroenterol. 20 (2014): 2237-2246 [1132] Rummel, M. J. et al.,
Leuk. Lymphoma 45 (2004): 49-54 [1133] Ryu, B. et al., PLoS. One. 2
(2007): e594 [1134] Ryu, S. J. et al., Expert. Opin. Ther. Targets.
13 (2009): 479-484 [1135] S3-Leitlinie Exokrines Pankreaskarzinom,
032-010OL, (2013) [1136] Saiki, R. K. et al., Science 239 (1988):
487-491 [1137] Saksena, S. et al., Am. J Physiol Gastrointest.
Liver Physiol 298 (2010): G159-G166 [1138] Salerno, C. et al., Ric.
Clin Lab 20 (1990): 85-93 [1139] Salman, B. et al., Oncoimmunology.
2 (2013): e26662 [1140] Samuel, A. M. et al., Cell Oncol (Dordr.)
37 (2014): 95-105 [1141] Sanchez, G. et al., Cell Cycle 7 (2008):
2299-2305 [1142] Sander, B., Semin. Diagn. Pathol. 28 (2011):
245-255 [1143] Sandset, P. M. et al., Haemostasis 21 (1991):
219-239 [1144] Santos, F. M. et al., Phytomedicine. 18 (2011):
1096-1101 [1145] Sasatomi, T. et al., Cancer 94 (2002): 1636-1641
[1146] Savitskaya, T. V. et al., Pediatr. Hematol. Oncol 29 (2012):
28-37 [1147] Scagliotti, G. V. et al., Ann. Oncol 10 Suppl 5
(1999): S83-S86 [1148] Scanlon, C. S. et al., J Dent. Res 92
(2013): 114-121 [1149] Scheffer, G. L. et al., Curr. Opin. Oncol 12
(2000): 550-556 [1150] Schiffner, S. et al., Carcinogenesis 32
(2011): 1176-1182 [1151] Schmid, R. et al., PLoS. One. 8 (2013):
e82166 [1152] Schramm, A. et al., Nat Genet. 47 (2015): 872-877
[1153] Schrier, S. A. et al., Curr. Opin. Ophthalmol. 22 (2011):
325-331 [1154] Schroder, W. A. et al., Cancer Med. 3 (2014):
500-513 [1155] Schwirzke, M. et al., Anticancer Res 18 (1998):
1409-1421 [1156] Seeger, F. H. et al., Immunogenetics 49 (1999):
571-576 [1157] Seftor, R. E. et al., Melanoma Res 1 (1991): 43-54
[1158] Sehrawat, A. et al., Breast Cancer Res Treat. 146 (2014):
543-555 [1159] Sekine, I. et al., Jpn. J Clin Oncol 37 (2007):
329-336 [1160] Serra, K. P. et al., Acta Histochem. (2016) [1161]
Sesen, J. et al., Int. J Mol. Sci. 15 (2014): 2172-2190 [1162]
Sethi, M. K. et al., J Proteomics. 126 (2015): 54-67 [1163]
Setoodeh, R. et al., Int. J Clin Exp. Pathol. 6 (2013): 155-167
[1164] Shahmoradgoli, M. et al., Int. J Cancer 132 (2013):
2714-2719 [1165] Shaker, M. et al., Pathobiology 78 (2011): 149-161
[1166] Shakhova, O., Curr. Opin. Oncol 26 (2014): 215-221 [1167]
Shao, N. et al., Mol. Biol Rep. 39 (2012): 10997-11004 [1168]
Sharma, A. et al., Mol. Cancer Res 12 (2014): 1205-1215 [1169]
Sharpe, L. J. et al., Traffic. 12 (2011): 19-27 [1170] Sher, Y. P.
et al., PLoS. One. 9 (2014): e94065 [1171] Sherman, F. et al.,
Laboratory Course Manual for Methods in Yeast Genetics (1986)
[1172] Shi, B. et al., BMC. Cancer 15 (2015): 380 [1173] Shimada,
H. et al., Br. J Haematol. 110 (2000): 210-213 [1174] Shimizu, S.
et al., Oncol Rep. 18 (2007): 1489-1497 [1175] Shimozono, N. et
al., Cancer Res 75 (2015): 4458-4465 [1176] Shintani, Y. et al.,
Cancer Res 64 (2004): 4190-4196 [1177] Shishkin, S. S. et al.,
Biochemistry (Mosc.) 78 (2013): 1415-1430 [1178] Shodeinde, A. et
al., J Mol Biochem. 2 (2013): 18-26 [1179] Shostak, K. et al., Nat
Commun. 5 (2014): 5232 [1180] Sierko, E. et al., Semin. Thromb.
Hemost. 33 (2007): 653-659 [1181] Silveira, S. M. et al., Head Neck
34 (2012): 485-492 [1182] Simpson, N. E. et al., Breast Cancer Res
Treat. 133 (2012): 959-968 [1183] Singh, S. et al., Tumour. Biol.
