U.S. patent application number 16/915797 was filed with the patent office on 2020-10-29 for peptides and combination thereof for use in the immunotherapy against cancers.
The applicant listed for this patent is Immatics Biotechnologies GmbH. Invention is credited to Moreno DI MARCO, Sebastian HAEN, Daniel Johannes KOWALEWSKI, Markus LOEFFLER, Annika NELDE, Hans-Georg RAMMENSEE, Stefan STEVANOVIC, Nico TRAUTWEIN, Juliane Sarah WALZ.
Application Number | 20200339656 16/915797 |
Document ID | / |
Family ID | 1000004954084 |
Filed Date | 2020-10-29 |
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United States Patent
Application |
20200339656 |
Kind Code |
A1 |
WALZ; Juliane Sarah ; et
al. |
October 29, 2020 |
PEPTIDES AND COMBINATION THEREOF FOR USE IN THE IMMUNOTHERAPY
AGAINST 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: |
WALZ; Juliane Sarah;
(Tuebingen, DE) ; KOWALEWSKI; Daniel Johannes;
(Kirchentellinsfurt, DE) ; LOEFFLER; Markus;
(Ammerbuch, DE) ; DI MARCO; Moreno; (Tuebingen,
DE) ; TRAUTWEIN; Nico; (Stuttgart, DE) ;
NELDE; Annika; (Tuebingen, DE) ; STEVANOVIC;
Stefan; (Tuebingen, DE) ; RAMMENSEE; Hans-Georg;
(Tuebingen, DE) ; HAEN; Sebastian; (Tuebingen,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Immatics Biotechnologies GmbH |
Tuebingen |
|
DE |
|
|
Family ID: |
1000004954084 |
Appl. No.: |
16/915797 |
Filed: |
June 29, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16804548 |
Feb 28, 2020 |
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16915797 |
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15949665 |
Apr 10, 2018 |
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16804548 |
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62483702 |
Apr 10, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/4748 20130101;
C07K 16/2833 20130101; C12N 15/115 20130101; C12N 5/0636 20130101;
C07K 2319/70 20130101; C07K 14/70539 20130101; A61K 39/0011
20130101; A61P 35/00 20180101; C07K 16/2818 20130101; G01N 33/505
20130101; A61K 35/17 20130101; C12N 2310/16 20130101; C07K 14/7051
20130101 |
International
Class: |
C07K 14/74 20060101
C07K014/74; C07K 16/28 20060101 C07K016/28; C07K 14/725 20060101
C07K014/725; C12N 15/115 20060101 C12N015/115; A61K 35/17 20060101
A61K035/17; A61P 35/00 20060101 A61P035/00; A61K 39/00 20060101
A61K039/00; C12N 5/0783 20060101 C12N005/0783; C07K 14/47 20060101
C07K014/47; G01N 33/50 20060101 G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 10, 2017 |
DE |
102017107697.2 |
Claims
1. A method of treating a patient who has cancer that presents a
peptide consisting of the amino acid sequence of SEQ ID NO: 8, 21,
231, 104, 100, 156, 170, 102, or 101 in a complex with an MHC class
I molecule on the surface of cancer cells, comprising administering
to said patient a population of activated CD8+ cytotoxic T cells
that recognize cancer cells that present a peptide consisting of
the amino acid sequence of SEQ ID NO: 8, 21, 231, 104, 100, 156,
170, 102, or 101, wherein said cancer is selected from the group
consisting of colorectal cancer, glioblastoma, gastric cancer,
hepatocellular carcinoma, renal cell carcinoma, chronic lymphocytic
leukemia, chronic myeloid leukemia and acute myeloid leukemia,
non-Hodgkin lymphoma, esophageal cancer, non-small cell lung
cancer, small cell lung cancer, pancreatic cancer, prostate cancer,
melanoma, breast cancer, gallbladder cancer and cholangiocarcinoma,
urinary bladder cancer, uterine cancer, head and neck squamous cell
carcinoma, and mesothelioma.
2. The method of claim 1, wherein the CD8+ cytotoxic T cells are
autologous to the patient.
3. The method of claim 1, wherein the CD8+ cytotoxic T cells are
obtained from a healthy donor.
4. The method of claim 1, wherein the CD8+ cytotoxic T cells are
obtained from tumor infiltrating lymphocytes or peripheral blood
mononuclear cells.
5. The method of claim 1, wherein the activated CD8+ cytotoxic T
cells are expanded in vitro.
6. The method of claim 1, further comprising administering to said
patient an adjuvant.
7. The method of claim 6, wherein the adjuvant is selected from
imiquimod, resiquimod, GM-CSF, interferon-alpha, interferon-beta,
CpG oligonucleotides, poly-(I:C), virosomes, interleukin (IL)-1,
IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.
8. The method of claim 1, further comprising administering said
patient cyclophosphamide, sunitinib, bevacizumab, or
sildenafil.
9. The method of claim 1, wherein the activated CD8+ cytotoxic T
cells are produced by contacting CD8+ cytotoxic T cells with an
antigen presenting cell that expresses the peptide in a complex
with an MHC class I molecule on the surface of the antigen
presenting cell, for a period of time sufficient to activate said
CD8+ cytotoxic T cell.
10. The method of claim 9, wherein the antigen presenting cell is
infected with a recombinant virus expressing the peptide.
11. A method of eliciting an immune response in a patient who has
cancer that presents a peptide consisting of the amino acid
sequence of SEQ ID NO: 8, 21, 231, 104, 100, 156, 170, 102, or 101
in a complex with an MHC class I molecule on the surface of cancer
cells, comprising administering to said patient a population of
activated CD8+ cytotoxic T cells that recognize cancer cells that
present a peptide consisting of the amino acid sequence of SEQ ID
NO: 8, 21, 231, 104, 100, 156, 170, 102, or 101, wherein said
cancer is selected from the group consisting of colorectal cancer,
glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell
carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia
and acute myeloid leukemia, non-Hodgkin lymphoma, esophageal
cancer, non-small cell lung cancer, small cell lung cancer,
pancreatic cancer, prostate cancer, melanoma, breast cancer,
gallbladder cancer and cholangiocarcinoma, urinary bladder cancer,
uterine cancer, head and neck squamous cell carcinoma, and
mesothelioma.
12. The method of claim 11, wherein the CD8+ cytotoxic T cells are
autologous to the patient.
13. The method of claim 11, wherein the CD8+ cytotoxic T cells are
obtained from a healthy donor.
14. The method of claim 11, wherein the CD8+ cytotoxic T cells are
obtained from tumor infiltrating lymphocytes or peripheral blood
mononuclear cells.
15. The method of claim 11, wherein the activated CD8+ cytotoxic T
cells are expanded in vitro.
16. The method of claim 11, further comprising administering to
said patient an adjuvant.
17. The method of claim 16, wherein the adjuvant is selected from
imiquimod, resiquimod, GM-CSF, interferon-alpha, interferon-beta,
CpG oligonucleotides, poly-(I:C), virosomes, interleukin (IL)-1,
IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.
