U.S. patent application number 17/710573 was filed with the patent office on 2022-07-28 for peptides and t cells for use in immunotherapeutic treatment of various cancers.
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 | 20220233663 17/710573 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-28 |
United States Patent
Application |
20220233663 |
Kind Code |
A1 |
MAHR; Andrea ; et
al. |
July 28, 2022 |
PEPTIDES AND T CELLS FOR USE IN IMMUNOTHERAPEUTIC TREATMENT OF
VARIOUS 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; (Tuebingen, DE) ;
SCHOOR; Oliver; (Tuebingen, DE) ; FRITSCHE; Jens;
(Tuebingen, DE) ; SINGH; Harpreet; (Tuebingen,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Immatics Biotechnologies GmbH |
Tuebingen |
|
DE |
|
|
Appl. No.: |
17/710573 |
Filed: |
March 31, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17320763 |
May 14, 2021 |
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17710573 |
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17017358 |
Sep 10, 2020 |
11065316 |
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17320763 |
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16887765 |
May 29, 2020 |
10898557 |
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17017358 |
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16673619 |
Nov 4, 2019 |
10695411 |
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16887765 |
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15982293 |
May 17, 2018 |
10576132 |
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16673619 |
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15249083 |
Aug 26, 2016 |
10335471 |
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15982293 |
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62211276 |
Aug 28, 2015 |
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International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 7/06 20060101 C07K007/06; G01N 33/574 20060101
G01N033/574; C07K 14/47 20060101 C07K014/47; C12Q 1/6886 20060101
C12Q001/6886; C12N 15/115 20060101 C12N015/115; C07K 7/08 20060101
C07K007/08; A61P 35/00 20060101 A61P035/00; C07K 14/74 20060101
C07K014/74; C07K 14/725 20060101 C07K014/725; C07K 16/28 20060101
C07K016/28; C07K 16/30 20060101 C07K016/30; C12N 5/0783 20060101
C12N005/0783 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 28, 2015 |
GB |
1515321.6 |
Claims
1. A peptide consisting of the amino acid sequence SYVKVLHHL (SEQ
ID NO: 241) in the form of a pharmaceutically acceptable salt.
2. The peptide of claim 1, wherein said peptide has the ability to
bind to an MHC class-I molecule, and wherein said peptide, when
bound to said MHC, is capable of being recognized by CD8 T
cells.
3. The peptide of claim 1, wherein the pharmaceutically acceptable
salt is chloride salt.
4. The peptide of claim 1, wherein the pharmaceutically acceptable
salt is acetate salt.
5. A composition comprising the peptide of claim 1, wherein the
composition comprises an adjuvant and a pharmaceutically acceptable
carrier.
6. The composition of claim 5, wherein the peptide is in the form
of a chloride salt.
7. The composition of claim 5, wherein the peptide is in the form
of an acetate salt.
8. The composition of claim 5 wherein the adjuvant is selected from
the group consisting of anti-CD40 antibody, imiquimod, resiquimod,
GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha,
interferon-beta, CpG oligonucleotides and derivatives, poly-(I:C)
and derivatives, RNA, sildenafil, particulate formulations with
poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1,
IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.
9. The composition of claim 8, wherein the adjuvant is IL-2.
10. The composition of claim 8, wherein the adjuvant is IL-7.
11. The composition of claim 8, wherein the adjuvant is IL-12.
12. The composition of claim 8, wherein the adjuvant is IL-15.
13. The composition of claim 8, wherein the adjuvant is IL-21.
14. A pegylated peptide consisting of the amino acid sequence of
SYVKVLHHL (SEQ ID NO: 241) or a pharmaceutically acceptable salt
thereof.
15. The peptide of claim 14, wherein the pharmaceutically
acceptable salt is chloride salt.
16. The peptide of claim 14, wherein the pharmaceutically
acceptable salt is acetate salt.
17. A composition comprising the pegylated peptide of claim 14 or
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier.
18. The composition of claim 5, wherein the pharmaceutically
acceptable carrier is selected from the group consisting of saline,
Ringer's solution, dextrose solution, and sustained release
preparation.
19. The peptide in the form of a pharmaceutically acceptable salt
of claim 1, wherein said peptide is produced by solid phase peptide
synthesis or produced by a yeast cell or bacterial cell expression
system.
20. A composition comprising the peptide of claim 1, wherein the
composition is a pharmaceutical composition and comprises water and
a buffer.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 17/320,763, filed May 14, 2021, which is a
continuation of U.S. patent application Ser. No. 17/017,358, filed
Sep. 10, 2020 (now U.S. Pat. No. 11,065,316, issued Jul. 20, 2021),
which is a continuation of U.S. patent application Ser. No.
16/887,765, filed May 29, 2020, (now U.S. Pat. No. 10,898,557,
issued Jan. 26, 2021), which is a continuation of U.S. patent
application Ser. No. 16/673,619, filed Nov. 4, 2019 (now U.S. Pat.
No. 10,695,411, issued Jun. 30, 2020), which is a continuation of
U.S. patent application Ser. No. 15/982,293, filed May 17, 2018
(now U.S. Pat. No. 10,576,132, issued Mar. 3, 2020), which is a
continuation of U.S. patent application Ser. No. 15/249,083, filed
Aug. 26, 2016 (now U.S. Pat. No. 10,335,471, issued Jul. 2, 2019),
which claims the benefit of U.S. Provisional Application Ser. No.
62/211,276, filed Aug. 28, 2015, and Great Britain Application No.
1515321.6, filed Aug. 28, 2015, the content of each of these
applications is herein incorporated by reference in their
entirety.
[0002] This application also is related to PCT/EP2016/070146 filed
26 Aug. 2016, the content of which is incorporated herein by
reference in its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT
FILE (.txt)
[0003] Pursuant to the EFS-Web legal framework and 37 CFR
.sctn..sctn. 1.821-825 (see MPEP .sctn. 2442.03(a)), a Sequence
Listing in the form of an ASCII-compliant text file (entitled
"Sequence_listing_2912919-054018_ST25.txt" created on Mar. 31,
2022, and 65,368 bytes in size) is submitted concurrently with the
instant application, and the entire contents of the Sequence
Listing are incorporated herein by reference.
FIELD
[0004] 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.
[0005] 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
[0006] According to the World Health Organization (WHO), cancer
ranged among the four major non-communicable deadly diseases
worldwide in 2012. For the same year, colorectal cancer, breast
cancer and respiratory tract cancers were listed within the top 10
causes of death in high income countries.
[0007] Epidemiology
[0008] In 2012, 14.1 million new cancer cases, 32.6 million
patients suffering from cancer (within 5 years of diagnosis) and
8.2 million cancer deaths were estimated worldwide (Ferlay et al.,
2013; Bray et al., 2013).
[0009] Within the groups of brain cancer, leukemia and lung cancer,
the present application particularly focuses on glioblastoma (GB),
chronic lymphocytic leukemia (CLL), and non-small cell and small
cell lung cancer (NSCLC and SCLC).
[0010] Lung cancer is the most common type of cancer worldwide and
the leading cause of death from cancer in many countries.
[0011] Breast cancer is an immunogenic cancer entity and different
types of infiltrating immune cells in primary tumors exhibit
distinct prognostic and predictive significance. A large number of
early phase immunotherapy trials have been conducted in breast
cancer patients. Most of the completed vaccination studies targeted
HER2 and carbohydrate antigens like MUC-1 and revealed rather
disappointing results. Clinical data on the effects of immune
checkpoint modulation with ipilimumab and other T cell-activating
antibodies in breast cancer patients are emerging (Emens,
2012).
[0012] Chronic Lymphocytic Leukemia
[0013] While CLL is not curable at present, many patients show only
slow progression of the disease or worsening of symptoms. As
patients do not benefit from an early onset of treatment, the
initial approach is "watch and wait" (Richards et al., 1999). For
patients with symptomatic or rapidly progressing disease, several
treatment options are available. These include chemotherapy,
targeted therapy, immune-based therapies like monoclonal
antibodies, chimeric antigen-receptors (CARs) and active
immunotherapy, and stem cell transplants.
[0014] Monoclonal antibodies are widely used in hematologic
malignancies. This is due to the knowledge of suitable antigens
based on the good characterization of immune cell surface molecules
and the accessibility of tumor cells in blood or bone marrow.
Common monoclonal antibodies used in CLL therapy target either CD20
or CD52. Rituximab, the first monoclonal anti-CD20 antibody
originally approved by the FDA for treatment of NHLs, is now widely
used in CLL therapy. Combinational treatment with
rituximab/fludarabine/cyclophosphamide leads to higher CR rates and
improved overall survival (OS) compared to the combination
fludarabine/cyclophosphamide and has become the preferred treatment
option (Hallek et al., 2008). Ofatumomab targets CD20 and is used
for therapy of refractory CLL patients (Wierda et al., 2011).
Obinutuzumab is another monoclonal anti-CD20 antibody used in
first-line treatment in combination with chlorambucil (Goede et
al., 2014).
[0015] Alemtuzumab is an anti-CD52 antibody used for treatment of
patients with chemotherapy-resistant disease or patients with poor
prognostic factors as del 17p or p53 mutations (Parikh et al.,
2011).
[0016] Novel monoclonal antibodies target CD37 (otlertuzumab, BI
836826, IMGN529 and (177)Lu-tetulomab) or CD40 (dacetuzumab and
lucatumumab) and are tested in pre-clinical settings (Robak and
Robak, 2014).
[0017] Several completed and ongoing trials are based on engineered
autologous chimeric antigen receptor (CAR)-modified T cells with
CD19 specificity (Maus et al., 2014). So far, only the minority of
patients showed detectable or persistent CARs. One partial response
(PR) and two complete responses (CR) have been detected in the CAR
T-cell trials by Porter et al. and Kalos et al. (Kalos et al.,
2011; Porter et al., 2011).
[0018] Active immunotherapy includes the following strategies: gene
therapy, whole modified tumor cell vaccines, DC-based vaccines and
tumor associated antigen (TAA)-derived peptide vaccines.
[0019] Approaches in gene therapy make use of autologous
genetically modified tumor cells. These B-CLL cells are transfected
with immuno-(co-)stimulatory genes like IL-2, IL-12, TNF-alpha,
GM-CSF, CD80, CD40L, LFA-3 and ICAM-1 to improve antigen
presentation and T cell activation (Carballido et al., 2012). While
specific T-cell responses and reduction in tumor cells are readily
observed, immune responses are only transient.
[0020] Several studies have used autologous DCs as antigen
presenting cells to elicit anti-tumor responses. DCs have been
loaded ex vivo with tumor associated peptides, whole tumor cell
lysate and tumor-derived RNA or DNA. Another strategy uses whole
tumor cells for fusion with DCs and generation of DC-B-CLL-cell
hybrids. Transfected DCs initiated both CD4+ and CD8+ T-cell
responses (Muller et al., 2004). Fusion hybrids and DCs loaded with
tumor cell lysate or apoptotic bodies increased tumor-specific CD8+
T-cell responses. Patients that showed a clinical response had
increased IL-12 serum levels and reduced numbers of Tregs (Palma et
al., 2008).
[0021] Different approaches use altered tumor cells to initiate or
increase CLL-specific immune responses. An example for this
strategy is the generation of trioma cells: B-CLL cells are fused
to anti-Fc receptor expressing hybridoma cells that have anti-APC
specificity. Trioma cells induced CLL-specific T-cell responses in
vitro (Kronenberger et al., 2008).
[0022] Another strategy makes use of irradiated autologous CLL
cells with Bacillus Calmette-Guerin as an adjuvant as a vaccine.
Several patients showed a reduction in leukocyte levels or stable
disease (Hus et al., 2008).
[0023] Besides isolated CLL cells, whole blood from CLL patients
has been used as a vaccine after preparation in a blood treatment
unit. The vaccine elicited CLL-specific T-cell responses and led to
partial clinical responses or stable disease in several patients
(Spaner et al., 2005).
[0024] Several TAAs are over-expressed in CLL and are suitable for
vaccinations. These include fibromodulin (Mayr et al., 2005),
RHAMM/CD168 (Giannopoulos et al., 2006), MDM2 (Mayr et al., 2006),
hTERT (Counter et al., 1995), the oncofetal antigen-immature
laminin receptor protein(OFAiLRP) (Siegel et al., 2003),
adipophilin (Schmidt et al., 2004), survivin (Granziero et al.,
2001), KW1 to KW14 (Krackhardt et al., 2002) and the tumor-derived
IgVHCDR3 region (Harig et al., 2001; Carballido et al., 2012). A
phase I clinical trial was conducted using the RHAMM-derived R3
peptide as a vaccine. 5 of 6 patients had detectable R3-specific
CD8+ T-cell responses (Giannopoulos et al., 2010).
[0025] Colorectal Cancer
[0026] Depending on the colorectal cancer (CRC) stage, different
standard therapies are available for colon and rectal cancer.
Standard procedures include surgery, radiation therapy,
chemotherapy and targeted therapy for CRC (Berman et al., 2015a;
Berman et al., 2015b).
[0027] In addition to chemotherapeutic drugs, several monoclonal
antibodies targeting the epidermal growth factor receptor (EGFR,
cetuximab, panitumumab) or the vascular endothelial growth factor-A
(VEGF-A, bevacizumab) are administered to patients with high stage
disease. For second-line and later treatment the inhibitor for VEGF
aflibercept, the tyrosine kinase inhibitor regorafenib and the
thymidylate-synthetase inhibitor TAS-102 and the dUTPase inhibitor
TAS-114 can be used (Stintzing, 2014; Wilson et al., 2014).
[0028] The most recent clinical trials analyze active immunotherapy
as a treatment option against CRC. Those strategies include the
vaccination with peptides from tumor-associated antigens (TAAs),
whole tumor cells, dendritic cell (DC) vaccines and viral vectors
(Koido et al., 2013).
[0029] Peptide vaccines have so far been directed against
carcinoembryonic antigen (CEA), mucin 1, EGFR, squamous cell
carcinoma antigen recognized by T cells 3 (SART3), beta-human
chorionic gonadotropin (beta-hCG), Wilms' Tumor antigen 1 (WT1),
Survivin-2B, MAGE3, p53, ring finger protein 43 and translocase of
the outer mitochondrial membrane 34 (TOMM34), or mutated KRAS. In
several phase I and II clinical trials patients showed
antigen-specific CTL responses or antibody production. In contrast
to immunological responses, many patients did not benefit from
peptide vaccines on the clinical level (Koido et al., 2013; Miyagi
et al., 2001; Moulton et al., 2002; Okuno et al., 2011).
[0030] Dendritic cell vaccines comprise DCs pulsed with either
TAA-derived peptides, tumor cell lysates, apoptotic tumor cells, or
tumor RNA or DC-tumor cell fusion products. While many patients in
phase I/II trials showed specific immunological responses, only the
minority had a clinical benefit (Koido et al., 2013).
[0031] Whole tumor cell vaccines consist of autologous tumor cells
modified to secrete GM-CSF, modified by irradiation or
virus-infected, irradiated cells. Most patients showed no clinical
benefit in several phase II/Ill trials (Koido et al., 2013).
[0032] Vaccinia virus or replication-defective avian poxvirus
encoding CEA as well as B7.1, ICAM-1 and LFA-3 have been used as
vehicles in viral vector vaccines in phase I clinical trials. A
different study used nonreplicating canarypox virus encoding CEA
and B7.1. Besides the induction of CEA-specific T cell responses
40% of patients showed objective clinical responses (Horig et al.,
2000; Kaufman et al., 2008).
[0033] Esophageal Cancer
[0034] The primary treatment strategy for esophageal cancer depends
on tumor stage and location, histological type and the medical
condition of the patient. Surgery alone is not sufficient, except
in a small subgroup of patients with squamous cell carcinoma.
[0035] Data on immunotherapeutic approaches in esophageal cancer
are scarce, as only a very limited number of early phase clinical
trials have been performed. A vaccine consisting of three peptides
derived from three different cancer-testis antigens (TTK protein
kinase, lymphocyte antigen 6 complex locus K and insulin-like
growth factor (IGF)-II mRNA binding protein 3) was administered to
patients with advanced esophageal cancer in a phase I trial with
moderate results. Intra-tumoral injection of activated T cells
after in vitro challenge with autologous malignant cells elicited
complete or partial tumor responses in four of eleven patients in a
phase I/II study (Toomey et al., 2013).
[0036] Gastric Cancer
[0037] Gastric cancer (GC) begins in the cells lining the mucosal
layer and spreads through the outer layers as it grows. Surgery is
the primary treatment and the only curative treatment for gastric
cancer. The efficacy of current therapeutic regimens for advanced
GC is poor, resulting in low 5-year survival rates. Immunotherapy
might be an alternative approach to ameliorate the survival of GC
patients. Adoptive transfer of tumor-associated lymphocytes and
cytokine induced killer cells, peptide-based vaccines targeting
HER2/neu, MAGE-3 or vascular endothelial growth factor receptor 1
and 2 and dendritic cell-based vaccines targeting HER2/neu showed
promising results in clinical GC trials. Immune checkpoint
inhibition and engineered T cells might represent additional
therapeutic options, which is currently evaluated in pre-clinical
and clinical studies (Matsueda and Graham, 2014).
[0038] Glioblastoma
[0039] The therapeutic options for glioblastoma (WHO grade IV) are
very limited. According to the guidelines released by the German
Society for Neurology the standard therapy in young patients
includes resection or biopsy of the tumor, focal radiation therapy
and chemotherapy with temozolomide or CCNU/lomustine or a
combination of procarbazine with CCNU and vincristine (PCV). In the
USA, Canada and Switzerland treatment with bevacizumab
(anti-VEGF-antibody) is also approved for relapse therapy
(Leitlinien fur Diagnostik and Therapie in der Neurologie,
2014).
[0040] Different immunotherapeutic approaches are investigated for
the treatment of GB, including immune-checkpoint inhibition,
vaccination and adoptive transfer of engineered T cells.
[0041] Antibodies directed against inhibitory T cell receptors or
their ligands were shown to efficiently enhance T cell-mediated
anti-tumor immune responses in different cancer types, including
melanoma and bladder cancer. The effects of T cell activating
antibodies like ipilimumab and nivolumab are therefore assessed in
clinical GB trials, but preliminary data indicate
autoimmune-related adverse events.
[0042] Different vaccination strategies for GB patients are
currently investigated, including peptide-based vaccines,
heat-shock protein vaccines, autologous tumor cell vaccines,
dendritic cell-based vaccines and viral protein-based vaccines. In
these approaches peptides derived from GB-associated proteins like
epidermal growth factor receptor variant III (EGFRvIII) or heat
shock proteins or dendritic cells pulsed with autologous tumor cell
lysate or cytomegalo virus components are applied to induce an
anti-tumor immune response in GB patients. Several of these studies
reveal good safety and tolerability profiles as well as promising
efficacy data.
[0043] Adoptive transfer of genetically modified T cells is an
additional immunotherapeutic approach for the treatment of GB.
Different clinical trials currently evaluate the safety and
efficacy of chimeric antigen receptor bearing T cells directed
against HER2, IL-13 receptor alpha 2 and EGFRvIII (Ampie et al.,
2015).
[0044] Liver Cancer
[0045] Disease management depends on the tumor stage at the time of
diagnosis and the overall condition of the liver. If surgery is not
a treatment option, different other therapies are available at
hand.
[0046] Lately, a limited number of immunotherapy trials for HCC
have been conducted. Cytokines have been used to activate subsets
of immune cells and/or increase the tumor immunogenicity (Reinisch
et al., 2002; Sangro et al., 2004). Other trials have focused on
the infusion of Tumor-infiltrating lymphocytes or activated
peripheral blood lymphocytes (Shi et al., 2004; Takayama et al.,
1991; Takayama et al., 2000b).
[0047] So far, a small number of therapeutic vaccination trials
have been executed. Butterfield et al. conducted two trials using
peptides derived from alpha-fetoprotein (AFP) as a vaccine or DCs
loaded with AFP peptides ex vivo (Butterfield et al., 2003;
Butterfield et al., 2006). In two different studies, autologous
dendritic cells (DCs) were pulsed ex vivo with autologous tumor
lysate (Lee et al., 2005) or lysate of the hepatoblastoma cell line
HepG2 (Palmer et al., 2009). So far, vaccination trials have only
shown limited improvements in clinical outcomes.
[0048] Melanoma
[0049] The standard therapy in melanoma is complete surgical
resection with surrounding healthy tissue. If resection is not
complete or not possible at all, patients receive primary radiation
therapy, which can be combined with interferon-alpha administration
in advanced stages (stages IIB/C and IIIA-C).
[0050] Enhancing the anti-tumor immune responses appears to be a
promising strategy for the treatment of advanced melanoma. In the
United States the immune checkpoint inhibitor ipilimumab as well as
the BRAF kinase inhibitors vemurafenib and dabrafenib and the MEK
inhibitor trametinib are already approved for the treatment of
advanced melanoma. Both approaches increase the patient's
anti-tumor immunity--ipilimumab directly by reducing T cell
inhibition and the kinase inhibitors indirectly by enhancing the
expression of melanocyte differentiation antigens. Additional
checkpoint inhibitors (nivolumab and lambrolizumab) are currently
investigated in clinical studies with first encouraging results.
Additionally, different combination therapies targeting the
anti-tumor immune response are tested in clinical trials including
ipilimumab plus nivolumab, ipilimumab plus a gp100-derived peptide
vaccine, ipilimumab plus dacarbazine, ipilimumab plus IL-2 and
iplimumab plus GM-CSF (Srivastava and McDermott, 2014).
[0051] Several different vaccination approaches have already been
evaluated in patients with advanced melanoma. So far, phase III
trials revealed rather disappointing results and vaccination
strategies clearly need to be improved. Therefore, new clinical
trials, like the OncoVEX GM-CSF trial or the DERMA trial, aim at
improving clinical efficacy without reducing tolerability.
[0052] Adoptive T cell transfer shows great promise for the
treatment of advanced stage melanoma. In vitro expanded autologous
tumor infiltrating lymphocytes as well as T cells harboring a high
affinity T cell receptor for the cancer-testis antigen NY-ESO-1 had
significant beneficial and low toxic effects upon transfer into
melanoma patients. Unfortunately, T cells with high affinity T cell
receptors for the melanocyte specific antigens MART1 and gp100 and
the cancer-testis antigen MAGEA3 induced considerable toxic effects
in clinical trials. Thus, adoptive T cell transfer has high
therapeutic potential, but safety and tolerability of these
treatments needs to be further increased (Phan and Rosenberg, 2013;
Hinrichs and Restifo, 2013).
[0053] Non-Small Cell Lung Cancer
[0054] Treatment options are determined by the type (small cell or
non-small cell) and stage of cancer and include surgery, radiation
therapy, chemotherapy, and targeted biological therapies such as
bevacizumab, erlotinib and gefitinib.
[0055] To expand the therapeutic options for NSCLC, different
immunotherapeutic approaches have been studied or are still under
investigation. While vaccination with L-BLP25 or MAGEA3 failed to
demonstrate an vaccine-mediated survival advantage in NSCLC
patients, an allogeneic cell line-derived vaccine showed promising
results in clinical studies. Additionally, further vaccination
trials targeting gangliosides, the epidermal growth factor receptor
and several other antigens are currently ongoing. An alternative
strategy to enhance the patient's anti-tumor T cell response
consists of blocking inhibitory T cell receptors or their ligands
with specific antibodies. The therapeutic potential of several of
these antibodies, including ipilimumab, nivolumab, pembrolizumab,
MPDL3280A and MEDI-4736, in NSCLC is currently evaluated in
clinical trials (Reinmuth et al., 2015).
[0056] Ovarian Cancer
[0057] Surgical resection is the primary therapy in early as well
as advanced stage ovarian carcinoma. Surgical removal is followed
by systemic chemotherapy with platinum analogs, except for very low
grade ovarian cancers (stage IA, grade 1), where post-operative
chemotherapy is not indicated.
[0058] Immunotherapy appears to be a promising strategy to
ameliorate the treatment of ovarian cancer patients, as the
presence of pro-inflammatory tumor infiltrating lymphocytes,
especially CD8-positive T cells, correlates with good prognosis and
T cells specific for tumor-associated antigens can be isolated from
cancer tissue.
[0059] Therefore, a lot of scientific effort is put into the
investigation of different immunotherapies in ovarian cancer. A
considerable number of pre-clinical and clinical studies has
already been performed and further studies are currently ongoing.
Clinical data are available for cytokine therapy, vaccination,
monoclonal antibody treatment, adoptive cell transfer and
immunomodulation.
[0060] Cytokine therapy with interleukin-2, interferon-alpha,
interferon-gamma or granulocyte-macrophage colony stimulating
factor aims at boosting the patient's own anti-tumor immune
response and these treatments have already shown promising results
in small study cohorts.
[0061] Phase I and II vaccination studies, using single or multiple
peptides, derived from several tumor-associated proteins (Her2/neu,
NY-ESO-1, p53, Wilms tumor-1) or whole tumor antigens, derived from
autologous tumor cells revealed good safety and tolerability
profiles, but only low to moderate clinical effects.
[0062] Monoclonal antibodies that specifically recognize
tumor-associated proteins are thought to enhance immune
cell-mediated killing of tumor cells. The anti-CA-125 antibodies
oregovomab and abagovomab as well as the anti-EpCAM antibody
catumaxomab achieved promising results in phase II and III studies.
In contrast, the anti-MUC1 antibody HMFG1 failed to clearly enhance
survival in a phase III study.
[0063] An alternative approach uses monoclonal antibodies to target
and block growth factor and survival receptors on tumor cells.
While administration of trastuzumab (anti-HER2/neu antibody) and
MOv18 and MORAb-003 (anti-folate receptor alpha antibodies) only
conferred limited clinical benefit to ovarian cancer patients,
addition of bevacizumab (anti-VEGF antibody) to the standard
chemotherapy in advanced ovarian cancer appears to be
advantageous.
[0064] Adoptive transfer of immune cells achieved heterogeneous
results in clinical trials. Adoptive transfer of autologous, in
vitro expanded tumor infiltrating T cells was shown to be a
promising approach in a pilot trial. In contrast, transfer of T
cells harboring a chimeric antigen receptor specific for folate
receptor alpha did not induce a significant clinical response in a
phase I trial. Dendritic cells pulsed with tumor cell lysate or
tumor-associated proteins in vitro were shown to enhance the
anti-tumor T cell response upon transfer, but the extent of T cell
activation did not correlate with clinical effects. Transfer of
natural killer cells caused significant toxicities in a phase II
study.
[0065] Intrinsic anti-tumor immunity as well as immunotherapy are
hampered by an immunosuppressive tumor microenvironment. To
overcome this obstacle immunomodulatory drugs, like
cyclophosphamide, anti-CD25 antibodies and pegylated liposomal
doxorubicin are tested in combination with immunotherapy. Most
reliable data are currently available for ipilimumab, an anti-CTLA4
antibody, which enhances T cell activity. Ipilimumab was shown to
exert significant anti-tumor effects in ovarian cancer patients
(Mantia-Smaldone et al., 2012).
[0066] Pancreatic Cancer
[0067] 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.
[0068] 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.
[0069] 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).
[0070] Prostate Cancer
[0071] The therapeutic strategy for prostate cancer mainly depends
on the cancer stage. The dendritic cell-based vaccine sipuleucel-T
was the first anti-cancer vaccine to be approved by the FDA. Due to
its positive effect on survival in patients with CRPC, much effort
is put into the development of further immunotherapies. Regarding
vaccination strategies, the peptide vaccine prostate-specific
antigen (PSA)-TRICOM, the personalized peptide vaccine PPV, the DNA
vaccine pTVG-HP and the whole cell vaccine expressing GM-CSF GVAX
showed promising results in different clinical trials. Furthermore,
dendritic cell-based vaccines other than sipuleucel-T, namely
BPX-101 and DCVAC/Pa were shown to elicited clinical responses in
prostate cancer patients. Immune checkpoint inhibitors like
ipilimumab and nivolumab are currently evaluated in clinical
studies as monotherapy as well as in combination with other
treatments, including androgen deprivation therapy, local radiation
therapy, PSA-TRICOM and GVAX. The immunomodulatory substance
tasquinimod, which significantly slowed progression and increased
progression free survival in a phase II trial, is currently further
investigated in a phase III trial. Lenalidomide, another
immunomodulator, induced promising effects in early phase clinical
studies, but failed to improve survival in a phase III trial.
Despite these disappointing results further lenalidomide trials are
ongoing (Quinn et al., 2015).
[0072] Renal Cell Carcinoma
[0073] Initial treatment is most commonly either partial or
complete removal of the affected kidney(s) and remains the mainstay
of curative treatment (Rini et al., 2008). The known immunogenity
of RCC has represented the basis supporting the use of
immunotherapy and cancer vaccines in advanced RCC. The interesting
correlation between lymphocytes PD-1 expression and RCC advanced
stage, grade and prognosis, as well as the selective PD-L1
expression by RCC tumor cells and its potential association with
worse clinical outcomes, have led to the development of new anti
PD-1/PD-L1 agents, alone or in combination with anti-angiogenic
drugs or other immunotherapeutic approaches, for the treatment of
RCC (Massari et al., 2015). In advanced RCC, a phase III cancer
vaccine trial called TRIST study evaluates whether TroVax (a
vaccine using a tumor-associated antigen, 5T4, with a pox virus
vector), added to first-line standard of care therapy, prolongs
survival of patients with locally advanced or mRCC. Median survival
had not been reached in either group with 399 patients (54%)
remaining on study however analysis of the data confirms prior
clinical results, demonstrating that TroVax is both immunologically
active and that there is a correlation between the strength of the
5T4-specific antibody response and improved survival. Further there
are several studies searching for peptide vaccines using epitopes
being over-expressed in RCC.
[0074] Various approaches of tumor vaccines have been under
investigation. Studies using whole-tumor approaches, including
tumor cell lysates, fusions of dendritic cells with tumor cells, or
whole-tumor RNA were done in RCC patients, and remissions of tumor
lesions were reported in some of these trials (Avigan et al., 2004;
Holtl et al., 2002; Marten et al., 2002; Su et al., 2003; Wittig et
al., 2001).
[0075] Small Cell Lung Cancer
[0076] The treatment and prognosis of SCLC depend strongly on the
diagnosed cancer stage. Immune therapy presents an excessively
investigated field of cancer therapy. Various approaches are
studded in the treatment of SCLC. One of the approaches targets the
blocking of CTLA-4, a natural human immune suppressor. The
inhibition of CTLA-4 intends to boost the immune system to combat
the cancer. Recently, the development of promising immune check
point inhibitors for treatment of SCLC has been started. Another
approach is based on anti-cancer vaccines which is currently
available for treatment of SCLC in clinical studies (American
Cancer Society, 2015; National Cancer Institute, 2015).
[0077] Acute Myeloid Leukemia
[0078] One treatment option for AML is targeting CD33 with
antibody-drug conjugates (anti-CD33+calechiamicin, SGN-CD33a,
anti-CD33+actinium-225), bispecific antibodies (recognition of
CD33+CD3 (AMG 330) or CD33+CD16) and chimeric antigen receptors
(CARs) (Estey, 2014).
[0079] Non-Hodgkin Lymphoma
[0080] Treatment of NHL depends on the histologic type and stage
(National Cancer Institute, 2015): Spontaneous tumor regression can
be observed in lymphoma patients. Therefore, active immunotherapy
is a therapy option (Palomba, 2012).
[0081] An important vaccination option includes Id vaccines. B
lymphocytes express surface immunoglobulins with a specific amino
acid sequence in the variable regions of their heavy and light
chains, unique to each cell clone (=idiotype, Id). The idiotype
functions as a tumor associated antigen.
[0082] Passive immunization includes the injection of recombinant
murine anti-Id monoclonal antibodies alone or in combination with
IFNalpha, IL2 or chlorambucil.
[0083] Active immunization includes the injection of recombinant
protein (Id) conjugated to an adjuvant (KLH), given together with
GM-CSF as an immune adjuvant. Tumor-specific Id is produced by
hybridoma cultures or using recombinant DNA technology (plasmids)
by bacterial, insect or mammalian cell culture.
[0084] Three phase III clinical trials have been conducted
(Biovest, Genitope, Favrille). In two trials patients had received
rituximab. GM-CSF was administered in all three trials. Biovest
used hybridoma-produced protein, Genitope and Favrille used
recombinant protein. In all three trials Id was conjugated to KLH.
Only Biovest had a significant result.
[0085] Vaccines other than Id include the cancer-testis antigens
MAGE, NY-ESO1 and PASD-1, the B-cell antigen CD20 or cellular
vaccines. The latest mentioned consist of DCs pulsed with apoptotic
tumor cells, tumor cell lysate, DC-tumor cell fusion or DCs pulsed
with tumor-derived RNA.
[0086] In situ vaccination involves the vaccination with
intra-tumoral CpG in combination with chemotherapy or irradiated
tumor cells grown in the presence of GM-CSF and
collection/expansion/re-infusion of T cells.
[0087] Vaccination with antibodies that alter immunologic
checkpoints are comprised of anti-CD40, anti-OX40, anti-41BB,
anti-CD27, anti-GITR (agonist antibodies that directly enhance
anti-tumor response) or anti-PD1, anti-CTLA-4 (blocking antibodies
that inhibit the checkpoint that would hinder the immune response).
Examples are ipilimumab (anti-CTLA-4) and CT-011 (anti-PD1)
(Palomba, 2012).
[0088] Uterine Cancer
[0089] Treatment of endometrial carcinomas is stage-dependent. The
majority of endometrical carcinomas comprises of well to moderately
differentiated endometrioid adenocarcinomas which are usually
confined to the corpus uteri at diagnosis and can be cured by
hysterectomy (World Cancer Report, 2014).
[0090] Also therapies for cervical cancer depend on the stage. In
early stages, excision is the standard therapy which might be
combined with radio-(chemo-)therapy. Primary radio-(chemo-)therapy
is chosen at late stages (Stage III and higher), in cases with
lymph node infiltration or in cases in which the tumor can not be
excised.
[0091] There are also some immunotherapeutic approaches that are
currently being tested. In a Phase I/II Clinical Trial patients
suffering from uterine cancer were vaccinated with autologous
dendritic cells (DCs) electroporated with Wilms' tumor gene 1 (WT1)
mRNA. Besides one case of local allergic reaction to the adjuvant,
no adverse side effects were observed and 3 out of 6 patients
showed an immunological response (Coosemans et al., 2013).
[0092] As stated above, HPV infections provoke lesions that may
ultimately lead to cervical cancer. Therefore, the HPV viral
oncoproteins E6 and E7 that are are constitutively expressed in
high-grade lesions and cancer and and are required for the onset
and maintenance of the malignant phenotype are considered promising
targets for immunotherapeutic approaches (Hung et al., 2008; Vici
et al., 2014). One study performed Adoptive T-cell therapy (ACT) in
patients with metastatic cervical cancer. Patients receive an
infusion with E6 and E7 reactive tumor-infiltrating T cells (TILs)
resulting in complete regression in 2 and a patial response in 1
out of 9 patients (Stevanovic et al., 2015). Furthermore, an
intracellular antibody targeting E7 was reported to block tumor
growth in mice (Accardi et al., 2014). Also peptide, DNA and
DC-based vaccines targing HPV E6 and E7 are in clinical trials
(Vici et al., 2014).
[0093] Gallbladder Adenocarcinoma and Cholangiocarcinoma
[0094] Cholangiocarcinoma is mostly identified in advanced stages
because it is difficult to diagnose. Cholangiocarcinoma is
difficult to treat and is usually lethal.
[0095] Gallbladder cancer is the most common and aggressive
malignancy of the biliary tract worldwide.
[0096] Urinary Bladder Cancer
[0097] The standard treatment for bladder cancer includes surgery,
radiation therapy, chemotherapy and immunotherapy.
[0098] At stage 0 and I, the bladder cancer is typically treated by
transurethral resection potentially followed by intravesical
chemotherapy and optionally combined with intravesical
immunotherapeutic treatment with BCG (bacillus
Calmette-Guerin).
[0099] An effective immunotherapeutic approach is established in
the treatment of aggressive non-muscle invasive bladder cancer
(NMIBC). Thereby, a weakened form of the bacterium Mycobacterium
bovis (bacillus Calmette-Guerin=BCG) is applied as an intravesical
solution. The major effect of BCG treatment is a significant
long-term (up to 10 years) protection from cancer recurrence and
reduced progression rate. In principle, the treatment with BCG
induces a local inflammatory response which stimulates the cellular
immune response. The immune response to BCG is based on the
following key steps: infection of urothelial and bladder cancer
cells by BCG, followed by increased expression of
antigen-presenting molecules, induction of immune response mediated
via cytokine release, induction of antitumor activity via
involvement of various immune cells (thereunder cytotoxic T
lymphocytes, neutrophils, natural killer cells, and macrophages)
(Fuge et al., 2015; Gandhi et al., 2013).
[0100] BCG treatment is in general well tolerated by patients but
can be fatal especially by the immunocompromised patients. BCG
refractory is observed in about 30-40% of patients (Fuge et al.,
2015; Steinberg et al., 2016a). The treatment of patients who
failed the BCG therapy is challenging. The patients who failed the
BCG treatment are at high risk for developing of muscle-invasive
disease. Radical cystectomy is the preferable treatment option for
non-responders (Steinberg et al., 2016b; von Rundstedt and Lerner,
2015). The FDA approved second line therapy of BCG-failed NMIBC for
patients who desire the bladder preservation is the
chemotherapeutic treatment with valrubicin. A number of other
second line therapies are available or being currently under
investigation as well, thereunder immunotherapeutic approaches like
combined BCG-interferon or BCG-check point inhibitor treatments,
pre-BCG transdermal vaccination, treatment with Mycobacterium phlei
cell wall-nucleic acid (MCNA) complex, mono- or combination
chemotherapy with various agents like mitomycin C, gemcitabine,
docetaxel, nab-paclitaxel, epirubicin, mitomycin/gemcitabine,
gemcitabine/docetaxel, and device-assisted chemotherapies like
thermochemo-, radiochemo-, electromotive or photodynamic therapies
(Fuge et al., 2015; Steinberg et al., 2016b; von Rundstedt and
Lerner, 2015). Further evaluation of available therapies in
clinical trials is still required.
[0101] The alternative treatment options for advanced bladder
cancer are being investigated in ongoing clinical trials. The
current clinical trials focused on the development of molecularly
targeted therapies and immunotherapies. The targeted therapies
investigate the effects of cancerogenesis related pathway
inhibitors (i.e. mTOR, vascular endothelial, fibroblast, or
epidermal growth factor receptors, anti-angiogenesis or cell cycle
inhibitors) in the treatment of bladder cancer. The development of
molecularly targeted therapies remains challenging due to high
degree of genetic diversity of bladder cancer. The main focus of
the current immunotherapy is the development of checkpoint blockage
agents like anti-PD1 monoclonal antibody and adoptive T-cell
transfer (Knollman et al., 2015b; Grivas et al., 2015; Jones et
al., 2016; Rouanne et al., 2016).
[0102] Head and Neck Squamous Cell Carcinoma
[0103] Head and neck squamous cell carcinomas (HNSCC) are
heterogeneous tumors with differences in epidemiology, etiology and
treatment (Economopoulou et al., 2016).
[0104] HNSCC is considered an immunosuppressive disease,
characterized by the dysregulation of immunocompetent cells and
impaired cytokine secretion (Economopoulou et al., 2016).
Immunotherapeutic strategies differ between HPV-negative and
HPV-positive tumors.
[0105] In HPV-positive tumors, the viral oncoproteins E6 and E7
represent good targets, as they are continuously expressed by tumor
cells and are essential to maintain the transformation status of
HPV-positive cancer cells. Several vaccination therapies are
currently under investigation in HPV-positive HNSCC, including DNA
vaccines, peptide vaccines and vaccines involving dendritic cells
(DCs). Additionally, an ongoing phase II clinical trial
investigates the efficacy of lymphodepletion followed by autologous
infusion of TILs in patients with HPV-positive tumors
(Economopoulou et al., 2016).
[0106] In HPV-negative tumors, several immunotherapeutic strategies
are currently used and under investigation. The chimeric IgG1
anti-EGFR monoclonal antibody cetuximab has been approved by the
FDA in combination with chemotherapy as standard first line
treatment for recurring/metastatic HNSCC. Other anti-EGFR
monoclonal antibodies, including panitumumab, nimotuzumab and
zalutumumab, are evaluated in HNSCC. Several immune checkpoint
inhibitors are investigated in clinical trials for their use in
HNSCC. They include the following antibodies: Ipilimumab
(anti-CTLA-4), tremelimumab (anti-CTLA-4), pembrolizumab
(anti-PD-1), nivolumab (anti-PD-1), durvalumab (anti-PD-1),
anti-KIR, urelumab (anti-CD137), and anti-LAG-3.