(2014) [1184] Singh, V. et al., OMICS. 19 (2015): 688-699 [1185]
Singh-Jasuja, H. et al., Cancer Immunol. Immunother. 53 (2004):
187-195 [1186] Skawran, B. et al., Mod. Pathol. 21 (2008): 505-516
[1187] Skene-Arnold, T. D. et al., Biochem. J 449 (2013): 649-659
[1188] Skondra, M. et al., Anticancer Res 34 (2014): 6691-6699
[1189] Skrzycki, M. et al., J Recept. Signal. Transduct. Res 33
(2013): 313-318 [1190] Slape, C. et al., Leuk. Lymphoma 45 (2004):
1341-1350 [1191] Small, E. J. et al., J Clin Oncol. 24 (2006):
3089-3094 [1192] Smith, K. A. et al., Proc. Natl. Acad. Sci. U.S.A
94 (1997): 1816-1821 [1193] Smith, K. A. et al., Cell 63 (1990):
1219-1227 [1194] Sollini, M. et al., Q. J Nucl. Med. Mol. Imaging
59 (2015): 168-183 [1195] Solomon, D. A. et al., Cancer Res 68
(2008): 8657-8660 [1196] Song, N. et al., J Zhejiang. Univ Sci. B
14 (2013): 451-459 [1197] Song, Z. et al., PLoS. One. 10 (2015):
e0128943 [1198] Sperlazza, J. et al., Blood 126 (2015): 1462-1472
[1199] Steen, H. C. et al., J Interferon Cytokine Res 32 (2012):
103-110 [1200] Stefansson, 0. A. et al., Breast Cancer Res 16
(2014): 307 [1201] Stein, U., Expert. Opin. Ther. Targets. 17
(2013): 1039-1052 [1202] Stewart, J. et al., Mod. Pathol. 28
(2015): 428-436 [1203] Strekalova, E. et al., Clin. Cancer Res.
(2015) [1204] Stremenova, J. et al., Int. J Oncol 36 (2010):
351-358 [1205] Stubbs, A. P. et al., Am. J Pathol. 154 (1999):
1335-1343 [1206] Sturm, M. et al., BMC. Bioinformatics. 9 (2008):
163 [1207] Sugimoto, T. et al., Genes Chromosomes. Cancer 48
(2009): 132-142 [1208] Sun, B. C. et al., Zhonghua Yi. Xue. Za Zhi.
86 (2006): 1808-1812 [1209] Sun, F. K. et al., J Gastroenterol.
Hepatol. 31 (2016a): 484-492 [1210] Sun, S. et al., Gene 584
(2016b): 90-96 [1211] Sun, S. Y., Cancer Lett. 340 (2013): 1-8
[1212] Sun, X. et al., Neoplasia. 14 (2012): 1122-1131 [1213]
Sundar, R. et al., Cell Cycle 14 (2015): 554-565 [1214] Szaflarski,
W. et al., Postepy Biochem. 57 (2011): 266-273 [1215] Szarvas, T.
et al., Int J Cancer 135 (2014): 1596-1604 [1216] Szczyrba, J. et
al., Int. J Cancer 132 (2013): 775-784 [1217] Taintor, A. R. et
al., J Am. Acad. Dermatol. 56 (2007): S73-S76 [1218] Takahashi, H.
et al., Urology 79 (2012): 240-248 [1219] Takeda, A. et al., Semin.
Cancer Biol 27 (2014): 3-10 [1220] Takei, H. et al., Anticancer Res
15 (1995): 1101-1105 [1221] Tamada, M. et al., Clin Cancer Res 18
(2012): 5554-5561 [1222] Tameda, M. et al., Int. J Oncol 45 (2014):
541-548 [1223] Tamura, K. et al., Cancer Res 67 (2007): 5117-5125
[1224] Tan, X. et al., Tumour. Biol 35 (2014): 12189-12200 [1225]
Tang, B. et al., Int. J Oncol 47 (2015): 2208-2216 [1226] Tang, W.
et al., Genet. Epidemiol. 37 (2013): 512-521 [1227] Tano, K. et
al., FEBS Lett. 584 (2010): 4575-4580 [1228] Tao, H. C. et al.,
Asian Pac. J Cancer Prev. 14 (2013): 5645-5650 [1229] Tavner, F. J.
et al., Mol. Cell Biol 18 (1998): 989-1002 [1230] Taylor, K. H. et
al., Cancer Res 67 (2007): 2617-2625 [1231] Tech, K. et al., Cancer
Lett. 356 (2015): 268-272 [1232] Teufel, R. et al., Cell Mol Life
Sci. 62 (2005): 1755-1762 [1233] Theiss, A. L. et al., Biochim.
Biophys. Acta 1813 (2011): 1137-1143 [1234] Thill, M. et al., Eur.
J Gynaecol. Oncol 35 (2014): 341-358 [1235] Thorsen, K. et al., Mol
Cell Proteomics. 7 (2008): 1214-1224 [1236] Tian, T. V. et al.,
Oncogene 33 (2014): 2204-2214 [1237] Tietz, O. et al., Curr. Med.
Chem 20 (2013): 4350-4369 [1238] Timme, S. et al., Oncogene 33
(2014): 3256-3266 [1239] Tiwari, R. V. et al., Exp. Biol Med.