18. The method of claim 11, further comprising administering said
patient cyclophosphamide, sunitinib, bevacizumab, or
sildenafil.
19. The method of claim 11, wherein the activated CD8+ cytotoxic T
cells are produced by contacting CD8+ cytotoxic T cells with an
antigen presenting cell that expresses the peptide in a complex
with an MHC class I molecule on the surface of the antigen
presenting cell, for a period of time sufficient to activate said
CD8+ cytotoxic T cell.
20. The method of claim 19, wherein the antigen presenting cell is
infected with a recombinant virus expressing the peptide.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/804,548, filed Feb. 28, 2020, which is a
continuation application of U.S. patent application Ser. No.
15/949,665, filed Apr. 10, 2018, which claims benefit to U.S.
Provisional Application No. 62/483,702, filed Apr. 10, 2017, and is
related to German Application No. 102017107697.2, filed Apr. 10,
2017, the content of which is incorporated herein by reference in
its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT
FILE (.TXT)
[0002] Pursuant to the EFS-Web legal framework and 37 CFR
.sctn..sctn. 1.821-825 (see MPEP .sctn. 2332.03(a)), a Sequence
Listing in the form of an ASCII-compliant text file (entitled
"Sequence_Listing_2912919-085005_ST25.txt" created on Jun. 29, 2020
and 41,839 bytes in size) is submitted concurrently with the
instant application, and the entire contents of the Sequence
Listing are incorporated herein by reference.
FIELD
[0003] 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.
[0004] 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
[0005] 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
(http://www.who.int/mediacentre/factsheets/fs310/en/).
[0006] 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).
[0007] Estimated incidences of different cancer types (adult
population, both sexes) world-wide in 2012 were (Ferlay et al.,
2013; Bray et al., 2013): For cancer of the brain and nervous
system: 256213, for colorectal cancer: 1360602, for kidney cancer:
337860, for liver cancer: 782451, and for gastric cancer: 951594
cases.
[0008] Estimated incidences of different cancer types (adult
population, both sexes) in the USA, EU-28, China and Japan in 2012
were (Ferlay et al., 2013; Bray et al., 2013): For cancer of the
brain and nervous system: 135884, for colorectal cancer: 845797,
for kidney cancer: 226733, for liver cancer: 513172, and for
gastric cancer: 615641 cases.
[0009] Estimated mortalities of different cancer types (adult
population, both sexes) world-wide in 2012 were (Ferlay et al.,
2013; Bray et al., 2013): For cancer of the brain and nervous
system: 189382, for colorectal cancer: 693933, for kidney cancer:
143406, for liver cancer: 745533, and for gastric cancer: 723073
cases.
[0010] Estimated mortalities of different cancer types (adult
population, both sexes) in the USA, EU-28, China and Japan in 2012
were (Ferlay et al., 2013; Bray et al., 2013): For cancer of the
brain and nervous system: 100865, for colorectal cancer: 396066,
for kidney cancer: 83741, for liver cancer: 488485, and for gastric
cancer: 447735 cases.
[0011] Within the groups of brain cancer, the current invention
specifically focuses on glioblastoma (GBM). GBM is the most common
central nervous system malignancy with an age-adjusted incidence
rate of 3.19 per 100,000 inhabitants within the United States. GBM
has a very poor prognosis with a 1-year survival rate of 35% and a
5-year survival rate lower than 5%. Male gender, older age and
ethnicity appear to be risk factors for GBM (Thakkar et al.,
2014).
[0012] Colorectal Cancer--
[0013] 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).
[0014] Removal of the tumor is essential for the treatment of CRC.
Anatomic conditions differ for rectal carcinomas from another CRC
as the rectum is located in the pelvis and the tumor can be
difficult to access. Well-differentiated small rectal tumors (stage
T1) require excision, but no further treatment with chemotherapy.
Patients with rectal tumors of higher T stages receive neoadjuvant
radio-chemotherapy with a fluoropyrimidine prior to total
mesorectal excision (TME) and adjuvant chemotherapy. For
chemotherapeutic treatment, the drugs capecitabine or
5-fluorouracil (5-FU) are used. For combinational chemotherapy, a
cocktail containing 5-FU, leucovorin and oxaliplatin (FOLFOX) is
recommended (Stintzing, 2014; Berman et al., 2015b).
[0015] Treatment of colon carcinomas involves radical hemicolectomy
and lymph node resection. Early stages (UICC stage 1) do not
require additional treatment. Patients with tumors of UICC stage II
receive 5-FU or capecitabine. Treatment for patients with UICC
stage III includes the drug combinations FOLFOX and XELOX
(capecitabine plus oxaliplatin) (Berman et al., 2015a; Stintzing,
2014).
[0016] Metastatic, unresectable CRC are treated with
chemotherapeutical cocktails such as FOLFIRI (5-FU, leucovorin,
irinotecan), FOLFOX, FOLFOXIRI (5-FU, irinotecan, oxaliplatin),
FOLFOX/capecitabine, FOLFOX/oxaliplatin, FOLFIRI/capecitabine and
irinotecan or UFT (5-FU, tegafur-uracil) (Stintzing, 2014).
[0017] 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).
[0018] Latest 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).
[0019] 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).
[0020] 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).
[0021] 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/III trials (Koido et al., 2013).
[0022] 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 non-replicating canary pox 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).
[0023] Gastric Cancer--
[0024] The wall of the stomach is made up of 3 layers of tissue:
the mucosal (innermost) layer, the muscularis (middle) layer, and
the serosal (outermost) layer. Gastric cancer (GC) begins in the
cells lining the mucosal layer and spreads through the outer layers
as it grows. Four types of standard treatment are used. Treatment
for gastric cancer may involve endoscopic or surgical resection,
chemotherapy, radiation therapy or chemoradiation. Surgery is the
primary treatment and the only curative treatment for gastric
cancer. Since the early stages of gastric cancer are mostly
asymptomatic, the disease is usually diagnosed in an advanced
stage. For metastatic gastric cancer, no globally accepted standard
chemotherapy combination regimen has yet been established. However,
the combination of 5-FU and a platinum analog is still the most
widely accepted reference regimen worldwide, although 5-FU can be
replaced by capecitabine or irinotecan and cisplatin can be
replaced by oxaliplatin. Additionally, triple-combination therapies
comprising cisplatin, 5-FU and docetaxel or, in the case of HER-2
over-expressing tumors, cisplatin, 5-FU and trastuzumab can be
applied (Leitlinie Magenkarzinom, 2012).
[0025] 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).
[0026] Glioblastoma--
[0027] 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. Alternative chemotherapeutic
regimens consist of CCNU/lomustine or a combination of procarbazine
with CCNU and vincristine (PCV). In elderly patients' resection or
biopsy of the tumor are not recommended. These patients receive
chemo- or radiation therapy, depending on the methylation state of
the 06-methylguanine-DNA-methyltransferase-(MGMT)-promoter.