[0107] Two clinical studies with HNSCC patients evaluated the use
of DCs loaded with p53 peptides or apoptotic tumor cells. The
immunological responses were satisfactory and side effects were
acceptable.
[0108] Several studies have been conducted using adoptive T cell
therapy (ACT). T cells were induced against either irradiated
autologous tumor cells or EBV. Results in disease control and
overall survival were promising (Economopoulou et al., 2016).
[0109] 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 hepatocellular
carcinoma (HCC), colorectal carcinoma (CRC), glioblastoma (GB),
gastric cancer (GC), esophageal cancer, non-small cell lung cancer
(NSCLC), pancreatic cancer (PC), renal cell carcinoma (RCC), benign
prostate hyperplasia (BPH), prostate cancer (PCA), ovarian cancer
(OC), melanoma, breast cancer, chronic lymphocytic leukemia (CLL),
Merkel cell carcinoma (MCC), small cell lung cancer (SCLC),
Non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML),
gallbladder cancer and cholangiocarcinoma (GBC, CCC), urinary
bladder cancer (UBC), uterine cancer (UEC), head and neck squamous
cell carcinoma (HNSCC), in particular. There is also a need to
identify factors representing biomarkers for cancer in general and
the above-mentioned cancer types in particular, leading to better
diagnosis of cancer, assessment of prognosis, and prediction of
treatment success.
[0110] 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.
[0111] The current classification of tumor associated antigens
(TAAs) comprises the following major groups:
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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.
[0122] 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.
[0123] 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).
[0124] 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).
[0125] Elongated peptides of the invention can act as MHC class II
active epitopes.
[0126] 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.
[0127] 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).
[0128] 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).
[0129] 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.
[0130] 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-1-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.
[0131] 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).
[0132] 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.
[0133] 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.
[0134] 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").
[0135] 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
[0136] 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:
388 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: 388, 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.
[0137] While the most important criterion for a peptide to function
as cancer therapy target is its over-presentation on primary tumor
tissues as compared to normal tissues, also the RNA expression
profile of the corresponding gene can help to select appropriate
peptides. Particularly, some peptides are hard to detect by mass
spectrometry, either due to their chemical properties or to their
low copy numbers on cells, and a screening approach focusing on
detection of peptide presentation may fail to identify these
targets. However, these targets may be detected by an alternative
approach starting with analysis of gene expression in normal
tissues and secondarily assessing peptide presentation and gene
expression in tumors. This approach was realized in this invention
using an mRNA database (Lonsdale, 2013) in combination with further
gene expression data (including tumor samples), as well as peptide
presentation data. If the mRNA of a gene is nearly absent in normal
tissues, especially in vital organ systems, targeting the
corresponding peptides by even very potent strategies (such as
bispecific affinity-optimized antibodies or T-cell receptors), is
more likely to be safe. Such peptides, even if identified on only a
small percentage of tumor tissues, represent interesting targets.
Routine mass spectrometry analysis is not sensitive enough to
assess target coverage on the peptide level. Rather, tumor mRNA
expression can be used to assess coverage. For detection of the
peptide itself, a targeted mass spectrometry approach with higher
sensitivity than in the routine screening may be necessary and may
lead to a better estimation of coverage on the level of peptide
presentation.
[0138] 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: 388 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: 388, 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.
[0139] 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
1A and Table 2A bind to HLA-A*02. Peptides in Table 1C and Table 2B
bind to HLA-A*24. Peptides in Table 1B bind to HLA class II
alleles. The peptides in Table 3 are additional peptides that are
HLA-A*24 binding and may be useful in combination with the other
peptides of the invention.
TABLE-US-00001 TABLE 1A Peptides according to the present
invention, HLA-A*02-binding. SEQ ID No. Sequence GeneID(s) Official
Gene Symbol(s) 1 PLWGKVFYL 10926 DBF4 2 ALYGKLLKL 157680 VPS13B 3
TLLGKQVTL 157680 VPS13B 4 ELAEIVFKV 203427, 349075, 51373 SLC25A43,
ZNF713, MRPS17 5 SLFGQEVYC 10840 ALDH1L1 6 FLDPAQRDL 57677 ZFP14 7
AAAAKVPEV 23382 AHCYL2 8 KLGPFLLNA 100508781, 653199, FAM115B,
FAM115A 9747 9 FLGDYVENL 54832 VPS13C 10 KTLDVFNIIL 54832 VPS13C 11
GVLKVFLENV 121504, 554313, 8294, HIST4H4, HIST2H4B, 12 GLIYEETRGV
8359, 8360, 8361, HIST1H41, HIST1H4A, 13 VLRDNIQGI 8362, 8363,
8364, 8365, HIST1H4D, HIST1H4F, 8366, 8367, 8368, HIST1H4K,
HIST1H4J, 8370 HIST1H4C, HIST1H4H, HIST1H4B, HIST1H4E, HIST1H4L,
HIST2H4A 14 LLDHLSFINKI 64863 METTL4 15 ALGDYVHAC 4588 MUC6 16
HLYNNEEQV 101060798, 1645, 8644 AKR1C1, AKR1C3 17 ILHEHHIFL 4233
MET 18 YVLNEEDLQKV 4233 MET 19 TLLPTVLTL 127707 KLHDC7A 20
ALDGHLYAI 127707 KLHDC7A 21 SLYHRVLLY 57221 KIAA1244 22 MLSDLTLQL
57221 KIAA1244 23 AQTVVVIKA 101059911, 4586, 727897 MUC5AC, MUC5B
24 FLWNGEDSAL 4586, 727897 MUC5AC, MUC5B 25 IQADDFRTL 101059911,
4586, 727897 MUC5AC, MUC5B 26 KVDGVVIQL 101059911, 4586, 727897
MUC5AC, MUC5B 27 KVFGDLDQV 169611 OLFML2A 28 TLYSMDLMKV 169611
OLFML2A 29 TLCNKTFTA 26137 ZBTB20 30 TVIDECTRI 26137 ZBTB20 31
ALSDETKNNWE 5591 PRKDC V 32 ILADEAFFSV 5591 PRKDC 33 LLLPLLPPLSPS
347252 IGFBPL1 LG 34 LLPKKTESHHKT 8330, 8331 HIST1H2AK, HIST1H2AJ
35 YVLPKLYVKL 100128168, 100996747, RP526P39, RPS26P11, 441502,
6231, 643003, RP526, RP526P28, 644166, 644928, RP526P20, RP526P15,
644934, 646753, RP526P50, RP526P2, 728937, 729188 RP526P25,
RP526P58 36 KLYGIEIEV 56107 PCDHGA9 37 ALINDILGELVKL 85463 ZC3H12C
38 KMQEDLVTL 781 CACNA2D1 39 ALMAVVSGL 55103 RALGPS2 40 SLLALPQDLQA
1364, 1365, 23562, CLDN4, CLDN3, CLDN14, 9074, 9080 CLDN6, CLDN9 41
FVLPLVVTL 2848 GPR25 42 VLSPFILTL 113730 KLHDC7B 43 LLWAGPVTA 28603
TRBV6-4 44 GLLWQIIKV 5357 PLS1 45 VLGPTPELV 100124692 46 SLAKHGIVAL
10693 CCT6B 47 GLYQAQVNL 89886 SLAMF9 48 TLDHKPVTV 203447 NRK 49
LLDESKLTL 64097 EPB41L4A 50 EYALLYHTL 26 ABP1 51 LLLDGDFTL 347051
SLC10A5 52 ELLSSIFFL 160418 TMTC3 53 SLLSHVIVA 545 ATR 54
FINPKGNWLL 3673 ITGA2 55 IASAIVNEL 57448 BIRC6 56 KILDLTRVL 79783
C7orf10 57 VLISSTVRL 166379 BBS12 58 ALDDSLTSL 2302 FOXJ1 59
ALTKILAEL 339766 MROH2A 60 FLIDTSASM 203522, 26512 DDX26B, INTS6 61
HLPDFVKQL 9857 CEP350 62 SLFNQEVQI 100528032, 22914, KLRK1, KLRC4
8302 63 TLSSERDFAL 100293534, 720, 721 C4A, C4B 64 GLSSSSYEL 89866
SEC16B 65 KLDGICWQV 733 C8G 66 FITDFYTTV 80055 PGAP1 67 GVIETVTSL
79895 ATP8B4 68 ALYGFFFKI 118663 BTBD16 69 GIYDGILHSI 158809,
392433 MAGEB6, MAGEB6P1 70 GLFSQHFNL 1789 DNMT3B 71 GLITVDIAL 84162
KIAA1109 72 GMIGFQVLL 6006, 6007 RHCE, RHD 73 GVPDTIATL 23120
ATP10B 74 ILDETLENV 167227 DCP2 75 ILDNVKNLL 4602 MYB 76
ILLDESNFNHFL 222584 FAM83B 77 IVLSTIASV 10559, 154313 SLC35A1,
C6orf165 78 LLWGHPRVA 25878 MXRA5 79 SLVPLQILL 101060288,
101060295, PRAMEF5, PRAMEF9, 101060308, 343068, PRAMEF4, PRAMEF11,
343070, 400735, PRAMEF6, PRAMEF15, 440560, 440561, 441873, PRAMEF23
645359, 653619, 729368 80 TLDEYLTYL 101060308, 343068, PRAMEF5,
PRAMEF9, 343070, 653619 PRAMEF15 81 VLFLGKLLV 204962 SLC44A5 82
VLLRVLIL 102 ADAM10 83 ELLEYLPQL 5288 PIK3C2G 84 FLEEEITRV 6570
SLC18A1 85 STLDGSLHAV 2081 ERNI 86 LLVTSLVVV 118471, 118472 PRAP1,
ZNF511 87 YLTEVFLHVV 55024 BANK1 88 ILLNTEDLASL 388015 RTL1 89
YLVAHNLLL 9365 KL 90 GAVAEEVLSSI 340273 ABCB5 91 SSLEPQIQPV 23029
RBM34 92 LLRGPPVARA 3486 IGFBP3 93 SLLTQPIFL 151295 SLC23A3
TABLE-US-00002 TABLE 1B Peptides according to the present
invention, HLA class II binding. SEQ Official ID Gene No. Sequence
GeneID(s) Symbol(s) 94 LKMENKEVLPQLVDAVTS 4547 MTTP 95
GLYLPLFKPSVSTSKAIGGGP 10165 SLC25A13
TABLE-US-00003 TABLE 1C Peptides according to the present
invention, HLA-A*24 binding. SEQ ID Official Gene No. Sequence
GeneID(s) Symbol(s) 96 YYTQYSQTI 25878 MXRA5 97 TYTFLKETF 203238
CCDC171 98 VFPRLHNVLF 9816 URB2 99 QYILAVPVL 91147 TMEM67 100
VYIESRIGT 10112 KIF20A STSF 101 IYIPVLPPHL 163486 DENND1B 102
VYPFENFEF 127700 OSCP1 103 NYIPVKNGKQF 3096 HIVEP1 104 SYLTWHQQI
125919 ZNF543 105 IYNETITDLL 1062 CENPE 106 IYNETVRDLL 3833 KIFC1
107 KYFPYLVVI 80131 LRRC8E 108 PYLVVIHTL 80131 LRRC8E 109 LFITGGQFF
114134 SLC2A13 110 SYPKIIEEF 2177 FANCD2 111 VYVQILQKL 4998 ORC1
112 IYNFVESKL 4998 ORC1 113 IYSFHTLSF 55183 RIF1 114 QYLDGTWSL
55083 KIF26B 115 RYLNKSFVL 63926 ANKRD5 116 AYVIAVHLF 10178 TENM1
117 IYLSDLTYI 55103 RALGPS2 118 KYLNSVQYI 55103 RALGPS2 119
VYRVYVTTF 57089 ENTPD7 120 GYIEHFSLW 5069 PAPPA 121 RYGLPAAWSTF
79713 IGFLR1 122 EYQARIPEF 55758 RCOR3 123 VYTPVLEHL 5591 PRKDC 124
TYKDYVDLF 5591 PRKDC 125 VFSRDFGLLVF 5591 PRKDC 126 PYDPALGSPSRLF
389058 SPS 127 QYFTGNPLF 3237 HOXD11 128 VYPFDWQYI 7941 PLA2G7 129
KYIDYLMTW 55233, 92597 MOB1A, MOB1B 130 VYAHIYHQHF 55233, 92597
MOB1A, MOB1B 131 EYLDRIGQLFF 51608 GET4 132 RYPALFPVL 11237 RNF24
133 KYLEDMKTYF 5273 SERPINB10 134 AYIPTPIYF 81796 SLCO5A1 135
VYEAMVPLF 85465 EPT1 136 IYPEWPVVFF 51146 A4GNT 137 EYLHNCSYF
25909, 285116 AHCTF1, AHCTF1P1 138 VYNAVSTSF 79915 ATAD5 139
IFGIFPNQF 79895 ATP8B4 140 RYLINSYDF 84002 B3GNT5 141 SYNGHLTIWF
56245 C21orf62 142 VYVDDIYVI 57082 CASC5 143 KYIFQLNEI 347475
CCDC160 144 VFASLPGFLF 1233 CCR4 145 VYALKVRTI 1237 CCR8 146
NYYERIHAL 8832 CD84 147 LYLAFPLAF 253782 CERS6 148 SYGTVSQIF 23601
CLEC5A 149 SYGTVSQI 23601 CLEC5A 150 IYITRQFVQF 81501 DCSTAMP 151
AYISGLDVF 8632 DNAH17 152 KFFDDLGDELLF 8632 DNAH17 153
VYVPFGGKSMITF 146754 DNAH2 154 VYGVPTPHF 151651 EFHB 155 IYKWITDNF
2302 FOXJ1 156 YYMELTKLLL 51659 GINS2 157 DYIPASGFALF 84059 GPR98
158 IYEETRGVL 121504, HIST4H4, KVF 554313, HIST2H4B, HIST1H41,
HIST1H4A, HIST1H4D, HIST1H4F, 159 IYEETRGVL 8294, 8359, HIST1H4K,
8360, HIST1H4J, 8361, 8362, HIST1H4C, 8363, 8364, HIST1H4H, 8365,
8366, HIST1H4B, 8367, 8368, HIST1H4E, 8370 HIST1H4L, HIST2H4A 160
RYGDGGSSF 3188 HNRNPH2 161 KYPDIVQQF 29851 ICOS 162 KYTSYILAF 3458
IFNG 163 RYLTISNLQF 28785 IGLV4-60 164 HYVPATKVF 259307 IL411 165
EYFTPLLSGQF 55175 KLHL11 166 FYTLPFHLI 55175 KLHL11 167 RYGFYYVEF
197021 LCTL 168 RYLEAALRL 10609 LEPREL4 169 NYITGKGDVF 84125 LRRIQ1
170 QYPFHVPLL 4049 LTA 171 NYEDHFPLL 4109 MAGEA10 172 VFIFKGNEF
4319 MMP10 173 QYLEKYYNL 4319 MMP10 174 VYEKNGYIYF 4322 MMP13 175
LYSPVPFTL 387521 TMEM189 176 FYINGQYQF 55728 N4BP2 177 VYFKAGLDVF
254827 NAALADL2 178 NYSSAVQKF 4983 OPHN1 179 TYIPVGLGRLL 58495
OVOL2 180 KYLQVVGMF 5021 OXTR 181 VYPPYLNYL 5241 PGR 182 AYAQLGYLLF
9033 PKD2L1 183 PYLQDVPRI 92340 C17orf72 184 IYSVGAFENF 389677
RBM12B 185 QYLVHVNDL 23322 RPGRIP1L 186 VFTTSSNIF 10371 SEMA3A 187
AYAANVHYL 151473 SLC16A14 188 GYKTFFNEF 64078 SLC28A3 189 AYFKQSSVF
54790 TET2 190 LYSELTETL 54790 TET2 191 TYPDGTYTG 201633 TIGIT RIF
192 RYSTFSEIF 8277 TKTL1 193 LYLENNAQTQF 8626 TP63 194 VYQSLSNSL
286827 TRIM59 195 AYIKGGWIL 125488 TTC39C 196 GYIRGSWQF 79465 ULBP3
197 IFTDIFHYL 54464 XRN1 198 DYVGFTLKI 19 ABCA1 199 SYLNHLNNL
154664 ABCA13 200 VFIHHLPQF 116285 ACSM1 201 GYNPNRVFF 158067 AK8
202 RYVEGIVSL 246 ALOX15 203 VYNVEVKNAEF 84250 ANKRD32 204
EYLSTCSKL 196528 ARID2 205 VYPVVLNQI 79798 ARMC5 206 NYLDVATFL
10973 ASCC3 207 LYSDAFKFIVF 344905 ATP13A5 208 TYLEKIDGF 100526740,
ATP5J2-PTCD1, 26024, 9551 PTCD1, ATP5J2
209 AFIETPIPLF 631 BFSP1 210 IYAGVGEFSF 701 BUB1B 211 VFKSEGAYF
375444 C5orf34 212 SYAPPSEDLF 100533105, SGK3 23678 213 SYAPPSEDLFL
100533105, SGK3 23678 214 KYLMELTLI 9133 CCNB2 215 SYVASFFLL 9398
CD101 216 FYVNVKEQF 79682 MLF1IP 217 IYISNSIYF 54967 CXorf48 218
LYSELNKWSF 1591 CYP24A1 219 SYLKAVFNL 163720, CYP4Z2P, CYP4Z1
199974 220 SYSEIKDFL 64421 DCLRE1C 221 KYIGNLDLL 8701 DNAH11 222
HYSTLVHMF 8701 DNAH11 223 TFITQSPLL 1767 DNAH5 224 PYFFANQEF 79843
FAM124B 225 TYTNTLERL 55719 FAM178A 226 MYLKLVQLF 2175 FANCA 227
IYRFITERF 2301 FOXE3 228 IYQYVADNF 2299 FOXI1 229 IYQFVADSF 344167
FOXI3 230 TYGMVMVTF 84059 GPR98 231 AFADVSVKF 84059 GPR98 232
YYLSDSPLL 51512 GTSE1 233 QYLTAAALHNL 3552 ILIA 234 SYLPAIWLL 3641
INSL4 235 VYKDSIYYI 84541 KBTBD8 236 VYLPKIPSW 157855 KCNU1 237
KYVGQLAVL 9928 KIF14 238 SYLEKVRQL 100653049, KRT31, KRT33A, 3881,
KRT33B, KRT34, 3883, 3884, 3885, 3886 KRT35 239 VYAIFRILL 987 LRBA
240 YYFFVQEKI 84944 MAEL 241 SYVKVLHHL 101060230, MAGEA12 4111 242
VYGEPRELL 392555, MAGEC2 51438 243 SYLELANTL 4163 MCC 244 VHFEDTGKT
4322 MMP13 LLF 245 LYPQLFVVL 377711, MR0H1 727957 246 KYLSVQLTL
339766 MR0H2A 247 SFTKTSPNF 200958 MUC20 248 AFPTFSVQL 4588 MUC6
249 RYHPTTCTI 4608 MYBPH 250 KYPDIASPTF 89795 NAV3 251 VYTKALSSL
64151 NCAPG 252 AFGQETNV 4695 NDUFA2 PLNNF 253 IYGFFNENF 10886
NPFFR2 254 KYLESSATF 91181 NUP210L 255 VYQKIILKF 139135 PASD1 256
VFGKSAYLF 118987 PDZD8 257 IFIDNSTQP 5288 PIK3C2G LHF 258 AYAQLGYLL
9033 PKD2L1 259 YFIKSPPSQLF 79949 PLEKHS1 260 VYMNVMTRL 5523
PPP2R3A 261 GYIKLINFI 10196 PRMT3 262 VYSSQFETI 23362 PSD3 263
RYILENHDF 442247 RFPL4B 264 LYTETRLQF 26150 RIBC2 265 SYLNEAFSF
286205 SCAI 266 KYTDVVTEFL 57713 SFMBT2 267 SFLNIEKTEI 347051
SLC10A5 LF 268 IFITKALQI 159371 SLC35G1 269 QYPYLQAFF 146857 SLFN13
270 YYSQESKVLYL 55181 SMG8 271 RFLMKSYSF 8435 SOAT2 272 RYVFPLPYL
8403 SOX14 273 IYGEKLQFIF 57405 SPC25 274 KQLDIANYELF 51430 SUCO
275 KYGTLDVTF 255928 SYT14 276 QYLDVLHAL 51256 TBC1D7 277 FYTFPFQQL
6996 TDG 278 KYVNLVMYF 116238 TLCD1 279 VWLPASVLF 85019 TMEM241 280
TYNPNLQDKL 5651 TMPRSS15 281 NYSPGLVSLIL 28677 TRAV9-2 282
NYLVDPVTI 129868, 653192 TRIM43, TRIM43B 283 EYQEIFQQL 129868,
653192 TRIM43, TRIM43B 284 DYLKDPVTI 391712, 653794 TRIM61,
TRIM60P14 285 VYVGDALLHAI 7223 TRPC4 286 SYGTILSHI 54986 ULK4 287
IYNPNLLTAS 81839 VANGL1 KF 288 VYPDTVALTF 284403 WDR62 289
FFHEGQYVF 389668 XKR9 290 KYGDFKLLEF 143570 XRRA1 291 YYLGSGRETF
152002 )0(YLT1 292 FYPQIINTF 79776 ZFHX4 293 VYPHFSTTNLI 79776
ZFHX4 294 RFPVQGTVTF 79818 ZNF552 295 SYLVIHERI 84775 ZNF607 296
SYQVIFQHF 344905 ATP13A5 297 TYIDTRTVF 827 CAPN6 298 AYKSEVVYF
441402, 728577, CNTNAP3B, 79937 CNTNAP3 299 KYQYVLNEF 400823
FAM177B 300 TYPSQLPSL 26290 GALNT8 301 KFDDVTMLF 2977 GUCY1A2 302
LYLPVHYGF 253012 HEPACAM2 303 LYSVIKEDF 285600 KIAA0825 304
EYNEVANLF 57097 PARP11 305 NYENKQYLF 144406 WDR66 306 VYPAEQPQI
2334 AFF2 307 GYAFTLPLF 440138 ALG11 308 TFDGHGVFF 29785 CYP2S1 309
KYYRQTLLF 27042 DIEXF 310 IYAPTLLVF 23341 DNAJC16 311 EYLQNLNHI
79659 DYNC2H1 312 SYTSVLSRL 57724 EPG5 313 KYTHFIQSF 26301 GBGT1
314 RYFKGDYSI 3709 ITPR2 315 FYIPHVPVSF 89866 SEC16B 316 VYFEGSDFKF
55164 SHQ1 317 VFDTSIAQLF 6477 SIAH1 318 TYSNSAFQYF 28672 TRAV12-3
319 KYSDVKNLI 57623 ZFAT 320 KFILALKVLF 6790 AURKA
TABLE-US-00004 TABLE 2A Additional peptides according to the
present invention, HLA-A*02-binding. SEQ ID Official Gene No.
Sequence GeneID(s) Symbol(s) 321 SLWFKPEEL 4831, 654364 NME2,
NME1-NME2 322 ALVSGGVAQA 64326 RFWD2 323 ILSVVNSQL 80183 KIAA0226L
324 AIFDFCPSV 23268 DNMBP 325 RLLPKVQEV 168417, 89958 ZNF679,
SAPCD2 326 SLLPLVWKI 1130 LYST 327 SIGDIFLKY 1894 ECT2 328
SVDSAPAAV 10635 RAD51AP1 329 FAWEPSFRD 1244 ABCC2 QV 330 FLWPKEVEL
146206 RLTPR 331 AIWKELISL 55183 RIF1 332 AVTKYTSAK 54145, 85236,
H2BFS, HIST1H2BK, 8970 HIST1H2BJ 333 GTFLEGVAK 126328 NDUFA11 334
GRADALRVL 79713 IGFLR1 335 VLLAAGPSAA 23225 NUP210 336 GLMDGSPHFL
157680 VPS13B 337 KVLGKIEKV 987 LRBA 338 LLYDGKLSSA 987 LRBA 339
VLGPGPPPL 254359 ZDHHC24 340 SVAKTILKR 55233, 92597 MOB1A,
MOB1B
TABLE-US-00005 TABLE 2B Additional peptides according to the
present invention, HLA-A*24-binding. SEQ ID Official Gene No.
Sequence GeneID(s) Symbol(s) 341 SYLTQHQRI 162655, ZNF519, ZNF264
344065, 9422 342 NYAFLHRTL 200316, 9582 APOBEC3F, APOBEC3B 343
NYLGGTSTI 367 AR 344 EYNSDLHQF 699 BUB1 345 EYNSDLHQFF 699 BUB1 346
IYVIPQPHF 57082 CASC5 347 VYAEVNSL 1459 CSNK2A2 348 IYLEHTESI 2177
FANCD2 349 QYSIISNVF 28982 FLVCR1 350 KYGNFIDKL 85865 GTPBP10 351
IFHEVPLKF 728432, 79664 NARG2 352 QYGGDLTNTF 3673 ITGA2 353
TYGKIDLGF 57650 KIAA1524 354 VYNEQIRDLL 81930 KIF18A 355 IYVTGGHLF
113730 KLHDC7B 356 NYMPGQLTI 346389 MACC1 357 QFITSTNTF 94025 MUC16
358 YYSEVPVKL 25878 MXRA5 359 NYGVLHVTF 204801 NLRP11 360 VFSPDGHLF
143471, 5688 PSMA8, PSMA7 361 TYADIGGLDNQI 5700 PSMC1 362 VYNYAEQTL
100526737, RBM14-RBM4, 10432, 5936 RBM14, RBM4 363 SYAELGTTI 23657
SLC7A11 364 KYLNENQLSQL 6491 STIL 365 VFIDHPVHL 26011 TENM4 366
QYLELAHSL 4796 TONSL 367 LYQDHMQYI 7474 WNT5A 368 KYQNVKHNL 79830
ZMYM1 369 VYTHEVVTL 983 CDK1 370 RFIGIPNQF 79659 DYNC2H1 371
AYSHLRYVF 2195 FAT1 372 VYVIEPHSMEF 23225 NUP210 373 GYISNGELF
116143, WDR92, PPP3R1, 5534, 5535 PPP3R2 374 VFLPRVTEL 5591 PRKDC
375 KYTDYILKI 374462 PTPRQ 376 VYTPVASRQSL 56852 RAD18 377
QYTPHSHQF 57521 RPTOR 378 VYIAELEKI 27127 SMC1B 379 VFIAQGYTL
160418 TMTC3 380 VYTGIDHHW 25879 DCAF13 381 KYPASSSVF 3217 HOXB7
382 AYLPPLQQVF 26523 ElF2C1 383 RYKPGEPITF 163486 DENND1B 384
RYFDVGLHNF 55733 HHAT 385 QYIEELQKF 55103 RALGPS2 386 TFSDVEAHF
55609 ZNF280C 387 KYTEKLEEI 95681 CEP41 388 IYGEKTYAF 5273
SERPINB10
TABLE-US-00006 TABLE 3 Peptides useful for cancer therapies
according to the invention, e.g. personalized cancer therapies. SEQ
ID Official Gene No. Sequence GeneID(s) Symbol(s) 389 EYLPEFLHTF
154664 ABCA13 390 RYLWATVTI 259266 ASPM 391 LYQILQGIVF 983 CDK1 392
RYLDSLKAIVF 55839 CENPN 393 KYIEAIQWI 81501 DCSTAMP 394 FYQPKIQQF
55215 FANCI 395 LYINKANIW 55632 G2E3 396 YYHFIFTTL 2899 GRIK3 397
IYNGKLFDL 11004 KIF2C 398 IYNGKLFDLL 11004 KIF2C 399 SYIDVLPEF 4233
MET 400 KYLEKYYNL 4312 MMP1 401 VFMKDGFFYF 4312 MMP1 402 VWSDVTPLTF
4320 MMP11 403 TYKYVDINTF 4321 MMP12 404 RYLEKFYGL 4321 MMP12 405
NYPKSIHSF 4321 MMP12 406 TYSEKTTLF 94025 MUC16 407 VYGIRLEHF 83540
NUF2 408 QYASRFVQL 10733 PLK4 409 YFISHVLAF 6241 RRM2 410 RFLSGIINF
83540 NUF2 411 VYIGHTSTI 23499, 93035 MACF1, PKHD1L1 412 SYNPLWLRI
259266 ASPM 413 NYLLYVSNF 4486 MST1R 414 MYPYIYHVL 54954 FAM120C
415 SYQKVIELF 55872 PBK 416 AYSDGHFLF 26011 TENM4 417 VYKVVGNLL
128239 IQGAP3
[0140] 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,
glioblastoma (GB), breast cancer (BRCA), colorectal cancer (CRC),
renal cell carcinoma (RCC), chronic lymphocytic leukemia (CLL),
hepatocellular carcinoma (HCC), non-small cell and small cell lung
cancer (NSCLC, SCLC), Non-Hodgkin lymphoma (NHL), acute myeloid
leukemia (AML), ovarian cancer (OC), pancreatic cancer (PC),
prostate cancer (PCA), esophageal cancer including cancer of the
gastric-esophageal junction (OSCAR), gallbladder cancer and
cholangiocarcinoma (GBC, CCC), melanoma (MEL), gastric cancer (GC),
testis cancer (TC), urinary bladder cancer (UBC), head-and neck
squamous cell carcinoma (HNSCC), and uterine cancer (UEC).
[0141] 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: 388. More preferred
are the peptides--alone or in combination--selected from the group
consisting of SEQ ID NO: 1 to SEQ ID NO: 295 (see Table 1A, B, C),
and their uses in the immunotherapy of glioblastoma, breast cancer,
colorectal cancer, renal cell carcinoma, chronic lymphocytic
leukemia, hepatocellular carcinoma, non-small cell and small cell
lung cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian
cancer, pancreatic cancer, prostate cancer, esophageal cancer
including cancer of the gastric-esophageal junction, gallbladder
cancer and cholangiocarcinoma, melanoma, gastric cancer, testis
cancer, urinary bladder cancer, head-and neck squamous cell
carcinoma, or uterine cancer.
[0142] Particularly preferred are the peptides--alone or in
combination--according to the present invention selected from the
group consisting of SEQ ID NO: 70, 80, 323, and 325. More preferred
are the peptides--alone or in combination--selected from the group
consisting of SEQ ID NO: 70, 80, 323, and 325, and their uses in
the immunotherapy of glioblastoma, breast cancer, colorectal
cancer, renal cell carcinoma, chronic lymphocytic leukemia,
hepatocellular carcinoma, non-small cell and small cell lung
cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian
cancer, pancreatic cancer, prostate cancer, esophageal cancer
including cancer of the gastric-esophageal junction, gallbladder
cancer and cholangiocarcinoma, melanoma, gastric cancer, testis
cancer, urinary bladder cancer, head- and neck squamous cell
carcinoma, or uterine cancer.
[0143] Also preferred are the peptides--alone or in
combination--according to the present invention selected from the
group consisting of SEQ ID NO: 391, and 403. More preferred are the
peptides--alone or in combination--selected from the group
consisting of SEQ ID NO: 391, and 403, and their uses in the
immunotherapy of glioblastoma, breast cancer, colorectal cancer,
renal cell carcinoma, chronic lymphocytic leukemia, hepatocellular
carcinoma, non-small cell and small cell lung cancer, Non-Hodgkin
lymphoma, acute myeloid leukemia, ovarian cancer, pancreatic
cancer, prostate cancer, esophageal cancer including cancer of the
gastric-esophageal junction, gallbladder cancer and
cholangiocarcinoma, melanoma, gastric cancer, testis cancer,
urinary bladder cancer, head-and neck squamous cell carcinoma, or
uterine cancer.
[0144] As shown in Example 1, many of the peptides according to the
present invention are found on various tumor types and can, thus,
also be used in the immunotherapy of a variety of indications.
Over-expression of the underlying polypeptides in a variety of
cancers, as shown in Example 2, hints towards the usefulness of
these peptides in various other oncological indications.
[0145] 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 glioblastoma, breast cancer, colorectal
cancer, renal cell carcinoma, chronic lymphocytic leukemia,
hepatocellular carcinoma, non-small cell and small cell lung
cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian
cancer, pancreatic cancer, prostate cancer, esophageal cancer
including cancer of the gastric-esophageal junction, gallbladder
cancer and cholangiocarcinoma, melanoma, gastric cancer, testis
cancer, urinary bladder cancer, head-and neck squamous cell
carcinoma, or uterine cancer.
[0146] 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.
[0147] 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: 388.
[0148] The present invention further relates to the peptides
according to the present invention, wherein said peptide is
modified and/or includes non-peptide bonds.
[0149] 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.
[0150] Another embodiment of the present invention relates to a
non-naturally occurring peptide wherein said peptide consists or
consists essentially of an amino acid sequence according to SEQ ID
No: 1 to SEQ ID No: 48 and has been synthetically produced (e.g.
synthesized) as a pharmaceutically acceptable salt. Methods to
synthetically produce peptides are well known in the art. The salts
of the peptides according to the present invention differ
substantially from the peptides in their state(s) in vivo, as the
peptides as generated in vivo are no salts. The non-natural salt
form of the peptide mediates the solubility of the peptide, in
particular in the context of pharmaceutical compositions comprising
the peptides, e.g. the peptide vaccines as disclosed herein. A
sufficient and at least substantial solubility of the peptide(s) is
required in order to efficiently provide the peptides to the
subject to be treated. Preferably, the salts are pharmaceutically
acceptable salts of the peptides. These salts according to the
invention include alkaline and earth alkaline salts such as salts
of the Hofmeister series comprising as anions PO.sub.4.sup.3-,
SO.sub.4.sup.2-, CH.sub.3COO.sup.-, Cl.sup.-, Br.sup.-,
NO.sub.3.sup.-, ClO.sub.4.sup.-, I.sup.-, SCN.sup.- and as cations
NH.sub.4.sup.+, Rb.sup.+, K.sup.+, Na.sup.+, Cs.sup.+, Li.sup.+,
Zn.sup.2+, Mg.sup.2+, Ca.sup.2+, Mn.sup.2+, Cu.sup.2+ and
Ba.sup.2+. Particularly salts are selected from
(NH.sub.4).sub.3PO.sub.4, (NH.sub.4).sub.2HPO.sub.4,
(NH.sub.4)H.sub.2PO.sub.4, (NH.sub.4).sub.2SO.sub.4,
NH.sub.4CH.sub.3COO, NH.sub.4C.sub.1, NH.sub.4Br, NH.sub.4NO.sub.3,
NH.sub.4ClO.sub.4, NH.sub.4I, NH.sub.4SCN, Rb.sub.3PO.sub.4,
Rb.sub.2HPO.sub.4, RbH.sub.2PO.sub.4, Rb.sub.2SO.sub.4,
Rb.sub.4CH.sub.3COO, Rb.sub.4Cl, Rb.sub.4Br, Rb.sub.4NO.sub.3,
Rb.sub.4ClO.sub.4, Rb.sub.4I, Rb.sub.4SCN, K.sub.3PO.sub.4,
K.sub.2HPO.sub.4, KH.sub.2PO.sub.4, K.sub.2SO.sub.4, KCH.sub.3COO,
KCl, KBr, KNO.sub.3, KClO.sub.4, KI, KSCN, Na.sub.3PO.sub.4,
Na.sub.2HPO.sub.4, NaH.sub.2PO.sub.4, Na.sub.2SO.sub.4,
NaCH.sub.3COO, NaCl, NaBr, NaNO.sub.3, NaClO.sub.4, NaI, NaSCN,
ZnCl.sub.2 Cs.sub.3PO.sub.4, Cs.sub.2HPO.sub.4, CsH.sub.2PO.sub.4,
Cs.sub.2SO.sub.4, CsCH.sub.3COO, CsCl, CsBr, CsNO.sub.3,
CsClO.sub.4, CsI, CsSCN, Li.sub.3PO.sub.4, Li.sub.2HPO.sub.4,
LiH.sub.2PO.sub.4, Li.sub.2SO.sub.4, LiCH.sub.3COO, LiCl, LiBr,
LiNO.sub.3, LiClO.sub.4, LiI, LiSCN, Cu.sub.2SO.sub.4,
Mg.sub.3(PO.sub.4).sub.2, Mg.sub.2HPO.sub.4,
Mg(H.sub.2PO.sub.4).sub.2, Mg.sub.2SO.sub.4, Mg(CH.sub.3COO).sub.2,
MgCl.sub.2, MgBr.sub.2, Mg(NO.sub.3).sub.2, Mg(ClO.sub.4).sub.2,
MgI.sub.2, Mg(SCN).sub.2, MnCl.sub.2, Ca.sub.3(PO.sub.4),
Ca.sub.2HPO.sub.4, Ca(H.sub.2PO.sub.4).sub.2, CaSO.sub.4,
Ca(CH.sub.3COO).sub.2, CaCl.sub.2, CaBr.sub.2, Ca(NO.sub.3).sub.2,
Ca(ClO.sub.4).sub.2, CaI.sub.2, Ca(SCN).sub.2,
Ba.sub.3(PO.sub.4).sub.2, Ba.sub.2HPO.sub.4,
Ba(H.sub.2PO.sub.4).sub.2, BaSO.sub.4, Ba(CH.sub.3COO).sub.2,
BaCl.sub.2, BaBr.sub.2, Ba(NO.sub.3).sub.2, Ba(ClO.sub.4).sub.2,
BaI.sub.2, and Ba(SCN).sub.2. Particularly preferred are NH
acetate, MgCl.sub.2, KH.sub.2PO.sub.4, Na.sub.2SO.sub.4, KCl, NaCl,
and CaCl.sub.2, such as, for example, the chloride or acetate
(trifluoroacetate) salts.
[0151] 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 ninhydrine, 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).
[0152] 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).
[0153] 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. acetonitril/water
gradient separation.
[0154] 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.
[0155] The present invention further relates to an expression
vector capable of expressing and/or expressing a nucleic acid
according to the present invention.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] The antibodies and TCRs are additional embodiments of the
immunotherapeutic use of the peptides according to the invention at
hand.
[0160] 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.
[0161] 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.
[0162] 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.
[0163] 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.: 388,
preferably containing SEQ ID No. 1 to SEQ ID No. 295, or a variant
amino acid sequence.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] Preferably, said medicament is a cellular therapy, a vaccine
or a protein based on a soluble TCR or antibody.
[0168] The present invention further relates to a use according to
the present invention, wherein said cancer cells are glioblastoma,
breast cancer, colorectal cancer, renal cell carcinoma, chronic
lymphocytic leukemia, hepatocellular carcinoma, non-small cell and
small cell lung cancer, Non-Hodgkin lymphoma, acute myeloid
leukemia, ovarian cancer, pancreatic cancer, prostate cancer,
esophageal cancer including cancer of the gastric-esophageal
junction, gallbladder cancer and cholangiocarcinoma, melanoma,
gastric cancer, testis cancer, urinary bladder cancer, head and
neck squamous cell carcinoma, or uterine cancer.
[0169] 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
of glioblastoma, breast cancer, colorectal cancer, renal cell
carcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma,
non-small cell and small cell lung cancer, Non-Hodgkin lymphoma,
acute myeloid leukemia, ovarian cancer, pancreatic cancer, prostate
cancer, esophageal cancer including cancer of the
gastric-esophageal junction, gallbladder cancer and
cholangiocarcinoma, melanoma, gastric cancer, testis cancer,
urinary bladder cancer, head and neck squamous cell carcinoma, or
uterine 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.
[0170] Optionally the antibody carries a further effector function
such as an immune stimulating domain or toxin.
[0171] The present invention also relates to the use of these novel
targets in the context of cancer treatment.
[0172] Both therapeutic and diagnostic uses against additional
cancerous diseases are disclosed in the following description of
the underlying expression products (polypeptides) of the peptides
according to the invention.
[0173] A4GNT is frequently expressed in pancreatic cancer cells but
not peripheral blood cells and quantitative analysis of A4GNT mRNA
expressed in the mononuclear cell fraction of peripheral blood will
contribute to the detection of pancreatic cancer (Ishizone et al.,
2006). A4GNT mRNA was detectable in 80% of patients with an early
stage of gastric cancer when the cancer cells were limited to the
gastric mucosa, and the expression levels of A4GNT mRNA were
increased in association with tumor progression (Shimizu et al.,
2003).
[0174] The up-regulated expression of ABCC2 in primary fallopian
tube carcinomas is associated with poor prognosis (Halon et al.,
2013).