(Maywood.) 239 (2014): 33-44 [1240] To, M. D. et al., Oncogene 25
(2006): 3557-3564 [1241] Tomasi, M. L. et al., Oncotarget. 6
(2015): 37706-37723 [1242] Tomasi, M. L. et al., Exp. Cell Res 319
(2013): 1902-1911 [1243] Tran, E. et al., Science 344 (2014):
641-645 [1244] Trotta, C. R. et al., Nature 441 (2006): 375-377
[1245] Tsofack, S. P. et al., Mol. Cancer 10 (2011): 145 [1246]
Tsuchiya, M. et al., Biochem. Biophys. Res Commun. 407 (2011):
378-382 [1247] Tucci, M. et al., Curr. Top. Med. Chem 9 (2009):
218-224 [1248] Vaillant, A. R. et al., Biochem. Cell Biol 73
(1995): 695-702 [1249] Valladares-Ayerbes, M. et al., Cancer
Epidemiol. Biomarkers Prev. 19 (2010): 1432-1440 [1250] van den
Heuvel-Eibrink M M et al., Int. J Clin Pharmacol. Ther. 38 (2000):
94-110 [1251] Van Ginkel, P. R. et al., Biochim. Biophys. Acta 1448
(1998): 290-297 [1252] van't Veer, M. B. et al., Haematologica 91
(2006): 56-63 [1253] Vasseur, S. et al., Mol. Cancer 4 (2005): 4
[1254] Vellanki, R. N. et al., PLoS. One. 8 (2013): e54060 [1255]
Verbeke, H. et al., Biochim. Biophys. Acta 1825 (2012): 117-129
[1256] Vilner, B. J. et al., Cancer Res 55 (1995): 408-413 [1257]
Vinayak, S. et al., Oncology (Williston. Park) 27 (2013): 38-44,
46, 48 [1258] Vincent, M. et al., PLoS. One. 5 (2010): e12941
[1259] Vincent-Chong, V. K. et al., PLoS. One. 8 (2013): e54705
[1260] Vogt, P. K. et al., Curr. Top. Microbiol. Immunol. 347
(2010): 79-104 [1261] Voloshanenko, O. et al., Nat Commun. 4
(2013): 2610 [1262] von Eyben, F. E., Cancer Genet. Cytogenet. 151
(2004): 93-138 [1263] Von Hoff, D. D. et al., N. Engl. J Med. 369
(2013): 1691-1703 [1264] Vrabel, D. et al., Klin. Onkol. 27 (2014):
340-346 [1265] Wagner, K. W. et al., Oncogene 23 (2004): 6621-6629
[1266] Walker, E. J. et al., World J Gastroenterol. 20 (2014):
2224-2236 [1267] Walter, S. et al., J Immunol 171 (2003): 4974-4978
[1268] Walter, S. et al., Nat Med. 18 (2012): 1254-1261 [1269] Wan,
Y. Y. et al., Zhonghua Zhong. Liu Za Zhi. 38 (2016): 28-34 [1270]
Wang, B. S. et al., Cell Stress. Chaperones. 18 (2013a): 359-366
[1271] Wang, D. et al., Biochem. Biophys. Res Commun. 458 (2015a):
313-320 [1272] Wang, G. et al., Tumour. Biol 36 (2015b): 1055-1065
[1273] Wang, H. et al., Tumour. Biol 34 (2013b): 1635-1639 [1274]
Wang, J. et al., J Clin Invest 112 (2003): 535-543 [1275] Wang, J.
L. et al., Gene 529 (2013c): 7-15 [1276] Wang, L. et al., World J
Gastroenterol. 17 (2011): 1434-1441 [1277] Wang, L. et al., Xi.
Bao. Yu Fen. Zi. Mian. Yi. Xue. Za Zhi. 31 (2015c): 1251-1254
[1278] Wang, L. et al., Cancer Cell 25 (2014a): 21-36 [1279] Wang,
L. J. et al., Oncotarget. 6 (2015d): 5932-5946 [1280] Wang, P. et
al., Med. Oncol 32 (2015e): 264 [1281] Wang, Q. et al., J Pathol.
236 (2015f): 278-289 [1282] Wang, S. et al., J Cell Sci. 120
(2007): 567-577 [1283] Wang, S. Y. et al., Eur. Rev Med. Pharmacol.
Sci. 19 (2015g): 1191-1197
[1284] Wang, T. et al., Neurobiol. Aging 36 (2015h): 527-535 [1285]
Wang, X. et al., BMC. Cancer 14 (2014b): 196 [1286] Wang, X. et
al., J Biot Chem 290 (2015i): 3925-3935 [1287] Wang, X. et al.,
PLoS. One. 8 (2013d): e72015 [1288] Wang, Y. et al., Cancer Lett.
360 (2015j): 171-176 [1289] Wang, Y. F. et al., Phytother. Res 29
(2015k): 674-679 [1290] Wang, Z. et al., J Cancer Res Clin Oncol
141 (2015l): 1353-1361 [1291] Wang, Z. et al., Gastroenterol. Res
Pract. 2014 (2014c): 132320 [1292] Wang, Z. et al., Oncotarget. 4
(2013e): 2476-2486 [1293] Wang, Z. et al., Science 304 (2004):
1164-1166 [1294] Wang, Z. et al., J Cell Physiol 224 (2010):
559-565 [1295] Wang, Z. et al., Med. Oncol 30 (2013f): 577 [1296]
Wang, Z. et al., Hum. Pathol. 46 (2015m): 1006-1014 [1297] Warner,
S. L. et al., Future. Med. Chem 6 (2014): 1167-1178 [1298] Wasa, M.
et al., Am. J Physiol Cell Physiol 282 (2002): C1246-C1253 [1299]
Watanabe, M. et al., Proteomics. Clin Appl. 2 (2008): 925-935
[1300] Waters, M. G. et al., Nature 349 (1991): 248-251 [1301]
Watson, P. J. et al., Traffic. 5 (2004): 79-88 [1302] Wehner, K. A.
et al., Mol. Cell 9 (2002): 329-339 [1303] Westin, G. et al., World
J Surg. 33 (2009): 2224-2233 [1304] Weston, R. et al., Genes Dev.
26 (2012): 1558-1572 [1305] Wheler, J. J. et al., BMC. Cancer 15
(2015): 442 [1306] Wiese, M. et al., Expert. Opin. Ther. Pat 24
(2014): 723-725 [1307] Willcox, B. E. et al., Protein Sci. 8
(1999): 2418-2423 [1308] Wilson, C. H. et al., Int. J Oncol 41
(2012): 919-932 [1309] Wojtukiewicz, M. Z. et al., Cancer
Metastasis Rev 34 (2015): 775-796 [1310] Woodburn, K. W. et al.,
Drug Metab Dispos. 41 (2013): 774-784 [1311] World Cancer Report,
(2014) [1312] Wu, H. C. et al., Nat Commun. 5 (2014): 3214 [1313]
Wu, S. et al., Acta Biochim. Biophys. Sin. (Shanghai) 45 (2013):
27-35 [1314] Wyatt, L. et al., Cell Cycle 7 (2008): 2290-2295
[1315] Wysocki, P. J., Expert. Rev Mol. Diagn. 9 (2009): 231-241
[1316] Xiao, H. et al., Biochem. Biophys. Res Commun. 460 (2015):
703-708 [1317] Xie, H. et al., PLoS. One. 7 (2012): e46990 [1318]
Xie, X. et al., Oncol Lett. 7 (2014): 1537-1543 [1319] Xing, X. et
al., Gene 344 (2005): 161-169 [1320] Xu, F. P. et al., Cancer Lett.