Negative methylation state is an indication for focal radiation
therapy, whereas positive methylation state is an indication for
temozolomide treatment with or without focal radiation therapy.
Relapse therapy comprises again resection as well as chemo- and
radiation therapy. In the USA, Canada and Switzerland treatment
with bevacizumab (anti-VEGF-antibody) is also approved for relapse
therapy (Leitlinien fur Diagnostik und Therapie in der Neurologie,
2014).
[0028] Different immunotherapeutic approaches are investigated for
the treatment of GB, including immune-checkpoint inhibition,
vaccination and adoptive transfer of engineered T cells.
[0029] 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.
[0030] 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.
[0031] 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).
[0032] Liver Cancer--
[0033] Disease management depends on the tumor stage at the time of
diagnosis and the overall condition of the liver. If possible,
parts of the liver (partial hepatectomy) or the whole organ (liver
resection) is removed by surgery. Especially patients with small or
completely resectable tumors are qualified to receive a liver
transplant.
[0034] If surgery is not a treatment option, different other
therapies are available at hand. For tumor ablation, a probe is
injected into the liver and the tumor is destroyed by radio or
microwaves or cryotherapy. In embolization procedures, the blood
supply of the tumor is blocked by mechanical or chemical means.
High energy radio waves can be used to destroy the tumor in
radiation therapy.
[0035] Chemotherapy against HCC includes combinations of
doxorubicin, 5-fluorouracil and cisplatin for systemic therapy and
doxorubicin, floxuridine and mitomycin C for hepatic artery
infusions. However, most HCC show a high resistance to
chemotherapeutics (Enguita-German and Fortes, 2014).
[0036] Therapeutic options in advanced non-resectable HCC are
limited to Sorafenib, a multi-tyrosine kinase inhibitor (Chang et
al., 2007; Wilhelm et al., 2004). Sorafenib is the only systemic
drug confirmed to increase survival by about 3 months and currently
represents the only experimental treatment option for such patients
(Chapiro et al., 2014; Llovet et al., 2008).
[0037] 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., 2000).
[0038] 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.
[0039] Renal Cell Carcinoma--
[0040] 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). For first-line treatment
of patients with poor prognostic score a guidance elaborated by
several cancer organizations and societies recommend the receptor
tyrosine kinase inhibitors (TKIs) sunitinib and pazopanib, the
monoclonal antibody bevacizumab combined with interferon-.alpha.
(IFN-.alpha.) and the mTOR inhibitor temsirolimus. Based on
guidelines elaborated by the US NCCN as well as the European EAU
and ESMO, the TKIs sorafenib, pazopanib or recently axitinib are
recommended as second-line therapy in RCC patients who have failed
prior therapy with cytokines (IFN-.alpha., IL-2). The NCCN
guidelines advise also sunitinib in this setting (high-level
evidence according to NCCN Category I).
[0041] Everolimus and axitinib are recommended as second-line
therapy of those patients who have not benefited from a
VEGF-targeted therapy with TKIs according to the established
guidelines.
[0042] 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.
[0043] 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).
[0044] 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 colorectal
cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, and
renal cell carcinoma. There is also a need to identify factors
representing biomarkers for cancer in general and colorectal
cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, and
renal cell carcinoma in particular, leading to better diagnosis of
cancer, assessment of prognosis, and prediction of treatment
success.
[0045] 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.
[0046] The current classification of tumor associated antigens
(TAAs) comprises the following major groups:
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] MHC class I molecules can be found on most nucleated cells.
They present peptides that result from proteolytic cleavage of
predominantly endogenous proteins, defective ribosomal products
(DRIPs) and larger peptides. However, peptides derived from
endosomal compartments or exogenous sources are also frequently
found on MHC class I molecules. This non-classical way of class I
presentation is referred to as cross-presentation in the literature
(Brossart and Bevan, 1997; Rock et al., 1990). MHC class II
molecules can be found predominantly on professional antigen
presenting cells (APCs), and primarily present peptides of
exogenous or transmembrane proteins that are taken up by APCs e.g.
during endocytosis, and are subsequently processed.
[0057] 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.
[0058] 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 immense
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).
[0059] 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).
[0060] Longer (elongated) peptides of the invention can act as MHC
class II active epitopes.
[0061] 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.
[0062] 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).
[0063] 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).
[0064] 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.
[0065] For an MHC class I peptide to trigger (elicit) a cellular
immune response, it also must bind to an MHC-molecule. This process
is dependent on the allele of the MHC-molecule and specific
polymorphisms of the amino acid sequence of the peptide.
MHC-class-I-binding peptides are usually 8-12 amino acid residues
in length and usually contain two conserved residues ("anchors") in
their sequence that interact with the corresponding binding groove
of the MHC-molecule. In this way, each MHC allele has a "binding
motif" determining which peptides can bind specifically to the
binding groove.
[0066] 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).
[0067] 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 und 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.
[0068] 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.
[0069] 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").
[0070] 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
[0071] 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:
268 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: 268, 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.
[0072] 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: 268 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: 268, wherein said peptide or variant thereof has an
overall length of between 8 and 100, preferably between 8 and 30,
and most preferred of between 8 and 14 amino acids.
BRIEF DESCRIPTION OF THE DRAWINGS
[0073] FIG. 1A to FIG. 9B all depict embodiments as described
herein.
[0074] FIGS. 1A through 1P show exemplary expression profile of
source genes of the present invention that are over-expressed in
different cancer samples. Tumor (black dots) and normal (grey dots)
samples are grouped according to organ of origin, and
box-and-whisker plots represent median, 25th and 75th percentile
(box), and minimum and maximum (whiskers) RPKM values. Normal
organs are ordered according to risk categories. RPKM=reads per
kilobase per million mapped reads. Normal samples: blood cells;
blood vessel; brain; heart; liver; lung; adipose: adipose tissue;
adren.gl.: adrenal gland; bile duct; bladder; BM: bone marrow;
cartilage; esoph: esophagus; eye; gallb: gallbladder; head and
neck; kidney; large_int: large intestine; LN: lymph node; nerve;
pancreas; parathyr: parathyroid; perit: peritoneum; pituit:
pituitary; skel.mus: skeletal muscle; skin; small_int: small
intestine; spleen; stomach; thyroid; trachea; ureter; breast;
ovary; placenta; prostate; testis; thymus; uterus. Tumor samples:
CRC: colorectal cancer; GBM: glioblastoma; GC: gastric cancer; HCC:
hepatocellular carcinoma; RCC: renal cell carcinoma. FIG. 1A) Gene
symbol: EGFR, Peptide: LPSPTDSNFY (SEQ ID No: 2), FIG. 1B) Gene
symbol: PTPRZ1, Peptide: LTDYINANY (SEQ ID No: 28), FIG. 1C) Gene
symbol: CCDC146, Peptide: KMMALVAEL (SEQ ID No: 42), FIG. 1D) Gene
symbol: MMP16, Peptide: YLPPTDPRMSV (SEQ ID No: 54), FIG. 1E) Gene
symbol: RP11-1220K2.2, Peptide: GLPDFVKEL (SEQ ID No: 60), FIG. 1F)
Gene symbol: GRM8, Peptide: ATMQSKLIQK (SEQ ID No: 99), FIG. 1G)
Gene symbol: HAVCR1, Peptide: GVIIAKKYFFK (SEQ ID No: 101), FIG.