[0175] In human cancer ADAM10 is up-regulated, with levels
generally correlating with parameters of tumor progression and poor
outcome. In preclinical models, a selective inhibitor against
ADAM10 has been shown to synergize with existing therapies in
decreasing tumor growth (Duffy et al., 2009).
[0176] AHCYL2 was shown to be down-regulated in colon carcinoma and
in a subset of lung carcinomas (Lleonart et al., 2006). mRNA
expression of AHCYL2 was described as being potentially associated
with the control of p53 function as well as the ras-MAPK pathway,
methylation and transcriptional cellular programs, and AHCYL2 may
thus be a regulatory suppressor gene involved in human colon and
lung tumors (Lleonart et al., 2006).
[0177] AKR1C1 .mu.lays a role in cisplatin resistance in cervical,
ovarian and lung cancer cells which includes mitochondrial membrane
depolarization, ROS production and activation of the JNK pathway
(Chen et al., 2015). Significantly higher intratumoral levels of
AKR1C1 were found in responders to neoadjuvant chemotherapy
compared with nonresponders (Hlavac et al., 2014).
[0178] Expression of AKR1C3 was shown to be positively correlated
with an elevated Gleason score in prostate cancer, indicating that
AKR1C3 can serve as a promising biomarker for the progression of
prostate cancer (Tian et al., 2014). AKR1C3 was shown to catalyze
the reduction of 4-androstene-3,17-dione to testosterone and
estrone to 17.beta.-estradiol, which promotes the proliferation of
hormone dependent prostate and breast cancers, respectively (Byrns
et al., 2011). AKR1C3 was shown to be up-regulated in breast
cancer, prostate cancer and skin squamous cell carcinoma (Byrns et
al., 2011; Mantel et al., 2014). AKR1C3 was shown to be a marker
within a gene signature which is able to discriminate responder
patients from non-responders upon chemo-radiotherapy treatment of
patients with locally advanced rectal cancer (Agostini et al.,
2015). AKR1C3 was shown to be associated with doxorubicin
resistance in human breast cancer by activation of anti-apoptosis
PTEN/Akt pathway via loss of the tumor suppressor PTEN (Zhong et
al., 2015). AKR1C3 was shown to be associated with a higher risk of
lung cancer among people from a Chinese county who were exposed to
coal emissions (Li et al., 2015a). AKR1C3 was described as a
potential therapeutic marker for choriocarcinoma which is also
associated with the development of methotrexate resistance in this
disease (Zhao et al., 2014a).
[0179] The expression of ALDH1L1 was shown to be down-regulated in
HCC and gliomas. The down-regulation of ALDH1L1 in those cancers
was associated with poorer prognosis and more aggressive phenotype
(Chen et al., 2012; Rodriguez et al., 2008).
[0180] ALOX15 is present at high levels in prostate cancer (PCa),
lung cancer, breast cancer, melanomas, and colonic adenocarcinomas
when compared with normal tissues (Kelavkar et al., 2002). ALOX15
enzyme activity contributes to PCa initiation and progression
(Kelavkar et al., 2007).
[0181] AR has been implicated in the development of various cancers
such as prostate, castrate-resistant prostate, breast, glioblastoma
multiforme, colon and gastric (Wang et al., 2009b; Yu et al.,
2015b; Mehta et al., 2015; Wang et al., 2015a; Sukocheva et al.,
2015). In addition to promoting prostate cancer proliferation,
androgen signaling through AR leads to apoptosis via inducing the
expression of p21 (WAF1/CIP1), a cyclin-dependent kinase inhibitor
(Yeh et al., 2000).
[0182] Mutations in ARMC5 cause macronodular cortisol-producing
neoplasias, bilateral macronodular hyperplasias, primary
macronodular adrenal hyperplasia, and meningioma (Espiard and
Bertherat, 2015; Kirschner and Stratakis, 2016; Elbelt et al.,
2015).
[0183] Pharmacogenomic studies reveal correlations between ATAD5
and anticancer agents (Abaan et al., 2013). ATAD5 is significantly
up-regulated in malignant peripheral nerve sheath tumors (Pasmant
et al., 2011). Hepatitis B virus protein X significantly enhances
the expression of ATAD5 in HBV-associated hepatocellular carcinoma
(Ghosh et al., 2016). Loss of ATAD5 is embryonically lethal in
mice, it acts as tumor suppressor in both mice and humans, and it
interacts with components of the human Fanconi Anemia pathway.
Furthermore, it may be responsible for some of the phenotypes
associated with neurofibromatosis, a hereditary disease with high
risk of tumor growth (Gazy et al., 2015; Jenne et al., 2001).
Variants of the ATAD5 gene locus are associated with epithelial
ovarian cancer risk (Kuchenbaecker et al., 2015). ATAD5 has a
bearing on at least one mammalian phenotype of non-small cell lung
cancer (Li et al., 2014).
[0184] The expression of ATP1OB is de-regulated in highly invasive
glioma cells and associated with the invasive behavior (Tatenhorst
et al., 2004).
[0185] ATP8B4 may be a prognostic marker and therapeutic target in
multiple myeloma patients and other entities (US Patent No.
20070237770 A1) (Ni et al., 2012).
[0186] ATR encodes ATR serine/threonine kinase, which belongs to
the PI3/PI4-kinase family. Copy number gain, amplification, or
translocation of the ATR gene were observed in oral squamous cell
carcinoma cell lines (Parikh et al., 2014). It has been
demonstrated that truncating ATR mutations in endometrial cancers
are associated with reduced disease-free and overall survival
(Zighelboim et al., 2009).
[0187] B3GNT5 is over-expressed in acute myeloid leukemia, and
mouse embryonal carcinoma and its expression is inversely
correlated with promotor methylation in glioblastoma (Ogasawara et
al., 2011; Etcheverry et al., 2010; Wang et al., 2012).
Down-regulation of B3GNT5 through miRNA-203 may contribute to the
malignancy of hypopharyngeal squamous cell carcinoma (Wang et al.,
2015g). B3GNT5 is associated with breast cancer patient survival
(Potapenko et al., 2015).
[0188] Bub1 expression is increased in subsets of lymphomas,
breast, gastric and prostate cancers. Bub1 over-expression
correlates with poor clinical prognosis (Ricke and van Deursen,
2011). Bub1 mutations can be found in colorectal carcinomas
exhibiting chromosomal instability (Williams et al., 2007).
[0189] C4A has been described as a biomarker for polycystic ovary
syndrome and endometrial cancer and experimental data suggest that
C4 can mediate cancer growth (Galazis et al., 2013; Rutkowski et
al., 2010).
[0190] In the acute myelomonocytic leukemia cell line JIH3 a
chromosome deletion includes C7orf10 (Pan et al., 2012).
[0191] C8 is constitutively expressed by the human hepatoma cell
line HepG2 and expression is strongly enhanced after stimulation
with the cytokines IL-6, IFN-gamma and IL-1 beta (Scheurer et al.,
1997).
[0192] Cancer-testis antigen specific primers can detect CASC5 in
glioblastoma multiforme, one of the most malignant and aggressive
tumors with very poor prognosis. CASC5 has specific binding motifs
at the N-terminus (for Bub1 and BubR1) and at the C-terminus (for
Zwint-1 and hMis14/hNsI1). Disruption of this connection may be
able to lead to tumorigenesis (Kiyomitsu et al., 2011; Jiang et
al., 2014c). CASC5 interacts with the tumor suppressor pRb
(Bogdanov and Takimoto, 2008). CASC5 is highly expressed in
proliferating somatic cells, tumors and healthy human testis
(Bogdanov and Takimoto, 2008; Sasao et al., 2004). CASC5 is linked
to cell growth suppression and maturation enhancement and its
disruption thus may be a key factor for leukemogenesis (Hayette et
al., 2000; Bogdanov and Takimoto, 2008; Chinwalla et al., 2003;
Kuefer et al., 2003; Yang et al., 2014a).
[0193] CCR4 has been described as a prognostic marker in various
tumors such as renal cell carcinoma, head and neck squamous cell
carcinoma, gastric cancer, breast cancer, colon cancer and Hodgkin
lymphoma (Ishida et al., 2006; Olkhanud et al., 2009; Yang et al.,
2011; Tsujikawa et al., 2013; Al-haidari et al., 2013; Liu et al.,
2014a). Studies have revealed that gastric cancer patients with
CCR4-positive tumors had significantly poorer prognosis compared to
those with CCR4-negative tumors (Lee et al., 2009a).
[0194] CCR8 expression is increased in monocytic and granulocytic
myeloid cell subsets in peripheral blood of patients with
urothelial and renal carcinomas. Up-regulated expression of CCR8 is
also detected within human bladder and renal cancer tissues and
primarily limited to tumor-associated macrophages. The CCL1/CCR8
axis is a component of cancer-related inflammation and may
contribute to immune evasion (Eruslanov et al., 2013).
[0195] A single nucleotide polymorphism in CD101 was shown to be
associated with pancreatic cancer risk, but results could not be
replicated in a prostate cancer case-control and cohort population,
thus, requiring future research in the possible role of CD101 in
pancreatic cancer (Reid-Lombardo et al., 2011). CD101 was
identified as one gene of a 6-gene signature that discriminated
chronic phase from blast crisis chronic myeloid leukemia using a
Bayesian model averaging approach (Oehler et al., 2009).
[0196] CD84 was described as a CD antigen which is differentially
abundant in progressive chronic lymphocytic leukemia as compared to
slow-progressive and stable chronic lymphocytic leukemia (Huang et
al., 2014). CD84 expression was shown to be significantly elevated
from the early stages of chronic lymphocytic leukemia
(Binsky-Ehrenreich et al., 2014).
[0197] CENPE expression significantly correlated with glioma grade
and might complement other parameters for predicting survival time
for glioma patients (Bie et al., 2011). CENPE is up-regulated in
chemo-resistant epithelial ovarian tumors compared to
chemo-sensitive tumors (Ju et al., 2009). CENPE is up-regulated in
invasive and aggressive-invasive prolactin pituitary tumors
(Wierinckx et al., 2007).
[0198] CLDN14 was shown to be up-regulated in gastric cancer (Gao
et al., 2013). CLDN14 expression was shown to be associated with
lymphatic metastasis in gastric cancer (Gao et al., 2013). CLDN14
was described to play a role in the regulation of tumor blood
vessel integrity and angiogenesis in mice (Baker et al., 2013).
[0199] CLDN3 is highly differentially expressed in many human
tumors and may provide an efficient molecular tool to specifically
identify and target biologically aggressive human cancer cells as
CLDN3 is a high affinity receptor of Clostridium perfringens
enterotoxin (Black et al., 2015). CLDN3 is frequently
over-expressed in several neoplasias, including ovarian, breast,
pancreatic, and prostate cancers (Morin, 2005). CLDN3 was
identified as prostate cancer biomarker as it is highly expressed
in prostate cancer (Amaro et al., 2014). Decreased expression of
CLDN3 is associated with a poor prognosis and EMT in completely
resected squamous cell lung carcinoma (Che et al., 2015). CLDN3
inhibits cancer aggressiveness via Wnt-EMT signaling and is a
potential prognostic biomarker for hepatocellular carcinoma (Jiang
et al., 2014b).
[0200] CLDN4 is highly differentially expressed in many human
tumors and may provide an efficient molecular tool to specifically
identify and target biologically aggressive human cancer cells as
CLDN4 is a high affinity receptor of Clostridium perfringens
enterotoxin (Black et al., 2015). CLDN4 is frequently
over-expressed in several neoplasias including ovarian, breast,
pancreatic, and prostate cancers (Morin, 2005). An antibody against
the extracellular domain of CLDN4 provides pro-chemotherapeutic
effects in bladder cancer (Kuwada et al., 2015). High expression of
CLDN4 was associated with the more differentiated intestinal-type
gastric carcinoma and lost in poorly differentiated diffuse type.
Low expression of CLDN4 was related to lymphangiogenesis (Shareef
et al., 2015).
[0201] CLDN6 expression was shown to be associated with lymph node
metastasis and TNM stage in non-small cell lung cancer (Wang et
al., 2015f). Furthermore, low expression of CLDN6 was shown to be
associated with significantly lower survival rates in patients with
non-small cell lung cancer (Wang et al., 2015f). Thus, low CLDN6
expression is an independent prognostic biomarker that indicates
worse prognosis in patients with non-small cell lung cancer (Wang
et al., 2015f). CLDN6 was shown to be down-regulated in cervical
carcinoma and gastric cancer (Zhang et al., 2015; Lin et al.,
2013). CLDN6 was shown to be up-regulated in BRCA1-related breast
cancer and ovarian papillary serous carcinoma (Wang et al., 2013b;
Heerma van Voss et al., 2014). CLDN6 was described as a tumor
suppressor for breast cancer (Zhang et al., 2015). Gain of CLDN6
expression in the cervical carcinoma cell lines HeLa and C33A was
shown to suppress cell proliferation, colony formation in vitro,
and tumor growth in vivo, suggesting that CLDN6 may function as a
tumor suppressor in cervical carcinoma cells (Zhang et al., 2015).
CLDN6 may play a positive role in the invasion and metastasis of
ovarian cancer (Wang et al., 2013b). CLDN6 was shown to be
consistently expressed in germ cell tumors and thus is a novel
diagnostic marker for primitive germ cell tumors (Ushiku et al.,
2012). CLDN6 expression was shown to be positive in most tumors of
an assessed set of atypical teratoid/rhabdoid tumors of the central
nervous system, with strong CLDN6 positivity being a potential
independent prognostic factor for outcome of the disease (Dufour et
al., 2012).
[0202] CLDN9 was shown to be up-regulated in the metastatic Lewis
lung carcinoma cell line p-3LL and in tumors derived from these
cells and in pituitary oncocytomas (Sharma et al., 2016; Hong et
al., 2014). Knock-down of CLDN9 expression in metastatic Lewis lung
carcinoma p-3LL cells was shown to result in significantly reduced
motility, invasiveness in vitro and metastasis in vivo, whereas
transient over-expression in these cells was shown to enhance their
motility (Sharma et al., 2016). Thus, CLDN9 may play an essential
role in promoting lung cancer metastasis (Sharma et al., 2016).
CLDN9 was shown to be down-regulated in cervical carcinoma tissues
(Zhu et al., 2015a). CLDN9 expression was observed to be correlated
with lymphatic metastasis of cervical carcinomas (Zhu et al.,
2015a). CLDN9 was described as the most significantly altered and
up-regulated gene in pituitary oncocytomas with higher expression
levels in invasive compared to non-invasive oncocytomas (Hong et
al., 2014). Thus, CLDN9 may be an important biomarker for invasive
pituitary oncocytomas (Hong et al., 2014). Over-expression of CLDN9
in the gastric adenocarcinoma cell line AGS was shown to enhance
invasive potential, cell migration and the proliferation rate and
is thus sufficient to enhance tumorigenic properties of a gastric
adenocarcinoma cell line (Zavala-Zendejas et al., 2011). Strong
CLDN9 expression was shown to be associated with a higher mortality
rate in diffuse-type gastric adenocarcinomas compared to the
intestinal type and its detection was described as a useful
prognostic marker in "intestinal-" and "diffuse-type" gastric
adenocarcinomas (Rendon-Huerta et al., 2010).
[0203] CLEC5A mRNA expression was shown to be significantly lower
in primary acute myeloid leukemia patients samples than in
macrophages and granulocytes from healthy donors (Batliner et al.,
2011). CLEC5A was described as a novel transcriptional target of
the tumor suppressor PU. 1 in monocytes/macrophages and
granulocytes (Batliner et al., 2011).
[0204] DCP2 was identified as miR-224 target that was
differentially expressed more than 2-fold in methotrexate resistant
human colon cancer cells (Mencia et al., 2011).
[0205] DCSTAMP expression is increased in papillary thyroid cancer
(Lee et al., 2009b; Kim et al., 2010b). Down-regulation of DCSTAMP
leads to a decreased colony formation of MCF-7 cells probably
because of decreased proliferation and cell cycle progression as
well as increased apoptosis (Zeng et al., 2015). DCSTAMP is
over-expressed in peripheral macrophages, and dendritic cells and
myeloma plasma cells show high susceptibility to DCSTAMP and are
able to transdifferentiate to osteoclasts. Malignant plasma cells
expressing cancer stem cell phenotype and high metastasizing
capability express osteoclast markers which activate the beta3
transcriptional pathway resulting in ERK1/2 phosphorylation and
initiation of bone resorbing activity (Silvestris et al., 2011).
Esculetin and parthenolide suppress c-Fos and nuclear factor of
activated T cell c1 signaling pathway resulting in suppressed
DCSTAMP expression, a marker gene for osteoclast differentiation
(Ihn et al., 2015; Baek et al., 2015; Cicek et al., 2011; Courtial
et al., 2012; Kim et al., 2014a).
[0206] SNPs in DENND1B were significantly associated with pancreas
cancer risk (Cotterchio et al., 2015).
[0207] DNAH17, also known as DNEL2, was described as a homologue to
a tumor-antigen identified in melanoma patients (Ehlken et al.,
2004). DNAH17 was described as one of several candidate genes
mapped to a small chromosome interval associated with sporadic
breast and ovarian tumorigenesis, and esophageal cancer in the
autosomal dominant disorders hereditary neuralgic amyotrophy and
tylosis (Kalikin et al., 1999).
[0208] DNAH2 is one of the genes mutated in .gtoreq.10% of patients
with chronic myelomonocytic leukemia (Mason et al., 2015). Genes
encoding microtubule-associated proteins, such as DNAH2, showed a
10% or higher incidence of genetic aberrations in CpG-island
methylator phenotype-positive clear renal cell carcinomas (Arai et
al., 2015).
[0209] Re-expression of methylation silenced tumor suppressor genes
by inhibiting DNMT3B has emerged as an effective strategy against
cancer (Singh et al., 2013).
[0210] FAM83B mRNA expression was significantly higher in squamous
cell carcinoma than in normal lung or adenocarcinoma and FAM83B
therefore is a novel biomarker for diagnosis and prognosis (Okabe
et al., 2015). FAM83B was identified as an oncogene involved in
activating CRAF/MAPK signaling and driving epithelial cell
transformation. Elevated expression is associated with elevated
tumor grade and decreased overall survival (Cipriano et al., 2014).
Elevated FAM83B expression also activates the PI3K/AKT signaling
pathway and confers a decreased sensitivity to PI3K, AKT, and mTOR
inhibitors (Cipriano et al., 2013).
[0211] Down-regulation or dysfunction of FANCD2 due to genetic
mutations has been reported in different cancer types including
breast cancer, acute lymphatic leukemia and testicular seminomas
and is associated with cancer development. Otherwise also
re-expression and up-regulation of FANCD2 was shown to be
associated with tumor progression and metastasis in gliomas and
colorectal cancer (Patil et al., 2014; Shen et al., 2015a; Shen et
al., 2015b; Ozawa et al., 2010; Rudland et al., 2010; Zhang et al.,
2010a; Smetsers et al., 2012). PI3K/mTOR/Akt pathway promotes
FANCD2 inducing the ATM/Chk2 checkpoint as DNA damage response and
monoubiquitinilated FANCD2 activates the transcription of the tumor
suppressor TAp63 (Shen et al., 2013; Park et al., 2013).
[0212] FOXJ1 expression is up-regulated, associated with tumor
stage, histologic grade and size and correlated with prognosis in
patients with clear cell renal cell carcinoma (Zhu et al., 2015b).
Decreased FOXJ1 expression was significantly correlated with clinic
stage, lymph node metastasis, and distant metastasis, and lower
FOXJ1 expression independently predicted shorter survival time in
gastric carcinoma (Wang et al., 2015c). Over-expression of FOXJ1
can promote tumor cell proliferation and cell-cycle transition in
hepatocellular carcinoma and is associated with histological grade
and poor prognosis (Chen et al., 2013).
[0213] High GINS2 transcript level predicts poor prognosis and
correlates with high histological grade and endocrine therapy
resistance through mammary cancer stem cells in breast cancer
patients (Zheng et al., 2014). GINS2 was reported to be present at
a high level in lung adenocarcinoma and associated with TNM stages
(Liu et al., 2013b). GINS2 express abundantly and abnormally in
many malignant solid tumors, such as breast cancer, melanoma and
hepatic carcinoma. Further, over-expression of GINS2 could promote
proliferation of leukemic cell lines (Zhang et al., 2013a).
[0214] GPR98 expression is increased in primary neuroendocrine
tumors relative to normal tissue (Sherman et al., 2013). GPR98 was
among the genes associated with survival of glioblastoma multiforme
(Sadeque et al., 2012). GPR98 displays a transcript regulated by
glucocorticoids which are used for the treatment of acute
lymphoblastic leukemia as they lead to the induction of apoptosis
(Rainer et al., 2012).
[0215] GTPBP10 is highly correlated with copy number variation,
gene expression, and patient outcome in glioblastoma (Kong et al.,
2016).
[0216] GTSE1 expression represses apoptotic signaling and confers
cisplatin resistance in gastric cancer cells (Subhash et al.,
2015). GTSE1 is over-expressed in uterine leiomyosarcoma (ULMS) and
participated in cell cycle regulation.
[0217] H2BFS was consistently expressed as a significant cluster
associated with the low-risk acute lymphoblastic leukemia subgroups
(Qiu et al., 2003).
[0218] HIST1H2BJ was shown to be down-regulated in brain tumors and
was described as potentially useful for developing molecular
markers of diagnostic or prognostic value (Luna et al., 2015).
[0219] Acetylation of HISTH4A might be a potential target to
inactivate embryonic kidney cancer (Wilms tumor) (Yan-Fang et al.,
2015).
[0220] HIST1H4C may act as risk distinguishing factor for the
development of treatment-related myeloid leukemia (Bogni et al.,
2006).
[0221] HIST1H4F was observed to be hyper-methylated in prostate
cancer which might also correlate with the aging of the patient
(Kitchen et al., 2016).
[0222] A high methylation rate of HIST1H4K was observed in
high-grade non-muscle invasive bladder cancer as well as in
prostate cancer and is therefore representing a potential biomarker
(Payne et al., 2009; Kachakova et al., 2013).
[0223] It was shown that HIST1H4L is significantly up-regulated in
ERG+ prostate carcinomas (Camoes et al., 2012). HIST1H4L encodes
the replication-dependent histone cluster 1, H4I that is a member
of the histone H4 family (RefSeq, 2002).
[0224] HIST2H4B was identified as novel protein in key cellular
pathogenic pathways in cells infected with a reovirus subtype that
is presently in clinical trials as an anti-cancer oncolytic agent
(Berard et al., 2015).
[0225] HIST4H4 was one of the genes which showed continuous
down-regulation in gastric cancer cells after treatment with
immune-conjugates composed of an alpha-emitter and the monoclonal
antibody d9MAb that specifically target cells expressing mutant
d9-E-cadherin (Seidl et al., 2010). Hyper-methylation of other
members of the histon H4 family was significantly associated with
shorter relapse-free survival in stage I non-small cell lung cancer
(Sandoval et al., 2013).
[0226] HIVEP1 was identified as cellular gene disrupted by human
T-lymphotropic virus type 1 integration in lymphoma cell lines (Cao
et al., 2015). HIVEP1 was associated with the unfavorable 11q
deletion and also with the unfavorable Binet stages B and C in
chronic lymphocytic leukemia (Aalto et al., 2001).
[0227] HNRNPH2 is up-regulated in different cancer types including
pancreatic, liver and gastric cancer (Honore et al., 2004; Zeng et
al., 2007). HNRNPH2 is involved in splicing of the beta-deletion
transcript of hTERT, which is highly expressed in cancer cells and
competes and thereby inhibits endogenous telomerase activity
(Listerman et al., 2013).
[0228] HOXD11 is dysregulated in head and neck squamous cell
carcinoma showing strikingly high levels in cell lines and patient
tumor samples. Knockdown of HOXD11 reduced invasion (Sharpe et al.,
2014). HOXD11 is significantly up-regulated in oral squamous cell
carcinoma (Rodini et al., 2012). HOXD11 is aberrantly methylated in
human breast cancers (Miyamoto et al., 2005). The HOXD11 gene is
fused to the NUP98 gene in acute myeloid leukemia with
t(2;11)(q31;p15) (Taketani et al., 2002).
[0229] ICOS acts as a ligand of programmed death-1 (PD-1) on T
cells, induces the immune escape of cancer cells and also acts as a
receptor mediating anti-apoptotic effects on cancer cells (Yang et
al., 2015c). Murine tumor models have provided significant support
for the targeting of multiple immune checkpoints involving ICOS
during tumor growth (Leung and Suh, 2014). ICOS+ cell infiltration
correlates with adverse patient prognosis, identifying ICOS as a
new target for cancer immunotherapy (Faget et al., 2013). ICOS can
enhance the cytotoxic effect of cytokine-induced killer cells
against cholangiocarcinoma both in vitro and in vivo (He et al.,
2011).
[0230] Intratumoral expression of IFNG was shown to be associated
with expression of MHC Class II molecules and a more aggressive
phenotype in human melanomas (Brocker et al., 1988). Autocrine IFNG
signaling was shown to enhance experimental metastatic ability of
IFNG gene-transfected mammary adenocarcinoma cells, and was
attributed to increased resistance to NK cells (Lollini et al.,
1993).
[0231] Triple-negative breast cancer has high tumor expression of
IGFBP3 associated with markers of poor prognosis (Marzec et al.,
2015). A novel cell death receptor that binds specifically to
IGFBP3 was identified and might be used in breast cancer treatment
(Mohanraj and Oh, 2011). IGFBP3 exhibits pro-survival and
growth-promoting properties in vitro (Johnson and Firth, 2014).
IGFBP3 is an independent marker of recurrence of the urothelial
cell carcinomas (Phe et al., 2009).
[0232] IGFBPL1 is a regulator of insulin-growth factors and is
down-regulated in breast cancer cell lines by aberrant
hypermethylation. Methylation in IGFBPL1 was clearly associated
with worse overall survival and disease-free survival (Smith et
al., 2007).
[0233] IGFLR1 is mutated in colorectal cancer (Donnard et al.,
2014). IGFLR1 has structural similarity with the tumor necrosis
factor receptor family (Lobito et al., 2011).
[0234] IL4I1 protein expression is very frequent in tumors. IL4I1
was detected in tumor-associated macrophages of different tumor
entities, in neoplastic cells from lymphomas and in rare cases of
solid cancers mainly mesothelioma (Carbonnelle-Puscian et al.,
2009). IL4I1 up-regulation in human Th17 cells limits their T-cell
receptor (TCR) -mediated expansion by blocking the molecular
pathway involved in the activation of the IL-2 promoter and by
maintaining high levels of Tob1, which impairs entry into the cell
cycle (Santarlasci et al., 2014).
[0235] 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).
[0236] IQGAP3 is modulating the EGFR/Ras/ERK signaling cascade and
interacts with Rac/Cdc42 (Yang et al., 2014b; Kunimoto et al.,
2009).
[0237] Elevated levels of ITGA2 were found in the highly invasive
and metastatic melanoma cell lines compared with normal cultured
melanocytes and non-metastatic melanoma cell lines (van Muijen et
al., 1995). The adhesion molecule ITGA2 was up-regulated by
IFN-gamma, TNF-alpha, and IL-1-beta in melanoma cells (Garbe and
Krasagakis, 1993). Transfection of ITGA2 into human
rhabdomyosarcoma cells which do not express ITGA2, potentiated the
frequency of metastases in various organs (Matsuura et al.,
1995).
[0238] KCNU1 is located on chromosome 8p in an area that is
frequently involved in complex chromosomal rearrangements in breast
cancer (Gelsi-Boyer et al., 2005). KCNU1 is one of the top 25
over-expressed extracellular membrane proteins in hepatoblastomas
of pediatric cancer samples (Orentas et al., 2012).
[0239] KIAA0226L is thought to be a tumor suppressor gene and is
hyper-methylated in cervical cancer. Re-activation of KIAA0226L
leads to decreased cell growth, viability, and colony formation
(Huisman et al., 2015; Eijsink et al., 2012; Huisman et al., 2013).
The methylation pattern of KIAA0226L can be used to differ between
precursor lesions and normal cervix cancer (Milutin et al., 2015).
The methylation pattern of KIAA0226L cannot be used as specific
biomarker for cervical cancer (Sohrabi et al., 2014). Re-activation
of KIAA0226L partially de-methylates its promotor region and also
decreases repressive histone methylations (Huisman et al.,
2013).
[0240] KIAA1244 over-expression is one of the important mechanisms
causing the activation of the estrogen/ERalpha signaling pathway in
the hormone-related growth of breast cancer cells (Kim et al.,
2009). Inhibiting the interaction between KIAA1244 and PHB2 may be
a new therapeutic strategy for the treatment of luminal-type breast
cancer (Yoshimaru et al., 2013).
[0241] KIAA1524 encodes Cancerous Inhibitor of Protein Phosphatase
2A (CIP2A). A critical role of CIP2A has been shown among other for
NSCLC, HCC, HNSCC, bladder, pancreatic, cervical, breast, prostate,
ovarian and colorectal cancers (Ventela et al., 2015; Ma et al.,
2014; Guo et al., 2015b; Rincon et al., 2015; Guo et al., 2015c; Wu
et al., 2015; Peng et al., 2015; Lei et al., 2014; Liu et al.,
2014c; Farrell et al., 2014; Fang et al., 2012; He et al., 2012;
Bockelman et al., 2012).
[0242] KIF18A was shown to be over-expressed in hepatocellular
cancer, which correlated with significantly shorter disease free
and overall survival. Thus, KIF18A might be a biomarker for
hepatocellular cancer diagnosis and an independent predictor of
disease free and overall survival after surgical resection (Liao et
al., 2014). KIF18A expression is up-regulated in specific subtypes
of synovial sarcoma (Przybyl et al., 2014). Estrogen strongly
induces KIF18A expression in breast cancer, which is associated
with increased proliferation and reduced apoptosis (Zou et al.,
2014).
[0243] Over-expression of KIF20A was detected in pancreatic ductal
adenocarcinoma, melanoma, bladder cancer, non-small cell lung
cancer and cholangiocellular carcinoma (Imai et al., 2011;
Yamashita et al., 2012; Stangel et al., 2015). Recently, it was
reported that patients with pancreatic ductal adenocarcinoma
vaccinated with a KIF20A-derived peptide exhibited better prognosis
compared to the control group (Asahara et al., 2013). In addition,
silencing of KIF20A resulted in an inhibition of proliferation,
motility, and invasion of pancreatic cancer cell lines (Stangel et
al., 2015).
[0244] High expression of KIF26B in breast cancer associates with
poor prognosis (Wang et al., 2013c). KIF26B up-regulation was
significantly correlated with tumor size analysing CRC tumor
tissues and paired adjacent normal mucosa. KIF26B plays an
important role in colorectal carcinogenesis and functions as a
novel prognostic indicator and a potential therapeutic target for
CRC (Wang et al., 2015d).
[0245] KIF2C was shown to be involved in directional migration and
invasion of tumor cells (Ritter et al., 2015). Over-expression of
KIF2C was shown to be associated with lymphatic invasion and lymph
node metastasis in gastric and colorectal cancer patients (Ritter
et al., 2015). KIF2C was shown to be up-regulated in oral tongue
cancer (Wang et al., 2014a). High expression of KIF2C was shown to
be associated with lymph node metastasis and tumor staging in
squamous cell carcinoma of the oral tongue (Wang et al., 2014a).
Silencing of KIF2C was shown to result in suppressed proliferation
and migration of the human oral squamous cell carcinoma cell line
Tca8113 (Wang et al., 2014a). Mutation of KIF2C was described as
being associated with colorectal cancer (Kumar et al., 2013).
[0246] KIFC1 was shown to be essential for proper spindle assembly,
stable pole-focusing and survival of cancer cells independently
from number of formed centrosomes (normal or supernumerary
centrisomes). KIFC1 expression was shown to be up-regulated in
breast cancer, particularly in estrogen receptor negative,
progesterone receptor negative and triple negative breast cancer,
and 8 human breast cancer cell lines. In estrogen receptor-positive
breast cancer cells, KIFC1 was one of 19 other kinesins whose
expression was strongly induced by estrogen. In breast cancer, the
overexpression of KIFC1 and its nuclear accumulation was shown to
correlate with histological grade and predict poor progression-free
and overall survival. In breast cancer cell lines, the
overexpression of KIFC1 was shown to mediate the resistance to
docetaxel. The KIFC1 silencing negatively affected the breast
cancer cell viability (Zou et al., 2014; Pannu et al., 2015; De et
al., 2009; Li et al., 2015b). KIFC1 was shown to be overexpressed
in ovarian cancer which was associated with tumor aggressiveness,
advanced tumor grade and stage. KIFC1 was identified as one of
three genes, whose higher expression in primary NSCLC tumors
indicated the higher risk for development of brain metastasis
(Grinberg-Rashi et al., 2009).
[0247] KL was described as a tumor suppressor which suppresses the
epithelial to mesenchymal transition in cervical cancer and which
functions as a tumor suppressor in several types of human cancers
by inhibiting insulin/IGF1, p53/p21, and Wnt signaling (Xie et al.,
2013; Qureshi et al., 2015). KL was described as an aberrantly
expressed gene in a number of cancers, including breast cancer,
lung cancer and hepatocellular carcinoma (Zhou and Wang, 2015). KL
was described to be down-regulated in pancreatic cancer,
hepatocellular carcinoma, and other tumors (Zhou and Wang, 2015).
KL was described as a novel biomarker for cancer whose
down-regulation was described to result in promoted proliferation
and reduced apoptosis of cancer cells. In this context
Wnt/.beta.-catenin signaling is one of several relevant signaling
pathways (Zhou and Wang, 2015). A KL gene polymorphism was shown to
be associated with increased risk of colorectal cancer (Liu et al.,
2015).
[0248] KLHDC7B is associated with cervical squamous cell carcinoma
and is a potential biomarker for cervical squamous cell carcinoma
(Guo et al., 2015a).
[0249] KLHL genes are responsible for several Mendelian diseases
and have been associated with cancer (Dhanoa et al., 2013).
[0250] Focal expression of KRT31 was observed in invasive
onychocytic carcinoma originating from nail matrix keratinocytes
(Wang et al., 2015e). Pilomatricomas are tumors that emulate the
differentiation of matrix cells of the hair follicle, showing
cortical differentiation, with sequential over-expression of KRT35
and KRT31 keratins (Battistella et al., 2014).
[0251] KRT35 was one of the most frequently and most strongly
expressed hair keratins in pilomatrixomas. Pilomatricomas are
tumors that emulate the differentiation of matrix cells of the hair
follicle, showing cortical differentiation, with sequential
over-expression of KRT35 and KRT31 keratins (Battistella et al.,
2014).
[0252] Knockdown of LCTL allowed hTERT to immortalize human colonic
epithelial cells (Kim et al., 2011).
[0253] Researchers have observed that inhibition of LRBA expression
by RNA interference, or by a dominant-negative mutant, resulted in
the growth inhibition of cancer cells. These findings imply that
deregulated expression of LRBA contributes to the altered growth
properties of a cancer cell (Wang et al., 2004).
[0254] LRRC8E is over-expressed in osteosarcoma and neuroblastoma
tissues in comparison to normal samples (Orentas et al., 2012).
[0255] LTA polymorphisms contributed to the increased risk of
cancers (Huang et al., 2013a). Bone resorbing factors like LTA are
produced by certain solid and hematologic cancers and have also
been implicated in tumour-induced hyper-calcemia (Goni and Tolis,
1993). There is a link between the LTA to LTbetaR signaling axis
and cancer (Drutskaya et al., 2010). B-cell-derived lymphotoxin
promotes castration-resistant prostate cancer (Ammirante et al.,
2010).
[0256] 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., 2013a; Stein, 2013; Wang et al., 2015b;
Wang et al., 2015h; 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).
[0257] MAGEA10 encodes MAGE family member A10, implicated in some
hereditary disorders, such as dyskeratosis congenital (RefSeq,
2002). By a vaccine directed against MAGEA10 and two other
cancer-testis antigens, all of which are known to be targets of
cytotoxic-T-lymphocyte responses, more than two-thirds of breast
cancers would be covered (Taylor et al., 2007). MAGEA10 was
expressed in 36.7% of the tumor tissues from hepatocellular
carcinoma patients; however, it was not expressed in the
para-cancer tissues (Chen et al., 2003). MAGEA10 was expressed in
14% of 79 lung cancer tissues (Kim et al., 2012).
[0258] MAGEB6 was identified as new MAGE gene not expressed in
normal tissues, except for testis, and expressed in tumors of
different histological origins (Lucas et al., 2000). MAGEB6 was
found frequently expressed in head and neck squamous cell carcinoma
and mRNA positivity presented significant associations with
recognized clinical features for poor outcome (Zamuner et al.,
2015).
[0259] MCC interacts with beta-catenin and re-expression of MCC in
colorectal cancer cells specifically inhibits Wnt signaling
(Fukuyama et al., 2008). The MCC gene is in close linkage with the
adenomatous polyposis coli gene on chromosome 5, in a region of
frequent loss of heterozygosity (LOH) in colorectal cancer
(Kohonen-Corish et al., 2007). LOH of MCC gene could be found in
both early and advanced stages of gastric, lung, esophageal and
breast cancers (Wang et al., 1999a; Medeiros et al., 1994).
[0260] MET was shown to be up-regulated in dedifferentiated
liposarcoma and is associated with melanocytic tumors,
hepatocellular carcinoma, non-small cell lung cancer, hereditary
papillary kidney cancers and gastric adenocarcinomas (Petrini,
2015; Finocchiaro et al., 2015; Steinway et al., 2015; Bill et al.,
2015; Yeh et al., 2015).
[0261] The expression of MMP10 in oral squamous cell carcinoma was
intensive and in verrucous carcinoma was moderate (Kadeh et al.,
2015). MMP10 contributes to hepatocarcinogenesis in a novel
crosstalk with the stromal derived factor 1/C-X-C chemokine
receptor 4 axis (Garcia-Irigoyen et al., 2015). Helicobacter pylori
infection promotes the invasion and metastasis of gastric cancer
through increasing the expression of MMP10 (Jiang et al., 2014a).
MMP10 promotes tumor progression through regulation of angiogenic
and apoptotic pathways in cervical tumors (Zhang et al., 2014).
[0262] The elevated level of preoperative MMP13 was found to
associate with tumor progression and poor survival in patients with
esophageal squamous cell carcinoma (Jiao et al., 2014). PAI-1, a
target gene of miR-143, regulates invasion and lung metastasis via
enhancement of MMP13 expression and secretion in human osteosarcoma
cells, suggesting that these molecules could be potential
therapeutic target genes for preventing lung metastasis in
osteosarcoma patients (Hirahata et al., 2016). MMP13 is already
upregulated in Oral lichen planus (OLP) which has been classified
as a pre-malignant condition for oral squamous cell carcinoma
(OSCC) (Agha-Hosseini and Mirzaii-Dizgah, 2015). MMP13 .mu.lays a
potentially unique physiological role in the regeneration of
osteoblast-like cells (Ozeki et al., 2016).
[0263] MUC5AC is de-regulated in a variety of cancer types
including colorectal, gastric, lung and pancreatic cancer.
Depletion or low expression in colorectal and gastric tumors is
associated with a more aggressive behavior and poor prognosis.
Over-expression in lung cancer results in an increased likelihood
of recurrence and metastases (Yonezawa et al., 1999; Kocer et al.,
2002; Kim et al., 2014b; Yu et al., 1996).
[0264] MUC5B is over-expressed in different cancer entities
including colorectal, lung and breast cancer and is associated with
tumor progression (Sonora et al., 2006; Valque et al., 2012; Walsh
et al., 2013; Nagashio et al., 2015). MUC5B can be repressed under
the influence of methylation and can be up-regulated by ATF-1,
c-Myc, NFkappaB, Sp1, CREB, TTF-11 and GCR (Perrais et al., 2001;
Van, I et al., 2000).
[0265] Genetic variants of MUC6 have been reported to modify the
risk of developing gastric cancer (Resende et al., 2011). In
salivary gland tumors the expression patterns of MUC6 appear to be
very closely correlated with the histopathological tumor type
indicating their potential use to improve diagnostic accuracy
(Mahomed, 2011). Studies have identified a differential expression
of MUC6 in breast cancer tissues when compared with the
non-neoplastic breast tissues (Mukhopadhyay et al., 2011).
[0266] A Chinese study identified MXRA5 as the second most
frequently mutated gene in non-small cell lung cancer (Xiong et
al., 2012). In colon cancer, MXRA5 was shown to be over-expressed
and might serve as a biomarker for early diagnosis and omental
metastasis (Zou et al., 2002; Wang et al., 2013a).