245 (2007): 69-74 [1321] Xu, L. et al., Nan. Fang Yi. Ke. Da. Xue.
Xue. Bao. 26 (2006): 231-233 [1322] Xu, Z. et al., Biomed. Res Int.
2015 (2015): 459170 [1323] Xue, J. et al., J Biol Chem 290 (2015):
18662-18670 [1324] Yagel, S. et al., Clin Exp. Metastasis 8 (1990):
305-317 [1325] Yan, L. et al., Nan. Fang Yi. Ke. Da. Xue. Xue. Bao.
35 (2015a): 767-71, 776 [1326] Yan, L. et al., Am. J Cancer Res 5
(2015b): 1447-1459 [1327] Yang, H. et al., Chem Biol Drug Des 84
(2014a): 578-584 [1328] Yang, J. et al., Am. J Pathol. 185 (2015):
2194-2205 [1329] Yang, J. et al., Cancer 113 (2008): 1532-1543
[1330] Yang, J. et al., Neurosurg. Clin N. Am. 23 (2012a): 451-458
[1331] Yang, L. et al., Cancer Lett. 336 (2013): 213-221 [1332]
Yang, S. et al., Gene 576 (2016): 421-428 [1333] Yang, Y. et al.,
PLoS. One. 7 (2012b): e36813 [1334] Yang, Y. et al., PLoS. One. 9
(2014b): e97578 [1335] Yao, T. W. et al., Mol. Cancer Res 9 (2011):
948-959 [1336] Yao, Y. et al., Cell Physiol Biochem. 35 (2015):
983-996 [1337] Yasui, W. et al., Gastric. Cancer 8 (2005): 86-94
[1338] Yasui, W. et al., Cancer Sci. 95 (2004): 385-392 [1339] Yi,
C. H. et al., Cancer Lett. 284 (2009): 149-156 [1340] Yong, Z. W.
et al., Sci. Rep. 4 (2014): 6073 [1341] Yoo, K. Y. et al., Breast
Cancer 10 (2003): 289-293 [1342] Yoon, S. Y. et al., Int. J Oncol
29 (2006): 315-327 [1343] Yoshihara, N. et al., J Dermatol. 41
(2014): 311-315 [1344] Younes, M. et al., Anticancer Res 20 (2000):
3775-3779 [1345] Yu, B. et al., Exp. Cell Res 315 (2009): 3086-3098
[1346] Yu, D. M. et al., FEBS J 277 (2010): 1126-1144 [1347] Yu, J.
et al., Tumour. Biol 36 (2015): 3221-3229 [1348] Yu, R. et al.,
Brain Pathol. 11 (2001): 328-341 [1349] Yu, Y. P. et al., Am. J
Pathol. 184 (2014): 2840-2849 [1350] Yuan, R. H. et al., Ann Surg.
Oncol 16 (2009): 1711-1719 [1351] Yuan, W. et al., Asian J Androl 7
(2005): 277-288 [1352] Yue, C. et al., Int. J Cancer 136 (2015):
117-126 [1353] Yun, H. M. et al., Oncogene 33 (2014): 5193-5200
[1354] Zajac-Kaye, M., Lung Cancer 34 Suppl 2 (2001): S43-S46
[1355] Zanfardino, M. et al., Int. J Oncol 43 (2013): 1763-1770
[1356] Zaremba, S. et al., Cancer Res. 57 (1997): 4570-4577 [1357]
Zekri, A. R. et al., Asian Pac. J Cancer Prev. 16 (2015): 3543-3549
[1358] Zhai, L. L. et al., Int. J Clin Exp. Pathol. 8 (2015a):
682-691 [1359] Zhai, L. L. et al., Onco. Targets. Ther. 8 (2015b):
2827-2834 [1360] Zhai, L. L. et al., Am. J Transl. Res 7 (2015c):
2412-2422 [1361] Zhang, B. et al., Br. J Cancer 109 (2013a): 14-23
[1362] Zhang, G. et al., FASEB J 27 (2013b): 2893-2901 [1363]
Zhang, H. et al., Onco. Targets. Ther. 8 (2015a): 2291-2301 [1364]
Zhang, J. et al., J Cancer Res Clin Oncol 140 (2014a): 1441-1449
[1365] Zhang, J. et al., BMC. Dev. Biol 8 (2008): 115 [1366] Zhang,
J. et al., Zhonghua Bing. Li Xue. Za Zhi. 42 (2013c): 810-814
[1367] Zhang, L. et al., Carcinogenesis 34 (2013d): 577-586 [1368]
Zhang, P. et al., Genome 57 (2014b): 253-257 [1369] Zhang, R. et
al., Mol. Carcinog 54 (2015b): 1554-1566 [1370] Zhang, T. et al.,
Acta Histochem. 115 (2013e): 48-55 [1371] Zhang, Y. et al., J
Cancer Res Clin Oncol 137 (2011): 1245-1253 [1372] Zhao, G. et al.,
Biochem. Biophys. Res Commun. 408 (2011): 154-159 [1373] Zhao, H.