1H) Gene symbol: SEMA5B, Peptide: GTESGTILK (SEQ ID No: 110), FIG.
1I) Gene symbol: PIWIL1, Peptide: SFDSNLLSF (SEQ ID No: 133), FIG.
1J) Gene symbol: UGT1A3, Peptide: KYLSIPTVF (SEQ ID No: 138), FIG.
1K) Gene symbol: FEZF1, Peptide: APAAVPSAPA (SEQ ID No: 153), FIG.
1L) Gene symbol: MMP11, Peptide: RPASSLRP (SEQ ID No: 163), FIG.
1M) Gene symbol: QRFPR, Peptide: SPMWHVQQL (SEQ ID No: 170), FIG.
1N) Gene symbol: REG4, Peptide: SRSMRLLLL (SEQ ID No: 190), FIG.
1O) Gene symbol: PTHLH, Peptide: AEIHTAEI (SEQ ID No: 231), FIG.
1P) Gene symbol: EGFR, Peptide: DEYLIPQQGF (SEQ ID No: 264).
[0075] FIG. 2 shows 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 267 peptide (KTLGKLWRL, Seq ID
NO: 267) (left panel). After three cycles of stimulation, the
detection of peptide-reactive cells was performed by 2D multimer
staining with A*02/Seq ID No 267. Right panel shows 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.
[0076] FIG. 3 shows 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: 268 peptide (left panel). After
three cycles of stimulation, the detection of peptide-reactive
cells was performed by 2D multimer staining with A*24/SEQ ID NO:
268 (DYIPYVFKL, SEQ ID NO: 268). Right panel shows 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.
[0077] FIGS. 4A and 4B show exemplary results of peptide-specific
in vitro CD8+ T cell responses of a healthy HLA-A*01+ donor. CD8+ T
cells were primed using artificial APCs coated with anti-CD28 mAb
and HLA-A*01 in complex with SEQ ID NO: 6 peptide (VLDLSTNVY; FIG.
4A, left panel) and SEQ ID NO: 245 peptide (ITEKNSGLY; FIG. 4B,
left panel), respectively. After three cycles of stimulation, the
detection of peptide-reactive cells was performed by 2D multimer
staining with A*01/SEQ ID NO: 6 (FIG. 4A) or A*01/SEQ ID NO: 245
(FIG. 4B). Right panels (FIGS. 4A and 4B) show control staining of
cells stimulated with irrelevant A*01/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.
[0078] FIGS. 5A and 5B 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: 42 peptide (KMMALVAEL; FIG.
5A, left panel) and SEQ ID NO: 250 peptide (KMILKMVQL; FIG. 5B,
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: 42 (FIG. 5A) or A*02/SEQ ID NO: 250
(FIG. 5B). Right panels (FIGS. 5A and 5B) 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.
[0079] FIGS. 6A and 6B show exemplary results of peptide-specific
in vitro CD8+ T cell responses of a healthy HLA-A*03+ donor. CD8+ T
cells were primed using artificial APCs coated with anti-CD28 mAb
and HLA-A*03 in complex with SEQ ID NO: 94 peptide (KTYVGHPVKM;
FIG. 6A, left panel) and SEQ ID NO: 110 peptide (GTESGTILK; FIG.
6B, left panel), respectively. After three cycles of stimulation,
the detection of peptide-reactive cells was performed by 2D
multimer staining with A*03/SEQ ID NO: 94 (FIG. 6A) or A*03/SEQ ID
NO: 110 (FIG. 6B). Right panels (FIGS. 6A and 6B) show control
staining of cells stimulated with irrelevant A*03/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.
[0080] FIG. 7 shows 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: 138 peptide (KYLSIPTVF; left
panel). After three cycles of stimulation, the detection of
peptide-reactive cells was performed by 2D multimer staining with
A*02/SEQ ID NO: 138. Right panel shows 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.
[0081] FIGS. 8A and 8B show exemplary results of peptide-specific
in vitro CD8+ T cell responses of a healthy HLA-B*07+ donor. CD8+ T
cells were primed using artificial APCs coated with anti-CD28 mAb
and HLA-B*07 in complex with SEQ ID NO: 170 peptide (SPMWHVQQL;
FIG. 8A, left panel) and SEQ ID NO: 155 peptide (FPYPYAERL; FIG.
8B, left panel), respectively. After three cycles of stimulation,
the detection of peptide-reactive cells was performed by 2D
multimer staining with B*07/SEQ ID NO: 170 (FIG. 8A) or B*07/SEQ ID
NO: 155 (FIG. 8B). Right panels (FIGS. 8A and 8B) show control
staining of cells stimulated with irrelevant B*07/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.
[0082] FIGS. 9A and 9B show exemplary results of peptide-specific
in vitro CD8+ T cell responses of a healthy HLA-B*44+ donor. CD8+ T
cells were primed using artificial APCs coated with anti-CD28 mAb
and HLA-B*44 in complex with SEQ ID NO: 225 peptide (SEAFPSRAL;
FIG. 9A, left panel) and SEQ ID NO: 236 peptide (EEKLIIQDF; FIG.
9B, left panel), respectively. After three cycles of stimulation,
the detection of peptide-reactive cells was performed by 2D
multimer staining with B*44/SEQ ID NO: 225 (FIG. 9A) or B*44/SEQ ID
NO: 236 (FIG. 9B). Right panels (FIGS. 9A and 9B) show control
staining of cells stimulated with irrelevant B*44/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.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
[0083] 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. In Table 1, peptides
with SEQ ID NO: 1 to SEQ ID NO: 37 bind to HLA-A*01, peptides with
SEQ ID NO: 38 to SEQ ID NO: 61 bind to HLA-A*02, peptides with SEQ
ID NO: 62 to SEQ ID NO: 112 bind to HLA-A*03, peptides with SEQ ID
NO: 113 to SEQ ID NO: 142 bind to HLA-A*24, peptides with SEQ ID
NO: 143 to SEQ ID NO: 175 bind to HLA-B*07, peptides with SEQ ID
NO: 176 to SEQ ID NO: 194 bind to HLA-B*08, peptides with SEQ ID
NO: 195 to SEQ ID NO: 241 bind to HLA-B*44. The peptides in Table 2
have been disclosed before in large listings as results of
high-throughput screenings with high error rates or calculated
using algorithms, but have not been associated with cancer at all
before. In Table 2, peptides with SEQ ID NO: 242 to SEQ ID NO: 248
bind to HLA-A*01, peptides with SEQ ID NO: 249 to SEQ ID NO: 251
bind to HLA-A*02, peptides with SEQ ID NO: 252 to SEQ ID NO: 254
bind to HLA-A*03, peptides with SEQ ID NO: 255 to SEQ ID NO: 259
bind to HLA-B*07, peptides with SEQ ID NO: 260 to SEQ ID NO: 266
bind to HLA-B*44. The peptides in Table 3 are additional peptides
that may be useful in combination with the other peptides of the
invention. In Table 3, peptide with SEQ ID NO: 267 binds to
HLA-A*02, peptide with SEQ ID NO: 268 binds to HLA-A*24.