[0267] MYB can be converted into an oncogenic transforming protein
through a few mutations (Zhou and Ness, 2011). MYB is known as
oncogene and is associated with apoptosis, cell cycle control, cell
growth/angiogenesis and cell adhesion by regulating expression of
key target genes such as cyclooxygenase-2, Bcl-2, BclX(L) and c-Myc
(Ramsay et al., 2003; Stenman et al., 2010). The oncogenic fusion
protein MYB-NFIB and MYB over-expression are found in adenoid
cystic carcinoma of the salivary gland and breast, pediatric
diffuse gliomas, acute myeloid leukemia and pancreatic cancer
(Wallrapp et al., 1999; Pattabiraman and Gonda, 2013; Nobusawa et
al., 2014; Chae et al., 2015; Marchio et al., 2010). By the synergy
between MYB and beta-Catenin during Wnt signaling, MYB is
associated with colon tumorigenesis (Burgess et al., 2011). Since
MYB is a direct target of estrogen signaling anti-MYB therapy is
considered for ER-positive breast tumors (Gonda et al., 2008).
[0268] N4BP2 has a potential role in the development of
nasopharyngeal carcinoma. There is a statistically relevant
difference in two different haplotype blocks which correlate with
the risk of sporadic nasopharyngeal carcinoma. Furthermore, N4BP2
is over-expressed in these tumor tissues relative to paired normal
tissues (Zheng et al., 2007).
[0269] In a multistage, case-only genome-wide association study of
12,518 prostate cancer cases, NAALADL2 was identified as a locus
associated with Gleason score, a pathological measure of disease
aggressiveness (Berndt et al., 2015). NAALADL2 is over-expressed in
prostate and colon cancer and promotes a pro-migratory and
pro-metastatic phenotype associated with poor survival (Whitaker et
al., 2014).
[0270] 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., 2012a). 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).
[0271] NRK encodes Nik related kinase, a protein kinase required
for JNK activation which may be involved in the induction of actin
polymerization in late embryogenesis (RefSeq, 2002). NRK activates
the c-Jun N-terminal kinase signaling pathway and may be involved
in the regulation of actin cytoskeletal organization in skeletal
muscle cells through cofilin phosphorylation (Nakano et al.,
2003).
[0272] NUP210 was shown to be a candidate gene carrying
polymorphisms associated with the risk of colorectal cancer (Landi
et al., 2012). NUP210 was shown to be up-regulated in cervical
cancer and suggested to play a role in the early phase of
tumorigenesis (Rajkumar et al., 2011).
[0273] ORC1 was shown to be over-expressed in tumor-derived cell
lines and is predicted to be a biomarker in prostate cancer as well
as in leukemia (Struyf et al., 2003; Zimmerman et al., 2013; Young
et al., 2014). Through its interaction with histone
acetyltranferases such as HBO1, ORC1 exerts oncogenic functions in
breast cancer (Wang et al., 2010).
[0274] Non-carrier of heterozygous mutations in two SNPs in OSCP1
might be a biomarker for susceptibility for non-viral liver
carcinoma (Toda et al., 2014).
[0275] OVOL2 induces mesenchymal-epithelial transition resulting in
decreased metastasis (Roca et al., 2013). OVOL2 inhibits c-Myc and
Notch1 (Wells et al., 2009). OVOL2 is hyper-methylated in
colorectal cancer resulting in its inability to inhibit Wnt
signaling (Ye et al., 2016). Over-expression of OVOL2 decreased
cell migration and invasion, reduced markers for
epithelial-mesenchymal transition, and suppressed metastasis (Ye et
al., 2016). OVOL2 is down-regulated in colorectal cancer and is
inversely correlated with tumor stage (Ye et al., 2016). OVOL2 is
regulated by Wnt signaling pathway (Ye et al., 2016).
[0276] OXTR is significantly over-expressed in primary small bowel
and pancreatic neuroendocrine tumors, small cell carcinoma of the
lung, ovarian carcinoma as well as in prostate cancer, mediating
cell migration and metastasis (Morita et al., 2004; Zhong et al.,
2010; Carr et al., 2012; Carr et al., 2013; Pequeux et al., 2002).
However, OXTR1 possesses also an inhibitory effect on proliferation
of neoplastic cells of either epithelial, nervous or bone origin,
which is thought to be dependent on the receptor localization on
the membrane (Cassoni et al., 2004).
[0277] PAPPA represents a metastasis-related gene occurring in a
range of cancer types such as NSCLC and hepatocellular carcinoma,
where it is positively associated to growth (VEGF and IGF-I) and
transcription factors (NF-kappaB p50, NF-kappaB p65, HIF-1alpha)
(Salim et al., 2013; lunusova et al., 2013; Engelmann et al.,
2015). PAPPA regulates mitotic progression through modulating the
IGF-1 signaling pathway in breast cancer and ovarian cancer cells,
where it is predominantly found at the primary site (Boldt and
Conover, 2011; Loddo et al., 2014; Becker et al., 2015; lunusova et
al., 2014).
[0278] PGAP1 is down-regulated in the adenocarcinoma cell line
AsPC-1 (Yang et al., 2016).
[0279] PGR is highly associated with breast cancer initiation and
progression, where it activates MAPK and PI3K/AKT pathways as well
as the expression of Growth Factors Receptors (GFR) (Jaiswal et
al., 2014; Piasecka et al., 2015). PGR (besides HER and estrogen
receptor) acts as a classification factor helping to distinguish
between three different subtypes of breast cancer (Safarpour and
Tavassoli, 2015).
[0280] PLA2G7 has strong influence on lipid metabolism in breast,
ovarian, melanoma and prostate cancer cells, where a blockage of
the enzyme leads to impaired cancer pathogenicity (Vainio et al.,
2011a; Massoner et al., 2013; Kohnz et al., 2015). PLA2G7 is highly
associated with prostate cancer and is therefore representing a
potential biomarker for this type of cancer (Vainio et al.,
2011b).
[0281] PPP3R1 is up-regulated in hepatocellular carcinoma cells
affecting up to 10 different signaling pathways (Zekri et al.,
2008).
[0282] 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., 2016).
[0283] An up-regulated expression of PSMA7T was found in metastatic
lung cancer, castration-recurrent prostate cancer (CRPC) as well as
in primary colorectal cancer, where it increases the risk of liver
metastasis (Hu et al., 2008; Hu et al., 2009; Romanuik et al.,
2010; Cai et al., 2010). It was also shown that the amount of
PSMA7T correlates with the transactivation of the androgen receptor
(AR) in androgen/AR-mediated prostate tumor growth (Ogiso et al.,
2002).
[0284] It was shown that PSMC1 is able to influence cell growth and
is therefore representing a potential anti-cancer target in
prostate cancer, multiple myeloma and glioblastoma cells (Dahlman
et al., 2012; Kim et al., 2008).
[0285] RAD18 is implicated in tumorigenesis due to its well-known
function in DNA damage bypass, post-replication repair and
homologous recombination (Ting et al., 2010). RAD18 Arg302Gln
polymorphism is associated with the risk for colorectal cancer and
non-small-cell lung cancer (Kanzaki et al., 2008; Kanzaki et al.,
2007). RAD18 mediates resistance to ionizing radiation in human
glioma cells and knockdown of RAD18 disrupts homologous
recombination-mediated repair, resulting in increased accumulation
of double strand breaks (Xie et al., 2014). Using melanoma tissue
microarray, it was shown that nuclear RAD18 expression was
up-regulated in primary and metastatic melanoma compared to
dysplastic nevi (Wong et al., 2012).
[0286] RAD51AP1 was shown to be associated with radiation exposure
papillary thyroid cancer (Handkiewicz-Junak et al., 2016).
Amplification of RAD51AP1 was shown to be correlated with cell
immortality and a shorter survival time in ovarian cancer
(Sankaranarayanan et al., 2015). RAD51AP1 was described as commonly
over-expressed in tumor cells and tissues and disruption of
RAD51AP1 function was suggested to be a promising approach in
targeted tumor therapy (Parplys et al., 2014). RAD51AP1
transcription was shown to be directly stimulated by the tumor
suppressor MEN1 (Fang et al., 2013). RAD51AP1 was shown to be
up-regulated in intrahepatic cholangiocarcinoma, human
papillomavirus-positive squamous cell carcinoma of the head and
neck and in BRCA1-deficient compared to sporadic breast tumors
(Martinez et al., 2007; Martin et al., 2007; Obama et al., 2008).
Suppression of RAD51AP1 was shown to result in growth suppression
in intrahepatic cholangiocarcinoma cells, suggesting its
involvement in the development and/or progression of intrahepatic
cholangiocarcinoma (Obama et al., 2008).
[0287] Knock-down of RBM14 was shown to block glioblastoma
multiforme re-growth after irradiation in vivo (Yuan et al., 2014).
RBM14 was shown to be down-regulated in renal cell carcinoma (Kang
et al., 2008). RBM14 was described as a potential tumor suppressor
in renal carcinoma which inhibits G(1)-S transition in human kidney
cells and suppresses anchorage-independent growth and xenograft
tumor formation in part by down-regulation of the proto-oncogene
c-myc (Kang et al., 2008). RBM14 was shown to be involved in the
migration-enhancing action of PEA3 and MCF7 human cancer cells
(Verreman et al., 2011). The RBM14 gene was shown to be amplified
in a subset of primary human cancers including non-small cell lung
carcinoma, squamous cell skin carcinoma and lymphoma (Sui et al.,
2007).
[0288] RBM4 is involved in regulatory splicing mechanisms of
pre-messenger RNA suppressing proliferation and migration of
various cancer cells (Lin et al., 2014; Wang et al., 2014c).
Dysregulations of BBM4 activity were found in cervical, breast,
lung, colon, ovarian and rectal cancers (Liang et al., 2015; Markus
et al., 2016).
[0289] Serum RCOR3 levels in liver cancer patients were
significantly lower than those in the patients with moderate
chronic hepatitis B and with mild chronic hepatitis B (Xue et al.,
2011).
[0290] Down-regulation of RFWD2 is correlated with poor prognosis
in gastric cancer (Sawada et al., 2013). RFWD2 directly interacts
with p27 and the de-regulation of this interaction is involved in
tumorigenesis (Choi et al., 2015b; Choi et al., 2015a; Marine,
2012). Up-regulation of RFWD2 is correlated with poor prognosis in
bladder cancer, gastric cancer, and triple-negative breast cancer
(Ouyang et al., 2015; Li et al., 2016; Li et al., 2012c).
[0291] RIF1 is highly expressed in human breast tumors, encodes an
anti-apoptotic factor required for DNA repair and is a potential
target for cancer treatment (Wang et al., 2009a). The role of RIF1
in the maintenance of genomic integrity has been expanded to
include the regulation of chromatin structure, replication timing
and intra-S phase checkpoint (Kumar and Cheok, 2014).
[0292] In patients diagnosed with visceral multicentric infantile
myofibromatosis novel homozygous variants in the RLTPR gene were
identified (Linhares et al., 2014).
[0293] RNF24 was shown to be up-regulated in esophageal
adenocarcinoma and plays a critical role in the progression of
Barrett's esophagus to esophageal adenocarcinoma (Wang et al.,
2014b). RNF24 was shown to be differentially expressed depending on
certain risk factors in oral squamous cell carcinoma (Cheong et
al., 2009).
[0294] RPGRIP1L suppresses anchorage-independent growth partly
through the mitotic checkpoint protein Mad2 and is a candidate
tumor suppressor gene in human hepatocellular carcinoma (Lin et
al., 2009).
[0295] Over-expression of Rtl1 in the livers of adult mice resulted
in highly penetrant tumor formation and over-expression of RTL1 was
detected in 30% of analyzed human hepatocellular carcinoma samples
(Riordan et al., 2013). Transcriptional activity of the imprinted
gene RTL1 was assessed in a panel of 32 Wilms tumors and a massive
over-expression was detected compared to normal renal tissue
(Hubertus et al., 2011).
[0296] SAPCD2 (also called p42.3 or C9orf140) encodes a protein
initially found to be expressed in gastric cancer, but not in
normal gastric mucosa (Xu et al., 2007). SAPCD2 is over-expressed
in different cancer entities including colorectal, gastric,
hepatocellular and brain cancer and high SAPCD2 levels are
associated with tumor progression (Sun et al., 2013; Weng et al.,
2014; Wan et al., 2014). The optimal pathway of SAPCD2 gene in
protein regulatory network in gastric cancer is Ras protein, Raf-1
protein, MEK, MAPK kinase, MAPK, tubulin, spindle protein,
centromere protein and tumor (Zhang et al., 2012a; Weng et al.,
2014).
[0297] Lower expression of SEMA3A was shown to be correlated with
shorter overall survival and had independent prognostic importance
in patients with head and neck squamous cell carcinoma (Wang et
al., 2016). Over-expression of SEMA3A was shown to suppress
migration, invasion and epithelial-to-mesenchymal transition due in
part to the inhibition of NF-kB and SNAI2 in head and neck squamous
cell carcinoma cell lines (Wang et al., 2016). Thus, SEMA3A serves
as a tumor suppressor in head and neck squamous cell carcinoma and
may be a new target for the treatment of this disease (Wang et al.,
2016). SEMA3A expression was shown to be significantly reverse
associated with metastasis in hepatocellular carcinoma (Yan-Chun et
al., 2015). SEMA3A was described as being down-regulated in
numerous types of cancer, including prostate cancer, breast cancer,
glioma, epithelial ovarian carcinoma and gastric cancer (Jiang et
al., 2015a; Tang et al., 2014). Low SEMA3A expression was shown to
be correlated with poor differentiation, vascular invasion, depth
of invasion, lymph node metastasis, distant metastasis, advanced
TNM stage and poor prognosis in gastric cancer (Tang et al., 2014).
SEMA3A was described as a candidate tumor suppressor and potential
prognostic biomarker in gastric carcinogenesis (Tang et al.,
2014).
[0298] It was shown that missense variations in the SERPINB10 gene
possess tumorigenic features leading to an increased risk of
prostate cancer (Shioji et al., 2005). In addition, SERPINB10
expression is significantly up-regulated in metastatic mammary
tumors (Klopfleisch et al., 2010).
[0299] Expression level of SLC16A14 is significantly associated
with progression-free survival and presents a novel putative marker
for the progression of epithelial ovarian cancer (Elsnerova et al.,
2016).
[0300] SLC18A1 was showing lower expression in unfavorable
neuroblastoma tumor types as compared to favorable ones (Wilzen et
al., 2009).
[0301] SLC25A43 was identified as a regulator of cell cycle
progression and proliferation through a putative mitochondrial
checkpoint in breast cancer cell lines (Gabrielson et al., 2016).
SLC25A43 affects drug efficacy and cell cycle regulation following
drug exposure in breast cancer cell lines (Gabrielson and Tina,
2013).
[0302] SLC28A3 was shown to be down-regulated in pancreatic ductal
adenocarcinoma (Mohelnikova-Duchonova et al., 2013). SLC28A3 is
associated with clinical outcome in metastatic breast cancer
treated with paclitaxel and gemcitabine chemotherapy, overall
survival in gemcitabine treated non-small cell lung cancer and
overall survival in gemcitabine-based chemoradiation treated
pancreatic adenocarcinoma (Li et al., 2012b; Lee et al., 2014b;
Marechal et al., 2009). SLC28A3 is associated with fludarabine
resistance in chronic lymphocytic leukemia and drug resistance in
T-cell leukemia (Karim et al., 2011; Fernandez-Calotti et al.,
2012).
[0303] SLC2A13 was consistently increased in the sphere-forming
cells in the primary cultures of oral squamous cell carcinoma
samples and confocal microscopy revealed that SLC2A13-expressing
cells were embedded in the limited areas of tumor tissue as a
cluster suggesting that SLC2A13 can be a potential marker for
cancer stem cells (Lee et al., 2011). SLC2A13 was identified as
gene associated with non-small-cell lung cancer promotion and
progression (Bankovic et al., 2010).
[0304] Inhibition of SLC35A1 was shown to reduce cancer cell
sialylation and decrease the metastatic potential of cancer cells
(Maggioni et al., 2014).
[0305] SLC7A11 was shown to be down-regulated in drug resistant
variants of the W1 ovarian cancer cell line and thus might play a
role in cancer cell drug resistance (Januchowski et al., 2013).
SLC7A11 was described to modulate tumor microenvironment, leading
to a growth advantage for cancer (Savaskan and Eyupoglu, 2010).
SLC7A11 was described to be involved in neurodegenerative processes
in glioma (Savaskan et al., 2015). SLC7A11 was shown to be
repressed by p53 in the context of ferroptosis, and the p53-SLC7A11
axis was described as preserved in the p53(3KR) mutant, and
contributes to its ability to suppress tumorigenesis in the absence
of the classical tumor suppression mechanisms (Jiang et al.,
2015b). SLC7A11 was described as the functional subunit of system
Xc- whose function is increased in aggressive breast cancer cells
(Linher-Melville et al., 2015). High membrane staining for SLC7A11
in cisplatin-resistant bladder cancer was shown to be associated
with a poorer clinical outcome and SLC7A11 inhibition was described
as a promising therapeutic approach to the treatment of this
disease (Drayton et al., 2014). SLC7A11 was shown to be
differentially expressed in the human promyelocytic leukemia cell
line HL-60 that had been exposed to benzene and its metabolites and
thus highlights a potential association of SLC7A11 with
leukemogenesis (Sarma et al., 2011). Disruption of SLC7A11 was
described to result in growth inhibition of a variety of
carcinomas, including lymphoma, glioma, prostate and breast cancer
(Chen et al., 2009). Inhibition of SLC7A11 was shown to inhibit
cell invasion in the esophageal cancer cell line KYSE150 in vitro
and its experimental metastasis in nude mice and thus establishes a
role of SLC7A11 in tumor metastasis (Chen et al., 2009).
[0306] SLCO5A1 is located at the plasma membrane and may contribute
to chemoresistance of small cell lung carcinoma by affecting the
intracellular transport of drugs (Olszewski-Hamilton et al., 2011).
SLCO5A1 is the most prominent organic anion transporting
polypeptide in metastatic small cell lung cancer and the mRNA level
of SLCO5A1 is highly increased in hepatic tumors and breast cancer
(Kindla et al., 2011; Wlcek et al., 2011; Brenner et al., 2015).
Gene fusions in oropharyngeal squamous cell carcinoma are
associated with down-regulation of SLCO5A1 (Guo et al., 2016).
[0307] SP5 was down-regulated after depletion of beta-catenin in
colorectal cancer cell lines and is a novel direct downstream
target in the Wnt signaling pathway (Takahashi et al., 2005). The
over-expression of SP5 demonstrated activation of the beta-catenin
pathway in rare human pancreatic neoplasms (Cavard et al., 2009).
In human colorectal carcinoma cells displaying de-regulated Wnt
signaling, monensin reduced the intracellular levels of
.beta.-catenin leading to a decrease in the expression of Wnt
signaling target genes such as SP5 and a decreased cell
proliferation rate (Tumova et al., 2014).
[0308] STIL is among the genes with copy number alterations and
copy-neutral losses of heterozygosity in 15 cortisol-secreting
adrenocortical adenomas (Ronchi et al., 2012). Chromosomal
deletions that fuse this gene and the adjacent locus commonly occur
in T cell leukemias, and are thought to arise through illegitimate
V-(D)-J recombination events (Karrman et al., 2009; Alonso et al.,
2012).
[0309] An elevated expression of TBC1D7 was found in the majority
of lung cancers and immunohistochemical staining suggested an
association of TBC1D7 expression with poor prognosis for NSCLC
patients (Sato et al., 2010). Over-expression of TBC7 enhanced
ubiquitination of TSC1 and increased phosphorylation of S6 protein
by S6 kinase, that is located in the mTOR-signaling pathway
(Nakashima et al., 2007).
[0310] TDG influences the Wnt signaling pathway in an up-regulating
manner via interaction with the transcription factor TCF4 and is
thought to be a potential biomarker for colorectal cancer (Xu et
al., 2014). On the other hand, a reduced expression of TDG leads to
an impaired base excision repair (BER) pathway with strong
oncogenic features (van de Klundert et al., 2012). A
down-regulation of the protein was observed in early breast cancer
esophageal squamous cell carcinoma (ESCC) as well as in gastric
cancer (Li et al., 2013; Du et al., 2015; Yang et al., 2015a).
[0311] Among the four most frequently mutated genes was TENM4
showing protein-changing mutations in primary CNS lymphomas (Vater
et al., 2015). MDA-MB-175 cell line contains a chromosomal
translocation that leads to the fusion of TENM4 and receptors of
the ErbB family. Chimeric genes were also found in neuroblastomas
(Wang et al., 1999b; Boeva et al., 2013).
[0312] TET2 is a critical regulator for hematopoietic stem cell
homeostasis whose functional impairment leads to hematological
malignancies (Nakajima and Kunimoto, 2014). TET2 mutations have an
adverse impact on prognosis and may help to justify risk-adapted
therapeutic strategies for patients with acute myeloid leukemia
(Liu et al., 2014b). Nuclear localization of TET2 was lost in a
significant portion of colorectal cancer tissues, in association
with metastasis (Huang et al., 2016).
[0313] TKTL1 is associated with the development and progression of
multiple tumor types such as esophageal squamous cell carcinoma,
oral squamous cell carcinoma, lung cancer, colorectal cancer and
non-small cell lung cancer (Kayser et al., 2011; Bentz et al.,
2013; Grimm et al., 2014).
[0314] TMEM67 functions in centriole migration to the apical
membrane and formation of the primary cilium. Defects in this gene
are a cause of Meckel syndrome type 3 (MKS3) and Joubert syndrome
type 6 (JBTS6) (RefSeq, 2002). TMEM67 is involved in cilia
formation and defective cilia may cause ocular coloboma, tongue
tumors, and medulloblastoma (Yang et al., 2015b; Parisi, 2009).
[0315] TONSL is involved in lung and esophageal carcinogenesis by
stabilizing the oncogenic protein MMS22L (Nguyen et al., 2012).
Further interactions were shown between TONSL and BRCA1, which acts
as a breast and ovarian tumor suppressor (Hill et al., 2014).
[0316] TP63 translocation was described as an event in a subset of
anaplastic lymphoma kinase-positive anaplastic large cell lymphomas
which is associated with an aggressive course of the disease
(Hapgood and Savage, 2015). TP63 was described to play a complex
role in cancer due to its involvement in epithelial
differentiation, cell cycle arrest and apoptosis (Lin et al.,
2015). The TP63 isoform TAp63 was described to be over-expressed in
hematological malignancies while TP63 missense mutations have been
reported in squamous cancers and TP63 translocations in lymphomas
and some lung adenocarcinomas (Orzol et al., 2015). Aberrant
splicing resulting in the over-expression of the TP63 isoform
DeltaNp63 was described to be frequently found in human cancers
such as cutaneous squamous cell carcinoma, where it is likely to
favor tumor initiation and progression (Missero and Antonini, 2014;
Inoue and Fry, 2014).
[0317] TRIM59 promotes proliferation and migration of non-small
cell lung cancer cells by up-regulating cell cycle related proteins
(Zhan et al., 2015). The putative ubiquitin ligase TRIM59 is
up-regulated in gastric tumors compared with non-tumor tissues and
levels of TRIM59 correlate with tumor progression and patient
survival times. TRIM59 interacts with P53, promoting its
ubiquitination and degradation, and TRIM59 might promote gastric
carcinogenesis via this mechanism (Zhou et al., 2014).
[0318] TRPC4 was found to be up-regulated in lung cancer, ovarian
cancer, head and neck cancer, kidney cancer and non-small cell lung
cancer (Zhang et al., 2010b; Zeng et al., 2013a; Jiang et al.,
2013; Park et al., 2016).
[0319] ULBP3 is expressed soluble and membrane bound isoform in
many tumor cells. Pediatric acute lymphoblastic leukemia blasts
express significantly higher levels of ULBP3 compared to adult
blasts (Torelli et al., 2014). Much higher expression levels of
ULBP3 were found in the leukemia cell line K562 compared to other
leukemia cell lines. Furthermore, it can be found in both leukemia
cell lines and primary malignant leukemic cells (Ma et al., 2013b).
The ULBP3 locus is methylated in colorectal cancer cell lines
(Bormann et al., 2011). Increased mRNA levels and surface
expression levels of ULBP3 have been detected in the human lung
cancer cell line SW-900 (Park et al., 2011). ULBP3 has a higher
surface expression in leukemic cells (Ogbomo et al., 2008). ULBP3
levels in different tumor cell lines correlate with NK cell
cytotoxicity, however, ULBP3 seems not to be suitable as biomarker
(Wang et al., 2008; Linkov et al., 2009). ULBP3 is not expressed in
the human nasopharyngeal carcinoma cell line CNE2 (Mei et al.,
2007). ULBP3 is expressed in ovarian cancer and inversely
correlated with patient survival (Carlsten et al., 2007; McGilvray
et al., 2010). B cells express ULBP3 in non-Hodgkin's lymphoma or
it can be found in peripheral blood, bone marrow, or lymph nodes
(Catellani et al., 2007). ULBP3 is expressed in breast cancer, the
glioblastoma cell line U251, human brain tumors, and in head and
neck squamous cell carcinoma (Eisele et al., 2006; Butler et al.,
2009; Bryant et al., 2011; de Kruijf et al., 2012). Tumor cells
express soluble and surface ULBP3 to regulate NK cell activity (Mou
et al., 2014). ULBP3 is over-expressed in certain epithelial
tumors. Furthermore, the ULBP3 level in cancer patient sera is
elevated compared to healthy donors (Mou et al., 2014).
[0320] VPS13B alleles are mutated in small cell lung cancers
(Iwakawa et al., 2015). Mutations of VPS13B were observed in
gastric and colorectal cancers (An et al., 2012).
[0321] Frameshift mutations of VPS13C were found in gastric and
colorectal cancers with microsatellite instability (An et al.,
2012).
[0322] WDR62 expression was significantly increased in gastric
cancer tissues and cell lines and was associated with poor
differentiation and prognosis. Further, WDR62 expression was
elevated in multidrug resistant cells (Zeng et al., 2013b). WDR62
over-expression is related to centrosome amplification and may be a
novel useful differentiation biomarker and a potential therapy
target for ovarian cancer (Zhang et al., 2013b).
[0323] Exosome-bound WDR92 inhibits breast cancer cell invasion by
degrading amphiregulin mRNA (Saeki et al., 2013). WDR92 potentiates
apoptosis induced by tumor necrosis factor-alpha and cycloheximide
(Ni et al., 2009).
[0324] WNT5A belongs to the WNT gene family that consists of
structurally related genes which encode secreted signaling
proteins. These proteins have been implicated in oncogenesis and in
several developmental processes, including regulation of cell fate
and patterning during embryogenesis. The WNT5A gene encodes a
member of the WNT family that signals through both the canonical
and non-canonical WNT pathways. This protein is a ligand for the
seven transmembrane receptor frizzled-5 and the tyrosine kinase
orphan receptor 2. This protein plays an essential role in
regulating developmental pathways during embryogenesis. This
protein may also play a role in oncogenesis (RefSeq, 2002). WNT5A
is over-expressed in CRC and had a concordance rate of 76% between
the primary tumor and metastatic site (Lee et al., 2014a). WNT5A is
up-regulated and a key regulator of the epithelial-to-mesenchymal
transition and metastasis in human gastric carcinoma cells,
nasopharyngeal carcinoma and pancreatic cancer (Kanzawa et al.,
2013; Zhu et al., 2014; Bo et al., 2013).
[0325] XRN1 is likely involved in a number of regulatory mRNA
pathways in astrocytes and astrocytoma cells (Moser et al., 2007).
Knockdown of XRN1 inhibited androgen receptor expression in
prostate cancer cells and plays an important role in miR-204/XRN1
axis in prostate adenocarcinoma (Ding et al., 2015).
[0326] Genome-wide association studies identified gene
polymorphisms in XXYLT1. It has been proposed that these
polymorphisms are susceptibility loci for non-small cell lung
cancer development (Zhang et al., 2012b).
[0327] ZBTB20 promotes cell proliferation in non-small cell lung
cancer through repression of FoxO1 (Zhao et al., 2014b). ZBTB20
expression is increased in hepatocellular carcinoma and associated
with poor prognosis (Wang et al., 2011c). Polymorphism in ZBTB20
gene is associated with gastric cancer (Song et al., 2013).
[0328] ZFHX4 is thought to regulate cell differentiation and its
suppression is linked to glioma-free survival (Chudnovsky et al.,
2014). Papillary tumors of the pineal region show high expression
levels of ZFHX4 (Fevre-Montange et al., 2006). ZFHX4 was found to
be a basal cell carcinoma susceptibility locus (Stacey et al.,
2015).
[0329] ZMYM1 is a major interactor of ZNF131 which acts in estrogen
signaling and breast cancer proliferation (Oh and Chung, 2012; Kim
et al., 2016).
DETAILED DESCRIPTION OF THE INVENTION
[0330] 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.
[0331] 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).
[0332] As used herein and except as noted otherwise all terms are
defined as given below.
[0333] The term "T-cell response" shall mean that 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.
[0334] 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, 13 or 14 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 15, 16, 17, 18, 19 or 20 or more
amino acids in length.
[0335] 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.
[0336] 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.
[0337] 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.
[0338] 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.
[0339] 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.
[0340] 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-00007 TABLE 4 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%
[0341] The peptides of the invention, preferably when included into
a vaccine of the invention as described herein preferably bind to
A*02, A*24 or class II alleles, as specified. 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.
[0342] 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%.
[0343] In a preferred embodiment, the term "nucleotide sequence"
refers to a heteropolymer of deoxyribonucleotides.
[0344] 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.
[0345] 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.
[0346] 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.
[0347] 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.
[0348] 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.
[0349] 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).
[0350] 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.
[0351] 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.
[0352] 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.
[0353] The term "promoter" means a region of DNA involved in
binding of RNA polymerase to initiate transcription.
[0354] 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.
[0355] 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.
[0356] 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.
[0357] 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.
[0358] 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)]
[0359] 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
[0360] (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
[0361] (ii) each gap in the Reference Sequence and
[0362] (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
[0363] (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.
[0364] 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.
[0365] 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: 388 or a variant thereof
which is 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 388, 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.
[0366] 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.
[0367] 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).
[0368] 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: 388. 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.
[0369] 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 388, 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.
[0370] 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. "
[0371] 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).
[0372] 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.
[0373] 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.
[0374] 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.
[0375] 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.
[0376] 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-00008 TABLE 5 Preferred variants and motifs of the
HLA-A*02-peptides according to SEQ ID NO: 2, 4, and 6. Position 1 2
3 4 5 6 7 8 9 SEQ ID NO. 4 E L A E I V F K V Variants I L A M I M L
M M A A I A L A A A V I V L V V A T I T L T T A Q I Q L Q Q A SEQ
ID NO 2 A L Y G K L L K 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 SEQ ID NO. 6 F L D P A
Q R D 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
TABLE-US-00009 TABLE 6 Preferred variants and motifs of the
HLA-A*24-binding peptides according to SEQ ID NO: 98, 114, and 158.
Position 1 2 3 4 5 6 7 8 9 10 11 12 SEQ ID 158 I Y E E T R G V L K
V F Variant I L F F I F L SEQ ID 114 Q Y L D G T W S L Variant I F
F F I F F SEQ ID 98 V F P R L H N V L F Variant Y I Y L Y I L
[0377] 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.
[0378] 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-00010 TABLE 6 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
[0379] 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.
[0380] 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.
[0381] In an alternative embodiment, the peptide is elongated on
either or both sides by more than four 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.
[0382] 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.
[0383] 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.
[0384] 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.
[0385] 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: 388.
[0386] "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 388 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.
[0387] 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.
[0388] 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.
[0389] 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.
[0390] 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.
[0391] 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 amido group may be added to the peptides'
carboxy termini.
[0392] 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.
[0393] 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.
[0394] 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 provide information on specific reagents.
[0395] 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-(dimethylamino)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 .alpha.-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.
[0396] 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.
[0397] 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).
[0398] 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.
[0399] 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).
[0400] 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).
[0401] 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.
[0402] 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.
[0403] For the identification of peptides of the present invention,
a database of RNA expression data (Lonsdale, 2013) from about 3000
normal (healthy) tissue samples was screened for genes with
near-absent expression in vital organ systems, and low expression
in other important organ systems. Then, cancer-associated peptides
derived from the protein products of these genes were identified by
mass spectrometry using the XPRESIDENT.TM. platform as described
herein.
[0404] In order to select over-presented peptides, a presentation
profile was 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) (cf.
Example 1).
[0405] 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 natural tumor-associated peptides (TUMAPs) recorded from
cancer samples (N=377 A*02-positive samples from 370 donors, N=204
A*24-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 primary tumors, these results provide direct evidence
for the natural processing and presentation of the identified
peptides on primary cancer tissue obtained from 574 cancer
patients.
[0406] 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.
[0407] 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.
[0408] HLA-peptide complexes from tissue 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 primary cancer samples, confirming
their presentation on primary glioblastoma, breast cancer,
colorectal cancer, renal cell carcinoma, chronic lymphocytic
leukemia, hepatocellular carcinoma, non-small cell and small cell
lung cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian
cancer, pancreatic cancer, prostate cancer, esophageal cancer
including cancer of the gastric-esophageal junction, gallbladder
cancer and cholangiocarcinoma, melanoma, gastric cancer, testis
cancer, urinary bladder cancer, or uterine cancer.
[0409] TUMAPs as identified on multiple 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.
[0410] Furthermore, the discovery pipeline XPRESIDENT.RTM. v2.1
allows the direct absolute quantitation of MHC-, preferably
HLA-restricted, peptide levels on cancer or other infected tissues.
Briefly, the total cell count was calculated from the total DNA
content of the analyzed tissue sample. The total peptide amount for
a TUMAP in a tissue sample was measured by nanoLC-MS/MS as the
ratio of the natural TUMAP and a known amount of an isotope-labeled
version of the TUMAP, the so-called internal standard. The
efficiency of TUMAP isolation was determined by spiking peptide:MHC
complexes of all selected TUMAPs into the tissue lysate at the
earliest possible point of the TUMAP isolation procedure and their
detection by nanoLC-MS/MS following completion of the peptide
isolation procedure. The total cell count and the amount of total
peptide were calculated from triplicate measurements per tissue
sample. The peptide-specific isolation efficiencies were calculated
as an average from 10 spike experiments each measured as a
triplicate (see Example 6 and Table 14).
[0411] This combined analysis of RNA expression and mass
spectrometry data resulted in the 417 peptides of the present
invention.
[0412] The present invention provides peptides that are useful in
treating cancers/tumors, preferably glioblastoma, breast cancer,
colorectal cancer, renal cell carcinoma, chronic lymphocytic
leukemia, hepatocellular carcinoma, non-small cell and small cell
lung cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian
cancer, pancreatic cancer, prostate cancer, esophageal cancer
including cancer of the gastric-esophageal junction, gallbladder
cancer and cholangiocarcinoma, melanoma, gastric cancer, testis
cancer, urinary bladder cancer, head and neck squamous cell
carcinoma, and uterine 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 primary
human cancer samples.
[0413] 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 cells or tissue derived
from the same organ as the tumor, 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 sample from a patient suffering from
cancer, but not on normal tissues (see, e.g., Example 1).
[0414] 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. glioblastoma,
breast cancer, colorectal cancer, renal cell carcinoma, chronic
lymphocytic leukemia, hepatocellular carcinoma, non-small cell and
small cell lung cancer, Non-Hodgkin lymphoma, acute myeloid
leukemia, ovarian cancer, pancreatic cancer, prostate cancer,
esophageal cancer including cancer of the gastric-esophageal
junction, gallbladder cancer and cholangiocarcinoma, melanoma,
gastric cancer, testis cancer, urinary bladder cancer, head and
neck squamous cell carcinoma, or uterine cancer cells presenting
the derived peptides.
[0415] 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). 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.
[0416] The present description further relates to T-cell receptors
(TCRs) comprising an alpha chain and a beta chain ("alpha/beta
TCRs"). Also provided are 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.
[0417] 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.
[0418] 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.
[0419] 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.
[0420] 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.
[0421] 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.
[0422] TCRs of the present description preferably bind to a
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.
[0423] 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 a
peptide-HLA molecule complex of 100 .mu.M or less.
[0424] 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.
[0425] 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.
[0426] 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.
[0427] 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.
[0428] 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, an 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 the peptides according tot
he invention can be enhanced by methods well known in the art.
[0429] 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/peptide monomers,
incubating the PBMCs with tetramer-phycoerythrin (PE) and isolating
the high avidity T-cells by fluo-rescence activated cell sorting
(FACS)--Calibur analysis.
[0430] 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 TCRap 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 peptide of
interest, 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.
[0431] 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. 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 sys-tems. 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.
[0432] 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)-1a and the spleen
focus-forming virus (SFFV) promoter. In a preferred embodiment, the
promoter is heterologous to the nucleic acid being expressed. 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).
[0433] 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.
[0434] 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).
[0435] 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).
[0436] 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).
[0437] 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).
[0438] 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.zeta. (CD3.zeta. fusion). (Schmitt et al.
2009).
[0439] 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.
[0440] 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.
[0441] 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.
[0442] In an especially preferred embodiment, the pharmaceutical
compositions comprise the peptides as salts of acetic acid
(acetates), trifluoro acetates or hydrochloric acid
(chlorides).
[0443] 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.
[0444] 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.
388, 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.
[0445] 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.
[0446] 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.
[0447] 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.
[0448] A desirable method of modifying the DNA encoding the
polypeptide of the invention employs the polymerase chain reaction
as disclosed by Saiki RK, 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.
[0449] 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.
[0450] 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.
[0451] 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.
[0452] Alternatively, the gene for such selectable trait can be on
another vector, which is used to co-transform the desired host
cell.
[0453] 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.
[0454] 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.
[0455] 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 (Ylps) and
incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3.
Plasmids 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.
[0456] 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 .about.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.
[0457] 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.
[0458] 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, YPH500 and YPH501, 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.
[0459] 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.
[0460] 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.
[0461] 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.
[0462] 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).
[0463] 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.
[0464] 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).
[0465] 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.
[0466] 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).
[0467] 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.
[0468] 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.
[0469] 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.
[0470] 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.
[0471] 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.
[0472] 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.
[0473] 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).
[0474] 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.
[0475] "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.
[0476] 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.
[0477] Each scaffold can be conjugated with a second active
molecule such as for example IL-21, anti-CD3, anti-CD28.
[0478] For further information on polypeptide scaffolds see for
example the background section of WO 2014/071978A1 and the
references cited therein.
[0479] 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.
[0480] 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.
[0481] 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.
[0482] Further, investigation of cell-binding behavior with flow
cytometry showed that the aptamers revealed very good apparent
affinities that are within the nanomolar range.
[0483] 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.
[0484] 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 388, according to the invention at hand with the MHC
molecule, using the cell-SELEX (Systematic Evolution of Ligands by
Exponential enrichment) technique.
[0485] 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.
[0486] 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.
[0487] 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.
[0488] 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.
[0489] 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.
[0490] 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: 388, or a variant thereof which is at least 88%
homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 388
or a variant thereof that induces T cells cross-reacting with said
peptide, wherein said peptide is not the underlying full-length
polypeptide.
[0491] 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: 388 or a variant thereof which is at
least 88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ
ID NO: 388, 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.
[0492] 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.
[0493] 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: 388.
[0494] The present invention further relates to the peptides
according to the invention, wherein the peptide is (chemically)
modified and/or includes non-peptide bonds.
[0495] 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.
[0496] 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.
[0497] The present invention further relates to the nucleic acid
according to the invention that is DNA, cDNA, PNA, RNA or
combinations thereof.
[0498] The present invention further relates to an expression
vector capable of expressing or expressing a nucleic acid according
to the present invention.
[0499] 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
cancers such as glioblastoma, breast cancer, colorectal cancer,
renal cell carcinoma, chronic lymphocytic leukemia, hepatocellular
carcinoma, non-small cell and small cell lung cancer, Non-Hodgkin
lymphoma, acute myeloid leukemia, ovarian cancer, pancreatic
cancer, prostate cancer, esophageal cancer including cancer of the
gastric-esophageal junction, gallbladder cancer and
cholangiocarcinoma, melanoma, gastric cancer (GC), testis cancer
(TC), urinary bladder cancer (UBC), head and neck squamous cell
carcinoma (HNSCC), or uterine cancer (UEC).
[0500] 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.
[0501] 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.
[0502] 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.
[0503] 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.
[0504] 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: 388 or said variant amino
acid sequence.
[0505] 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.