et al., Mol. Biol Cell 15 (2004): 506-519 [1374] Zhao, Z. et al.,
RNA. Biol 12 (2015): 538-554 [1375] Zhen, T. et al., Oncotarget. 5
(2014): 3756-3769 [1376] Zheng, L. H. et al., Climacteric. 17
(2014): 522-528 [1377] Zhou, J. et al., Oncol Rep. 30 (2013):
2229-2237 [1378] Zhou, J. et al., Carcinogenesis 36 (2015a):
441-451 [1379] Zhou, K. et al., Med. Oncol 31 (2014): 17 [1380]
Zhou, T. B. et al., J Recept. Signal. Transduct. Res 33 (2013):
28-36 [1381] Zhou, W. et al., Mol. Cell Biochem. 398 (2015b): 11-19
[1382] Zhu, J. et al., Int. J Clin Exp. Pathol. 8 (2015a): 702-710
[1383] Zhu, J. et al., Oncotarget. 6 (2015b): 16757-16765 [1384]
Zhu, Y. P. et al., Oncotarget. 6 (2015c): 14488-14496 [1385]
Zhuang, Y. J. et al., Cancer Biol Ther. 16 (2015): 88-96 [1386]
Zubel, A. et al., Gynecol. Oncol 114 (2009): 332-336
Sequence CWU 1
1
18018PRTHomo sapiens 1Phe Val Asp Thr Arg Thr Leu Leu 1 5
210PRTHomo sapiens 2Phe Gly Tyr Asp Gly Asp Phe Tyr Arg Ala 1 5 10
39PRTHomo sapiens 3Ile Leu Ile Gly Glu Thr Ile Lys Ile 1 5
411PRTHomo sapiens 4Ala Leu Asp Pro Ala Ala Gln Ala Phe Leu Leu 1 5
10 510PRTHomo sapiens 5Ala Leu Leu Thr Gly Ile Ile Ser Lys Ala 1 5
10 610PRTHomo sapiens 6Ala Leu Thr Gly Ile Pro Leu Pro Leu Ile 1 5
10 79PRTHomo sapiens 7Ala Leu Val Asp Ile Val Arg Ser Leu 1 5
811PRTHomo sapiens 8Ala Leu Tyr Thr Gly Ser Ala Leu Asp Phe Val 1 5
10 99PRTHomo sapiens 9Gln Ile Ile Asp Ala Ile Asn Lys Val 1 5
109PRTHomo sapiens 10Val Leu Leu Asp Lys Ile Lys Asn Leu 1 5
119PRTHomo sapiens 11Ala Leu Tyr Tyr Asn Pro His Leu Leu 1 5
129PRTHomo sapiens 12Ala Gln Tyr Lys Phe Val Tyr Gln Val 1 5
139PRTHomo sapiens 13Phe Ile Asp Ser Ser Asn Pro Gly Leu 1 5
1411PRTHomo sapiens 14Phe Ile Ile Asp Asn Pro Gln Asp Leu Lys Val 1
5 10 159PRTHomo sapiens 15Phe Ile Leu Ala Asn Glu His Asn Val 1 5
169PRTHomo sapiens 16Gly Leu Ile Asp Tyr Asp Thr Gly Ile 1 5
1711PRTHomo sapiens 17Gly Leu Ile Asp Tyr Asp Thr Gly Ile Arg Leu 1
5 10 189PRTHomo sapiens 18Ala Leu Phe Val Arg Leu Leu Ala Leu 1 5
1912PRTHomo sapiens 19Ala Leu Trp His Asp Ala Glu Asn Gln Thr Val
Val 1 5 10 2011PRTHomo sapiens 20Gly Leu Ile Asp Ile Glu Asn Pro
Asn Arg Val 1 5 10 2111PRTHomo sapiens 21Gly Leu Val Asp Gly Arg
Asp Leu Val Ile Val 1 5 10 229PRTHomo sapiens 22Ile Leu Ser Thr Glu
Ile Phe Gly Val 1 5 2311PRTHomo sapiens 23Lys Leu Asp Ser Ser Gly
Gly Ala Val Gln Leu 1 5 10 2411PRTHomo sapiens 24Lys Leu Ser Glu
Asn Ala Gly Ile Gln Ser Leu 1 5 10 259PRTHomo sapiens 25Leu Ile Asn
Pro Asn Ile Ala Thr Val 1 5 269PRTHomo sapiens 26Ser Leu Tyr Thr
Ala Leu Thr Glu Ala 1 5 279PRTHomo sapiens 27Thr Leu Leu Ala His
Pro Val Thr Leu 1 5 289PRTHomo sapiens 28Val Leu Asp Glu Phe Tyr
Ser Ser Leu 1 5 299PRTHomo sapiens 29Tyr Ile Leu Pro Phe Ser Glu
Val Leu 1 5 309PRTHomo sapiens 30Tyr Ile Tyr Lys Asp Thr Ile Gln
Val 1 5 319PRTHomo sapiens 31Tyr Leu Asp Ser Met Tyr Ile Met Leu 1
5 329PRTHomo sapiens 32Tyr Val Asp Asp Gly Leu Ile Ser Leu 1 5
3311PRTHomo sapiens 33Phe Leu Ala Asp Pro Asp Thr Val Asn His Leu 1
5 10 3410PRTHomo sapiens 34Phe Leu Glu Asp Asp Asp Ile Ala Ala Val
1 5 10 3510PRTHomo sapiens 35Phe Leu Phe Pro Ser Gln Tyr Val Asp
Val 1 5 10 369PRTHomo sapiens 36Phe Leu Gly Asp Leu Ser His Leu Leu
1 5 3710PRTHomo sapiens 37Phe Leu Asn Pro Asp Glu Val His Ala Ile 1