TABLE-US-00001 TABLE 1 Peptides according to the present invention.
Seq ID Official Gene No Sequence Symbol(s) HLA allotype 1 RSDPVTLDV
CEACAM5 A*01 2 LPSPTDSNFY EGFR A*01 3 ASSTDSASYY APOB A*01 4
NSDLKYNAL APOB A*01 5 SILGSDVRVPSY APOB A*01 6 VLDLSTNVY APOB A*01
7 LITGDPKAAYDY COL11A1 A*01/A*03 8 TPVTEFSLNTY COL6A3 A*01 9
FITAQNHGY CPS1 A*01 10 ITAQNHGY CPS1 A*01 11 LSAGSGPGQY CPT2 A*01
12 ITFGERFEY CYP2J2 A*01 13 GSTMVEHNY DCBLD2 A*01/A*03 14
YTERDGSAMVY DCLK2 A*01 15 LTDYLKNTY DPP4 A*01 16 LSLIDRLVLY EGLN3
A*01 17 YTDKLQHY EPHB2 A*01 18 EVSNGKWLLY ITGA3 A*01/A*03 19
VSNGKWLLY ITGA3 A*01/A*03 20 STDEITTRY KLB A*01 21 STDIGALMY MMP1
A*01 22 TLEQVQLYY MYO7B A*01 23 TASEDVFQY NOX1 A*01 24 YTHHLFIFY
NOX1 A*01 25 LMKEVMEHY PLOD2 A*01/B*15 26 EVLDSHIHAY PTPRZ1 A*01 27
LDSHIHAY PTPRZ1 A*01 28 LTDYINANY PTPRZ1 A*01 29 SVTDLEMPHY PTPRZ1
A*01/B*18 30 VLDSHIHAY PTPRZ1 A*01 31 VTDLEMPHY PTPRZ1 A*01 32
ATVGYFIFY RNF128 A*01 33 FADKIHLAY RNF128 A*01 34 ITDFNNIRY
RP11-1220K2.2 A*01 35 FASDLLHLY SLC16A11 A*01 36 YAAYIIHAY TLR3
A*01/A*29 37 LTDSFPLKV TTPA A*01 38 VMLNSNVLL AC010879.1, A*02
NLGN4X, NLGN4Y 39 YLLPSVVLL AGPAT5 A*02 40 KIDDIWNLEV APOB A*02 41
SLQDTKITL APOB A*02 42 KMMALVAEL CCDC146 A*02 43 GLMTIVTSL CCL24
A*02 44 SQTGFVVLV CHI3L1 A*02 45 KLLDEVTYL CYP2J2 A*02 46 VLITGLPLI
CYP2J2 A*02 47 YQDSWFQQL CYP2J2 A*02 48 NLTFIIILI F13B A*02 49
NLASRPYSL F5 A*02 50 ELMPRVYTL FAT1 A*02 51 ALAAELNQL GFAP A*02 52
YVSSGEMMV GFAP A*02 53 LLMTSLTES LRRN1 A*02 54 YLPPTDPRMSV MMP16
A*02 55 RLWQIQHHL MTCP1 A*02 56 FLNQIYTQL MUC5AC A*02 57 GLTGVIMTI
NOX1 A*02 58 MLCLLLTL PAEP A*02 59 KLHEIYIQA PCDHGC3 A*02 60
GLPDFVKEL RP11-1220K2.2 A*02 61 RLFGLFLNNV TLR3 A*02 62 GSYSALLAKK
ABCC2 A*03/A*11 63 KVLGPNGLLK ABCC2 A*03 64 STTKLYLAK ABCC2
A*03/A*11 65 VLGPNGLLK ABCC2 A*03/A*68 66 ATYEGIQKK ALDH1L1,
A*03/A*11 ALDH1L2 67 ATALSLSNK APOB A*03 68 ATAYGSTVSK APOB
A*03/A*11 69 ATAYGSTVSKR APOB A*03 70 ATWSASLKNK APOB A*03 71
KLGNNPVSK APOB A*03 72 KQVFPGLNY APOB A*03 73 KSFDRHFEK APOB
A*03/A*11 74 QLYSKFLLK APOB A*03 75 QVPTFTIPK APOB A*03 76
SAFGYVFPK APOB A*03/A*11 77 SSASLAHMK APOB A*03/A*68 78 STKSTSPPK
APOB A*03 79 STNNEGNLK APOB A*03/A*11 80 STSHHLVSR APOB A*03/A*68
81 SVKLQGTSK APOB A*03/A*68 82 TAYGSTVSK APOB A*03 83 TAYGSTVSKR
APOB A*03/A*68 84 TVASLHTEK APOB A*03/A*68 85 KMAAWPFSR C4BPA A*03
86 KTPSGALHRK C4BPA A*03/A*11 87 SSYSRSSAVK DCLK2 A*03 88 MLLQQPLIY
DNAH11 A*03 89 KITDFGLAK EGFR A*03 90 GSRLGKYYVK EGLN3 A*03 91
SLIDRLVLY EGLN3 A*03 92 AVLDLGSLLAK FAM149A A*03 93 ALDKPGKSK
FAM181B A*03 94 KTYVGHPVKM FAT1 A*03 95 RLFESSFHY GAL3ST1 A*03/A*29
96 FSLAGALNAGFK GFAP A*03 97 RMPPPLPTR GFAP A*03 98 KLYPTYSTK GPLD1
A*03 99 ATMQSKLIQK GRM8 A*03 100 ALLGVIIAK HAVCR1 A*03 101
GVIIAKKYFFK HAVCR1 A*03/A*11 102 IIAKKYFFK HAVCR1 A*03 103
KSWTASSSY LOXL2 A*03 104 STQDTLLIK MXRA5 A*03 105 GSAALYLLR
NDUFA4L2 A*03 106 RLSPNDQYK NDUFA4L2 A*03 107 EIYGGHHAGF OLIG2 A*03
108 LLKSSVGNFY PCDHB8 A*03 109 KIIAPLVTR PLOD2 A*03/A*11 110
GTESGTILK SEMA5B A*03/A*11 111 KIKEHVRSK UBD A*03 112 KMMADYGIRK
UBD A*03 113 VWAKILSAF ABCB4 A*24 114 KFLDSNIKF APOB A*24/A*23 115
YFEEAANFL BAAT A*24 116 LVLDYSKDYNHW CPS1 A*24 117 NFLPPIIARF
DCBLD2 A*24 118 TYISKTIAL EXOC3L2 A*24 119 YMKALGVGF FABP7
A*24/B*15 120 MYAKEFDLL FMO5 A*24 121 SYIEKVRFL GFAP A*24
122 KLYGMPTDFGF GRB7 A*24/A*32 123 RQYLAINQI ITPR2 A*24 124
EVYSPEADQW KLHDC8A A*24/A*25 125 IYGPKYIHPSF MACC1 A*24/A*23 126
TFQDKTLNF MACC1 A*24 127 IFINLSPEF MUC5AC A*24 128 SYTKVEARL MUC5AC
A*24 129 VFLNQIYTQL MUC5AC A*24 130 VYGDGHYLTF MUC5AC A*24 131
KQLDHNLTF NOX1 A*24/B*15 132 VYNPVIYVF OPN3 A*24 133 SFDSNLLSF
PIWIL1 A*24 134 TYLTGRQF PLCB4 A*24 135 VIAPIISNF SLC12A2 A*24/B*15
136 EYNNIQHLF TLR3 A*24 137 KYLSLSNSF TLR3 A*24 138 KYLSIPTVF