[0506] 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.
[0507] 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.
[0508] 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.
[0509] The present invention further relates to a use according to
the invention, wherein said cancer cells are solid or hematological
tumor cells such as glioblastoma, breast cancer, colorectal cancer,
renal cell carcinoma, chronic lymphocytic leukemia, hepatocellular
carcinoma, non-small cell and small cell lung cancer, Non-Hodgkin
lymphoma, acute myeloid leukemia, ovarian cancer, pancreatic
cancer, prostate cancer, esophageal cancer including cancer of the
gastric-esophageal junction, gallbladder cancer and
cholangiocarcinoma, melanoma, gastric cancer, testis cancer,
urinary bladder cancer, head and neck squamous cell carcinoma
(HNSCC), or uterine cancer.
[0510] 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 glioblastoma, breast cancer,
colorectal cancer, renal cell carcinoma, chronic lymphocytic
leukemia, hepatocellular carcinoma, non-small cell and small cell
lung cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian
cancer, pancreatic cancer, prostate cancer, esophageal cancer
including cancer of the gastric-esophageal junction, gallbladder
cancer and cholangiocarcinoma, melanoma, gastric cancer, testis
cancer, urinary bladder cancer, head and neck squamous cell
carcinoma (HNSCC), or uterine cancer. The present invention also
relates to the use of these novel targets for cancer treatment.
[0511] 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
glioblastoma, breast cancer, colorectal cancer, renal cell
carcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma,
non-small cell and small cell lung cancer, Non-Hodgkin lymphoma,
acute myeloid leukemia, ovarian cancer, pancreatic cancer, prostate
cancer, esophageal cancer including cancer of the
gastric-esophageal junction, gallbladder cancer and
cholangiocarcinoma, melanoma, gastric cancer, testis cancer,
urinary bladder cancer, head and neck squamous cell carcinoma
(HNSCC), or uterine cancer marker (poly)peptide, delivery of a
toxin to a cancer cell expressing a cancer marker gene at an
increased level, and/or inhibiting the activity of a cancer marker
polypeptide) according to the invention.
[0512] 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.
[0513] The person of skill will understand that either full length
glioblastoma, breast cancer, colorectal cancer, renal cell
carcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma,
non-small cell and small cell lung cancer, Non-Hodgkin lymphoma,
acute myeloid leukemia, ovarian cancer, pancreatic cancer, prostate
cancer, esophageal cancer including cancer of the
gastric-esophageal junction, gallbladder cancer and
cholangiocarcinoma, melanoma, gastric cancer, testis cancer,
urinary bladder cancer, head and neck squamous cell carcinoma
(HNSCC), or uterine 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.
[0514] 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: 388 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 marker polypeptide for above-mentioned cancers used to
generate the antibody according to the invention.
[0515] 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.
[0516] 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 (US 4,816,567, which is hereby
incorporated in its entirety).
[0517] 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.
[0518] 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).
[0519] 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.
[0520] 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.
[0521] 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.
[0522] 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.
[0523] 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.
[0524] 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.
[0525] 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.
[0526] 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
glioblastoma, breast cancer, colorectal cancer, renal cell
carcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma,
non-small cell and small cell lung cancer, Non-Hodgkin lymphoma,
acute myeloid leukemia, ovarian cancer, pancreatic cancer, prostate
cancer, esophageal cancer including cancer of the
gastric-esophageal junction, gallbladder cancer and
cholangiocarcinoma, melanoma, gastric cancer, testis cancer,
urinary bladder cancer, head and neck squamous cell carcinoma
(HNSCC), or uterine 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 occurs in the absence of antibody
administration, is an efficacious antibody for treatment of
cancer.
[0527] 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), 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.
[0528] 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.
[0529] 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.
[0530] 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.
[0531] 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.
[0532] 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.
[0533] 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.
[0534] 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).
[0535] 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.
[0536] In case of a MHC class I epitope being used as an antigen,
the T cells are CD8-positive T cells.
[0537] 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: 388, or a variant amino acid sequence thereof.
[0538] 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 aAPC-based systems often require the
addition of appropriate soluble factors, e.g. cytokines, like
interleukin-12.
[0539] 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, 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.
[0540] 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.
[0541] 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 388.
[0542] 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.
[0543] 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)).
[0544] 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.
[0545] By "aberrantly expressed" the inventors also mean that the
polypeptide is over-expressed compared to levels of expression in
normal tissues 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.
[0546] T cells may be obtained by methods known in the art, e.g.
those described above.
[0547] 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).
[0548] 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.
[0549] 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).
[0550] The present invention is further directed at a kit
comprising:
[0551] (a) a container containing a pharmaceutical composition as
described above, in solution or in lyophilized form;
[0552] (b) optionally a second container containing a diluent or
reconstituting solution for the lyophilized formulation; and
[0553] (c) optionally, instructions for (i) use of the solution or
(ii) reconstitution and/or use of the lyophilized formulation.
[0554] 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.
[0555] 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.
[0556] 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).
[0557] 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.
[0558] 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.
[0559] 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.
[0560] 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.
[0561] 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.
[0562] Since the peptides of the invention were isolated from
glioblastoma, breast cancer, colorectal cancer, renal cell
carcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma,
non-small cell and small cell lung cancer, Non-Hodgkin lymphoma,
acute myeloid leukemia, ovarian cancer, pancreatic cancer, prostate
cancer, esophageal cancer including cancer of the
gastric-esophageal junction, gallbladder cancer and
cholangiocarcinoma, melanoma, gastric cancer, testis cancer,
urinary bladder cancer, or uterine cancer, the medicament of the
invention is preferably used to treat glioblastoma, breast cancer,
colorectal cancer, renal cell carcinoma, chronic lymphocytic
leukemia, hepatocellular carcinoma, non-small cell and small cell
lung cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian
cancer, pancreatic cancer, prostate cancer, esophageal cancer
including cancer of the gastric-esophageal junction, gallbladder
cancer and cholangiocarcinoma, melanoma, gastric cancer, testis
cancer, urinary bladder cancer, head and neck squamous cell
carcinoma (HNSCC), or uterine cancer.
[0563] 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.
[0564] 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.
[0565] 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 glioblastoma, breast
cancer, colorectal cancer, renal cell carcinoma, chronic
lymphocytic leukemia, hepatocellular carcinoma, non-small cell and
small cell lung cancer, Non-Hodgkin lymphoma, acute myeloid
leukemia, ovarian cancer, pancreatic cancer, prostate cancer,
esophageal cancer including cancer of the gastric-esophageal
junction, gallbladder cancer and cholangiocarcinoma, melanoma,
gastric cancer, testis cancer, urinary bladder cancer, head and
neck squamous cell carcinoma (HNSCC), or uterine 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 cancer tissues, 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.
[0566] TUMAPs for the warehouse are identified by using an
integrated functional genomics approach combining gene expression
analysis, mass spectrometry, and T-cell immunology
(XPresident.RTM.). 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, glioblastoma, breast cancer, colorectal
cancer, renal cell carcinoma, chronic lymphocytic leukemia,
hepatocellular carcinoma, non-small cell and small cell lung
cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian
cancer, pancreatic cancer, prostate cancer, esophageal cancer
including cancer of the gastric-esophageal junction, gallbladder
cancer and cholangiocarcinoma, melanoma, gastric cancer, testis
cancer, urinary bladder cancer, head and neck squamous cell
carcinoma (HNSCC), and uterine cancer samples from patients and
blood from healthy donors were analyzed in a stepwise approach:
[0567] 1. HLA ligands from the malignant material were identified
by mass spectrometry
[0568] 2. Genome-wide messenger ribonucleic acid (mRNA) expression
analysis was used to identify genes over-expressed in the malignant
tissue compared with a range of normal organs and tissues
[0569] 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.
[0570] 4. Literature research was performed in order to identify
additional evidence supporting the relevance of the identified
peptides as TUMAPs
[0571] 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.
[0572] 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 cancer patients.
[0573] 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.
[0574] 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.
[0575] 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.
[0576] 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.
[0577] 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: (al) 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.
[0578] 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.
[0579] 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.
[0580] 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.
[0581] 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.
[0582] 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.
[0583] 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 .about.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.
[0584] 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.
[0585] 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 glioblastoma, breast cancer,
colorectal cancer, renal cell carcinoma, chronic lymphocytic
leukemia, hepatocellular carcinoma, non-small cell and small cell
lung cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian
cancer, pancreatic cancer, prostate cancer, esophageal cancer
including cancer of the gastric-esophageal junction, gallbladder
cancer and cholangiocarcinoma, melanoma, gastric cancer, testis
cancer, urinary bladder cancer, head and neck squamous cell
carcinoma (HNSCC), or uterine 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.
[0586] 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 glioblastoma, breast cancer,
colorectal cancer, renal cell carcinoma, chronic lymphocytic
leukemia, hepatocellular carcinoma, non-small cell and small cell
lung cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian
cancer, pancreatic cancer, prostate cancer, esophageal cancer
including cancer of the gastric-esophageal junction, gallbladder
cancer and cholangiocarcinoma, melanoma, gastric cancer, testis
cancer, urinary bladder cancer, head and neck squamous cell
carcinoma (HNSCC), or uterine cancer. Presence of groups of
peptides can enable classification or sub-classification of
diseased tissues.
[0587] 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.
[0588] 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.
[0589] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0590] FIGS. 1A-1Z show the over-presentation of various peptides
in normal tissues (white bars) and different cancers (black bars).
FIG. 1A) Gene symbol: IGFBPL1, Peptide: LLPLLPPLSPSLG (SEQ ID NO:
33)--Tissues from left to right: 1 cell line (1 pancreatic), 1
normal tissue (1 thyroid gland), 22 cancer tissues (5 brain
cancers, 1 breast cancer, 1 colon cancer, 1 esophageal cancer, 1
gallbladder cancer, 1 liver cancer, 10 lung cancers, 1 pancreas
cancer, 1 stomach cancer); FIG. 1B) Gene symbol: HIVEP1, Peptide:
NYIPVKNGKQF (SEQ ID NO: 103)--Tissues from left to right: 11 cancer
tissues (1 brain cancer, 1 liver cancer, 8 lung cancers, 1 prostate
cancer); FIG. 1C) Gene symbol: GET4, Peptide: EYLDRIGQLFF (SEQ ID
NO: 131)--Tissues from left to right: 2 normal tissues (1 kidney, 1
lung), 41 cancer tissues (2 brain cancers, 1 kidney cancer, 3 liver
cancers, 29 lung cancers, 2 prostate cancers, 4 stomach cancers);
FIG. 1D) Gene symbol: N4BP2, Peptide: FYINGQYQF (SEQ ID NO:
176)--Tissues from left to right: 1 cell line (1 prostate), 3
normal tissues (1 kidney, 1 pituitary gland, 1 skin), 67 cancer
tissues (4 brain cancers, 2 liver cancers, 42 lung cancers, 12
prostate cancers, 7 stomach cancers). FIGS. 1E) to Z) show the
over-presentation of various peptides in different cancer tissues
compared to normal tissues. The analyses included data from more
than 440 normal tissue samples, and 526 cancer samples. Shown are
only samples where the peptide was found to be presented. FIG. 1E)
Gene symbol: AKR1C1, AKR1C3, Peptide: HLYNNEEQV (SEQ ID NO:
16)--Tissues from left to right: 1 cell line (pancreas), 15 cancer
tissues (1 bile duct cancer, 1 esophageal cancer, 6 liver cancers,
5 lung cancers, 2 urinary bladder cancers); FIG. 1F) Gene symbol:
RPS26P39, RPS26P11, RPS26, RPS26P28, RPS26P20,RPS26P15, RPS26P50,
RPS26P2, RPS26P25, RPS26P58, Peptide: YVLPKLYVKL (SEQ ID NO:
35)--Tissues from left to right: 1 normal tissue (1 leukocyte
sample), 8 cancer tissues (1 head-and-neck cancer, 3 leukocytic
leukemia cancers, 1 myeloid cells cancer, 1 gallbladder cancer, 1
colon cancer, 1 lymph node cancer); FIG. 1G) Gene symbol: CLDN4,
CLDN3, CLDN14, CLDN6, CLDN9, Peptide: SLLALPQDLQA (SEQ ID NO:
40)--Tissues from left to right: 21 cancer tissues (1 bile duct
cancer, 1 breast cancer, 3 colon cancers, 1 rectum cancer, 6 lung
cancers, 2 ovarian cancers, 1 prostate cancer, 3 urinary bladder
cancers, 3 uterus cancers); FIG. 1H) Gene symbol: KLHDC7B, Peptide:
VLSPFILTL (SEQ ID NO: 42)--Tissues from left to right: 18 cancer
tissues (1 leukocytic leukemia cancer, 1 myeloid cells cancer, 1
breast cancer, 1 kidney cancer, 6 lung cancers, 3 lymph node
cancers, 2 ovarian cancers, 2 urinary bladder cancers, 1 uterus
cancer); FIG. 1I) Gene symbol: ATR, Peptide: SLLSHVIVA (SEQ ID NO:
53)--Tissues from left to right: 3 cell lines (1 blood cell, 2
pancreas), 21 cancer tissues (1 head-and-neck cancer, 1 bile duct
cancer, 2 leukocytic leukemia cancers, 1 breast cancer, 2
esophageal cancers, 1 gallbladder cancer, 1 kidney cancer, 1 liver
cancer, 2 lung cancers, 4 lymph node cancers, 1 ovarian cancer, 3
skin cancers, 1 urinary bladder cancer); FIG. 1J) Gene symbol:
PGAP1, Peptide: FITDFYTTV (SEQ ID NO: 66)--Tissues from left to
right: 1 cell line (skin), 1 normal tissue (1 colon), 13 cancer
tissues (1 head-and-neck cancer, 6 brain cancers, 1 colon cancer, 1
liver cancer, 2 skin cancers, 2 urinary bladder cancers); FIG. 1K)
Gene symbol: ZNF679, SAPCD2, Peptide: RLLPKVQEV (SEQ ID NO:
325)--Tissues from left to right: 4 cell lines (2 blood cells, 1
kidney, 1 large intestine), 22 cancer tissues (1 myeloid cells
cancer, 1 breast cancer, 1 esophageal cancer, 4 colon cancers, 1
rectum cancer, 10 lung cancers, 2 ovarian cancers, 1 stomach
cancer, 1 urinary bladder cancer); FIG. 1L) Gene symbol: ZDHHC24,
Peptide: VLGPGPPPL (SEQ ID NO: 339)--Tissues from left to right: 2
cell lines (1 kidney, 1 pancreas), 19 cancer tissues (4 leukocytic
leukemia cancers, 1 myeloid cells cancer, 1 bone marrow cancer, 2
brain cancers, 1 liver cancer, 2 lung cancers, 6 lymph node
cancers, 1 skin cancer, 1 uterus cancer); FIG. 1M) Gene symbol:
ORC1, Peptide: VYVQILQKL (SEQ ID NO: 111)--Tissues from left to
right: 1 normal tissue (1 liver), 32 cancer tissues (2 liver
cancers, 24 lung cancers, 6 stomach cancers); FIG. 1N) Gene symbol:
RIF1, Peptide: IYSFHTLSF (SEQ ID NO: 113)--Tissues from left to
right: 28 cancer tissues (1 prostate, 1 brain cancer, 25 lung
cancers, 2 stomach cancers); FIG. 1O) Gene symbol: ANKRD5, Peptide:
RYLNKSFVL (SEQ ID NO: 115)--Tissues from left to right: 1 normal
tissue (1 stomach), 25 cancer tissues (1 brain cancer, 2 liver
cancers, 17 lung cancers, 2 prostate cancers, 3 stomach cancers);
FIG. 1P) Gene symbol: IGFLR1, Peptide: RYGLPAAWSTF (SEQ ID NO:
121)--Tissues from left to right: 20 cancer tissues (2 liver
cancers, 17 lung cancers, 1 stomach cancer); FIG. 1Q) Gene symbol:
CCR8, Peptide: VYALKVRTI (SEQ ID NO: 145)--Tissues from left to
right: 25 cancer tissues (25 lung cancers); FIG. 1R) Gene symbol:
CLEC5A, Peptide: SYGTVSQIF (SEQ ID NO: 148)--Tissues from left to
right: 5 normal tissues (1 liver, 3 lungs, 1 pituitary gland), 100
cancer tissues (10 brain cancers, 4 liver cancers, 74 lung cancers,
1 prostate cancer, 11 stomach cancers); FIG. 1S) Gene symbol:
FOXJ1, Peptide: IYKWITDNF (SEQ ID NO: 155)--Tissues from left to
right: 4 normal tissues (4 kidneys), 53 cancer tissues (10 brain
cancers, 1 liver cancer, 26 lung cancers, 1 prostate cancer, 15
stomach cancers); FIG. 1T) Gene symbol: IFNG, Peptide: KYTSYILAF
(SEQ ID NO: 162)--Tissues from left to right: 3 cell lines (3
prostates), 4 normal tissues (1 liver, 1 lung, 1 pancreas, 1
stomach), 95 cancer tissues (1 kidney cancer, 5 liver cancers, 71
lung cancers, 2 prostate cancers, 16 stomach cancers); FIG. 1U)
Gene symbol: KLHL11, Peptide: EYFTPLLSGQF (SEQ ID NO: 165)--Tissues
from left to right: 10 cancer tissues (10 lung cancers); FIG. 1V)
Gene symbol: TMEM189, Peptide: LYSPVPFTL (SEQ ID NO: 175)--Tissues
from left to right: 42 cancer tissues (4 brain cancers, 1 liver
cancer, 30 lung cancers, 7 stomach cancers); FIG. 1W) Gene symbol:
BUB1, Peptide: EYNSDLHQFF (SEQ ID NO: 345)--Tissues from left to
right: 13 cancer tissues (3 brain cancers, 10 lung cancers); FIG.
1X) Gene symbol: CASC5, Peptide: IYVIPQPHF (SEQ ID NO:
346)--Tissues from left to right: 21 cancer tissues (3 brain
cancers, 1 kidney cancer, 1 liver cancer, 14 lung cancers, 2
stomach cancers); FIG. 1Y) Gene symbol: KIF18A, Peptide: VYNEQIRDLL
(SEQ ID NO: 354)--Tissues from left to right: 13 cancer tissues (1
brain cancer, 11 lung cancers, 1 stomach cancer); and FIG. 1Z) Gene
symbol: PSMA8, PSMA7, Peptide: VFSPDGHLF (SEQ ID NO: 360)--Tissues
from left to right: 33 cancer tissues (4 liver cancers, 27 lung
cancers, 1 prostate cancer, 1 stomach cancer).
[0591] FIGS. 2A-2D show exemplary expression profiles of source
genes of the present invention that are highly over-expressed or
exclusively expressed in different cancers in a panel of normal
tissues (white bars) and different cancer samples (black bars).
FIG. 2A) Gene symbol: MXRA5--Tissues from left to right: 61 normal
tissue samples (6 arteries, 1 brain, 1 heart, 2 livers, 2 lungs, 2
veins, 1 adipose tissue, 1 adrenal gland, 5 bone marrows, 1
cartilage, 1 colon, 1 esophagus, 2 gallbladders, 1 kidney, 6 lymph
nodes, 1 pancreas, 1 pituitary gland, 1 rectum, 1 skeletal muscle,
1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thymus, 1 thyroid
gland, 5 tracheas, 1 urinary bladder, 1 breast, 5 ovaries, 3
.mu.lacentas, 1 prostate, 1 testis, 1 uterus) and 70 cancer samples
(10 breast cancers, 11 lung cancers, 12 ovary cancers, 11
esophageal cancers, 26 pancreas cancers); FIG. 2B) Gene symbol:
KIF26B--Tissues from left to right: 61 normal tissue samples (6
arteries, 1 brain, 1 heart, 2 livers, 2 lungs, 2 veins, 1 adipose
tissue, 1 adrenal gland, 5 bone marrows, 1 cartilage, 1 colon, 1
esophagus, 2 gallbladders, 1 kidney, 6 lymph nodes, 1 pancreas, 1
pituitary gland, 1 rectum, 1 skeletal muscle, 1 skin, 1 small
intestine, 1 spleen, 1 stomach, 1 thymus, 1 thyroid gland, 5
tracheas, 1 urinary bladder, 1 breast, 5 ovaries, 3 .mu.lacentas, 1
prostate, 1 testis, 1 uterus) and 58 cancer samples (10 breast
cancers, 11 lung cancers, 11 esophageal cancers, 26 pancreas
cancers); FIG. 2C) Gene symbol: IL4I1--Tissues from left to right:
61 normal tissue samples (6 arteries, 1 brain, 1 heart, 2 livers, 2
lungs, 2 veins, 1 adipose tissue, 1 adrenal gland, 5 bone marrows,
1 cartilage, 1 colon, 1 esophagus, 2 gallbladders, 1 kidney, 6
lymph nodes, 1 pancreas, 1 pituitary gland, 1 rectum, 1 skeletal
muscle, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thymus, 1
thyroid gland, 5 tracheas, 1 urinary bladder, 1 breast, 5 ovaries,
3 .mu.lacentas, 1 prostate, 1 testis, 1 uterus) and 34 cancer
samples (11 lung cancers, 12 ovary cancers, 11 esophageal cancers);
FIG. 2D) Gene symbol: TP63--Tissues from left to right: 61 normal
tissue samples (6 arteries, 1 brain, 1 heart, 2 livers, 2 lungs, 2
veins, 1 adipose tissue, 1 adrenal gland, 5 bone marrows, 1
cartilage, 1 colon, 1 esophagus, 2 gallbladders, 1 kidney, 6 lymph
nodes, 1 pancreas, 1 pituitary gland, 1 rectum, 1 skeletal muscle,
1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thymus, 1 thyroid
gland, 5 tracheas, 1 urinary bladder, 1 breast, 5 ovaries, 3
.mu.lacentas, 1 prostate, 1 testis, 1 uterus) and 11 esophageal
cancer samples
[0592] FIGS. 3A-3B show exemplary immunogenicity data: flow
cytometry results after peptide-specific multimer staining.
[0593] FIGS. 4A-4C show exemplary results of peptide-specific in
vitro CD8+ T cell responses of a healthy HLA-A*02+ donor. CD8+ T
cells were primed using artificial APCs coated with anti-CD28 mAb
and HLA-A*02 in complex with SEQ ID NO: 2 peptide (FIG. 4A, left
panel), SEQ ID NO: 9 peptide (FIG. 4B, left panel) and SEQ ID NO:
331 peptide (FIG. 4C, left panel), respectively. After three cycles
of stimulation, the detection of peptide-reactive cells was
performed by 2D multimer staining with A*02/SEQ ID NO: 2 (FIG. 4A),
A*02/SEQ ID NO: 9 (FIG. 4B) or A*02/SEQ ID NO: 331 (FIG. 4C). Right
panels (FIGS. 4A, 4B and 4C) 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.
[0594] FIGS. 5A-5D show exemplary results of peptide-specific in
vitro CD8+ T cell responses of a healthy HLA-A*24+ donor. CD8+ T
cells were primed using artificial APCs coated with anti-CD28 mAb
and HLA-A*24 in complex with SEQ ID NO: 99 peptide (FIG. 5A, left
panel), SEQ ID NO: 119 peptide (FIG. 5B, left panel), SEQ ID NO:
142 peptide (FIG. 5C, left panel) and SEQ ID NO: 174 peptide (FIG.
5D, left panel), respectively. After three cycles of stimulation,
the detection of peptide-reactive cells was performed by 2D
multimer staining with A*24/SEQ ID NO: 99 (FIG. 5A), A*24/SEQ ID
NO: 119 (B), A*24/SEQ ID NO: 142 (C) or A*24/SEQ ID NO: 174 (FIG.
5D). Right panels (FIGS. 5A, 5B, 5C and 5D) show control staining
of cells stimulated with irrelevant A*24/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
[0595] Identification and Quantitation of Tumor Associated Peptides
Presented on the Cell Surface
[0596] Tissue Samples
[0597] Patients' tumor tissues were obtained under written informed
consents of all patients 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.
[0598] Peptides were selected if two conditions were true: (1) Its
underlying transcript(s) and/or exon(s) are expressed at very low
levels, i.e. the median reads per kilobase per million reads (RPKM)
was required to be less than two, and the 75% quartile was required
to be less than 5 for the following organs: brain, blood vessel,
heart, liver, lung, blood. In addition, the median RPKM was
required to be less than 10 for the following organs: urinary
bladder, salivary gland, stomach, adrenal gland, colon, small
intestine, spleen, bone marrow, pancreas, muscle, adipose tissue,
skin, esophagus, kidney, thyroid gland, pituitary gland, nerve. (2)
The peptide was tumor-associated, i.e. found specifically or on
tumors or over-expressed compared to a baseline of normal tissues
(cf. Example 1).
[0599] Sample numbers for HLA-A*02 TUMAP selection were: for
pancreatic cancer N=16, for renal cancer N=20, for colorectal
cancer N=28, for esophageal carcinoma including cancer of the
gastric-esophageal junction N=15, for prostate tumors N=35, for
hepatocellular carcinoma N=16, for non-small cell lung cancer N=88,
for gastric cancer N=29, for breast cancer N=9, for melanoma N=3,
for ovarian cancer N=20, for chronic lymphocytic leukemia N=13 (of
12 donors), for urinary bladder cancer N=5, for testis cancer N=1,
for small-cell lung cancer N=18 (of 13 donors), for gallbladder
cancer and cholangiocarcinoma N=3, for acute myeloid leukemia N=5
(of 4 donors), for glioblastoma N=40, and for uterine cancer
N=5.
[0600] Sample numbers for HLA-A*24 TUMAP selection were: for
gastric cancer N=44, for prostate tumors N=37, for non-small cell
lung cancer N=88, for hepatocellular carcinoma N=15, for renal
cancer N=2, for colorectal cancer N=1, and for glioblastoma
N=17.
[0601] Isolation of HLA Peptides from Tissue Samples
[0602] HLA peptide pools from shock-frozen tissue samples were
obtained by immune precipitation from solid tissues according to
published protocols (Falk et al., 1991; Seeger et al., 1999) with
minor modifications using the HLA-A*02-specific antibody BB7.2, the
HLA-A, -B, -C-specific antibody W6/32, the HLA class II-specific
antibody L243, CNBr-activated sepharose, acid treatment, and
ultrafiltration.
[0603] Mass Spectrometry Analyses
[0604] 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.
[0605] 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 different cancer samples to a
baseline of normal tissue samples. Presentation profiles of
exemplary over-presented peptides are shown in FIGS. 1A-1Z.
[0606] Table 8 (A and B) and 9 (A and B) show the presentation on
various cancer entities for selected peptides, and thus the
particular relevance of the peptides as mentioned for the diagnosis
and/or treatment of the cancers as indicated (e.g. peptide SEQ ID
No. 1 for urinary bladder cancer, esophageal cancer, including
cancer of the gastric-esophageal junction, hepatocellular
carcinoma, non-small cell lung cancer, and pancreatic cancer,
peptide SEQ ID No. 2 for renal cancer, esophageal cancer, including
cancer of the gastric-esophageal, glioblastoma, . . . etc. ).
TABLE-US-00011 TABLE 8A Overview of presentation of selected
HLA-A*02-binding tumor-associated peptides of the present invention
across entities. GB = glioblastoma, BRCA = breast cancer, CRC =
colorectal cancer, RCC = renal cell carcinoma, CLL = chronic
lymphocytic leukemia, HCC = hepatocellular carcinoma, NSCLC =
non-small cell lung cancer, SCLC = small cell lung cancer, NHL =
non-Hodgkin lymphoma (8 samples), AML = acute myeloid leukemia, OC
= ovarian cancer, PC = pancreatic cancer, cIPC = pancreatic cancer
cell lines, PCA = prostate cancer and benign prostate hyperplasia,
OSCAR = esophageal cancer, including cancer of the gastric-
esophageal junction, GBC_CCC = gallbladder adenocarcinoma and
cholangiocarcinoma, MEL = melanoma, GC = gastric cancer, TC =
testis cancer, UBC = urinary bladder cancer, UEC = uterine cancer.
SEQ ID Peptide Presentation No. Sequence on cancer entities 1
HLYNNEEQV UBC, OSCAR, HCC, NSCLC, cIPC 2 ALYGKLLKL RCC, OSCAR, GB,
BRCA, CLL, UBC, HCC, SCLC, NSCLC, CRC, OC, GC, NHL 4 ELAEIVFKV CRC,
CLL, NSCLC, GB 5 SLFGQEVYC HCC, GB, CRC 6 FLDPAQRDL UBC, NSCLC, GB,
AML, cIPC 7 AAAAKVPEV NSCLC, GB, CRC 8 KLGPFLLNA GC, GB, cIPC,
NSCLC 9 FLGDYVENL CLL, CRC, UBC 10 KTLDVFNII CLL, GC, GBC_CCC,
OSCAR L 11 GVLKVFLEN HCC, NSCLC, GC, OC, OSCAR V 12 GLIYEETRG GC,
OSCAR, NSCLC, NHL V 13 VLRDNIQGI NSCLC, GBC_CCC, OC, GC, BRCA,
OSCAR, CRC, GB, UEC, CLL, RCC, UBC, HCC, MEL, SCLC, NHL 15
ALGDYVHAC HCC, GC 16 PLWGKVFYL GBC_CCC, NSCLC, GB, cIPC, CRC 17
ILHEHHIFL RCC, NSCLC 19 TLLPTVLTL RCC, UBC, SCLC 20 ALDGHLYAI RCC,
UBC, GC 21 SLYHRVLLY RCC, OSCAR, NSCLC 22 MLSDLTLQL CRC, PCA, RCC,
NSCLC, BRCA, GC, SCLC, PC, OSCAR 23 AQTVVVIKA GC 24 FLWNGEDS PC,
CRC AL 25 IQADDFRTL GC 26 KVDGVVIQL OSCAR, GC 27 KVFGDLDQV OSCAR,
GC 29 TLCNKTFTA PCA, GB 30 TVIDECTRI GC 31 ALSDETKNN AML, CLL WEV
32 ILADEAFF CLL, PC, GB, UBC, PCA, SV CRC, SCLC, HCC, RCC, OC,
NSCLC, MEL, OSCAR, cIPC, GC, BRCA, NHL 34 LLPKKTESH CRC, HCC,
NSCLC, GB HKT 35 YVLPKLYVK CLL, CRC, NSCLC, L GBC_CCC, UBC, OSCAR
36 KLYGIEIEV NSCLC, GB, RCC 37 ALINDILGE cIPC, CRC, RCC, HCC LVKL
38 KMQEDLVTL NSCLC, RCC, OC, GC, GBC_CCC, OSCAR, cIPC, GB, BRCA,
PCA, PC, UEC, HCC, CRC, SCLC, NHL 39 ALMAWSGL OSCAR, GBC_CCC, CLL,
BRCA, HCC, UBC, NSCLC, OC, GC, MEL 40 SLLALPQD PCA, NSCLC, CRC,
UBC, LQA OC 41 FVLPLWTL OC, OSCAR, CLL, PCA, SCLC, NSCLC, NHL 42
VLSPFILTL NSCLC, RCC, BRCA, UBC, OC, NHL 43 LLWAGPVTA CLL, HCC,
CRC, NSCLC, RCC, UBC, NHL, TC 44 GLLWQIIKV CRC, NSCLC, GC 45
VLGPTPELV CRC, SCLC, GC, cIPC, PC 46 SLAKHGIVAL PC, NSCLC, CRC,
RCC, OC, cIPC, PCA, NHL 47 GLYQAQVNL NSCLC, SCLC 48 TLDHKPVTV OC,
PCA, NSCLC 49 LLDESKLTL UBC, OC, PCA, NSCLC, RCC 50 EYALLYHTL CRC,
GC 51 LLLDGDFTL SCLC, HCC 52 ELLSSIFFL UBC, NSCLC, CRC, RCC 53
SLLSHVIVA cIPC, RCC, GBC_CCC, UBC, NSCLC 54 FINPKGNWLL UBC, NSCLC,
cIPC 55 IASAIVNEL BRCA, GC 56 KILDLTRVL CRC, NSCLC 57 VLISSTVRL
cIPC, RCC, CLL, NHL 58 ALDDSLTSL cIPC, PC, GB 59 ALTKILAEL UBC,
NSCLC 60 FLIDTSASM UBC, SCLC, RCC 61 HLPDFVKQL BRCA, CLL, GBC_CCC
62 SLFNQEVQI CLL, NHL 63 TLSSERDFAL NSCLC, GC 66 FITDFYTTV GB 67
GVIETVTSL NSCLC, GC, CRC 68 ALYGFFFKI UBC 69 GIYDGILHSI UEC, GB 70
GLFSQHFNL HCC, NSCLC 71 GLITVDIAL SCLC, PCA 72 GMIGFQVLL UBC, OC,
CLL, GB, GBC_CCC 74 ILDETLENV UBC, SCLC, NHL 76 ILLDESNFNHFL NSCLC
77 IVLSTIASV cIPC, CRC, PCA 81 VLFLGKLLV CRC, UBC 82 VLLRVLIL
NSCLC, TC 83 ELLEYLPQL PC, GBC_CCC 84 FLEEEITRV CRC, GC 85
STLDGSLHAV OSCAR, PCA, GC 87 YLTEVFLHW CLL, NHL 89 YLVAHNLLL RCC 90
GAVAEEVLSSI GB 92 LLRGPPVARA cIPC, PC, RCC, UBC, OSCAR 93 SLLTQPIFL
RCC, HCC 321 SLWFKPEEL GC, BRCA, CLL, PC, GB, UBC, PCA, CRC, SCLC,
HCC, MEL, OC, NSCLC, GBC_CCC, OSCAR, cIPC, NHL 322 ALVSGGVAQA
GBC_CCC, OSCAR, BRCA, CLL, UBC, HCC, PC, SCLC, NSCLC, CRC, OC, NHL
323 ILSVVNSQL CRC, GC, BRCA, OC, CLL, NSCLC, NHL 324 AIFDFCPSV
NSCLC, CRC, GC, MEL, GB, OSCAR, CLL 325 RLLPKVQEV OSCAR, NSCLC,
CRC, SCLC, OC 326 SLLPLVWKI NSCLC, CRC, GB, RCC, MEL, CLL, TC 327
SIGDIFLKY GC, GB, CRC, RCC, NSCLC 328 SVDSAPAAV SCLC, OC, PC,
OSCAR, RCC, NSCLC, UBC
329 FAWEPSFRDQV SCLC, HCC 330 FLWPKEVEL NSCLC, BRCA, SCLC, OC, CLL,
NHL 331 AIWKELISL GB, CRC 332 AVTKYTSAK CLL, NSCLC, MEL, NHL 333
GTFLEGVAK RCC, CLL, HCC 334 GRADALRVL BRCA, SCLC, CLL 335
VLLAAGPSAA UBC, CLL, NSCLC, GC, cIPC 336 GLMDGSPHFL PC, NSCLC 337
KVLGKIEKV RCC, CRC 339 VLGPGPPPL NSCLC, cIPC, CLL, NHL 340
SVAKTILKR NSCLC, OSCAR
[0607] Table 8B shows the presentation on additional cancer
entities for selected peptides, and thus the particular relevance
of the peptides as mentioned for the diagnosis and/or treatment of
the cancers as indicated.