5 10 3810PRTHomo sapiens 38Phe Leu Thr Glu Ala Ala Leu Gly Asp Ala
1 5 10 399PRTHomo sapiens 39Phe Leu Thr Pro Ser Ile Phe Ile Ile 1 5
409PRTHomo sapiens 40Gly Leu Ala Pro Gln Ile His Asp Leu 1 5
4111PRTHomo sapiens 41Gly Leu Leu Ala Gly Asn Glu Lys Leu Thr Met 1
5 10 4211PRTHomo sapiens 42Ile Leu Ser Asp Met Arg Ser Gln Tyr Glu
Val 1 5 10 439PRTHomo sapiens 43His Leu Gly Val Lys Val Phe Ser Val
1 5 449PRTHomo sapiens 44Ile Leu Ala Gln Val Gly Phe Ser Val 1 5
4512PRTHomo sapiens 45Ile Leu Tyr Ser Asp Asp Gly Gln Lys Trp Thr
Val 1 5 10 469PRTHomo sapiens 46Thr Met Val Glu His Asn Tyr Tyr Val
1 5 479PRTHomo sapiens 47Leu Ile Tyr Lys Asp Leu Val Ser Val 1 5
4810PRTHomo sapiens 48Leu Leu Asp Glu Asn Gly Val Leu Lys Leu 1 5
10 499PRTHomo sapiens 49Leu Leu Asp Gly Phe Pro Arg Thr Val 1 5
5010PRTHomo sapiens 50Leu Leu Phe Gly Ser Asp Gly Tyr Tyr Val 1 5
10 519PRTHomo sapiens 51Leu Leu Gly Pro Ala Gly Ala Arg Ala 1 5
529PRTHomo sapiens 52Leu Leu Ser Asp Pro Ile Pro Glu Val 1 5
5311PRTHomo sapiens 53Leu Leu Trp Asp Pro Ser Thr Gly Lys Gln Val 1
5 10 5410PRTHomo sapiens 54Leu Thr Gln Pro Gly Pro Ile Ala Ser Ala
1 5 10 559PRTHomo sapiens 55Asn Leu Ala Pro Ala Pro Leu Asn Ala 1 5
569PRTHomo sapiens 56Asn Leu Ile Gly Val Thr Ala Glu Leu 1 5
5710PRTHomo sapiens 57Arg Leu Ser Glu Leu Gly Ile Thr Gln Ala 1 5
10 5810PRTHomo sapiens 58Arg Gln Tyr Pro Trp Gly Val Val Gln Val 1
5 10 599PRTHomo sapiens 59Ser Leu Ser Glu Ser Phe Phe Met Val 1 5
609PRTHomo sapiens 60Ser Leu Trp Glu Asp Tyr Pro His Val 1 5
619PRTHomo sapiens 61Ser Met Tyr Asp Gly Leu Leu Gln Ala 1 5
629PRTHomo sapiens 62Ser Val Phe Pro Gly Ala Arg Leu Leu 1 5
639PRTHomo sapiens 63Ser Val Thr Gly Ile Ile Val Gly Val 1 5
6411PRTHomo sapiens 64Thr Leu Phe Ser Glu Pro Lys Phe Ala Gln Val 1
5 10 659PRTHomo sapiens 65Thr Leu Asn Glu Lys Leu Thr Ala Leu 1 5
6610PRTHomo sapiens 66Thr Val Asp Asp Pro Tyr Ala Thr Phe Val 1 5
10 679PRTHomo sapiens 67Val Ile Trp Gly Thr Asp Val Asn Val 1 5
689PRTHomo sapiens 68Val Leu Phe Asp Val Thr Gly Gln Val 1 5
699PRTHomo sapiens 69Val Leu Phe Ser Gly Ser Leu Arg Leu 1 5
708PRTHomo sapiens 70Val Leu Gly Val Ile Trp Gly Val 1 5
7111PRTHomo sapiens 71Val Leu Leu Pro Glu Gly Gly Ile Thr Ala Ile 1
5 10 7211PRTHomo sapiens 72Val Met Ala Ser Pro Gly Gly Leu Ser Ala
Val 1 5 10 7310PRTHomo sapiens 73Val Met Val Asp Gly Lys Pro Val
Asn Leu 1 5 10 749PRTHomo sapiens 74Tyr Ile Asp Lys Asp Leu Glu Tyr
Val 1 5 759PRTHomo sapiens 75Phe Ser Phe Val Asp Leu Arg Leu Leu 1
5 7611PRTHomo sapiens 76Leu Val Ser Glu Ser Ser Asp Val Leu Pro Lys
1 5 10 779PRTHomo sapiens 77Arg Leu Phe Pro Gly Ser Ser Phe Leu 1 5
7811PRTHomo sapiens 78Ser Leu Gln Asp Thr Glu Glu Lys Ser Arg Ser 1
5 10 7910PRTHomo sapiens 79Val Val Tyr Glu Gly Gln Leu Ile Ser Ile
1 5 10 8011PRTHomo sapiens 80Leu Leu Pro Gly Thr Glu Tyr Val Val
Ser Val 1 5 10 8112PRTHomo sapiens 81Val Val Tyr Asp Asp Ser Thr
Gly Leu Ile Arg Leu 1 5 10 829PRTHomo sapiens 82Ala Leu Ile Ala Glu
Gly Ile Ala Leu 1 5 839PRTHomo sapiens 83Ala Leu Ser Lys Glu Ile
Tyr Val Ile 1 5 849PRTHomo sapiens 84Phe Ile Leu Pro Ile Gly Ala
Thr Val 1 5 8510PRTHomo sapiens 85Phe Leu Ser Asp Gly Thr Ile Ile
Ser Val 1 5 10 8610PRTHomo sapiens 86Gly Leu Gly Asp Phe Ile Phe