UGT1A3 A*24 139 PYASLASELF UGT1A3, A*24 UGT1A4, UGT1A5 140
KYLSIPAVF UGT1A4, A*24 UGT1A5 141 KYLSIPAVFF UGT1A4, A*24 UGT1A5
142 SSFPGAGNTW WSCD1 A*24/A*25 143 FELPTGAGLQL APOB B*07 144
IPEPSAQQL APOB B*07 145 RVPSYTLIL APOB B*07 146 SPGDKRLAA APOB B*07
147 SPIKVPLLL APOB B*07 148 VPDGVSKVL APOB B*07 149 YPLTGDTRL APOB
B*07 150 KPSSKALGTSL ATP10B B*07 151 VVHPRTLLL CYP2J2 B*07/B*15 152
IPSRLLAIL EFNA5 B*07 153 APAAVPSAPA FEZF1 B*07 154 GPGTRLSL GFAP
B*07 155 FPYPYAERL GRIN2D B*07/B*35 156 HPQVVILSL HAVCR1 B*07/B*35
157 SPSPGKDPTL HSF4 B*07 158 VPERGEPEL HSF4 B*07 159 FPAHPSLLL
ITGA3 B*07 160 RPAPADSAL KISS1R B*07 161 NPYEGRVEV LOXL2 B*07/B*51
162 MPMISIPRV LPPR5 B*07/B*51 163 RPASSLRP MMP11 B*07 164
ISTPSEVSTPL MUC17 B*07 165 TPIAKVSEL NKD1 B*07 166 HDPDVGSNSL
PCDHGC3 B*07 167 YPSEVEHMF PGF B*07/B*35 168 IPTDKLLVI PLOD2 B*07
169 FPTEVTPHAF PTPRZ1 B*07 170 SPMWHVQQL QRFPR B*07 171 APKLFAVAF
SEC14L6 B*07 172 KPAHYPLIAL TEX11 B*07 173 MVPSAGQLALF TGFA B*07
174 VPSLQRLML TLR3 B*07 175 HPIETLVDIF VEGFA B*07/B*35 176 AAMSRYEL
APOB B*08 177 DLKYNALDL APOB B*08 178 HAKEKLTAL APOB B*08 179
IQIYKKLRTSSF APOB B*08 180 LLKAEPLAF APOB B*08/B*15 181 YKKLRTSSF
APOB B*08 182 LPFLRENDL ASTN1 B*08/B*07 183 FQKLKLLSL ATP10B B*08
184 EPVKKSRL CCND1 B*08 185 NPNLKTLL CHI3L1 B*08 186 SLIDRLVL EGLN3
B*08/B*07 187 YVKERSKAM EGLN3 B*08 188 SALDHVTRL EXOC3L2 B*08 189
HIFLRTTL ITPR2 B*08 190 SRSMRLLLL REG4 B*08 191 LINLKYLSL TLR3 B*08
192 LPMLKVLNL TLR3 B*08 193 LSYNKYLQL TLR3 B*08 194 EAKRHLLQV UBD
B*08 195 AEAVLKTLQEL APOB B*44/B*40 196 AEQTGTWKL APOB B*44 197
EEAKQVLFL APOB B*44 198 FELPTGAGL APOB B*44/B*40 199 GEATLQRIY APOB
B*44 200 GEELGFASL APOB B*44 201 GEHTSKATL APOB B*44 202 KEFNLQNMGL
APOB B*44 203 KENFAGEATL APOB B*44 204 KESQLPTVM APOB B*44 205
QEVLLQTFL APOB B*44 206 SEPINIIDAL APOB B*44/B*40 207 TEATMTFKY
APOB B*44 208 AEHDAVRNAL ASCL2 B*44 209 YEVDTVLRY BCAN B*44 210
SENIVIQVY C5 B*44 211 TEKEMIQKL CCDC146 B*44 212 AEETCAPSV CCND1
B*44/B*51 213 TTMDQKSLW CHI3L2 B*44 214 AEQPDGLIL CPS1 B*44 215
AFITAQNHGY CPS1 B*44 216 LQEEKVPAIY CPS1 B*44 217 NEINEKIAPSF CPS1
B*44 218 AEGGKVPIKW EGFR B*44 219 AENAEYLRV EGFR B*44 220 KEITGFLLI
EGFR B*44 221 AEERAEAKKKF EGLN3 B*44 222 NEISTFHNL GPC3 B*44 223
SEVPVARVW IGFBP1 B*44 224 SESAVFHGF ITGA3 B*44 225 SEAFPSRAL KISS1R
B*44 226 EELLHGQLF MUC5AC B*44 227 TEHTQSQAAW NXPH4 B*44 228
AEKQTPDGRKY PCDHGB2 B*44 229 KESDGFHRF PLOD2 B*44 230 AENLFRAFL
PRKDC B*44 231 AEIHTAEI PTHLH B*44 232 AEKDGKLTDY PTPRZ1 B*44 233
DESEKTTKSF PTPRZ1 B*44 234 EEESLLTSF PTPRZ1 B*44 235 EEFETLKEF
PTPRZ1 B*44 236 EEKLIIQDF PTPRZ1 B*44 237 LEMPHYSTF PTPRZ1 B*44 238
SENPETITY PTPRZ1 B*44 239 TEVLDSHIHAY PTPRZ1 B*44 240 HELENHSMY
TRIM9 B*44 241 REAEPIPKM TRIO B*44
TABLE-US-00002 TABLE 2 Additional peptides according to the present
invention with no prior known cancer association. Seq ID Official
Gene No Sequence Symbol(s) HLA allotype 242 FSDKELAAY ABCB4 A*01
243 RSPNNFLSY CCND1 A*01/A*03 244 RSDPVTLNV CEACAM1, A*01 CEACAM6,
CEACAM7, PSG1, PSG4, PSG5, PSG7 245 ITEKNSGLY CEACAM5 A*01 246
YSDLHAFYY MANEAL A*01 247 RSDPGGGGLAY MEX3B A*01 248 YSHAAGQGTGLY
SOX9 A*01 249 ALFPERITV ATAT1 A*02 250 KMILKMVQL PRAME A*02 251
RLASRPLLL PTGFRN A*02 252 RIYNGIGVSR DCBLD2 A*03 253 KLFGTSGQK EGFR
A*03 254 AVATKFVNK TRIO A*03 255 LPDGSRVEL ACTL8 B*07 256 LPALPQQLI
COL6A3 B*07 257 SPLRGGSSL EFNA3, EFNA4 B*07 258 APSGTRVVQVL PCDHGC3
B*07 259 RPAVGHSGL ZC3H3 B*07 260 EEAPLVTKAF ASPSCR1 B*44 261
IEALLESSL CCND1 B*44 262 MELLLVNKL CCND1 B*44 263 QQATPGPAY CEA,
CEACAM5, B*44 CEACAM6 264 DEYLIPQQGF EGFR B*44 265 EEVDVPIKLY
EPHB1, EPHB2 B*44 266 ARLTPIPFGL TMEM64 B*44
TABLE-US-00003 TABLE 3 Peptides of the invention useful for e.g.