TABLE-US-00012 TABLE 8B Overview of presentation of selected
HLA-A*02 peptides across entities. GB = glioblastoma, BRCA = breast
cancer, CRC = colorectal cancer, RCC = renal cell carcinoma, CLL =
chronic lymphocytic leukemia, HCC = hepatocellular carcinoma, NSCLC
= non-smallcell lung cancer, SCLC = small cell lung cancer, NHL =
non-Hodgkin lymphoma, AML = acutemyeloid leukemia, OC = ovarian
cancer, PC = pancreatic cancer, BPH = prostate cancer and benign
prostate hyperplasia, OSCAR = esophageal cancer, including cancer
of the gastric- oesophageal junction, GBC_CCC = gallbladder
adenocarcinoma and cholangiocarcinoma, MEL = melanoma, GC = gastric
cancer, UBC = urinary bladder cancer, UTC = uterine cancer, HNSCC =
head and neck squamous cell carcinoma. SEQ ID Peptide Presentation
No. Sequence on cancer entities 1 HLYNNEEQV GBC_CCC 2 ALYGKLLKL
cIPC, UTC, PCA, MEL, AML 3 TLLGKQVTL CLL, NSCLC, NHL, AML 5
SLFGQEVYC GBC_CCC, PCA 6 FLDPAQRDL MEL 7 AAAAKVPEV MEL, HNSCC, NHL
8 KLGPFLLNA UTC, HCC 9 FLGDYVENL UTC, AML, OC, cIPC 12 GLIYEETRGV
AML, UTC, HNSCC 13 VLRDNIQGI AML, HNSCC 17 ILHEHHIFL UTC 18
YVLNEEDLQKV UTC, NSCLC 19 TLLPTVLTL GBC_CCC, BRCA, UTC 22 MLSDLTLQL
MEL 24 FLWNGEDSAL NSCLC, GC, UTC 28 TLYSMDLMKV HNSCC, RCC 32
ILADEAFFSV HNSCC, UTC, AML, GBC_CCC 33 LLLPLLPPLSPSL MEL, NSCLC,
GBC_CCC, G GC, cIPC, SCLC, GB, PC, MCC, CRC, HCC 34 LLPKKTESHHKT
UTC 35 YVLPKLYVKL HNSCC, NHL, AML 36 KLYGIEIEV UTC 37 ALINDILGELVKL
MEL, UTC 38 KMQEDLVTL MEL, AML 39 ALMAWSGL HNSCC, NHL, UTC, AML 40
SLLALPQDLQA GBC_CCC, BRCA, UTC 41 FVLPLWTL AML, CRC, BRCA, HNSCC,
UTC 42 VLSPFILTL AML, CLL, UTC 43 LLWAGPVTA HNSCC 45 VLGPTPELV
OSCAR, GBC_CCC, BRCA 46 SLAKHGIVAL UBC, HNSCC, GB, CLL, MEL, UTC,
HCC 47 GLYQAQVNL OSCAR 50 EYALLYHTL GBC_CCC 51 LLLDGDFTL OSCAR 53
SLLSHVIVA HCC, AML, OC, OSCAR, HNSCC, MEL, CLL, NHL, BRCA 54
FINPKGNWLL UTC, HNSCC 55 IASAIVNEL HCC, GBC_CCC 56 KILDLTRVL
GBC_CCC 57 VLISSTVRL MEL 59 ALTKILAEL HCC 60 FLIDTSASM AML, CLL,
BRCA, HNSCC, UTC, NHL 61 HLPDFVKQL MEL, AML 64 GLSSSSYEL GBC_CCC,
HCC 65 KLDGICWQV GBC_CCC, HCC 66 FITDFYTTV MEL, UBC, HCC, HNSCC,
CRC 67 GVIETVTSL AML 70 GLFSQHFNL BRCA, UTC, HNSCC, UBC, AML,
OSCAR, cIPC 71 GLITVDIAL AML, MEL, UTC 72 GMIGFQVLL HNSCC, AML 73
GVPDTIATL GC 74 ILDETLENV AML, BRCA 75 ILDNVKNLL AML 77 IVLSTIASV
AML 78 LLWGHPRVA NSCLC 79 SLVPLQILL HCC 80 TLDEYLTYL HCC 81
VLFLGKLLV HNSCC 86 LLVTSLVW HCC, GBC_CCC 88 ILLNTEDLASL RCC 91
SSLEPQIQPV MEL, CLL 93 SLLTQPIFL GBC_CCC 321 SLWFKPEEL UTC, HNSCC,
AML 322 ALVSGGVAQA AML, GC, cIPC, UTC, MEL 323 ILSWNSQL MEL,
GBC_CCC, AML, OSCAR 324 AIFDFCPSV BRCA, NHL, UTC, AML, HNSCC 325
RLLPKVQEV AML, BRCA, UBC, GC 326 SLLPLVWKI AML 327 SIGDIFLKY MEL,
AML 328 SVDSAPAAV NHL, BRCA, AML, UTC, CLL, HNSCC, MEL 329
FAWEPSFRDQV GBC_CCC 330 FLWPKEVEL AML 331 AIWKELISL MEL, CLL, NSCLC
333 GTFLEGVAK MEL, NSCLC 334 GRADALRVL MEL, GBC_CCC, AML 335
VLLAAGPSAA AML, CRC, UTC, NHL 336 GLMDGSPHFL MEL 338 LLYDGKLSSA
CRC, UBC, OC 339 VLGPGPPPL MEL, GB, UTC, AML, HCC 340 SVAKTILKR
NHL
TABLE-US-00013 TABLE 9A Overview of presentation of selected
HLA-A*24-binding tumor-associated peptides of the present invention
across entities, GB = glioblastoma, HCC = hepatocellular carcinoma,
NSCLC = non-small cell lung cancer, PCA = prostate cancer, GC =
gastric cancer, CRC = colorectal cancer, RCC = renal cell
carcinoma, SEQ ID NO. Sequence ENTITIES 96 LYSPVPFTL HCC, NSCLC,
GC, GB 97 TYTFLKETF PCA, HCC, NSCLC, GB 98 VFPRLHNVLF HCC, NSCLC,
GC 99 QYILAVPVL NSCLC, GC, GB, PCA 100 VYIESRIGTS GB, HCC, NSCLC,
GC TSF 101 IYIPVLPPHL HCC, NSCLC 102 VYPFENFEF GC, NSCLC 103
NYIPVKNGK PCA, HCC, NSCLC, GB QF 104 SYLTWHQQI PCA, HCC, NSCLC 105
IYNETITDLL GC, GB, HCC, NSCLC 106 IYNETVRDLL GC, GB, NSCLC 107
KYFPYLVVI HCC, NSCLC, GC 109 LFITGGQFF HCC, NSCLC, GC 110 SYPKIIEEF
GB, HCC, NSCLC, GC 111 VYVQILQKL GC, HCC, NSCLC 112 IYNFVESKL
NSCLC, GC 113 IYSFHTLSF NSCLC, GC, GB 114 QYLDGTWSL NSCLC, GC, GB
115 RYLNKSFVL NSCLC, GC, GB, PCA, HCC 116 AYVIAVHLF GB, PCA, HCC,
NSCLC 117 IYLSDLTYI HCC, NSCLC, GC, PCA 118 KYLNSVQYI HCC, NSCLC,
GC, GB, PCA 119 VYRVYVTTF NSCLC, GC 120 GYIEHFSLW HCC, NSCLC, GC
121 RYGLPAAWS HCC, NSCLC, GC TF 122 EYQARIPEF NSCLC, GC, GB, PCA,
HCC 123 VYTPVLEHL NSCLC, GC, GB, HCC 124 TYKDYVDLF GC, RCC, GB,
PCA, HCC, NSCLC 125 VFSRDFGLL GC, HCC, NSCLC VF 126 PYDPALGSPS
NSCLC, GC, PCA, HCC RLF 127 QYFTGNPLF NSCLC, GC, GB, RCC, PCA 128
VYPFDWQYI GB, PCA, HCC, NSCLC, GC 129 KYIDYLMTW NSCLC, GC, GB, PCA,
HCC 130 VYAHIYHQHF NSCLC, GC, PCA HCC 131 EYLDRIGQL NSCLC, GC, RCC,
GB, PCA, FF HCC 132 RYPALFPVL HCC, NSCLC, GC, GB, PCA 133
KYLEDMKTYF HCC, NSCLC, GC, GB 134 AYIPTPIYF PCA, NSCLC, GB 135
VYEAMVPLF GC, NSCLC 136 IYPEWPVVFF GC 137 EYLHNCSYF GC, PCA, HCC,
NSCLC 138 VYNAVSTSF NSCLC, GC 139 IFGIFPNQF PCA, NSCLC 142
VYVDDIYVI NSCLC, GC 143 KYIFQLNEI GB, NSCLC 144 VFASLPGFLF NSCLC,
GC 145 VYALKVRTI NSCLC 147 LYLAFPLAF NSCLC, GC, PCA, HCC 148
SYGTVSQIF PCA, HCC, NSCLC, GC, GB 149 SYGTVSQI HCC, NSCLC, GB 150
IYITRQFVQF PCA, HCC, NSCLC, GB 151 AYISGLDVF HCC, NSCLC, PCA 153
VYVPFGGKS NSCLC MITF 154 VYGVPTPHF GB, NSCLC 155 IYKWITDNF HCC,
NSCLC, GC, GB 156 YYMELTKLLL NSCLC, GC, HCC 157 DYIPASGFA NSCLC, GB
LF 158 IYEETRGVL HCC, NSCLC, GC KVF 159 IYEETRGVL HCC, NSCLC 160
RYGDGGSSF PCA, NSCLC, GC 161 KYPDIVQQF PCA, HCC, NSCLC, GC 162
KYTSYILAF NSCLC, GC, PCA, HCC 163 RYLTISNLQF NSCLC 165 EYFTPLLSG
NSCLC QF 166 FYTLPFHLI HCC, NSCLC 168 RYLEAALRL NSCLC, GC, GB, PCA,
HCC 169 NYITGKGDVF NSCLC, PCA 170 QYPFHVPLL GC, PCA, HCC, NSCLC 174
VYEKNGYIYF NSCLC, GB 175 YYTQYSQTI GB, NSCLC 176 FYINGQYQF GB, PCA,
HCC, NSCLC, GC 177 VYFKAGLDVF PCA, NSCLC 178 NYSSAVQKF PCA, HCC,
NSCLC, GB 179 TYIPVGLGR NSCLC, GC LL 180 KYLQVVGMF NSCLC, GB 182
AYAQLGYLLF NSCLC 183 PYLQDVPRI NSCLC, GB 186 VFTTSSNIF NSCLC, GB
187 AYAANVHYL NSCLC 188 GYKTFFNEF NSCLC 192 RYSTFSEIF HCC, NSCLC,
GC 194 VYQSLSNSL NSCLC 195 AYIKGGWIL RCC, HCC, NSCLC, GC 196
GYIRGSWQF NSCLC, GC 197 IFTDIFHYL HCC, NSCLC, GC, GB 199 SYLNHLNNL
NSCLC 201 GYNPNRVFF GB, NSCLC 202 RYVEGIVSL NSCLC 204 EYLSTCSKL
NSCLC, HCC 206 NYLDVATFL NSCLC, GC, GB, PCA, HCC 207 LYSDAFKFI
NSCLC VF 209 AFIETPIPLF NSCLC 210 IYAGVGEFSF NSCLC, GC 215
SYVASFFLL GC, NSCLC 217 IYISNSIYF NSCLC, GC 221 KYIGNLDLL NSCLC, GB
223 TFITQSPLL NSCLC 225 TYTNTLERL NSCLC 226 MYLKLVQLF HCC, GC 228
IYQYVADNF NSCLC 229 IYQFVADSF NSCLC 232 YYLSDSPLL NSCLC, GC 234
SYLPAIWLL GC 235 VYKDSIYYI GB, PCA, HCC, NSCLC, GC 236 VYLPKIPSW
HCC, NSCLC 238 SYLEKVRQL NSCLC 240 YYFFVQEKI HCC, NSCLC 243
SYLELANTL PCA, NSCLC 248 AFPTFSVQL NSCLC 249 RYHPTTCTI NSCLC 250
KYPDIASPT HCC
F 251 VYTKALSSL NSCLC, HCC 252 AFGQETNVP HCC LNNF 253 IYGFFNENF HCC
254 KYLESSATF NSCLC 255 VYQKIILKF HCC 257 IFIDNSTQPLHF HCC 259
YFIKSPPSQLF NSCLC, GC 260 VYMNVMTRL NSCLC 261 GYIKLINFI GC 262
VYSSQFETI GB 264 LYTETRLQF NSCLC 265 SYLNEAFSF PCA 266 KYTDWTEFL
HCC, NSCLC, GC, GB, PCA 268 IFITKALQI GC 269 QYPYLQAFF NSCLC 271
RFLMKSYSF HCC 274 KQLDIANYELF NSCLC, GB, HCC 275 KYGTLDVTF NSCLC
276 QYLDVLHAL GC, RCC 277 FYTFPFQQL GC, RCC, PCA, HCC, NSCLC 280
TYNPNLQDKL HCC 281 NYSPGLVSLIL NSCLC 284 DYLKDPVTI NSCLC 285
VYVGDALLHAI PCA 286 SYGTILSHI NSCLC 288 VYPDTVALTF NSCLC, GC 289
FFHEGQYVF GC 290 KYGDFKLLEF PCA, GB 295 SYLVIHERI NSCLC, GC 296
SYQVIFQHF NSCLC, GC 297 TYIDTRTVF PCA, HCC, NSCLC, GC 298 AYKSEWYF
NSCLC, GB 299 KYQYVLNEF NSCLC, GC, GB 300 TYPSQLPSL CRC, GC 301
KFDDVTMLF NSCLC, GC, HCC 303 LYSVIKEDF GB, PCA, HCC, NSCLC, GC 304
EYNEVANLF HCC, NSCLC, GC, RCC, GB, PCA 305 NYENKQYLF NSCLC, GB, HCC
306 VYPAEQPQI NSCLC 307 GYAFTLPLF NSCLC, GC 308 TFDGHGVFF NSCLC, GC
309 KYYRQTLLF PCA, HCC, NSCLC, GC, GB 310 IYAPTLLVF GC, GB, RCC,
HCC, NSCLC 311 EYLQNLNHI PCA 312 SYTSVLSRL PCA, HCC, NSCLC 313
KYTHFIQSF NSCLC, GC, RCC, GB, PCA, HCC 314 RYFKGDYSI GB, HCC 315
FYIPHVPVSF HCC, NSCLC 316 VYFEGSDFKF GB, PCA, HCC, NSCLC 317
VFDTSIAQLF GB, RCC, HCC, NSCLC, GC 318 TYSNSAFQYF GC, RCC, PCA,
HCC, NSCLC 319 KYSDVKNLI PCA, HCC, NSCLC, GB 320 KFILALKVLF HCC,
NSCLC 341 SYLTQHQRI PCA, NSCLC 343 NYLGGTSTI PCA, HCC, GB 344
EYNSDLHQF GB, RCC, HCC, NSCLC, GC 345 EYNSDLHQFF GB, NSCLC 346
IYVIPQPHF NSCLC, GC, GB, HCC 347 VYAEVNSL GB, NSCLC, GC 348
IYLEHTESI GC, HCC, NSCLC 349 QYSIISNVF GC, HCC, NSCLC 350 KYGNFIDKL
NSCLC, GC, HCC 351 IFHEVPLKF HCC, NSCLC 352 QYGGDLTNTF NSCLC, GB
353 TYGKIDLGF HCC, NSCLC, GC, GB 354 VYNEQIRDLL NSCLC, GC, GB 355
IYVTGGHLF HCC, NSCLC, GC, RCC, GB, PCA 356 NYMPGQLTI RCC, NSCLC, GC
357 QFITSTNTF NSCLC 358 YYSEVPVKL NSCLC, GB 359 NYGVLHVTF RCC, HCC,
NSCLC 360 VFSPDGHLF GB, PCA, HCC, NSCLC 361 TYADIGGLD PCA, NSCLC,
GC, NQI GB, RCC 362 VYNYAEQTL GC, GB, NSCLC 363 SYAELGTTI GB,
NSCLC, GC 365 VFIDHPVHL NSCLC, GB 366 QYLELAHSL HCC, NSCLC, GC 367
LYQDHMQYI HCC, NSCLC, GC, GB, PCA 368 KYQNVKHNL NSCLC, HCC 369
VYTHEVVTL NSCLC 370 RFIGIPNQF PCA 371 AYSHLRYVF GB, PCA, HCC, NSCLC
373 GYISNGELF PCA, HCC 375 KYTDYILKI NSCLC 376 VYTPVASRQSL HCC,
NSCLC, GC, GB, PCA 377 QYTPHSHQF HCC, NSCLC 378 VYIAELEKI HCC,
NSCLC 380 VYTGIDHHW NSCLC, GC, RCC, GB, PCA, HCC 382 AYLPPLQQVF
PCA, HCC, NSCLC, GC, RCC, GB 383 RYKPGEPITF GB, PCA, HCC, NSCLC 384
RYFDVGLHNF GC, GB, PCA, HCC, NSCLC 385 QYIEELQKF NSCLC, HCC 386
TFSDVEAHF HCC, NSCLC, GC, GB 387 KYTEKLEEI HCC, NSCLC, GB, PCA 388
IYGEKTYAF HCC, NSCLC, GC, RCC, GB, PCA 389 EYLPEFLHTF NSCLC 390
RYLWATVTI GC, HCC, NSCLC 391 LYQILQGIVF NSCLC, GC, GB, RCC, HCC 392
RYLDSLKAIVF NSCLC, GC, RCC, HCC 393 KYIEAIQWI HCC, NSCLC 394
FYQPKIQQF GB, PCA, HCC, NSCLC, GC 395 LYINKANIW NSCLC, GC, HCC 396
YYHFIFTTL GB 397 IYNGKLFDL GB, NSCLC, GC 398 IYNGKLFDLL CRC, GC,
RCC, GB, PCA, HCC, NSCLC 399 SYIDVLPEF HCC, NSCLC, GC, RCC, GB, PCA
400 KYLEKYYNL NSCLC 401 VFMKDGFFYF NSCLC, GC, PCA 402 VWSDVTPLTF
NSCLC, CRC, GC, RCC, GB, PCA, HCC 403 TYKYVDINTF NSCLC, GC 404
RYLEKFYGL NSCLC, GC, HCC 405 NYPKSIHSF NSCLC 406 TYSEKTTLF NSCLC,
GC
407 VYGIRLEHF HCC, NSCLC, GC, GB 408 QYASRFVQL GC, GB, HCC, NSCLC
409 YFISHVLAF GC, NSCLC 410 RFLSGIINF NSCLC, GC, GB, HCC 411
VYIGHTSTI NSCLC 412 SYNPLWLRI GB, RCC, HCC, NSCLC, GC 413 NYLLYVSNF
HCC, NSCLC, GC 414 MYPYIYHVL HCC, NSCLC, GC, GB, PCA 415 SYQKVIELF
PCA, HCC, NSCLC, CRC, GC, RCC, GB 416 AYSDGHFLF NSCLC, GC, RCC, GB,
PCA, HCC 417 VYKVVGNLL GB, RCC, HCC, NSCLC, GC
[0608] Table 9B show the presentation on additional cancer entities
for selected peptides, and thus the particular relevance of the
peptides as mentioned for the diagnosis and/or treatment of the
cancers as indicated.
TABLE-US-00014 TABLE 9B Overview of presentation of selected
HLA-A*24 peptides across cancer entities. GB = glioblastoma, CRC =
colorectal cancer, RCC = renal cell carcinoma, HCC = hepatocellular
carcinoma, NSCLC = non-small cell lung cancer, PCA = prostate
cancer and benign prostate hyperplasia, GC = gastric cancer. SEQ
Peptide Presentation ID No. Sequence on cancer entities 50
EYALLYHTL PCA, HCC, NSCLC, CRC, GC, RCC 104 SYLTWHQQI GB 108
PYLVVIHTL NSCLC 110 SYPKIIEEF PCA 132 RYPALFPVL RCC 135 VYEAMVPLF
HCC 140 RYLINSYDF NSCLC 141 SYNGHLTIWF GB 146 NYYERIHAL NSCLC 148
SYGTVSQIF CRC 152 KFFDDLGDELLF NSCLC 155 IYKWITDNF PCA 164
HYVPATKVF NSCLC 167 RYGFYYVEF GB 171 NYEDHFPLL NSCLC 172 VFIFKGNEF
NSCLC 173 QYLEKYYNL NSCLC 181 VYPPYLNYL PCA 184 IYSVGAFENF NSCLC
185 QYLVHVNDL GB 189 AYFKQSSVF NSCLC 190 LYSELTETL NSCLC 191
TYPDGTYTGRIF NSCLC 193 LYLENNAQTQF NSCLC 198 DYVGFTLKI NSCLC 200
VFIHHLPQF HCC 203 VYNVEVKNAEF NSCLC 204 EYLSTCSKL PCA 205 VYPVVLNQI
NSCLC 208 TYLEKIDGF NSCLC 211 VFKSEGAYF NSCLC 212 SYAPPSEDLF NSCLC
213 SYAPPSEDLFL NSCLC 214 KYLMELTLI NSCLC 216 FYVNVKEQF NSCLC 218
LYSELNKWSF NSCLC 219 SYLKAVFNL NSCLC 220 SYSEIKDFL NSCLC 222
HYSTLVHMF NSCLC 224 PYFFANQEF HCC 227 IYRFITERF NSCLC 230 TYGMVMVTF
NSCLC 231 AFADVSVKF NSCLC 233 QYLTAAALHNL NSCLC 237 KYVGQLAVL HCC
239 VYAIFRILL GC 241 SYVKVLHHL HCC 242 VYGEPRELL HCC 244
VHFEDTGKTLLF NSCLC 245 LYPQLFVVL GC 246 KYLSVQLTL NSCLC 247
SFTKTSPNF HCC 256 VFGKSAYLF NSCLC 258 AYAQLGYLL NSCLC 263 RYILENHDF
HCC 267 SFLNIEKTEILF HCC 270 YYSQESKVLYL HCC 272 RYVFPLPYL NSCLC
273 IYGEKLQFIF NSCLC 278 KYVNLVMYF NSCLC 279 VWLPASVLF NSCLC 282
NYLVDPVTI NSCLC 283 EYQEIFQQL NSCLC 287 IYNPNLLTASKF NSCLC 291
YYLGSGRETF NSCLC, GB, PCA, HCC 292 FYPQIINTF NSCLC 293 VYPHFSTTNLI
HCC 294 RFPVQGTVTF PCA 302 LYLPVHYGF NSCLC 342 NYAFLHRTL NSCLC 344
EYNSDLHQF PCA 348 IYLEHTESI GB 350 KYGNFIDKL PCA 364 KYLNENQLSQL
NSCLC 372 VYVIEPHSMEF NSCLC 374 VFLPRVTEL NSCLC 379 VFIAQGYTL NSCLC
381 KYPASSSVF RCC, NSCLC
Example 2
[0609] Expression Profiling of Genes Encoding the Peptides of the
Invention
[0610] 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, and a high tumor-to-normal ratio of gene expression
indicates a therapeutic window. Moreover, over-expression of source
genes in tumor entities that were not yet analyzed for peptide
presentation indicates that a certain peptide may be of importance
in the respective entity.
[0611] For HLA class I-binding peptides of this invention, normal
tissue expression of all source genes was shown to be minimal based
on a database of RNASeq data covering around 3000 normal tissue
samples (Lonsdale, 2013). In addition, gene expression data from
tumors vs normal tissues were analyzed to assess target coverage in
various tumor entities.
[0612] RNA Sources and Preparation
[0613] 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.
[0614] Total RNA from healthy human tissues for RNASeq experiments
was obtained from: Asterand (Detroit, Mich., USA and Royston,
Herts, UK); Bio-Options Inc. (Brea, Calif., USA); ProteoGenex Inc.
(Culver City, Calif., USA); Geneticist Inc. (Glendale, Calif.,
USA); Istituto Nazionale Tumori "Pascale" (Naples, Italy);
University Hospital of Heidelberg (Heidelberg, Germany); BioCat
GmbH (Heidelberg, Germany), BioServe (Beltsville, Md., USA),
Capital BioScience Inc. (Rockville, Md., USA).
[0615] Total RNA from tumor tissues for RNASeq experiments was
obtained from: Asterand (Detroit, Mich., USA & Royston, Herts,
UK), Bio-Options Inc. (Brea, Calif., USA), BioServe (Beltsville,
Md., USA), Geneticist Inc. (Glendale, Calif., USA), ProteoGenex
Inc. (Culver City, Calif., USA), Tissue Solutions Ltd (Glasgow,
UK), University Hospital Bonn (Bonn, Germany), University Hospital
Heidelberg (Heidelberg, Germany), University Hospital Tubingen
(Tubingen, Germany)
[0616] 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).
[0617] RNAseq Experiments
[0618] Gene expression analysis of tumor and normal tissue RNA
samples was performed by next generation sequencing (RNAseq) by
CeGaT (Tubingen, Germany). Briefly, sequencing libraries are
prepared using the Illumina HiSeq v4 reagent kit according to the
provider's protocol (Illumina Inc, San Diego, Calif., USA), which
includes RNA fragmentation, cDNA conversion and addition of
sequencing adaptors. Libraries derived from multiple samples are
mixed equimolar and sequenced on the Illumina HiSeq 2500 sequencer
according to the manufacturer's instructions, generating 50 bp
single end reads. Processed reads are mapped to the human genome
(GRCh38) using the STAR software. Expression data are provided on
transcript level as RPKM (Reads Per Kilobase per Million mapped
reads, generated by the software Cufflinks) and on exon level
(total reads, generated by the software Bedtools), based on
annotations of the ensembl sequence database (Ensembl77). Exon
reads are normalized for exon length and alignment size to obtain
RPKM values.
[0619] Exemplary expression profiles of source genes of the present
invention that are highly over-expressed or exclusively expressed
in different cancers are shown in FIGS. 2A-2D. Expression scores
for further exemplary targets are shown in Table 10 (A and B),
based on in-house RNASeq analyses. Expression data for other
entities and further exemplary peptides are summarized in Table 11,
based on data generated by the TCGA Research Network:
cancergenome.nih.gov/.
TABLE-US-00015 TABLE 10A Target coverage within various tumor
entities, for expression of source genes of selected peptides.
Over-expression was defined as more than 1.5-fold higher expression
on a tumor compared to the relevant normal tissue that showed
highest expression of the gene. <19% over-expression = I, 20-49%
= II, 50-69% = III, >70% = IV. If a peptide could be derIVed
from several source genes, the gene with minimal coverage was
decisIVe. The baseline included the following relevant normal
tissues: adipose tissue, adrenal gland, artery, bone marrow, brain,
cartilage, colon, esophagus, gallbladder, heart, kidney, lIVer,
lung, lymph node, pancreas, pituitary, rectum, skeletal muscle,
skin, small intestine, spleen, stomach, thymus, thyroid gland,
trachea, urinary bladder and vein. In case expression data for
several samples of the same tissue type were available, the
arithmetic mean of all respectIVe samples was used for the
calculation. SEQ AML BRCA CLL CRC GB HCC OC PC RCC ID (N = (N = (N
= (N = (N = (N = pNSCLC (N = OSCAR (N = (N = SCLC NO. Sequence 7)
10) 10) 20) 24) 15) (N = 11) 12) (N = 11) 26) 10) (N = 10) 2
ALYGKLLKL I II I I I I I I I I I I 3 TLLGKQVTL I II I I I I I I I I
I I 5 SLFGQEVYC I I I I I II I I I I I I 9 FLGDYVENL I I IV I III I
I I I I I II 10 KTLDVFNIIL I I IV I III I I I I I I II 11
GVLKVFLENV I II I I I I I II I I I I 12 GLIYEETRGV I II I I I I I
II I I I I 13 VLRDNIQGI I II I I I I I II I I I I 14 LLDHLSFINKI I
I III I I I I I I I I III 16 HLYNNEEQV I I I I I IV I I II I II I
17 ILHEHHIFL I I I II I II II II I II IV I 18 YVLNEEDLQKV I I I II
I II II II I II IV I 19 TLLPTVLTL I I I I I I I I I I IV I 20
ALDGHLYAI I I I I I I I I I I IV I 27 KVFGDLDQV I I I I I I I I I I
II I 28 TLYSMDLMKV I I I I I I I I I I II I 31 ALSDETKNNWEV I III I
II III I III III II I I IV 32 ILADEAFFSV I III I II III I III III
II I I IV 33 LLLPLLPPLSPSLG I I I I I I I I I I I II 36 KLYGIEIEV I
I I I II I I I I I I I 39 ALMAVVSGL I I IV I I I I I I I I I 42
VLSPFILTL I I I I I I I I II I I I 44 GLLWQIIKV I I I III I I I I I
I I I 45 VLGPTPELV I I I I I I I I I II I I 46 SLAKHGIVAL I II I I
I I I I I I I I 47 GLYQAQVNL I I I II II II III I IV II I II 49
LLDESKLTL I I I I I I I I I I III I 50 EYALLYHTL I I I II I I I I I
I II I 51 LLLDGDFTL I I I I I III I I I I I I 52 ELLSSIFFL I I I I
I I II I II I II II 53 SLLSHVIVA I II I II I I II II II I I II 54
FINPKGNWLL I I I I I I II I I II I I 55 IASAIVNEL I II II I II I II
II I I I II 59 ALTKILAEL I I I I I I I I I I I II 63 TLSSERDFAL I I
I I I III I I I I II I 64 GLSSSSYEL I I I I I III I I I I I I 65
KLDGICWQV I I I I I II I I I I I I 67 GVIETVTSL IV I I I I I I I I
I I I 69 GIYDGILHSI I I I II II II II II II I I II 70 GLFSQHFNL II
I I I I I I I II I I I 73 GVPDTIATL I I I II I I I I I I I I 75
ILDNVKNLL IV I I I I I I I I I I I 78 LLWGHPRVA I IV I II I I III
III IV III I I 79 SLVPLQILL I I I I I II I I I I I I 80 TLDEYLTYL I
I I I I II I II I I I I 81 VLFLGKLLV I I II II III II I II II II II
IV 84 FLEEEITRV I I I I I I I I I I I II 86 LLVTSLVVV I I I I I III
I I I I I I 88 ILLNTEDLASL I I I I I I I I I I II II 91 SSLEPQIQPV
I II II I II I I II III I II I 322 ALVSGGVAQA I III I I I I I III
III II I I 323 ILSVVNSQL I I IV II I I I I I I I I 324 AIFDFCPSV I
I IV I I I I I I I I I 325 RLLPKVQEV I I I I II I I I I I I I 327
SIGDIFLKY I II I III II I IV III III II I IV 328 SVDSAPAAV I I I I
I I I I I I I II 329 FAWEPSFRDQV I I I I I III I I I I I I 331
AIWKELISL I I I I II I I I I I I III 332 AVTKYTSAK I II I I I II I
I I I I II 334 GRADALRVL I I IV I I I I I I I I I 335 VLLAAGPSAA I
I II I I I I I I I I II 336 GLMDGSPHFL I II I I I I I I I I I I 337
KVLGKIEKV I II I I I I I I I I I II 338 LLYDGKLSSA I II I I I I I I
I I I II 96 YYTQYSQTI I IV I II I I III III IV III I I 98
VFPRLHNVLF I I I I I I I I I I I II 100 VYIESRIGTSTSF I II I I I I
I II II I I II 101 IYIPVLPPHL I IV I I I I IV III III I I I 103
NYIPVKNGKQF I I III I I I I I I I I I 106 IYNETVRDLL I I I I I I I
II I I I I 107 KYFPYLVVI I II I I I I I II III I II I 108 PYLVVIHTL
I II I I I I I II III I II I 110 SYPKIIEEF I I III I I I I I I I I
I 113 IYSFHTLSF I I I I II I I I I I I III 114 QYLDGTWSL I IV I II
II I IV II IV III I II 115 RYLNKSFVL I II I II I I I III I I I II
117 IYLSDLTYI I I IV I I I I I I I I I 118 KYLNSVQYI I I IV I I I I
I I I I I 119 VYRVYVTTF I I I I I I II I II I I I 121 RYGLPAAWSTF I
I IV I I I I I I I I I 123 VYTPVLEHL I III I II III I III III II I
I IV 124 TYKDYVDLF I III I II III I III III II I I IV 125
VFSRDFGLLVF I III I II III I III III II I I IV 126 PYDPALGSPSRLF I
I I II I III I II I I I I 127 QYFTGNPLF I I I I I I I I I I I II
132 RYPALFPVL I I I I I I I II I I I I 135 VYEAMVPLF I I I I I I I
I I I I II 138 VYNAVSTSF I I I I I I I I I I I II 139 IFGIFPNQF IV
I I I I I I I I I I I 140 RYLINSYDF I I I I I I II I I I I II 141
SYNGHLTIWF I I I I III I I I I I I I 146 NYYERIHAL III I IV I II I
II II I I I I 147 LYLAFPLAF I II I I I I I I I I I I 151 AYISGLDVF
I I I I I I II I IV I I II 152 KFFDDLGDELLF I I I I I I II I IV I I
II 156 YYMELTKLLL I I I I I I I I III I I IV 157 DYIPASGFALF I I I
I II I I I I I I I 158 IYEETRGVLKVF I II I I I I I II I I I I 159
IYEETRGVL I II I I I I I II I I I I 162 KYTSYILAF I I I I I I I II
I I II I 164 HYVPATKVF I II I I I I IV IV III I II II 167 RYGFYYVEF
I I I I IV I III II II I I I 171 NYEDHFPLL I I I I I I I II II I I
II 172 VFIFKGNEF I I I I I I II I II I I I 173 QYLEKYYNL I I I I I
I II I II I I I 174 VYEKNGYIYF I II I I I I II I III I I I 175
LYSPVPFTL I I I I I I I I II I I I 176 FYINGQYQF III I III I II I
II I II I I II 177 VYFKAGLDVF I II I II I I I II I I I I 178
NYSSAVQKF I I I I III I I I I I I I
179 TYIPVGLGRLL I I I I I I I III II I I II 181 VYPPYLNYL I II I I
I I I I I I I I 182 AYAQLGYLLF I I I I I I I I I I I II 184
IYSVGAFENF I II I I I I I I I I I II 185 QYLVHVNDL I I I I II I I I
I I II II 189 AYFKQSSVF II I II I I I I I I I I I 190 LYSELTETL IV
III IV I II I IV I I II I IV 191 TYPDGTYTGRIF I I II I I I I I I I
I I 193 LYLENNAQTQF I I I I I I II I IV I I I 195 AYIKGGWIL I I I I
I II I I I I I I 198 DYVGFTLKI I I I I III I II I I I I I 203
VYNVEVKNAEF I I I I I I I I I I I II 204 EYLSTCSKL II II I I I I I
II I I I III 205 VYPVVLNQI I I I I I I I I I I I II 206 NYLDVATFL I
I I I II I II I I I I II 208 TYLEKIDGF I I I I I I I I II I I I 210
IYAGVGEFSF I I I I I I I I I I I II 211 VFKSEGAYF I I I I I I I I I
I I II 212 SYAPPSEDLF I II I I I I I I I I I I 213 SYAPPSEDLFL I II
I I I I I I I I I I 214 KYLMELTLI I I I I I I I I I I I II 216
FYVNVKEQF I I I I I I I I I I I II 218 LYSELNKWSF I I I I I I II I
I I I I 219 SYLKAVFNL I III I I I I I I I I I I 220 SYSEIKDFL I I
IV I I I I I I I I I 221 KYIGNLDLL I I I I I I I I I I II I 222
HYSTLVHMF I I I I I I I I I I II I 224 PYFFANQEF II I I I I I I I I
I I I 226 MYLKLVQLF I I I I I I I I I I I II 227 IYRFITERF I I I I
I I I I I I I II 230 TYGMVMVTF I I I I II I I I I I I I 231
AFADVSVKF I I I I II I I I I I I I 233 QYLTAAALHNL I I I I I I I I
I I I I 235 VYKDSIYYI II I IV I I I I I I I I I 236 VYLPKIPSW I I I
I I II I I I I I I 237 KYVGQLAVL I I I I I I I I I I I II 239
VYAIFRILL I II I I I I I I I I I II 240 YYFFVQEKI I I I I II I I I
I I I I 241 SYVKVLHHL I I I I I I I I I I I II 242 VYGEPRELL I II I
I I II I I I I I II 243 SYLELANTL I I I I I I I I II I I I 244
VHFEDTGKTLLF I II I I I I II I III I I I 245 LYPQLFVVL I I I I I I
I II I I I I 246 KYLSVQLTL I I I I I I I I I I I II 247 SFTKTSPNF I
I I II I I I I I I I I 251 VYTKALSSL I I I I I I I I I I I III 256
VFGKSAYLF II I I I I I I I I I I I 258 AYAQLGYLL I I I I I I I I I
I I II 263 RYILENHDF I I I I I II I I I I I I 267 SFLNIEKTEILF I I
I I I III I I I I I I 270 YYSQESKVLYL I II II I II I I III II I I
II 272 RYVFPLPYL I I I II I II I I I I I I 273 IYGEKLQFIF I I I I I
I I I I I I II 274 KQLDIANYELF I III I I II I I II I I I II 276
QYLDVLHAL I II I I I I I II II I II II 277 FYTFPFQQL I I I I I I I
I I I I II 278 KYVNLVMYF I I I I I I I II I I I I 279 VWLPASVLF I I
I I I I I I I I I II 282 NYLVDPVTI I II I I I I I III I I I I 283
EYQEIFQQL I II I I I I I III I I I I 287 IYNPNLLTASKF I III I I I I
I III III I I I 291 YYLGSGRETF I I I I II I II II III I I II 292
FYPQIINTF I I I I II I I I I I I I 293 VYPHFSTTNLI I I I I II I I I
I I I I 294 RFPVQGTVTF I IV I I I I I II I I I I 295 SYLVIHERI I I
II I II I I III I I I II 300 TYPSQLPSL I I I II I I I I I I I I 302
LYLPVHYGF I I I II I I I I I I I II 304 EYNEVANLF I I I I II I I I
I I I I 307 GYAFTLPLF I II I II II I I II I I II II 308 TFDGHGVFF I
I I II I I I I II I I I 309 KYYRQTLLF I I I I I I I III IV I II II
310 IYAPTLLVF I II II I I I I I I I I I 314 RYFKGDYSI I I I I II I
I I I I I I 315 FYIPHVPVSF I I I I I III I I I I I I 320 KFILALKVLF
I I I I I I I I I I I II 342 NYAFLHRTL II I I I I I I II III I I I
343 NYLGGTSTI I II I I I II I I I I I II 344 EYNSDLHQF I I I I I I
I I I I I II 345 EYNSDLHQFF I I I I I I I I I I I II 348 IYLEHTESI
I I II I I I I I I I I I 350 KYGNFIDKL I I I I I I I II I I I I 351
IFHEVPLKF I I I I I I I I I I I II 352 QYGGDLTNTF I I I I I I II I
I II I I 353 TYGKIDLGF I I I I I I I I I I I II 354 VYNEQIRDLL I I
I I I I I I I I I II 355 IYVTGGHLF I I I I I I I I II I I I 356
NYMPGQLTI I I I II I I II II I II II I 358 YYSEVPVKL I IV I II I I
III III IV III I I 359 NYGVLHVTF II I II II II III I I I I II II
360 VFSPDGHLF I II I I I I I I I I I I 361 TYADIGGLDNQI II I I I I
I I I I I I I 363 SYAELGTTI I I I I II I I I II I I I 364
KYLNENQLSQL I I I I I I I I I I I III 365 VFIDHPVHL I I I I II I I
I I I I II 366 QYLELAHSL I I I I I I I II I I I II 367 LYQDHMQYI I
I I I I I I I II I I I 368 KYQNVKHNL I I I I I I I I I I I II 371
AYSHLRYVF I I I IV III I IV II IV III III II 372 VYVIEPHSMEF I I II
I I I I I I I I II 374 VFLPRVTEL I III I II III I III III II I I IV
376 VYTPVASRQSL I I I I II I I I I I I II 377 QYTPHSHQF I I I I II
I II I I I I III 378 VYIAELEKI I II I I I I I I I I I II 379
VFIAQGYTL I I I I I I II I II I II II 380 VYTGIDHHW I II I II I I
II III IV I I III 381 KYPASSSVF I I I I I I I II I I III I 382
AYLPPLQQVF I I I I I I I I I I I II 383 RYKPGEPITF II IV I II I I
IV II II I I II 385 QYIEELQKF I I IV I I I I I I I I I 386
TFSDVEAHF I I I I I I I I I I I II 387 KYTEKLEEI I I I I IV I I II
I I I III
TABLE-US-00016 TABLE 10B Target coverage for source genes of
selected peptides. Over-expression was defined as more than
1.5-fold higher expression on a tumor compared to the relevant
normal tissue that showed highest expression of the gene. <19%
over-expression = I, 20-49% = II, 50-69% = III, >70% = IV. If a
peptide could be derIVed from several source genes, the gene with
minimal coverage was decisIVe. The baseline included the following
relevant normal tissues: adipose tissue, adrenal gland, artery,
bone marrow, brain, cartilage, colon, esophagus, gallbladder,
heart, kidney, lIVer, lung, lymph node, pancreas, pituitary,
rectum, skeletal muscle, skin, small intestine, spleen, stomach,
thymus, thyroid gland, trachea, urinary bladder and vein. In case
expression data for several samples of the same tissue type were
available, the arithmetic mean of all respectIVe samples was used
for the calculation. NHL = non-Hodgkin lymphoma, PCA = prostate
cancer and benign prostate hyperplasia, GC = gastric cancer,
GBC_CCC = gallbladder adenocarcinoma and cholangiocarcinoma, MEL =
melanoma, UBC = urinary bladder cancer, UTC = uterine cancer, HNSCC
= head and neck small cell carcinoma. SEQ NHL PCA GC GBC_CCC MEL
UBC UTC HNSCC ID NO. Sequence (N = 10) (N = 10) (N = 11) (N = 10)
(N = 10) (N = 10) (N = 10) (N = 10) 2 ALYGKLLKL I I I I I I I I 3
TLLGKQVTL I I I I I I I II 4 ELAEIVFKV I I I I I I I I 9 FLGDYVENL
I I I I I I I I 12 GLIYEETRGV II II I I II I I I 13 VLRDNIQGI II II
I I II I I I 19 TLLPTVLTL I I I I I III I I 32 ILADEAFFSV II I I I
II I I I 34 LLPKKTESHHKT II I I I I I I I 35 YVLPKLYVKL I I I I I I
I I 37 ALINDILGELVKL I I I I I I I I 39 ALMAVVSGL II I I I I I I I
41 FVLPLVVTL I I I I I I I I 42 VLSPFILTL I I I II I II I II 45
VLGPTPELV I I II II I I I I 46 SLAKHGIVAL I I I I I I II I 47
GLYQAQVNL I I I II IV II I III 51 LLLDGDFTL I I I I I I I I 53
SLLSHVIVA III I I II I I II I 55 IASAIVNEL II I I II I I I I 60
FLIDTSASM I I I I I I I I 61 HLPDFVKQL I I I I I I I I 70 GLFSQHFNL
I I I II I II III II 96 YYTQYSQTI I I I II I I I II 98 VFPRLHNVLF I
I I I I I I I 99 QYILAVPVL I I I I I I I I 100 VYIESRIGTSTSF I I I
I II I I I 101 IYIPVLPPHL III III I III I II III III 103
NYIPVKNGKQF I I I I I I I I 104 SYLTWHQQI I I I I I I I I 105
IYNETITDLL I I I I I I I I 110 SYPKIIEEF II I I I I I I I 113
IYSFHTLSF I I I I I I I II 114 QYLDGTWSL II I II IV IV III IV III
116 AYVIAVHLF I I I I I I I I 117 IYLSDLTYI II I I I I I I I 118
KYLNSVQYI II I I I I I I I 119 VYRVYVTTF I I I I I I II I 121
RYGLPAAWSTF I I I I I I I I 123 VYTPVLEHL II I I I II I I I 124
TYKDYVDLF II I I I II I I I 126 PYDPALGSPSRLF I II II II I I III I
127 QYFTGNPLF I I I I I I I I 128 VYPFDWQYI I I I I I I I I 132
RYPALFPVL I I I I I I I I 135 VYEAMVPLF I I I I I I I I 144
VFASLPGFLF III I I I I I I I 145 VYALKVRTI II I I II II II I II 147
LYLAFPLAF I I I I I I I I 150 IYITRQFVQF I I I I II I I I 151
AYISGLDVF I I I I I I I III 155 IYKWITDNF I I I I I I II I 156
YYMELTKLLL II I I II I I I I 158 IYEETRGVLKVF II II I I II I I I
161 KYPDIVQQF II I I I I I I I 162 KYTSYILAF II I I II I I I I 163
RYLTISNLQF I I I I I I I I 165 EYFTPLLSGQF I I I I I I I I 166
FYTLPFHLI I I I I I I I I 168 RYLEAALRL I I I I II I I I 170
QYPFHVPLL III I I I I I I I 171 NYEDHFPLL I I II I IV II I II 172
VFIFKGNEF I I I I I I I I 175 LYSPVPFTL I I I II I I I II 176
FYINGQYQF I II I I I I I II 177 VYFKAGLDVF I III I II I II II I 178
NYSSAVQKF I III I I II I I I 179 TYIPVGLGRLL I I I I I I III II 180
KYLQVVGMF I I I I I I I 191 TYPDGTYTGRIF II I I I I I I I 195
AYIKGGWIL I I I I I I I I 197 IFTDIFHYL I I I I I I I I 204
EYLSTCSKL II II I I I I IV II 206 NYLDVATFL I I I I I I II I 235
VYKDSIYYI IV I I I I I I I 277 FYTFPFQQL I I I I I I I II 291
YYLGSGRETF I I I II II II III III 296 SYQVIFQHF I I I I I I I II
297 TYIDTRTVF I I I I I I III I 304 EYNEVANLF I III I I I I I I 307
GYAFTLPLF I II I I I I II I 309 KYYRQTLLF I I I I I I II I 312
SYTSVLSRL II I I I II I I I 316 VYFEGSDFKF II I I I I I I I 317
VFDTSIAQLF I I I I I I I I 318 TYSNSAFQYF I I I I I I I I 319
KYSDVKNLI I I I I I I I I 326 SLLPLVWKI I I I I I I I I 327
SIGDIFLKY I I II III I I III II 328 SVDSAPAAV II I I I I I I I 330
FLWPKEVEL II I I I I I I I 331 AIWKELISL I I I I I I I II 332
AVTKYTSAK I I I I I I I I 333 GTFLEGVAK I I I I I I I I 334
GRADALRVL I I I I I I I I 335 VLLAAGPSAA III I I I I I I I 342
NYAFLHRTL I I I I I II I I 343 NYLGGTSTI I IV I I I I II I 344
EYNSDLHQF II I I I I I I I 345 EYNSDLHQFF II I I I I I I I 347
VYAEVNSL I I I I I I I I 348 IYLEHTESI I I I I I I I I 350
KYGNFIDKL I I I I I I I I 352 QYGGDLTNTF I I I II I II I I 353
TYGKIDLGF II I I I I I I I 354 VYNEQIRDLL I I I I I I I I 355
IYVTGGHLF I I I II I II I II 356 NYMPGQLTI I I II II I I I I 359
NYGVLHVTF IV I I I II I I I
360 VFSPDGHLF II I I I I I I I 361 TYADIGGLDNQI I I I I I I I I 362
VYNYAEQTL I I I I I I I I 363 SYAELGTTI I I I I I I I I 365
VFIDHPVHL I I I I I I I I 366 QYLELAHSL I I I I I I I I 367
LYQDHMQYI I I I I I I I I 371 AYSHLRYVF I I III II I IV I II 376
VYTPVASRQSL II I I II I II I I 380 VYTGIDHHW II I I II I I I II 383
RYKPGEPITF II III I III I II III II 386 TFSDVEAHF II I I I I I I I
387 KYTEKLEEI I I I I I I I I
TABLE-US-00017 TABLE 8 Target coverage within various tumor
entities, for expression of source genes of selected peptides. A
gene was considered over-expressed if its expression level in a
tumor sample was more than 2-fold above the highest 75% percentile
of expression levels determined from samples of the following
normal organs (adjacent to tumors): rectum (n = 10), esophagus (n =
11), bladder (n = 19), kidney (n = 129), stomach (n = 35), colon (n
= 41), head and neck (n = 43), liver (n = 50), lung (n = 51),
thyroid (n = 59), lung (n = 59). Over-expression categories are
indicated as "A" (>=50% of tumors above the cutoff), "B"
(>=20% of tumors above the cutoff, but <50%), and "C"
(>=5% of tumors above the cutoff, but <20%). SEQ ACC BLCA
CESC CHOL DLBC HNSC KICH KIRP LGG MESO PCPG PRAD SARC SKCM STAD
TGCT THCA THYM UCEC UCS UVM ID (N = (N = (N = (N = (N = (N = (N =
(N = (N = (N = (N = (N = (N = (N = (N = (N = (N = (N = (N = (N = (N
= NO. 79) 408) 307) 36) 48) 521) 66) 291) 534) 87) 184) 498) 263)
473) 415) 156) 513) 120) 546) 57) 80) 16 C C C C C 2 3 4 B C B B C
C A C C 5 6 8 C C C 9 10 11 C B B B A C C C C B A C B B B C A A B C
12 C B B B A C C C C B A C B B B C A A B C 13 C B B B A C C C C B A
C B B B C A A B C 1 C B B C C C B A B C B 17 B A C C C A B 18 B A C
C C A B 21 22 24 C C C 27 C 28 C 29 C 30 C 31 C C B C C B B B C 32
C C B C C B B B C 34 C C B C B B B C 35 B 36 C C B C C C 37 38 C 39
C B C 40 C C A C C C C A A C B A 42 A A B B B C C C C C C A C 45 B
47 C C C C B C C B C C C 48 C C C 49 51 C C C 52 C 53 54 C C C C C
C C 55 C 56 C 58 C B C 60 C C B C C C C 61 62 B C C C C 63 64 C 65
66 C B A C C C C C 67 B C B 68 C 69 C 70 B B C B B C C C A B A A 72
C C B C B C C B C C C 74 A A A 75 B 76 C 78 C C C B B B C C C 79 C
80 81 C C C C C C C C C C 84 A 86 87 C C 88 C A B 90 C C B A C A 91
C C C C 92 B C C C C C C 321 B B C C B B C A B B C B B C B B B B
323 B 325 C B C B A C B C B C 326 A C C C 327 B A C A C B C A A B B
328 C B C C C B A C C B 329 330 C C C A C C A C C A A C C 331 B 332
C B A B B B C B A B B B A C B A B B 334 A C C C C 335 B A C A C C C
C B B C A B C 336 340 B B C 175 C C B C C 97 C B 98 C C B C B 100 C
B A C B B B B B A B B A 101 103 104 105 B A B B B C A B B C B 106 B
A A B B B C A A B A 107 C B 108 C B 110 B A A C C C B C A B B B 111
C A B B C C C A B C 112 C A B B C C C A B C 113 B 114 C B C C C C B
B B C C C C C 115 C 116 B B A B C B A B C C 117 C B C 118 C B C 119
C 121 A C C C C 122 123 C C B C C B B B C 124 C C B C C B B B C 125
C C B C C B B B C 126 C C C B C B B B 127 C 128 C B C 129 B B C 130
B B C 131 B 132 C C 133 C 134 B C B C C B A C B B 135 C C C C 136 C
137 138 B A C B B C C C A A B C B 139 B C B 140 141 142 C B B B C B
C A A C C 144 C C 145 C C C C B 146 B C 147 C 148 C 149 C 150 C A
151 C A B 152 C A B 153 155 C B C 156 C C A A B C C C B B C C 157 B
B C C 158 C B B B A C C C C B A C B B B C A A B C 159 C B B B A C C
C C B A C B B B C A A B C 161 C B 162 C B A C C C B C B C C 164 B B
C A B B B B B B A C C A B 165 C 166 C 167 C C B 168 C C B C C C A
169 170 C C A C C C C C B B 171 B C C B C C C C 172 C B A C C C 173
C B A C C C 174 B B C A C B B C C C C C 96 C C C B B B C C C 176
177 B 178 A C C 180 C A C B A C C C C C C C 181 C B C A C 182 C 183
C C 184 185 C C C C C 186 C C C 187 C C C A C C C C C C C 188 C B B
B B B B 189 190 191 A C C C 192 B C B C A C B C 193 C B 194 B A C A
B C B B B A A B B 195 196 197 C 198 C 199 C B B C C 200 A 202 C 203
C C A C C C C C B C A C C B 204 205 C C 206 C C 207 C C C C 208 C C
B C A C 209 C C 210 B A B B C C B B B A B B B 211 B B A B C C C A B
B A B B A 212 C C 213 C C 214 B A B B C B C B A A C B 216 C B A B B
C B B C B B C B 217 C 218 C C 219 C C C C 220 C B 221 B C C 222 B C
C 223 C 224 B 225 C C C C C 226 C B A C A B B B A A C B B 227 C C C
228 A 229 C A 230 B B C C 231 B B C C 232 C B A C A B C C B A B A A
B A 233 B C B 234 C C C C C 235 A C 236 237 C A A C A A C B B A A A
A B A 238 C C B C C B 240 C C C B 241 C B C C C A B C C C 242 C C C
C C B C B C 243 C C 244 B B C A C B B C C C C C 247 C C C A 249 C B
250 C A C C B B C C B 251 C B A C A B C C A B B A A B A 253 C B B C
C C C C 255 C B 256 C B C 258 C 259 C C C B B 262 C B C B
263 B 264 C A C C C B C 265 C 266 C B 267 C C C 268 269 C A A C B C
B B C B C C 270 C C 271 C A 272 B C C C 273 C C B A C C C C B A A C
B 274 C C C 276 A C C A 277 C C C C 278 B C C C 279 C 282 C C C 283
C C C 284 C 285 C 286 B C C B 287 288 C C B B C C B C B A A C B 289
C C 291 C C C C C B 292 C C C B 293 C C C B 294 C C C 295 B C 296 C
C C C 297 C C C B C C C 298 C 300 A A B A B 301 C C 302 B 304 305 C
B C A B C 306 C C C B B C C C B C C 307 C C C C C C 309 B C C C 312
C 313 C C 314 B 315 C 316 C 317 B C 319 B 320 C B B B B B B B A C C
A 341 B C C 342 C C C C C 343 C B 344 B B A B C B B A A B B A 345 B
B A B C B B A A B B A 346 C B B B C B C A A C C 347 C 348 B A A C C
C B C A B B B 349 C B 350 C C 351 352 C C C C C C C 353 C B A C A A
C B B B A A B B A 354 C B A C B B B B B A A B B A 355 A A B B B C C
C C C C A C 356 C B C 357 C B C C C B C A C 358 C C C B B B C C C
359 C B C C 360 C C B C C C B 361 C C C C C B C B C 363 C C C C B C
364 B A C C B B C A A C B C 365 C 366 C B A C B B C B A A C B A C
367 368 C 369 C B A B B B C B C B B B 371 C B C C 372 B A C A C C C
C B B C A B C 373 C 374 C C B C C B B B C 375 B C 376 C C C C C C B
C B 377 C C 378 C A C C C C C C 379 C 380 C C C C C C B C B C 382 C
383 385 C B C 386 C C C C B B C C 387 B B C C C 388 C 389 C B B C C
390 C B A C B B A B B A A B C B 391 C B A B B B C B C B B B 392 B B
A C B B C A B B B A B C B 393 C A 394 B A C A B C C B B B A A B B
395 C C B 396 A B C C C 397 A A C A A C C B B B A A B A 398 A A C A
A C C B B B A A B A 399 B A C C C A B 400 B B B A C C C A C C 401 B
B B A C C C A C C 402 C A A A C A C A A C C A B A B B B A A 403 C B
B B B B 404 C B B B B B 405 C B B B B B 406 C B C C C B C A C 407 C
A A C A B C C B B B A A B A 408 B A B B B C B A B B B 409 B A B B C
C B B B C 410 C A A C A B C C B B B A A B A 412 C B A C B B A B B A
A B C B 413 C 414 A C C 415 C C B C C C C B B C C C C 416 C 417 C B
A B B C B B A A B B ACC = Adrenocortical carcinoma (N = 79), BLCA =
Bladder urothelial carcinoma (N = 408), LGG = Lower grade glioma (N
= 534), CESC = Cervical squamous cell carcinoma and endocervical
adenocarcinoma (N = 307), STAD = Stomach adenocarcinoma (N = 415),
CHOL = Cholangiocarcinoma (N = 36), MESO = Mesothelioma (N = 87),
KICH = Kidney chromophobe (N = 66), PRAD = Prostate adenocarcinoma
(N = 498), DLBC = Lymphoid neoplasm diffuse large B-cell lymphoma
(N = 48), PCPG = Pheochromocytoma and paraganglioma (N = 184), KIRP
= Kidney renal papillary cell carcinoma (N = 291), SKCM = Skin
cutaneous melanoma (N = 473), SARC = Sarcoma (N = 263), THCA =
Thyroid carcinoma (N = 513), THYM = Thymoma (N = 120), UCS =
Uterine carcinosarcoma (N = 57), UCEC = Uterine corpus endometrial
carcinoma (N = 546), UVM = Uveal melanoma (N = 80), TGCT =
Testicular germ cell tumors (N = 156), HNSC = Head and neck
squamous cell carcinoma (N = 521)
Example 3
[0620] In Vitro Immunogenicity for MHC Class I Presented
Peptides
[0621] 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 HLA-A*02:01 restricted TUMAPs of the invention,
demonstrating that these peptides are T-cell epitopes against which
CD8+ precursor T cells exist in humans (Table 9).