Tyr Ser Val 1 5 10 879PRTHomo sapiens 87Gly Leu Leu Pro Ala Leu Val
Ala Leu 1 5 889PRTHomo sapiens 88Ile Ile Asp Asp Thr Ile Phe Asn
Leu 1 5 8910PRTHomo sapiens 89Lys Leu Ala Asp Ile Gln Ile Glu Gln
Leu 1 5 10 909PRTHomo sapiens 90Lys Leu Leu Thr Pro Ile Thr Thr Leu
1 5 919PRTHomo sapiens 91Leu Leu Phe Asn Asp Val Gln Thr Leu 1 5
9210PRTHomo sapiens 92Tyr Leu Thr Asn Glu Gly Ile Ala His Leu 1 5
10 939PRTHomo sapiens 93Ser Ile Asp Ser Glu Pro Ala Leu Val 1 5
949PRTHomo sapiens 94Val Met Met Glu Glu Phe Val Gln Leu 1 5
9510PRTHomo sapiens 95Ala Leu Ala Asp Asp Asp Phe Leu Thr Val 1 5
10 9613PRTHomo sapiens 96Ala Leu Ala Pro Ala Thr Gly Gly Gly Ser
Leu Leu Leu 1 5 10 979PRTHomo sapiens 97Ala Leu Asp Asp Met Ile Ser
Thr Leu 1 5 989PRTHomo sapiens 98Ala Leu Asp Gln Lys Val Arg Ser
Val 1 5 999PRTHomo sapiens 99Ala Leu Glu Ser Phe Leu Lys Gln Val 1
5 10010PRTHomo sapiens 100Ala Leu Phe Gly Ala Gly Pro Ala Ser Ile 1
5 10 10111PRTHomo sapiens 101Ala Leu Val Glu Glu Asn Gly Ile Phe
Glu Leu 1 5 10 10210PRTHomo sapiens 102Ala Leu Tyr Pro Gly Thr Asp
Tyr Thr Val 1 5 10 1039PRTHomo sapiens 103Ala Val Ala Ala Val Leu
Thr Gln Val 1 5 1049PRTHomo sapiens 104Phe Leu Gln Pro Asp Leu Asp
Ser Leu 1 5 1059PRTHomo sapiens 105Phe Leu Ser Glu Val Phe His Gln
Ala 1 5 1069PRTHomo sapiens 106Phe Val Trp Ser Gly Thr Ala Glu Ala
1 5 10710PRTHomo sapiens 107Phe Val Tyr Gly Gly Pro Gln Val Gln Leu
1 5 10 1089PRTHomo sapiens 108Ile Ala Asp Gly Gly Phe Thr Glu Leu 1
5 1099PRTHomo sapiens 109Ile Leu Ala Ser Val Ile Leu Asn Val 1 5
1109PRTHomo sapiens 110Ile Leu Leu Thr Gly Thr Pro Ala Leu 1 5
11111PRTHomo sapiens 111Leu Leu Leu Ala Ala Ala Arg Leu Ala Ala Ala
1 5 10 1129PRTHomo sapiens 112Leu Leu Ser Asp Val Arg Phe Val Leu 1
5 11310PRTHomo sapiens 113Leu Met Met Ser Glu Asp Arg Ile Ser Leu 1
5 10 11410PRTHomo sapiens 114Ser Leu Phe Pro His Asn Pro Gln Phe
Ile 1 5 10 11511PRTHomo sapiens 115Ser Leu Met Asp Pro Asn Lys Phe
Leu Leu Leu 1 5 10 1169PRTHomo sapiens 116Ser Met Met Asp Pro Asn
His Phe Leu 1 5 1179PRTHomo sapiens 117Ser Val Asp Gly Val Ile Lys
Glu Val 1 5 1189PRTHomo sapiens 118Thr Leu Trp Tyr Arg Pro Pro Glu
Leu 1 5 11911PRTHomo sapiens 119Val Leu Gly Asp Asp Pro Gln Leu Met
Lys Val 1 5 10 1209PRTHomo sapiens 120Val Leu Val Asn Asp Phe Phe
Leu Val 1 5 12110PRTHomo sapiens 121Tyr Leu Asp Glu Asp Thr Ile Tyr
His Leu 1 5 10 12211PRTHomo sapiens 122Met Gln Ala Pro Arg Ala Ala
Leu Val Phe Ala 1 5 10 1239PRTHomo sapiens 123Lys Ile Ser Thr Ile
Thr Pro Gln Ile 1 5 12411PRTHomo sapiens 124Ala Leu Phe Glu Glu Ser
Gly Leu Ile Arg Ile 1 5 10 12511PRTHomo sapiens 125Ala Leu Leu Gly
Lys Leu Asp Ala Ile Asn Val 1 5 10 12610PRTHomo sapiens 126Ala Leu
Leu Ser Leu Asp Pro Ala Ala Val 1 5 10 12710PRTHomo sapiens 127Ala
Leu Ser Asp Leu Ala Leu His Phe Leu 1 5 10 12810PRTHomo sapiens
128Ala Leu Tyr Asp Val Arg Thr Ile Leu Leu 1 5 10 12910PRTHomo
sapiens 129Ala Leu Tyr Glu Lys Asp Asn Thr Tyr Leu 1 5 10
13010PRTHomo sapiens 130Phe Leu Phe Gly Glu Glu Pro Ser Lys Leu 1 5
10 13110PRTHomo sapiens 131Phe Leu Ile Glu Glu Gln Lys Ile Val Val
1 5 10 13212PRTHomo sapiens 132Phe Leu Trp Ala Gly Gly Arg Ala Ser
Tyr Gly Val 1 5 10 13310PRTHomo sapiens 133Ile Leu Asp Asp Val Ser