personalized cancer therapies Seq ID Official Gene No Sequence
Symbol(s) HLA allotype 267 KTLGKLWRL SOX10, SOX8, SOX9 A*02 268
DYIPYVFKL APOB A*24
[0084] The present invention generally relates to the peptides
according to the present invention for use in the treatment of
proliferative diseases, such as, for example, chronic lymphocytic
leukemia, chronic myeloid leukemia and acute myeloid leukemia, and
other lymphoid neoplasms, for example, Non-Hodgkin lymphoma,
post-transplant lymphoproliferative disorders (PTLD) as well as
other myeloid neoplasms, such as primary myelofibrosis, essential
thrombocytopenia, polycythemia vera, as well as other neoplasms
such as esophageal cancer, non-small cell lung cancer, small cell
lung cancer, pancreatic cancer, prostate cancer, melanoma, breast
cancer, gallbladder cancer and cholangiocarcinoma, urinary bladder
cancer, uterine cancer, head and neck squamous cell carcinoma,
mesothelioma.
[0085] 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: 268. More preferred
are the peptides--alone or in combination--selected from the group
consisting of SEQ ID NO: 1 to SEQ ID NO: 241 (see Table 1), and
their uses in the immunotherapy of colorectal cancer, glioblastoma,
gastric cancer, hepatocellular carcinoma, and renal cell carcinoma,
chronic lymphocytic leukemia, chronic myeloid leukemia and acute
myeloid leukemia, and other lymphoid neoplasms, for example,
Non-Hodgkin lymphoma, post-transplant lymphoproliferative disorders
(PTLD) as well as other myeloid neoplasms, such as primary
myelofibrosis, essential thrombocytopenia, polycythemia vera, as
well as other neoplasms such as esophageal cancer, non-small cell
lung cancer, small cell lung cancer, pancreatic cancer, prostate
cancer, melanoma, breast cancer, gallbladder cancer and
cholangiocarcinoma, urinary bladder cancer, uterine cancer, head
and neck squamous cell carcinoma, mesothelioma, and preferably
colorectal cancer, glioblastoma, gastric cancer, hepatocellular
carcinoma, and renal cell carcinoma.
[0086] 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 colorectal cancer, glioblastoma, gastric
cancer, hepatocellular carcinoma, and renal cell carcinoma, chronic
lymphocytic leukemia, chronic myeloid leukemia and acute myeloid
leukemia, and other lymphoid neoplasms, for example, Non-Hodgkin
lymphoma, post-transplant lymphoproliferative disorders (PTLD) as
well as other myeloid neoplasms, such as primary myelofibrosis,
essential thrombocytopenia, polycythemia vera, as well as other
neoplasms such as esophageal cancer, non-small cell lung cancer,
small cell lung cancer, pancreatic cancer, prostate cancer,
melanoma, breast cancer, gallbladder cancer and cholangiocarcinoma,
urinary bladder cancer, uterine cancer, head and neck squamous cell
carcinoma, mesothelioma.
[0087] 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.
[0088] 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: 268.
[0089] The present invention further relates to the peptides
according to the present invention, wherein said peptide is
modified and/or includes non-peptide bonds.
[0090] 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 (li), or fused to
(or into the sequence of) an antibody, such as, for example, an
antibody that is specific for dendritic cells.
[0091] 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. The present invention further relates to an expression
vector capable of expressing and/or expressing a nucleic acid
according to the present invention.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] The antibodies and TCRs are additional embodiments of the
immunotherapeutic use of the peptides according to the invention at
hand.
[0096] 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.
[0097] 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.
[0098] 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.
[0099] 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: 268,
preferably containing SEQ ID No. 1 to SEQ ID No. 241, or a variant
amino acid sequence.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] Preferably, said medicament is a cellular therapy, a vaccine
or a protein based on a soluble TCR or antibody.
[0104] The present invention further relates to a use according to
the present invention, wherein said cancer cells are colorectal
cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, and
renal cell carcinoma, chronic lymphocytic leukemia, chronic myeloid
leukemia and acute myeloid leukemia, and other lymphoid neoplasms,
for example, Non-Hodgkin lymphoma, post-transplant
lymphoproliferative disorders (PTLD) as well as other myeloid
neoplasms, such as primary myelofibrosis, essential
thrombocytopenia, polycythemia vera, as well as other neoplasms
such as esophageal cancer, non-small cell lung cancer, small cell
lung cancer, pancreatic cancer, prostate cancer, melanoma, breast
cancer, gallbladder cancer and cholangiocarcinoma, urinary bladder
cancer, uterine cancer, head and neck squamous cell carcinoma,
mesothelioma, and preferably colorectal cancer, glioblastoma,
gastric cancer, hepatocellular carcinoma, and renal cell carcinoma
cells.
[0105] 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
colorectal cancer, glioblastoma, gastric cancer, hepatocellular
carcinoma, and renal cell carcinoma. 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.
[0106] Optionally the antibody carries a further effector function
such as an immune stimulating domain or toxin.
[0107] The present invention also relates to the use of these novel
targets in the context of cancer treatment.
[0108] 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.
[0109] 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).
[0110] The term "T-cell response" means the specific proliferation
and activation of effector functions induced by a peptide in vitro
or in vivo. For MHC class I restricted cytotoxic T cells, effector
functions may be lysis of peptide-pulsed, peptide-precursor pulsed
or naturally peptide-presenting target cells, secretion of
cytokines, preferably Interferon-gamma, TNF-alpha, or IL-2 induced
by peptide, secretion of effector molecules, preferably granzymes
or perforins induced by peptide, or degranulation.
[0111] 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, or 12 or longer, and in case of MHC class II peptides
(elongated variants of the peptides of the invention) they can be
as long as 13, 14, 15, 16, 17, 18, 19 or 20 or more amino acids in
length.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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, HLAA*02, and HLAB*07 are examples of different MHC
class I alleles that can be expressed from these loci.