[0622] In Vitro Priming of CD8+ T Cells
[0623] 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.
[0624] 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.
[0625] 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.
[0626] 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).
[0627] pMHC used for positive and negative control stimulations
were A*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 418) from
modified Melan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5,
SEQ ID NO. 419), respectively.
[0628] 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, Oreg., 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).
[0629] In Vitro Immunogenicity for Different Cancer Peptides
[0630] 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
FIGS. 3A-3B together with corresponding negative controls. Results
for 5 peptides from the invention are summarized in Table12A.
Exemplary flow cytometry results after TUMAP-specific multimer
staining for 7 peptides of the invention are shown in FIGS. 4A-4C
and FIGS. 5A-5D together with corresponding negative controls.
Results for 74 peptides from the invention are summarized in Table
12B.
TABLE-US-00018 TABLE 9A in vitro immunogenicity of HLA class I
peptides of the invention Exemplary results of in vitro
immunogenicity experiments conducted by the applicant for the
peptides of the invention. <20% = +; 20%-49% = ++; 50%-69% =
+++; >= 70% = ++++ Seq ID Sequence wells 393 KYIEAIQWI ++ 399
SYIDVLPEF ++ 400 KYLEKYYNL ++ 407 VYGIRLEHF +++ 414 MYPYIYHVL
++
TABLE-US-00019 TABLE 12B in vitro immunogenicity of HLA class I
peptides of the invention Exemplary results of in vitro
immunogenicity experiments conducted by the applicant for the
peptides of the invention. <20% = +; 20%-49% = ++; 50%-69% =
+++; >= 70% = ++++ Seq ID Sequence Wells positIVe [%] 2
ALYGKLLKL ++++ 7 AAAAKVPEV + 8 KLGPFLLNA +++ 9 FLGDYVENL + 17
ILHEHHIFL + 43 LLWAGPVTA ++++ 322 ALVSGGVAQA + 331 AIWKELISL ++ 96
YYTQYSQTI + 98 VFPRLHNVLF + 99 QYILAVPVL +++ 102 VYPFENFEF +++ 103
NYIPVKNGKQF + 104 SYLTWHQQI + 105 IYNETITDLL + 106 IYNETVRDLL + 107
KYFPYLVVI ++ 109 LFITGGQFF ++ 110 SYPKIIEEF ++ 111 VYVQILQKL + 112
IYNFVESKL +++ 114 QYLDGTWSL +++ 115 RYLNKSFVL + 119 VYRVYVTTF +++
120 GYIEHFSLW ++ 122 EYQARIPEF ++ 132 RYPALFPVL + 137 EYLHNCSYF +
139 IFGIFPNQF ++ 140 RYLINSYDF +++ 142 VYVDDIYVI ++++ 144
VFASLPGFLF ++ 155 IYKWITDNF ++ 156 YYMELTKLLL + 157 DYIPASGFALF +
158 IYEETRGVLKVF + 160 RYGDGGSSF + 161 KYPDIVQQF + 162 KYTSYILAF +
163 RYLTISNLQF + 164 HYVPATKVF + 166 FYTLPFHLI ++++ 167 RYGFYYVEF
++++ 168 RYLEAALRL +++ 170 QYPFHVPLL +++ 171 NYEDHFPLL ++ 172
VFIFKGNEF + 174 VYEKNGYIYF ++++ 175 LYSPVPFTL + 177 VYFKAGLDVF +
179 TYIPVGLGRLL +++ 180 KYLQVVGMF + 181 VYPPYLNYL ++++ 182
AYAQLGYLLF +++ 186 VFTTSSNIF + 190 LYSELTETL ++++ 277 FYTFPFQQL +++
344 EYNSDLHQF + 345 EYNSDLHQFF ++ 349 QYSIISNVF ++ 350 KYGNFIDKL
+++ 351 IFHEVPLKF ++ 353 TYGKIDLGF + 354 VYNEQIRDLL + 356 NYMPGQLTI
+ 358 YYSEVPVKL ++++ 359 NYGVLHVTF + 360 VFSPDGHLF ++ 363 SYAELGTTI
+ 365 VFIDHPVHL + 366 QYLELAHSL ++ 367 LYQDHMQYI ++ 371 AYSHLRYVF
++ 380 VYTGIDHHW +
Example 4
[0631] Synthesis of Peptides
[0632] 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
[0633] MHC Binding Assays
[0634] 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).
[0635] 96 well MAXISorp plates (NUNC) were coated over night with 2
ug/ml streptavidin in PBS at room temperature, washed 4.times. and
blocked for1 h 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 1 h at
37.degree. C., washed again and detected with TMB solution that is
stopped with NH.sub.2SO.sub.4. 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.
TABLE-US-00020 TABLE 13 MHC class I binding scores. Binding of
HLA-class I restricted peptides to HLA-A*02 was ranged by peptide
exchange yield: <20% = +; 20%-49% = ++; 50%-75% +++; >= 75% =
++++ Seq ID Sequence Peptide exchange 1 PLWGKVFYL ++ 2 ALYGKLLKL
+++ 3 TLLGKQVTL +++ 4 ELAEIVFKV +++ 5 SLFGQEVYC +++ 6 FLDPAQRDL +++
7 AAAAKVPEV +++ 8 KLGPFLLNA +++ 9 FLGDYVENL ++ 10 KTLDVFNIIL ++ 11
GVLKVFLENV ++ 12 GLIYEETRGV ++ 13 VLRDNIQGI +++ 14 LLDHLSFINKI ++
16 HLYNNEEQV ++ 17 ILHEHHIFL +++ 18 YVLNEEDLQKV +++ 19 TLLPTVLTL
+++ 20 ALDGHLYAI +++ 21 SLYHRVLLY ++++ 22 MLSDLTLQL ++++ 23
AQTVVVIKA + 24 FLWNGEDSAL +++ 25 IQADDFRTL ++ 26 KVDGVVIQL +++ 27
KVFGDLDQV +++ 28 TLYSMDLMKV +++ 29 TLCNKTFTA +++ 31 ALSDETKNNWEV
++++ 32 ILADEAFFSV +++ 33 LLLPLLPPLSPSLG +++ 35 YVLPKLYVKL ++ 36
KLYGIEIEV ++++ 37 ALINDILGELVKL +++ 38 KMQEDLVTL +++ 39 ALMAVVSGL
+++ 40 SLLALPQDLQA +++ 41 FVLPLVVTL +++ 42 VLSPFILTL +++ 43
LLWAGPVTA +++ 44 GLLWQIIKV ++ 45 VLGPTPELV +++ 46 SLAKHGIVAL +++ 47
GLYQAQVNL +++ 48 TLDHKPVTV ++ 49 LLDESKLTL +++ 50 EYALLYHTL ++ 51
LLLDGDFTL +++ 52 ELLSSIFFL +++ 53 SLLSHVIVA +++ 54 FINPKGNWLL +++
55 IASAIVNEL ++ 56 KILDLTRVL ++ 57 VLISSTVRL ++ 58 ALDDSLTSL ++ 59
ALTKILAEL +++ 60 FLIDTSASM ++ 61 HLPDFVKQL ++ 62 SLFNQEVQI +++ 63
TLSSERDFAL + 64 GLSSSSYEL ++ 65 KLDGICWQV +++ 66 FITDFYTTV +++ 67
GVIETVTSL ++ 69 GIYDGILHSI +++ 70 GLFSQHFNL +++ 71 GLITVDIAL +++ 72
GMIGFQVLL +++ 74 ILDETLENV ++ 75 ILDNVKNLL +++ 76 ILLDESNFNHFL +++
77 IVLSTIASV +++ 78 LLWGHPRVA +++ 79 SLVPLQILL ++++ 80 TLDEYLTYL
+++ 81 VLFLGKLLV ++ 82 VLLRVLIL ++ 83 ELLEYLPQL +++ 84 FLEEEITRV
+++ 85 STLDGSLHAV +++ 87 YLTEVFLHVV +++ 88 ILLNTEDLASL +++ 89
YLVAHNLLL +++ 90 GAVAEEVLSSI + 91 SSLEPQIQPV + 92 LLRGPPVARA ++ 93
SLLTQPIFL +++ 321 SLWFKPEEL +++ 322 ALVSGGVAQA +++ 323 ILSVVNSQL
+++ 324 AIFDFCPSV ++++ 325 RLLPKVQEV ++ 326 SLLPLVWKI +++ 327
SIGDIFLKY +++ 328 SVDSAPAAV ++ 329 FAWEPSFRDQV ++ 330 FLWPKEVEL +++
331 AIWKELISL +++ 333 GTFLEGVAK +++ 334 GRADALRVL +++ 335
VLLAAGPSAA ++ 336 GLMDGSPHFL ++ 337 KVLGKIEKV +++ 338 LLYDGKLSSA ++
339 VLGPGPPPL ++ 340 SVAKTILKR ++
TABLE-US-00021 TABLE 14 MHC class 1 binding scores. Binding of
HLA-class I restricted peptides to HLA-A*24 was ranged by peptide
exchange yield: <20% = +; 20%-49% = ++; 50%-75% = +++; >= 75%
= ++++ Seq ID Sequence Peptide exchange 96 YYTQYSQTI ++++ 97
TYTFLKETF ++++ 98 VFPRLHNVLF +++ 99 QYILAVPVL ++++ 100
VYIESRIGTSTSF +++ 102 VYPFENFEF +++ 103 NYIPVKNGKQF +++ 104
SYLTWHQQI ++++ 105 IYNETITDLL +++ 106 IYNETVRDLL +++ 107 KYFPYLVVI
+++ 108 PYLVVIHTL +++ 109 LFITGGQFF ++++ 110 SYPKIIEEF +++ 111
VYVQILQKL +++ 112 IYNFVESKL +++ 113 IYSFHTLSF +++ 114 QYLDGTWSL
++++ 115 RYLNKSFVL +++ 116 AYVIAVHLF ++++ 117 IYLSDLTYI +++ 118
KYLNSVQYI +++ 119 VYRVYVTTF +++ 120 GYIEHFSLW ++++ 121 RYGLPAAWSTF
+++ 122 EYQARIPEF +++ 123 VYTPVLEHL ++ 124 TYKDYVDLF + 125
VFSRDFGLLVF +++ 127 QYFTGNPLF +++ 128 VYPFDWQYI ++++ 129 KYIDYLMTW
++++ 131 EYLDRIGQLFF +++ 132 RYPALFPVL ++++ 133 KYLEDMKTYF +++ 134
AYIPTPIYF +++ 135 VYEAMVPLF ++++ 136 IYPEWPVVFF +++ 137 EYLHNCSYF
++++ 138 VYNAVSTSF ++ 139 IFGIFPNQF +++ 140 RYLINSYDF ++++ 141
SYNGHLTIWF +++ 142 VYVDDIYVI +++ 143 KYIFQLNEI +++ 144 VFASLPGFLF
++++ 145 VYALKVRTI +++ 146 NYYERIHAL +++ 147 LYLAFPLAF +++ 148
SYGTVSQIF ++++ 149 SYGTVSQI ++++ 152 KFFDDLGDELLF ++ 153
VYVPFGGKSMITF ++++ 154 VYGVPTPHF ++++ 155 IYKWITDNF ++++ 156
YYMELTKLLL ++++ 157 DYIPASGFALF +++ 158 IYEETRGVLKVF +++ 159
IYEETRGVL +++ 160 RYGDGGSSF +++ 161 KYPDIVQQF +++ 162 KYTSYILAF ++
163 RYLTISNLQF ++++ 164 HYVPATKVF +++ 165 EYFTPLLSGQF +++ 166
FYTLPFHLI ++++ 167 RYGFYYVEF +++ 168 RYLEAALRL +++ 169 NYITGKGDVF
+++ 170 QYPFHVPLL ++++ 171 NYEDHFPLL +++ 172 VFIFKGNEF ++++ 173
QYLEKYYNL ++++ 174 VYEKNGYIYF +++ 175 LYSPVPFTL +++ 176 FYINGQYQF
+++ 177 VYFKAGLDVF +++ 178 NYSSAVQKF +++ 179 TYIPVGLGRLL +++ 180
KYLQVVGMF +++ 181 VYPPYLNYL +++ 182 AYAQLGYLLF ++++ 183 PYLQDVPRI
+++ 184 IYSVGAFENF ++++ 185 QYLVHVNDL ++++ 186 VFTTSSNIF ++++ 187
AYAANVHYL ++++ 188 GYKTFFNEF +++ 190 LYSELTETL +++ 191 TYPDGTYTGRIF
+++ 192 RYSTFSEIF +++ 193 LYLENNAQTQF +++ 194 VYQSLSNSL +++ 195
AYIKGGWIL +++ 196 GYIRGSWQF ++++ 197 IFTDIFHYL ++++ 198 DYVGFTLKI
++ 199 SYLNHLNNL +++ 200 VFIHHLPQF +++ 201 GYNPNRVFF +++ 202
RYVEGIVSL +++ 204 EYLSTCSKL +++ 205 VYPVVLNQI +++ 206 NYLDVATFL
++++ 207 LYSDAFKFIVF +++ 208 TYLEKIDGF ++++ 209 AFIETPIPLF ++++ 210
IYAGVGEFSF ++++ 211 VFKSEGAYF ++++ 212 SYAPPSEDLF ++ 213
SYAPPSEDLFL ++ 214 KYLMELTLI +++ 215 SYVASFFLL ++ 216 FYVNVKEQF +++
217 IYISNSIYF ++++ 218 LYSELNKWSF +++ 219 SYLKAVFNL +++ 220
SYSEIKDFL ++++ 221 KYIGNLDLL ++++ 223 TFITQSPLL ++++ 224 PYFFANQEF
+++
225 TYTNTLERL +++ 226 MYLKLVQLF ++ 227 IYRFITERF +++ 228 IYQYVADNF
+++ 229 IYQFVADSF +++ 230 TYGMVMVTF +++ 231 AFADVSVKF ++++ 232
YYLSDSPLL +++ 233 QYLTAAALHNL +++ 234 SYLPAIWLL +++ 235 VYKDSIYYI
+++ 236 VYLPKIPSW +++ 237 KYVGQLAVL +++ 239 VYAIFRILL +++ 240
YYFFVQEKI +++ 241 SYVKVLHHL +++ 242 VYGEPRELL +++ 243 SYLELANTL +++
244 VHFEDTGKTLLF +++ 245 LYPQLFVVL +++ 246 KYLSVQLTL ++ 247
SFTKTSPNF +++ 248 AFPTFSVQL ++++ 249 RYHPTTCTI ++++ 250 KYPDIASPTF
++ 251 VYTKALSSL +++ 252 AFGQETNVPLNNF ++++ 253 IYGFFNENF +++ 254
KYLESSATF +++ 255 VYQKIILKF +++ 256 VFGKSAYLF +++ 257 IFIDNSTQPLHF
+++ 258 AYAQLGYLL +++ 259 YFIKSPPSQLF ++ 260 VYMNVMTRL ++++ 261
GYIKLINFI ++++ 262 VYSSQFETI ++++ 263 RYILENHDF +++ 264 LYTETRLQF
++++ 265 SYLNEAFSF ++++ 266 KYTDVVTEFL +++ 267 SFLNIEKTEILF ++ 268
IFITKALQI ++ 269 QYPYLQAFF +++ 270 YYSQESKVLYL +++ 271 RFLMKSYSF
++++ 272 RYVFPLPYL ++++ 273 IYGEKLQFIF +++ 274 KQLDIANYELF ++++ 275
KYGTLDVTF ++++ 276 QYLDVLHAL ++++ 277 FYTFPFQQL +++ 279 VWLPASVLF
+++ 280 TYNPNLQDKL ++++ 281 NYSPGLVSLIL +++ 282 NYLVDPVTI +++ 283
EYQEIFQQL +++ 284 DYLKDPVTI +++ 285 VYVGDALLHAI +++ 286 SYGTILSHI
++++ 287 IYNPNLLTASKF +++ 288 VYPDTVALTF ++ 289 FFHEGQYVF ++++ 290
KYGDFKLLEF ++++ 291 YYLGSGRETF +++ 292 FYPQIINTF ++++ 293
VYPHFSTTNLI ++++ 294 RFPVQGTVTF +++ 295 SYLVIHERI +++ 296 SYQVIFQHF
++++ 297 TYIDTRTVF ++++ 298 AYKSEVVYF ++++ 299 KYQYVLNEF +++ 300
TYPSQLPSL +++ 301 KFDDVTMLF ++++ 302 LYLPVHYGF +++ 303 LYSVIKEDF
+++ 304 EYNEVANLF +++ 305 NYENKQYLF ++++ 306 VYPAEQPQI +++ 307
GYAFTLPLF +++ 308 TFDGHGVFF +++ 309 KYYRQTLLF ++ 310 IYAPTLLVF +++
311 EYLQNLNHI ++++ 312 SYTSVLSRL +++ 313 KYTHFIQSF ++++ 314
RYFKGDYSI +++ 315 FYIPHVPVSF +++ 316 VYFEGSDFKF +++ 317 VFDTSIAQLF
+++ 318 TYSNSAFQYF +++ 319 KYSDVKNLI ++++ 341 SYLTQHQRI +++ 342
NYAFLHRTL +++ 343 NYLGGTSTI +++ 344 EYNSDLHQF +++ 345 EYNSDLHQFF
+++ 347 VYAEVNSL +++ 348 IYLEHTESI +++ 349 QYSIISNVF +++ 350
KYGNFIDKL +++ 351 IFHEVPLKF +++ 352 QYGGDLTNTF +++ 353 TYGKIDLGF
+++ 354 VYNEQIRDLL +++ 355 IYVTGGHLF +++ 356 NYMPGQLTI ++++ 357
QFITSTNTF ++++ 358 YYSEVPVKL +++ 359 NYGVLHVTF ++++ 360 VFSPDGHLF
+++ 361 TYADIGGLDNQI +++ 362 VYNYAEQTL ++ 363 SYAELGTTI ++ 364
KYLNENQLSQL +++ 365 VFIDHPVHL ++++ 366 QYLELAHSL +++ 367 LYQDHMQYI
++ 368 KYQNVKHNL +++ 369 VYTHEVVTL +++ 370 RFIGIPNQF +++ 371
AYSHLRYVF ++ 372 VYVIEPHSMEF +++ 373 GYISNGELF +++ 374 VFLPRVTEL
++
375 KYTDYILKI +++ 376 VYTPVASRQSL +++ 377 QYTPHSHQF +++ 378
VYIAELEKI +++ 379 VFIAQGYTL ++++ 380 VYTGIDHHW ++++ 381 KYPASSSVF
+++ 382 AYLPPLQQVF +++ 383 RYKPGEPITF +++ 384 RYFDVGLHNF +++ 385
QYIEELQKF +++ 386 TFSDVEAHF +++ 387 KYTEKLEEI +++ 388 IYGEKTYAF
+++
Example 6
[0636] Absolute Quantitation of Tumor Associated Peptides Presented
on the Cell Surface
[0637] The generation of binders, such as antibodies and/or TCRs,
is a laborious process, which may be conducted only for a number of
selected targets. In the case of tumor-associated and -specific
peptides, selection criteria include but are not restricted to
exclusiveness of presentation and the density of peptide presented
on the cell surface. In addition to the isolation and relative
quantitation of peptides as described in Example 1, the inventors
did analyze absolute peptide copies per cell as described. The
quantitation of TUMAP copies per cell in solid tumor samples
requires the absolute quantitation of the isolated TUMAP, the
efficiency of TUMAP isolation, and the cell count of the tissue
sample analyzed.
[0638] Peptide Quantitation by NanoLC-MS/MS
[0639] For an accurate quantitation of peptides by mass
spectrometry, a calibration curve was generated for each peptide
using the internal standard method. The internal standard is a
double-isotope-labeled variant of each peptide, i.e. two
isotope-labeled amino acids were included in TUMAP synthesis. It
differs from the tumor-associated peptide only in its mass but
shows no difference in other physicochemical properties (Anderson
et al., 2012). The internal standard was spiked to each MS sample
and all MS signals were normalized to the MS signal of the internal
standard to level out potential technical variances between MS
experiments.
[0640] The calibration curves were prepared in at least three
different matrices, i.e. HLA peptide eluates from natural samples
similar to the routine MS samples, and each preparation was
measured in duplicate MS runs. For evaluation, MS signals were
normalized to the signal of the internal standard and a calibration
curve was calculated by logistic regression.
[0641] For the quantitation of tumor-associated peptides from
tissue samples, the respective samples were also spiked with the
internal standard, the MS signals were normalized to the internal
standard and quantified using the peptide calibration curve.
[0642] Efficiency of Peptide/MHC Isolation
[0643] As for any protein purification process, the isolation of
proteins from tissue samples is associated with a certain loss of
the protein of interest. To determine the efficiency of TUMAP
isolation, peptide/MHC complexes were generated for all TUMAPs
selected for absolute quantitation. To be able to discriminate the
spiked from the natural peptide/MHC complexes,
single-isotope-labeled versions of the TUMAPs were used, i.e. one
isotope-labeled amino acid was included in TUMAP synthesis. These
complexes were spiked into the freshly prepared tissue lysates,
i.e. at the earliest possible point of the TUMAP isolation
procedure, and then captured like the natural peptide/MHC complexes
in the following affinity purification. Measuring the recovery of
the single-labeled TUMAPs therefore allows conclusions regarding
the efficiency of isolation of individual natural TUMAPs.
[0644] The efficiency of isolation was analyzed in a low number of
samples and was comparable among these tissue samples. In contrast,
the isolation efficiency differs between individual peptides. This
suggests that the isolation efficiency, although determined in only
a limited number of tissue samples, may be extrapolated to any
other tissue preparation. However, it is necessary to analyze each
TUMAP individually as the isolation efficiency may not be
extrapolated from one peptide to others.
[0645] Determination of the Cell Count in Solid, Frozen Tissue
[0646] In order to determine the cell count of the tissue samples
subjected to absolute peptide quantitation, the inventors applied
DNA content analysis. This method is applicable to a wide range of
samples of different origin and, most importantly, frozen samples
(Alcoser et al., 2011; Forsey and Chaudhuri, 2009; Silva et al.,
2013). During the peptide isolation protocol, a tissue sample is
processed to a homogenous lysate, from which a small lysate aliquot
is taken. The aliquot is divided in three parts, from which DNA is
isolated (QiaAmp DNA Mini Kit, Qiagen, Hilden, Germany). The total
DNA content from each DNA isolation is quantified using a
fluorescence-based DNA quantitation assay (Qubit dsDNA HS Assay
Kit, Life Technologies, Darmstadt, Germany) in at least two
replicates.
[0647] In order to calculate the cell number, a DNA standard curve
from aliquots of single healthy blood cells, with a range of
defined cell numbers, has been generated. The standard curve is
used to calculate the total cell content from the total DNA content
from each DNA isolation. The mean total cell count of the tissue
sample used for peptide isolation is extrapolated considering the
known volume of the lysate aliquots and the total lysate
volume.
[0648] Peptide Copies Per Cell
[0649] With data of the aforementioned experiments, the inventors
calculated the number of TUMAP copies per cell by dividing the
total peptide amount by the total cell count of the sample,
followed by division through isolation efficiency. Copy cell number
for selected peptides is shown in Table 15.