Leu Thr His Leu 1 5 10 13411PRTHomo sapiens 134Ile Leu Leu Ala Glu
Gly Arg Leu Val Asn Leu 1 5 10 1359PRTHomo sapiens 135Lys Leu Asp
Asp Thr Tyr Ile Lys Ala 1 5 13611PRTHomo sapiens 136Lys Leu Phe Pro
Gly Phe Glu Ile Glu Thr Val 1 5 10 1379PRTHomo sapiens 137Lys Leu
Gly Pro Glu Gly Glu Leu Leu 1 5 13811PRTHomo sapiens 138Asn Ile Phe
Pro Asn Pro Glu Ala Thr Phe Val 1 5 10 1399PRTHomo sapiens 139Ser
Ile Asp Arg Asn Pro Pro Gln Leu 1 5 14011PRTHomo sapiens 140Ser Leu
Leu Asn Pro Pro Glu Thr Leu Asn Leu 1 5 10 1419PRTHomo sapiens
141Ser Leu Thr Glu Gln Val His Ser Leu 1 5 1429PRTHomo sapiens
142Ser Leu Tyr Gly Tyr Leu Arg Gly Ala 1 5 1439PRTHomo sapiens
143Thr Ala Asp Pro Leu Asp Tyr Arg Leu 1 5 1449PRTHomo sapiens
144Thr Ala Val Ala Leu Leu Arg Leu Leu 1 5 1459PRTHomo sapiens
145Thr Thr Phe Pro Arg Pro Val Thr Val 1 5 14610PRTHomo sapiens
146Val Leu Ile Ser Gly Val Val His Glu Ile 1 5 10 1479PRTHomo
sapiens 147Tyr Ala Phe Pro Lys Ala Val Ser Val 1 5 1489PRTHomo
sapiens 148Tyr Leu His Asn Gln Gly Ile Gly Val 1 5 14911PRTHomo
sapiens 149Ile Leu Gly Thr Glu Asp Leu Ile Val Glu Val 1 5 10
15010PRTHomo sapiens 150Ala Leu Phe Gln Pro His Leu Ile Asn Val 1 5
10 1519PRTHomo sapiens 151Ala Leu Leu Asp Ile Ile Arg Ser Leu 1 5
15211PRTHomo sapiens 152Ala Leu Leu Glu Pro Glu Phe Ile Leu Lys Ala
1 5 10 15310PRTHomo sapiens 153Ala Leu Pro Lys Glu Asp Pro Thr Ala
Val 1 5 10 1549PRTHomo sapiens 154Lys Val Ala Asp Leu Val Leu Met
Leu 1 5 15512PRTHomo sapiens 155Leu Leu Leu Asp Pro Asp Thr Ala Val
Leu Lys Leu 1 5 10 15610PRTHomo sapiens 156Leu Leu Leu Pro Pro Pro
Pro Cys Pro Ala 1 5 10 15710PRTHomo sapiens 157Met Leu Leu Glu Ile
Pro Tyr Met Ala Ala 1 5 10 1589PRTHomo sapiens 158Ser Leu Ile Glu
Lys Tyr Phe Ser Val 1 5 1599PRTHomo sapiens 159Ser Leu Leu Asp Leu
His Thr Lys Val 1 5 16011PRTHomo sapiens 160Val Leu Leu Pro Asp Glu
Arg Thr Ile Ser Leu 1 5 10 16111PRTHomo sapiens 161Tyr Leu Pro Asp
Ile Ile Lys Asp Gln Lys Ala 1 5 10 1629PRTHomo sapiens 162Asn Ala
Asp Pro Gln Ala Val Thr Met 1 5 1639PRTHomo sapiens 163Val Met Ala
Pro Arg Thr Leu Val Leu 1 5 1649PRTHomo sapiens 164Tyr Leu Gly Arg
Leu Ala His Glu Val 1 5 1659PRTHomo sapiens 165Tyr Leu Leu Ser Tyr
Ile Gln Ser Ile 1 5 1669PRTHomo sapiens 166Ser Leu Phe Pro Gly Gln
Val Val Ile 1 5 16711PRTHomo sapiens 167Met Leu Phe Gly His Pro Leu
Leu Val Ser Val 1 5 10 16811PRTHomo sapiens 168Ser Glu Trp Gly Ser
Pro His Ala Ala Val Pro 1 5 10 1699PRTHomo sapiens 169Phe Met Leu
Pro Asp Pro Gln Asn Ile 1 5 17011PRTHomo sapiens 170Ile Leu Ala Pro
Ala Gly Ser Leu Pro Lys Ile 1 5 10 17110PRTHomo sapiens 171Leu Leu
Leu Asp Val Thr Pro Leu Ser Leu 1 5 10 1729PRTHomo sapiens 172Thr
Met Met Ser Arg Pro Pro Val Leu 1 5 17311PRTHomo sapiens 173Ser Leu
Ala Gly Asp Val Ala Leu Gln Gln Leu 1 5 10 1749PRTHomo sapiens
174Thr Leu Asp Pro Arg Ser Phe Leu Leu 1 5 17510PRTHomo sapiens
175Ala Leu Leu Glu Ser Ser Leu Arg Gln Ala 1 5 10 1769PRTHomo
sapiens 176Tyr Leu Met Pro Gly Phe Ile His Leu 1 5 1779PRTHomo
sapiens 177Ser Leu Tyr Lys Gly Leu Leu Ser Val 1 5 1789PRTHomo
sapiens 178Lys Ile Gln Glu Ile Leu Thr Gln Val 1 5 17910PRTHomo
sapiens 179Glu Leu Ala Gly Ile Gly Ile Leu Thr Val 1 5 10
1809PRTHomo sapiens 180Tyr Leu Leu Pro Ala Ile Val His Ile 1 5
* * * * *
References