TABLE-US-00022 TABLE 15 Absolute copy numbers. The table lists the
results of absolute peptide quantitation in tumor samples. The
median number of copies per cell are indicated for each peptide:
<100 = +; > = 100 = ++; > = 1,000 +++; > = 10,000 =
++++. The number of samples, in which evaluable, high quality MS
data are available is indicated. SEQ ID Number of No. Peptide Code
Copies per cell (median) samples 70 DNMT3B-001 ++ 16 323
KIAA0226L-002 ++ 19 325 ZNF-003 ++ 14
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Sequence CWU 1
1
41919PRTHomo sapiens 1Pro Leu Trp Gly Lys Val Phe Tyr Leu1
529PRTHomo sapiens 2Ala Leu Tyr Gly Lys Leu Leu Lys Leu1 539PRTHomo
sapiens 3Thr Leu Leu Gly Lys Gln Val Thr Leu1 549PRTHomo sapiens
4Glu Leu Ala Glu Ile Val Phe Lys Val1 559PRTHomo sapiens 5Ser Leu
Phe Gly Gln Glu Val Tyr Cys1 569PRTHomo sapiens 6Phe Leu Asp Pro
Ala Gln Arg Asp Leu1 579PRTHomo sapiens 7Ala Ala Ala Ala Lys Val
Pro Glu Val1 589PRTHomo sapiens 8Lys Leu Gly Pro Phe Leu Leu Asn
Ala1 599PRTHomo sapiens 9Phe Leu Gly Asp Tyr Val Glu Asn Leu1
51010PRTHomo sapiens 10Lys Thr Leu Asp Val Phe Asn Ile Ile Leu1 5
101110PRTHomo sapiens 11Gly Val Leu Lys Val Phe Leu Glu Asn Val1 5
101210PRTHomo sapiens 12Gly Leu Ile Tyr Glu Glu Thr Arg Gly Val1 5
10139PRTHomo sapiens 13Val Leu Arg Asp Asn Ile Gln Gly Ile1
51411PRTHomo sapiens 14Leu Leu Asp His Leu Ser Phe Ile Asn Lys Ile1
5 10159PRTHomo sapiens 15Ala Leu Gly Asp Tyr Val His Ala Cys1
5169PRTHomo sapiens 16His Leu Tyr Asn Asn Glu Glu Gln Val1
5179PRTHomo sapiens 17Ile Leu His Glu His His Ile Phe Leu1
51811PRTHomo sapiens 18Tyr Val Leu Asn Glu Glu Asp Leu Gln Lys Val1
5 10199PRTHomo sapiens 19Thr Leu Leu Pro Thr Val Leu Thr Leu1
5209PRTHomo sapiens 20Ala Leu Asp Gly His Leu Tyr Ala Ile1
5219PRTHomo sapiens 21Ser Leu Tyr His Arg Val Leu Leu Tyr1
5229PRTHomo sapiens 22Met Leu Ser Asp Leu Thr Leu Gln Leu1
5239PRTHomo sapiens 23Ala Gln Thr Val Val Val Ile Lys Ala1
52410PRTHomo sapiens 24Phe Leu Trp Asn Gly Glu Asp Ser Ala Leu1 5
10259PRTHomo sapiens 25Ile Gln Ala Asp Asp Phe Arg Thr Leu1
5269PRTHomo sapiens 26Lys Val Asp Gly Val Val Ile Gln Leu1
5279PRTHomo sapiens 27Lys Val Phe Gly Asp Leu Asp Gln Val1
52810PRTHomo sapiens 28Thr Leu Tyr Ser Met Asp Leu Met Lys Val1 5
10299PRTHomo sapiens 29Thr Leu Cys Asn Lys Thr Phe Thr Ala1
5309PRTHomo sapiens 30Thr Val Ile Asp Glu Cys Thr Arg Ile1
53112PRTHomo sapiens 31Ala Leu Ser Asp Glu Thr Lys Asn Asn Trp Glu
Val1 5 103210PRTHomo sapiens 32Ile Leu Ala Asp Glu Ala Phe Phe Ser
Val1 5 103314PRTHomo sapiens 33Leu Leu Leu Pro Leu Leu Pro Pro Leu
Ser Pro Ser Leu Gly1 5 103412PRTHomo sapiens 34Leu Leu Pro Lys Lys
Thr Glu Ser His His Lys Thr1 5 103510PRTHomo sapiens 35Tyr Val Leu
Pro Lys Leu Tyr Val Lys Leu1 5 10369PRTHomo sapiens 36Lys Leu Tyr
Gly Ile Glu Ile Glu Val1 53713PRTHomo sapiens 37Ala Leu Ile Asn Asp
Ile Leu Gly Glu Leu Val Lys Leu1 5 10389PRTHomo sapiens 38Lys Met
Gln Glu Asp Leu Val Thr Leu1 5399PRTHomo sapiens 39Ala Leu Met Ala
Val Val Ser Gly Leu1 54011PRTHomo sapiens 40Ser Leu Leu Ala Leu Pro
Gln Asp Leu Gln Ala1 5 10419PRTHomo sapiens 41Phe Val Leu Pro Leu
Val Val Thr Leu1 5429PRTHomo sapiens 42Val Leu Ser Pro Phe Ile Leu
Thr Leu1 5439PRTHomo sapiens 43Leu Leu Trp Ala Gly Pro Val Thr Ala1
5449PRTHomo sapiens 44Gly Leu Leu Trp Gln Ile Ile Lys Val1
5459PRTHomo sapiens 45Val Leu Gly Pro Thr Pro Glu Leu Val1
54610PRTHomo sapiens 46Ser Leu Ala Lys His Gly Ile Val Ala Leu1 5
10479PRTHomo sapiens 47Gly Leu Tyr Gln Ala Gln Val Asn Leu1
5489PRTHomo sapiens 48Thr Leu Asp His Lys Pro Val Thr Val1
5499PRTHomo sapiens 49Leu Leu Asp Glu Ser Lys Leu Thr Leu1
5509PRTHomo sapiens 50Glu Tyr Ala Leu Leu Tyr His Thr Leu1
5519PRTHomo sapiens 51Leu Leu Leu Asp Gly Asp Phe Thr Leu1
5529PRTHomo sapiens 52Glu Leu Leu Ser Ser Ile Phe Phe Leu1
5539PRTHomo sapiens 53Ser Leu Leu Ser His Val Ile Val Ala1
55410PRTHomo sapiens 54Phe Ile Asn Pro Lys Gly Asn Trp Leu Leu1 5
10559PRTHomo sapiens 55Ile Ala Ser Ala Ile Val Asn Glu Leu1
5569PRTHomo sapiens 56Lys Ile Leu Asp Leu Thr Arg Val Leu1
5579PRTHomo sapiens 57Val Leu Ile Ser Ser Thr Val Arg Leu1
5589PRTHomo sapiens 58Ala Leu Asp Asp Ser Leu Thr Ser Leu1
5599PRTHomo sapiens 59Ala Leu Thr Lys Ile Leu Ala Glu Leu1
5609PRTHomo sapiens 60Phe Leu Ile Asp Thr Ser Ala Ser Met1
5619PRTHomo sapiens 61His Leu Pro Asp Phe Val Lys Gln Leu1
5629PRTHomo sapiens 62Ser Leu Phe Asn Gln Glu Val Gln Ile1
56310PRTHomo sapiens 63Thr Leu Ser Ser Glu Arg Asp Phe Ala Leu1 5
10649PRTHomo sapiens 64Gly Leu Ser Ser Ser Ser Tyr Glu Leu1
5659PRTHomo sapiens 65Lys Leu Asp Gly Ile Cys Trp Gln Val1
5669PRTHomo sapiens 66Phe Ile Thr Asp Phe Tyr Thr Thr Val1
5679PRTHomo sapiens 67Gly Val Ile Glu Thr Val Thr Ser Leu1
5689PRTHomo sapiens 68Ala Leu Tyr Gly Phe Phe Phe Lys Ile1
56910PRTHomo sapiens 69Gly Ile Tyr Asp Gly Ile Leu His Ser Ile1 5
10709PRTHomo sapiens 70Gly Leu Phe Ser Gln His Phe Asn Leu1
5719PRTHomo sapiens 71Gly Leu Ile Thr Val Asp Ile Ala Leu1
5729PRTHomo sapiens 72Gly Met Ile Gly Phe Gln Val Leu Leu1
5739PRTHomo sapiens 73Gly Val Pro Asp Thr Ile Ala Thr Leu1
5749PRTHomo sapiens 74Ile Leu Asp Glu Thr Leu Glu Asn Val1
5759PRTHomo sapiens 75Ile Leu Asp Asn Val Lys Asn Leu Leu1
57612PRTHomo sapiens 76Ile Leu Leu Asp Glu Ser Asn Phe Asn His Phe
Leu1 5 10779PRTHomo sapiens 77Ile Val Leu Ser Thr Ile Ala Ser Val1
5789PRTHomo sapiens 78Leu Leu Trp Gly His Pro Arg Val Ala1
5799PRTHomo sapiens 79Ser Leu Val Pro Leu Gln Ile Leu Leu1
5809PRTHomo sapiens 80Thr Leu Asp Glu Tyr Leu Thr Tyr Leu1
5819PRTHomo sapiens 81Val Leu Phe Leu Gly Lys Leu Leu Val1
5828PRTHomo sapiens 82Val Leu Leu Arg Val Leu Ile Leu1 5839PRTHomo
sapiens 83Glu Leu Leu Glu Tyr Leu Pro Gln Leu1 5849PRTHomo sapiens
84Phe Leu Glu Glu Glu Ile Thr Arg Val1 58510PRTHomo sapiens 85Ser
Thr Leu Asp Gly Ser Leu His Ala Val1 5 10869PRTHomo sapiens 86Leu
Leu Val Thr Ser Leu Val Val Val1 58710PRTHomo sapiens 87Tyr Leu Thr
Glu Val Phe Leu His Val Val1 5 108811PRTHomo sapiens 88Ile Leu Leu
Asn Thr Glu Asp Leu Ala Ser Leu1 5 10899PRTHomo sapiens 89Tyr Leu
Val Ala His Asn Leu Leu Leu1 59011PRTHomo sapiens 90Gly Ala Val Ala
Glu Glu Val Leu Ser Ser Ile1 5 109110PRTHomo sapiens 91Ser Ser Leu
Glu Pro Gln Ile Gln Pro Val1 5 109210PRTHomo sapiens 92Leu Leu Arg
Gly Pro Pro Val Ala Arg Ala1 5 10939PRTHomo sapiens 93Ser Leu Leu
Thr Gln Pro Ile Phe Leu1 59418PRTHomo sapiens 94Leu Lys Met Glu Asn
Lys Glu Val Leu Pro Gln Leu Val Asp Ala Val1 5 10 15Thr
Ser9521PRTHomo sapiens 95Gly Leu Tyr Leu Pro Leu Phe Lys Pro Ser
Val Ser Thr Ser Lys Ala1 5 10 15Ile Gly Gly Gly Pro 20969PRTHomo
sapiens 96Tyr Tyr Thr Gln Tyr Ser Gln Thr Ile1 5979PRTHomo sapiens
97Thr Tyr Thr Phe Leu Lys Glu Thr Phe1 59810PRTHomo sapiens 98Val
Phe Pro Arg Leu His Asn Val Leu Phe1 5 10999PRTHomo sapiens 99Gln
Tyr Ile Leu Ala Val Pro Val Leu1 510013PRTHomo sapiens 100Val Tyr
Ile Glu Ser Arg Ile Gly Thr Ser Thr Ser Phe1 5 1010110PRTHomo
sapiens 101Ile Tyr Ile Pro Val Leu Pro Pro His Leu1 5 101029PRTHomo
sapiens 102Val Tyr Pro Phe Glu Asn Phe Glu Phe1 510311PRTHomo
sapiens 103Asn Tyr Ile Pro Val Lys Asn Gly Lys Gln Phe1 5
101049PRTHomo sapiens 104Ser Tyr Leu Thr Trp His Gln Gln Ile1
510510PRTHomo sapiens 105Ile Tyr Asn Glu Thr Ile Thr Asp Leu Leu1 5
1010610PRTHomo sapiens 106Ile Tyr Asn Glu Thr Val Arg Asp Leu Leu1
5 101079PRTHomo sapiens 107Lys Tyr Phe Pro Tyr Leu Val Val Ile1
51089PRTHomo sapiens 108Pro Tyr Leu Val Val Ile His Thr Leu1
51099PRTHomo sapiens 109Leu Phe Ile Thr Gly Gly Gln Phe Phe1
51109PRTHomo sapiens 110Ser Tyr Pro Lys Ile Ile Glu Glu Phe1
51119PRTHomo sapiens 111Val Tyr Val Gln Ile Leu Gln Lys Leu1
51129PRTHomo sapiens 112Ile Tyr Asn Phe Val Glu Ser Lys Leu1
51139PRTHomo sapiens 113Ile Tyr Ser Phe His Thr Leu Ser Phe1
51149PRTHomo sapiens 114Gln Tyr Leu Asp Gly Thr Trp Ser Leu1
51159PRTHomo sapiens 115Arg Tyr Leu Asn Lys Ser Phe Val Leu1
51169PRTHomo sapiens 116Ala Tyr Val Ile Ala Val His Leu Phe1
51179PRTHomo sapiens 117Ile Tyr Leu Ser Asp Leu Thr Tyr Ile1
51189PRTHomo sapiens 118Lys Tyr Leu Asn Ser Val Gln Tyr Ile1
51199PRTHomo sapiens 119Val Tyr Arg Val Tyr Val Thr Thr Phe1
51209PRTHomo sapiens 120Gly Tyr Ile Glu His Phe Ser Leu Trp1
512111PRTHomo sapiens 121Arg Tyr Gly Leu Pro Ala Ala Trp Ser Thr
Phe1 5 101229PRTHomo sapiens 122Glu Tyr Gln Ala Arg Ile Pro Glu
Phe1 51239PRTHomo sapiens 123Val Tyr Thr Pro Val Leu Glu His Leu1
51249PRTHomo sapiens 124Thr Tyr Lys Asp Tyr Val Asp Leu Phe1
512511PRTHomo sapiens 125Val Phe Ser Arg Asp Phe Gly Leu Leu Val
Phe1 5 1012613PRTHomo sapiens 126Pro Tyr Asp Pro Ala Leu Gly Ser
Pro Ser Arg Leu Phe1 5 101279PRTHomo sapiens 127Gln Tyr Phe Thr Gly
Asn Pro Leu Phe1 51289PRTHomo sapiens 128Val Tyr Pro Phe Asp Trp
Gln Tyr Ile1 51299PRTHomo sapiens 129Lys Tyr Ile Asp Tyr Leu Met
Thr Trp1 513010PRTHomo sapiens 130Val Tyr Ala His Ile Tyr His Gln
His Phe1 5 1013111PRTHomo sapiens 131Glu Tyr Leu Asp Arg Ile Gly
Gln Leu Phe Phe1 5 101329PRTHomo sapiens 132Arg Tyr Pro Ala Leu Phe
Pro Val Leu1 513310PRTHomo sapiens 133Lys Tyr Leu Glu Asp Met Lys
Thr Tyr Phe1 5 101349PRTHomo sapiens 134Ala Tyr Ile Pro Thr Pro Ile
Tyr Phe1 51359PRTHomo sapiens 135Val Tyr Glu Ala Met Val Pro Leu
Phe1 513610PRTHomo sapiens 136Ile Tyr Pro Glu Trp Pro Val Val Phe
Phe1 5 101379PRTHomo sapiens 137Glu Tyr Leu His Asn Cys Ser Tyr
Phe1 51389PRTHomo sapiens 138Val Tyr Asn Ala Val Ser Thr Ser Phe1
51399PRTHomo sapiens 139Ile Phe Gly Ile Phe Pro Asn Gln Phe1
51409PRTHomo sapiens 140Arg Tyr Leu Ile Asn Ser Tyr Asp Phe1
514110PRTHomo sapiens 141Ser Tyr Asn Gly His Leu Thr Ile Trp Phe1 5
101429PRTHomo sapiens 142Val Tyr Val Asp Asp Ile Tyr Val Ile1
51439PRTHomo sapiens 143Lys Tyr Ile Phe Gln Leu Asn Glu Ile1
514410PRTHomo sapiens 144Val Phe Ala Ser Leu Pro Gly Phe Leu Phe1 5
101459PRTHomo sapiens 145Val Tyr Ala Leu Lys Val Arg Thr Ile1
51469PRTHomo sapiens 146Asn Tyr Tyr Glu Arg Ile His Ala Leu1
51479PRTHomo sapiens 147Leu Tyr Leu Ala Phe Pro Leu Ala Phe1
51489PRTHomo sapiens 148Ser Tyr Gly Thr Val Ser Gln Ile Phe1
51498PRTHomo sapiens 149Ser Tyr Gly Thr Val Ser Gln Ile1
515010PRTHomo sapiens 150Ile Tyr Ile Thr Arg Gln Phe Val Gln Phe1 5
101519PRTHomo sapiens 151Ala Tyr Ile Ser Gly Leu Asp Val Phe1
515212PRTHomo sapiens 152Lys Phe Phe Asp Asp Leu Gly Asp Glu Leu
Leu Phe1 5 1015313PRTHomo sapiens 153Val Tyr Val Pro Phe Gly Gly
Lys Ser Met Ile Thr Phe1 5 101549PRTHomo sapiens 154Val Tyr Gly Val
Pro Thr Pro His Phe1 51559PRTHomo sapiens 155Ile Tyr Lys Trp Ile
Thr Asp Asn Phe1 515610PRTHomo sapiens 156Tyr Tyr Met Glu Leu Thr
Lys Leu Leu Leu1 5 1015711PRTHomo sapiens 157Asp Tyr Ile Pro Ala
Ser Gly Phe Ala Leu Phe1 5 1015812PRTHomo sapiens 158Ile Tyr Glu
Glu Thr Arg Gly Val Leu Lys Val Phe1 5 101599PRTHomo sapiens 159Ile
Tyr Glu Glu Thr Arg Gly Val Leu1 51609PRTHomo sapiens 160Arg Tyr
Gly Asp Gly Gly Ser Ser Phe1 51619PRTHomo sapiens 161Lys Tyr Pro
Asp Ile Val Gln Gln Phe1 51629PRTHomo sapiens 162Lys Tyr Thr Ser
Tyr Ile Leu Ala Phe1 516310PRTHomo sapiens 163Arg Tyr Leu Thr Ile
Ser Asn Leu Gln Phe1 5 101649PRTHomo sapiens 164His Tyr Val Pro Ala
Thr Lys Val Phe1 516511PRTHomo sapiens 165Glu Tyr Phe Thr Pro Leu
Leu Ser Gly Gln Phe1 5 101669PRTHomo sapiens 166Phe Tyr Thr Leu Pro
Phe His Leu Ile1 51679PRTHomo sapiens 167Arg Tyr Gly Phe Tyr Tyr
Val Glu Phe1 51689PRTHomo sapiens 168Arg Tyr Leu Glu Ala Ala Leu
Arg Leu1 516910PRTHomo sapiens 169Asn Tyr Ile Thr Gly Lys Gly Asp
Val Phe1 5 101709PRTHomo sapiens 170Gln Tyr Pro Phe His Val Pro Leu
Leu1 51719PRTHomo sapiens 171Asn Tyr Glu Asp His Phe Pro Leu Leu1
51729PRTHomo sapiens 172Val Phe Ile Phe Lys Gly Asn Glu Phe1
51739PRTHomo sapiens 173Gln Tyr Leu Glu Lys Tyr Tyr Asn Leu1
517410PRTHomo sapiens 174Val Tyr Glu Lys Asn Gly Tyr Ile Tyr Phe1 5
101759PRTHomo sapiens 175Leu Tyr Ser Pro Val Pro Phe Thr Leu1
51769PRTHomo sapiens 176Phe Tyr Ile Asn Gly Gln Tyr Gln Phe1
517710PRTHomo sapiens 177Val Tyr Phe Lys Ala Gly Leu Asp Val Phe1 5
101789PRTHomo sapiens 178Asn Tyr Ser Ser Ala Val Gln Lys Phe1
517911PRTHomo sapiens 179Thr Tyr Ile Pro Val Gly Leu Gly Arg Leu
Leu1 5 101809PRTHomo sapiens 180Lys Tyr Leu Gln Val Val Gly Met
Phe1 51819PRTHomo sapiens 181Val Tyr Pro Pro Tyr Leu Asn Tyr Leu1
518210PRTHomo sapiens 182Ala Tyr Ala Gln Leu Gly Tyr Leu Leu Phe1 5
101839PRTHomo sapiens 183Pro Tyr Leu Gln Asp Val Pro Arg Ile1
518410PRTHomo sapiens 184Ile Tyr Ser Val Gly Ala Phe Glu Asn Phe1 5
101859PRTHomo sapiens 185Gln Tyr Leu Val His Val Asn Asp Leu1
51869PRTHomo sapiens 186Val Phe Thr Thr Ser Ser Asn Ile Phe1
51879PRTHomo sapiens 187Ala Tyr Ala Ala Asn Val His Tyr Leu1
51889PRTHomo sapiens 188Gly Tyr Lys Thr Phe Phe Asn Glu Phe1
51899PRTHomo sapiens 189Ala Tyr Phe Lys Gln Ser Ser Val Phe1
51909PRTHomo sapiens 190Leu Tyr Ser Glu Leu Thr Glu Thr Leu1
519112PRTHomo sapiens 191Thr Tyr Pro Asp Gly Thr Tyr Thr Gly Arg
Ile Phe1 5 101929PRTHomo sapiens 192Arg Tyr Ser Thr Phe Ser Glu Ile
Phe1 519311PRTHomo sapiens 193Leu Tyr Leu Glu Asn Asn Ala Gln Thr
Gln Phe1 5 101949PRTHomo sapiens 194Val Tyr Gln Ser Leu Ser Asn Ser
Leu1 51959PRTHomo sapiens 195Ala Tyr Ile Lys Gly Gly Trp Ile Leu1
51969PRTHomo sapiens 196Gly Tyr Ile Arg Gly Ser Trp Gln Phe1
51979PRTHomo sapiens 197Ile Phe Thr Asp Ile Phe His Tyr Leu1
51989PRTHomo sapiens 198Asp Tyr Val Gly Phe Thr Leu Lys Ile1
51999PRTHomo sapiens 199Ser Tyr Leu Asn His Leu Asn Asn Leu1
52009PRTHomo sapiens 200Val Phe Ile His His Leu Pro Gln Phe1
52019PRTHomo sapiens 201Gly Tyr Asn Pro Asn Arg Val Phe Phe1
52029PRTHomo sapiens 202Arg Tyr Val Glu Gly Ile Val Ser Leu1
520311PRTHomo sapiens 203Val Tyr Asn Val Glu Val Lys Asn Ala Glu
Phe1 5 102049PRTHomo sapiens 204Glu Tyr Leu Ser Thr Cys Ser Lys
Leu1 52059PRTHomo sapiens 205Val Tyr Pro Val Val Leu Asn Gln Ile1
52069PRTHomo sapiens 206Asn Tyr Leu Asp Val Ala Thr Phe Leu1
520711PRTHomo sapiens 207Leu Tyr Ser Asp Ala Phe Lys Phe Ile Val
Phe1 5 102089PRTHomo sapiens 208Thr Tyr Leu Glu Lys Ile Asp Gly
Phe1 520910PRTHomo sapiens 209Ala Phe Ile Glu Thr Pro Ile Pro Leu
Phe1 5 1021010PRTHomo sapiens 210Ile Tyr Ala Gly Val Gly Glu Phe
Ser Phe1 5 102119PRTHomo sapiens 211Val Phe Lys Ser Glu Gly Ala Tyr
Phe1 521210PRTHomo sapiens 212Ser Tyr Ala Pro Pro Ser Glu Asp Leu
Phe1 5 1021311PRTHomo sapiens 213Ser Tyr Ala Pro Pro Ser Glu Asp
Leu Phe Leu1 5 102149PRTHomo sapiens 214Lys Tyr Leu Met Glu Leu Thr
Leu Ile1 52159PRTHomo sapiens 215Ser Tyr Val Ala Ser Phe Phe Leu
Leu1 52169PRTHomo sapiens 216Phe Tyr Val Asn Val Lys Glu Gln Phe1
52179PRTHomo sapiens 217Ile Tyr Ile Ser Asn Ser Ile Tyr Phe1
521810PRTHomo sapiens 218Leu Tyr Ser Glu Leu Asn Lys Trp Ser Phe1 5
102199PRTHomo sapiens 219Ser Tyr Leu Lys Ala Val Phe Asn Leu1
52209PRTHomo sapiens 220Ser Tyr Ser Glu Ile Lys Asp Phe Leu1
52219PRTHomo sapiens 221Lys Tyr Ile Gly Asn Leu Asp Leu Leu1
52229PRTHomo sapiens 222His Tyr Ser Thr Leu Val His Met Phe1
52239PRTHomo sapiens 223Thr Phe Ile Thr Gln Ser Pro Leu Leu1
52249PRTHomo sapiens 224Pro Tyr Phe Phe Ala Asn Gln Glu Phe1
52259PRTHomo sapiens 225Thr Tyr Thr Asn Thr Leu Glu Arg Leu1
52269PRTHomo sapiens 226Met Tyr Leu Lys Leu Val Gln Leu Phe1
52279PRTHomo sapiens 227Ile Tyr Arg Phe Ile Thr Glu Arg Phe1
52289PRTHomo sapiens 228Ile Tyr Gln Tyr Val Ala Asp Asn Phe1
52299PRTHomo sapiens 229Ile Tyr Gln Phe Val Ala Asp Ser Phe1
52309PRTHomo sapiens 230Thr Tyr Gly Met Val Met Val Thr Phe1
52319PRTHomo sapiens 231Ala Phe Ala Asp Val Ser Val Lys Phe1
52329PRTHomo sapiens 232Tyr Tyr Leu Ser Asp Ser Pro Leu Leu1
523311PRTHomo sapiens 233Gln Tyr Leu Thr Ala Ala Ala Leu His Asn
Leu1 5 102349PRTHomo sapiens 234Ser Tyr Leu Pro Ala Ile Trp Leu
Leu1 52359PRTHomo sapiens 235Val Tyr Lys Asp Ser Ile Tyr Tyr Ile1
52369PRTHomo sapiens 236Val Tyr Leu Pro Lys Ile Pro Ser Trp1
52379PRTHomo sapiens 237Lys Tyr Val Gly Gln Leu Ala Val Leu1
52389PRTHomo sapiens 238Ser Tyr Leu Glu Lys Val Arg Gln Leu1
52399PRTHomo sapiens 239Val Tyr Ala Ile Phe Arg Ile Leu Leu1
52409PRTHomo sapiens 240Tyr Tyr Phe Phe Val Gln Glu Lys Ile1
52419PRTHomo sapiens 241Ser Tyr Val Lys Val Leu His His Leu1
52429PRTHomo sapiens 242Val Tyr Gly Glu Pro Arg Glu Leu Leu1
52439PRTHomo sapiens 243Ser Tyr Leu Glu Leu Ala Asn Thr Leu1
524412PRTHomo sapiens 244Val His Phe Glu Asp Thr Gly Lys Thr Leu
Leu Phe1 5 102459PRTHomo sapiens 245Leu Tyr Pro Gln Leu Phe Val Val
Leu1 52469PRTHomo sapiens 246Lys Tyr Leu Ser Val Gln Leu Thr Leu1
52479PRTHomo sapiens 247Ser Phe Thr Lys Thr Ser Pro Asn Phe1
52489PRTHomo sapiens 248Ala Phe Pro Thr Phe Ser Val Gln Leu1
52499PRTHomo sapiens 249Arg Tyr His Pro Thr Thr Cys Thr Ile1
525010PRTHomo sapiens 250Lys Tyr Pro Asp Ile Ala Ser Pro Thr Phe1 5
102519PRTHomo sapiens 251Val Tyr Thr Lys Ala Leu Ser Ser Leu1
525213PRTHomo sapiens 252Ala Phe Gly Gln Glu Thr Asn Val Pro Leu
Asn Asn Phe1 5 102539PRTHomo sapiens 253Ile Tyr Gly Phe Phe Asn Glu
Asn Phe1 52549PRTHomo sapiens 254Lys Tyr Leu Glu Ser Ser Ala Thr
Phe1 52559PRTHomo sapiens 255Val Tyr Gln Lys Ile Ile Leu Lys Phe1
52569PRTHomo sapiens 256Val Phe Gly Lys Ser Ala Tyr Leu Phe1
525712PRTHomo sapiens 257Ile Phe Ile Asp Asn Ser Thr Gln Pro Leu
His Phe1 5 102589PRTHomo sapiens 258Ala Tyr Ala Gln Leu Gly Tyr Leu
Leu1 525911PRTHomo sapiens 259Tyr Phe Ile Lys Ser Pro Pro Ser Gln
Leu Phe1 5 102609PRTHomo sapiens 260Val Tyr Met Asn Val Met Thr Arg
Leu1 52619PRTHomo sapiens 261Gly Tyr Ile Lys Leu Ile Asn Phe Ile1
52629PRTHomo sapiens 262Val Tyr Ser Ser Gln Phe Glu Thr Ile1
52639PRTHomo sapiens 263Arg Tyr Ile Leu Glu Asn His Asp Phe1
52649PRTHomo sapiens 264Leu Tyr Thr Glu Thr Arg Leu Gln Phe1
52659PRTHomo sapiens 265Ser Tyr Leu Asn Glu Ala Phe Ser Phe1
52669PRTHomo sapiens 266Lys Tyr Thr Asp Trp Thr Glu Phe Leu1
526712PRTHomo sapiens 267Ser Phe Leu Asn Ile Glu Lys Thr Glu Ile
Leu Phe1 5 102689PRTHomo sapiens 268Ile Phe Ile Thr Lys Ala Leu Gln
Ile1 52699PRTHomo sapiens 269Gln Tyr Pro Tyr Leu Gln Ala Phe Phe1
527011PRTHomo sapiens 270Tyr Tyr Ser Gln Glu Ser Lys Val Leu Tyr
Leu1 5 102719PRTHomo sapiens 271Arg Phe Leu Met Lys Ser Tyr Ser
Phe1 52729PRTHomo sapiens 272Arg Tyr Val Phe Pro Leu Pro Tyr Leu1
527310PRTHomo sapiens 273Ile Tyr Gly Glu Lys Leu Gln Phe Ile Phe1 5
1027411PRTHomo sapiens 274Lys Gln Leu Asp Ile Ala Asn Tyr Glu Leu
Phe1 5 102759PRTHomo sapiens 275Lys Tyr Gly Thr Leu Asp Val Thr
Phe1 52769PRTHomo sapiens 276Gln Tyr Leu Asp Val Leu His Ala Leu1
52779PRTHomo sapiens 277Phe Tyr Thr Phe Pro Phe Gln Gln Leu1
52789PRTHomo sapiens 278Lys Tyr Val Asn Leu Val Met Tyr Phe1
52799PRTHomo sapiens 279Val Trp Leu Pro Ala Ser Val Leu Phe1
528010PRTHomo sapiens 280Thr Tyr Asn Pro Asn Leu Gln Asp Lys Leu1 5
1028111PRTHomo sapiens 281Asn Tyr Ser Pro Gly Leu Val Ser Leu Ile
Leu1 5 102829PRTHomo sapiens 282Asn Tyr Leu Val Asp Pro Val Thr
Ile1 52839PRTHomo sapiens 283Glu Tyr Gln Glu Ile Phe Gln Gln Leu1
52849PRTHomo sapiens 284Asp Tyr Leu Lys Asp Pro Val Thr Ile1
528511PRTHomo sapiens 285Val Tyr Val Gly Asp Ala Leu Leu His Ala
Ile1 5 102869PRTHomo sapiens 286Ser Tyr Gly Thr Ile Leu Ser His
Ile1 528712PRTHomo sapiens 287Ile Tyr Asn Pro Asn Leu Leu Thr Ala
Ser Lys Phe1 5 1028810PRTHomo sapiens 288Val Tyr Pro Asp Thr Val
Ala Leu Thr Phe1 5 102899PRTHomo sapiens 289Phe Phe His Glu Gly Gln
Tyr Val Phe1 529010PRTHomo sapiens 290Lys Tyr Gly Asp Phe Lys Leu
Leu Glu Phe1 5 1029110PRTHomo sapiens 291Tyr Tyr Leu Gly Ser Gly
Arg Glu Thr Phe1 5 102929PRTHomo sapiens 292Phe Tyr Pro Gln Ile Ile
Asn Thr Phe1 529311PRTHomo sapiens 293Val Tyr Pro His Phe Ser Thr
Thr Asn Leu Ile1 5 1029410PRTHomo sapiens 294Arg Phe Pro Val Gln
Gly Thr Val Thr Phe1 5 102959PRTHomo sapiens 295Ser Tyr Leu Val Ile
His Glu Arg Ile1 52969PRTHomo sapiens 296Ser Tyr Gln Val Ile Phe
Gln His Phe1 52979PRTHomo sapiens 297Thr Tyr Ile Asp Thr Arg Thr
Val Phe1 52989PRTHomo sapiens 298Ala Tyr Lys Ser Glu Val Val Tyr
Phe1 52999PRTHomo sapiens 299Lys Tyr Gln Tyr Val Leu Asn Glu Phe1
53009PRTHomo sapiens 300Thr Tyr Pro Ser Gln Leu Pro Ser Leu1
53019PRTHomo sapiens 301Lys Phe Asp Asp Val Thr Met Leu Phe1
53029PRTHomo sapiens 302Leu Tyr Leu Pro Val His Tyr Gly Phe1
53039PRTHomo sapiens 303Leu Tyr Ser Val Ile Lys Glu Asp Phe1
53049PRTHomo sapiens 304Glu Tyr Asn Glu Val Ala Asn Leu Phe1
53059PRTHomo sapiens 305Asn Tyr Glu Asn Lys Gln Tyr Leu Phe1
53069PRTHomo sapiens 306Val Tyr Pro Ala Glu Gln Pro Gln Ile1
53079PRTHomo sapiens 307Gly Tyr Ala Phe Thr Leu Pro Leu Phe1
53089PRTHomo sapiens 308Thr Phe Asp Gly His Gly Val Phe Phe1
53099PRTHomo sapiens 309Lys Tyr Tyr Arg Gln Thr Leu Leu Phe1
53109PRTHomo sapiens 310Ile Tyr Ala Pro Thr Leu Leu Val Phe1
53119PRTHomo sapiens 311Glu Tyr Leu Gln Asn Leu Asn His Ile1
53129PRTHomo sapiens 312Ser Tyr Thr Ser Val Leu Ser Arg Leu1
53139PRTHomo sapiens 313Lys Tyr Thr His Phe Ile Gln Ser Phe1
53149PRTHomo sapiens 314Arg Tyr Phe Lys Gly Asp Tyr Ser Ile1
531510PRTHomo sapiens 315Phe Tyr Ile Pro His Val Pro Val Ser Phe1 5
1031610PRTHomo sapiens 316Val Tyr Phe Glu Gly Ser Asp Phe Lys Phe1
5 1031710PRTHomo sapiens 317Val Phe Asp Thr Ser Ile Ala Gln Leu
Phe1 5 1031810PRTHomo sapiens 318Thr Tyr Ser Asn Ser Ala Phe Gln
Tyr Phe1 5 103199PRTHomo sapiens 319Lys Tyr Ser Asp Val Lys Asn Leu
Ile1 532010PRTHomo sapiens 320Lys Phe Ile Leu Ala Leu Lys Val Leu
Phe1 5 103219PRTHomo sapiens 321Ser Leu Trp Phe Lys Pro Glu Glu
Leu1 532210PRTHomo sapiens 322Ala Leu Val Ser Gly Gly Val Ala Gln
Ala1 5 103239PRTHomo sapiens 323Ile Leu Ser Val Val Asn Ser Gln
Leu1 53249PRTHomo sapiens 324Ala Ile Phe Asp Phe Cys Pro Ser Val1
53259PRTHomo sapiens 325Arg Leu Leu Pro Lys Val Gln Glu Val1
53269PRTHomo sapiens 326Ser Leu Leu Pro Leu Val Trp Lys Ile1
53279PRTHomo sapiens 327Ser Ile Gly Asp Ile Phe Leu Lys Tyr1
53289PRTHomo sapiens 328Ser Val Asp Ser Ala Pro Ala Ala Val1
532911PRTHomo sapiens 329Phe Ala Trp Glu Pro Ser Phe Arg Asp Gln
Val1 5 103309PRTHomo sapiens 330Phe Leu Trp Pro Lys Glu Val Glu
Leu1 53319PRTHomo sapiens 331Ala Ile Trp Lys Glu Leu Ile Ser Leu1
53329PRTHomo sapiens 332Ala Val Thr Lys Tyr Thr Ser Ala Lys1
53339PRTHomo sapiens 333Gly Thr Phe Leu Glu Gly Val Ala Lys1
53349PRTHomo sapiens 334Gly Arg Ala Asp Ala Leu Arg Val Leu1
533510PRTHomo sapiens 335Val Leu Leu Ala Ala Gly Pro Ser Ala Ala1 5
1033610PRTHomo sapiens 336Gly Leu Met Asp Gly Ser Pro His Phe Leu1
5 103379PRTHomo sapiens 337Lys Val Leu Gly Lys Ile Glu Lys Val1
533810PRTHomo sapiens 338Leu Leu Tyr Asp Gly Lys Leu Ser Ser Ala1 5
103399PRTHomo sapiens 339Val Leu Gly Pro Gly Pro Pro Pro Leu1
53409PRTHomo sapiens 340Ser Val Ala Lys Thr Ile Leu Lys Arg1
53419PRTHomo sapiens 341Ser Tyr Leu Thr Gln His Gln Arg Ile1
53429PRTHomo sapiens 342Asn Tyr Ala Phe Leu His Arg Thr Leu1
53439PRTHomo sapiens 343Asn Tyr Leu Gly Gly Thr Ser Thr Ile1
53449PRTHomo sapiens 344Glu Tyr Asn Ser Asp Leu His Gln Phe1
534510PRTHomo sapiens 345Glu Tyr Asn Ser Asp Leu His Gln Phe Phe1 5
103469PRTHomo sapiens 346Ile Tyr Val Ile Pro Gln Pro His Phe1
53478PRTHomo sapiens 347Val Tyr Ala Glu Val Asn Ser Leu1
53489PRTHomo sapiens 348Ile Tyr Leu Glu His Thr Glu Ser Ile1
53499PRTHomo sapiens 349Gln Tyr Ser Ile Ile Ser Asn Val Phe1
53509PRTHomo sapiens 350Lys Tyr Gly Asn Phe Ile Asp Lys Leu1
53519PRTHomo sapiens 351Ile Phe His Glu Val Pro Leu Lys Phe1
535210PRTHomo sapiens 352Gln Tyr Gly Gly Asp Leu Thr Asn Thr Phe1 5
103539PRTHomo sapiens 353Thr Tyr Gly Lys Ile Asp Leu Gly Phe1
535410PRTHomo sapiens 354Val Tyr Asn Glu Gln Ile Arg Asp Leu Leu1 5
103559PRTHomo sapiens 355Ile Tyr Val Thr Gly Gly His Leu Phe1
53569PRTHomo sapiens 356Asn Tyr Met Pro Gly Gln Leu Thr Ile1
53579PRTHomo sapiens 357Gln Phe Ile Thr Ser Thr Asn Thr Phe1
53589PRTHomo sapiens 358Tyr Tyr Ser Glu Val Pro Val Lys Leu1
53599PRTHomo sapiens 359Asn Tyr Gly Val Leu His Val Thr Phe1
53609PRTHomo sapiens 360Val Phe Ser Pro Asp Gly His Leu Phe1
536112PRTHomo sapiens 361Thr Tyr Ala Asp Ile Gly Gly Leu Asp Asn
Gln Ile1 5 103629PRTHomo sapiens 362Val Tyr Asn Tyr Ala Glu Gln Thr
Leu1 53639PRTHomo sapiens 363Ser Tyr Ala Glu Leu Gly Thr Thr Ile1
536411PRTHomo sapiens 364Lys Tyr Leu Asn Glu Asn Gln Leu Ser Gln
Leu1 5 103659PRTHomo sapiens 365Val Phe Ile Asp His Pro Val His
Leu1 53669PRTHomo sapiens 366Gln Tyr Leu Glu Leu Ala His Ser Leu1
53679PRTHomo sapiens 367Leu Tyr Gln Asp His Met Gln Tyr Ile1
53689PRTHomo sapiens 368Lys Tyr Gln Asn Val Lys His Asn Leu1
53699PRTHomo sapiens 369Val Tyr Thr His Glu Val Val Thr Leu1
53709PRTHomo sapiens 370Arg Phe Ile Gly Ile Pro Asn Gln Phe1
53719PRTHomo sapiens 371Ala Tyr Ser His Leu Arg Tyr Val Phe1
537211PRTHomo sapiens 372Val Tyr Val Ile Glu Pro His Ser Met Glu
Phe1 5 103739PRTHomo sapiens 373Gly Tyr Ile Ser Asn Gly Glu Leu
Phe1 53749PRTHomo sapiens 374Val Phe Leu Pro Arg Val Thr Glu Leu1
53759PRTHomo sapiens 375Lys Tyr Thr Asp Tyr Ile Leu Lys Ile1
537611PRTHomo sapiens 376Val Tyr Thr Pro Val Ala Ser Arg Gln Ser
Leu1 5 103779PRTHomo sapiens 377Gln Tyr Thr Pro His Ser His Gln
Phe1 53789PRTHomo sapiens 378Val Tyr Ile Ala Glu Leu Glu Lys Ile1
53799PRTHomo sapiens 379Val Phe Ile Ala Gln Gly Tyr Thr Leu1
53809PRTHomo sapiens 380Val Tyr Thr Gly Ile Asp His His Trp1
53819PRTHomo sapiens 381Lys Tyr Pro Ala Ser Ser Ser Val Phe1
538210PRTHomo sapiens 382Ala Tyr Leu Pro Pro Leu Gln Gln Val Phe1 5
1038310PRTHomo sapiens 383Arg Tyr Lys Pro Gly Glu Pro Ile Thr Phe1
5 1038410PRTHomo sapiens 384Arg Tyr Phe Asp Val Gly Leu His Asn
Phe1 5 103859PRTHomo sapiens 385Gln Tyr Ile Glu Glu Leu Gln Lys
Phe1 53869PRTHomo sapiens 386Thr Phe Ser Asp Val Glu Ala His Phe1
53879PRTHomo sapiens 387Lys Tyr Thr Glu Lys Leu Glu Glu Ile1
53889PRTHomo sapiens 388Ile Tyr Gly Glu Lys Thr Tyr Ala Phe1
538910PRTHomo sapiens 389Glu Tyr Leu Pro Glu Phe Leu His Thr Phe1 5
103909PRTHomo sapiens 390Arg Tyr Leu Trp Ala Thr Val Thr Ile1
539110PRTHomo sapiens 391Leu Tyr Gln Ile Leu Gln Gly Ile Val Phe1 5
1039211PRTHomo sapiens 392Arg Tyr Leu Asp Ser Leu Lys Ala Ile Val
Phe1 5 103939PRTHomo sapiens 393Lys Tyr Ile Glu Ala Ile Gln Trp
Ile1 53949PRTHomo sapiens 394Phe Tyr Gln Pro Lys Ile Gln Gln Phe1
53959PRTHomo sapiens 395Leu Tyr Ile Asn Lys Ala Asn Ile Trp1
53969PRTHomo sapiens 396Tyr Tyr His Phe Ile Phe Thr Thr Leu1
53979PRTHomo sapiens 397Ile Tyr Asn Gly Lys Leu Phe Asp Leu1
539810PRTHomo sapiens 398Ile Tyr Asn Gly Lys Leu Phe Asp Leu Leu1 5
103999PRTHomo sapiens 399Ser Tyr Ile Asp Val Leu Pro Glu Phe1
54009PRTHomo sapiens 400Lys Tyr Leu Glu Lys Tyr Tyr Asn Leu1
540110PRTHomo sapiens 401Val Phe Met Lys Asp Gly Phe Phe Tyr Phe1 5
1040210PRTHomo sapiens 402Val Trp Ser Asp Val Thr Pro Leu Thr Phe1
5 1040310PRTHomo sapiens 403Thr Tyr Lys Tyr Val Asp Ile Asn Thr
Phe1 5 104049PRTHomo sapiens 404Arg Tyr Leu Glu Lys Phe Tyr Gly
Leu1 54059PRTHomo sapiens 405Asn Tyr Pro Lys Ser Ile His Ser Phe1
54069PRTHomo sapiens 406Thr Tyr Ser Glu Lys Thr Thr Leu Phe1
54079PRTHomo sapiens 407Val Tyr Gly Ile Arg Leu Glu His Phe1
54089PRTHomo sapiens 408Gln Tyr Ala Ser Arg Phe Val Gln Leu1
54099PRTHomo sapiens 409Tyr Phe Ile Ser His Val Leu Ala Phe1
54109PRTHomo sapiens 410Arg Phe Leu Ser Gly Ile Ile Asn Phe1
54119PRTHomo sapiens 411Val Tyr Ile Gly His Thr Ser Thr Ile1
54129PRTHomo sapiens 412Ser Tyr Asn Pro Leu Trp Leu Arg Ile1
54139PRTHomo sapiens 413Asn Tyr Leu Leu Tyr Val Ser Asn Phe1
54149PRTHomo sapiens 414Met Tyr Pro Tyr Ile Tyr His Val Leu1
54159PRTHomo sapiens 415Ser Tyr Gln Lys Val Ile Glu Leu Phe1
54169PRTHomo sapiens 416Ala Tyr Ser Asp Gly His Phe Leu Phe1
54179PRTHomo sapiens 417Val Tyr Lys Val Val Gly Asn Leu Leu1
541810PRTHomo sapiens 418Glu Leu Ala Gly Ile Gly Ile Leu Thr Val1 5
104199PRTHomo sapiens 419Tyr Leu Leu Pro Ala Ile Val His Ile1 5
* * * * *