TABLE-US-00004 TABLE 4 Expression frequencies F of HLA-A*02,
HLA-A*01, HLA-A*03, HLA-A*24, HLA-B*07, HLA-B*08 and HLA-B*44
serotypes. Haplotype frequencies Gf are derived from a study which
used HLA-typing data from a registry of more than 6.5 million
volunteer donors in the U.S. (Gragert et al., 2013). The haplotype
frequency is the frequency of a distinct allele on an individual
chromosome. Due to the diploid set of chromosomes within mammalian
cells, the frequency of genotypic occurrence of this allele is
higher and can be calculated employing the Hardy-Weinberg principle
(F = 1 - (1 - Gf).sup.2). Calculated phenotype Allele Population
from allele frequency (F) A*02 African (N = 28557) 32.3% European
Caucasian (N = 1242890) 49.3% Japanese (N = 24582) 42.7% Hispanic,
S + Cent Amer. (N = 146714) 46.1% Southeast Asian (N = 27978) 30.4%
A*01 African (N = 28557) 10.2% European Caucasian (N = 1242890)
30.2% Japanese (N = 24582) 1.8% Hispanic, S + Cent Amer. (N =
146714) 14.0% Southeast Asian (N = 27978) 21.0% A*03 African (N =
28557) 14.8% European Caucasian (N = 1242890) 26.4% Japanese (N =
24582) 1.8% Hispanic, S + Cent Amer. (N = 146714) 14.4% Southeast
Asian (N = 27978) 10.6% A*24 African (N = 28557) 2.0% European
Caucasian (N = 1242890) 8.6% Japanese (N = 24582) 35.5% Hispanic, S
+ Cent Amer. (N = 146714) 13.6% Southeast Asian (N = 27978) 16.9%
B*07 African (N = 28557) 14.7% European Caucasian (N = 1242890)
25.0% Japanese (N = 24582) 11.4% Hispanic, S + Cent Amer. (N =
146714) 12.2% Southeast Asian (N = 27978) 10.4% B*08 African (N =
28557) 6.0% European Caucasian (N = 1242890) 21.6% Japanese (N =
24582) 1.0% Hispanic, S + Cent Amer. (N = 146714) 7.6% Southeast
Asian (N = 27978) 6.2% B*44 African (N = 28557) 10.6% European
Caucasian (N = 1242890) 26.9% Japanese (N = 24582) 13.0% Hispanic,
S + Cent Amer. (N = 146714) 18.2% Southeast Asian (N = 27978)
13.1%
[0118] The peptides of the invention, preferably when included into
a vaccine of the invention as described herein bind to A*02, A*01,
A*03, A*24, B*07, B*08 or B*44. 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-,
A*01-, A*03-, A*24-, B*07-, B*08- or B*44-positive, whereas no
selection for MHC class II allotypes is necessary due to the
pan-binding nature of these peptides.
[0119] If A*02 peptides of the invention are combined with peptides
binding to another allele, for example A*24, a higher percentage of
any patient population can be treated compared with addressing
either MHC class I allele alone. While in most populations less
than 50% of patients could be addressed by either allele alone, a
vaccine comprising HLA-A*24 and HLA-A*02 epitopes can treat at
least 60% of patients in any relevant population. Specifically, the
following percentages of patients will be positive for at least one
of these alleles in various regions: USA 61%, Western Europe 62%,
China 75%, South Korea 77%, Japan 86% (calculated from
www.allelefrequencies.net).
TABLE-US-00005 TABLE 5 HLA alleles coverage in European Caucasian
population (calculated from (Gragert et al., 2013)). coverage
combined (at least one combined combined with B*07 A-allele) with
B*07 with B*44 and B*44 A*02/A*01 70% 78% 78% 84% A*02/A*03 68% 76%
76% 83% A*02/A*24 61% 71% 71% 80% A*'01/A*03 52% 64% 65% 75%
A*01/A*24 44% 58% 59% 71% A*03/A*24 40% 55% 56% 69% A*02/A*01/A*03
84% 88% 88% 91% A*02/A*01/A*24 79% 84% 84% 89% A*02/A*03/A*24 77%
82% 83% 88% A*01/A*03/A*24 63% 72% 73% 81% A*02/A*01/A*03/A*24 90%
92% 93% 95%
[0120] In a preferred embodiment, the term "nucleotide sequence"
refers to a heteropolymer of deoxyribonucleotides.
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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).
[0127] 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.
[0128] 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.
[0129] 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.
[0130] The term "promoter" means a region of DNA involved in
binding of RNA polymerase to initiate transcription.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] In accordance with the present invention, the term "percent
identity" or "percent identical", when referring to a sequence,
means that a sequence is compared to a claimed or described
sequence after alignment of the sequence to be compared (the
"Compared Sequence") with the described or claimed sequence (the
"Reference Sequence"). The percent identity is then determined
according to the following formula:
percent identity=100[1-(C/R)]
wherein C is the number of differences between the Reference
Sequence and the Compared Sequence over the length of alignment
between the Reference Sequence and the Compared Sequence,
wherein
[0136] (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
[0137] (ii) each gap in the Reference Sequence and
[0138] (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
[0139] (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.
[0140] 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.
[0141] 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: 268 or a variant thereof
which is 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 268, 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.
[0142] 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.
[0143] 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).
[0144] 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: 268. 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.
[0145] 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 268 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.
[0146] 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." 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).
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] The amino acid residues that do not substantially contribute
to interactions with the T-cell receptor can be modified by
replacement with other amino acid whose incorporation does 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-00006 TABLE 6 Variants and motif of the peptides according
to SEQ ID NO: 1, 21, 48, 250, 91, 253, 117, 125, 160, 255, 178,
183, 229, and 232. Position 1 2 3 4 5 6 7 8 9 SEQ ID No 1 R S D P V
T L D V Variant Y A E Y E A T Y T A T E Y T E A SEQ ID No 21 S T D
I G A L M Y Variant S S A S E S E A A E E A SEQ ID No 48 N L T F I
I I L I Variant V L A M V M M L M A A V A A L A A V V V V L V A T V
T T L T A Q V Q Q L Q A SEQ ID No 250 K M I L K M V Q L Variant L V
L I L L A V I 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 91 S L I D R L V L Y Variant K R F I K I I R I F M K
M M R M F V K V V R V F T K T T R T F SEQ ID No 253 K L F G T S G Q
K Variant Y R F I I Y I R I F M M Y M R M F V V Y V R V F T T Y T R
T F Position 1 2 3 4 5 6 7 8 9 10 SEQ ID No 117 N F L P P I I A R F
Variant Y I Y L Y I L Position 1 2 3 4 5 6 7 8 9 10 11 SEQ ID No
125 I Y G P K Y I H P S F Variant I L F I F L F Position 1 2 3 4 5
6 7 8 9 SEQ ID No 160 R P A P A D S A L Variant F V M A I SEQ ID No
255 L P D G S R V E L Variant F V M A I SEQ ID No 178 H A K E K L T
A L Variant V I M F R R V R I R M R F H H V H I H M H F R R V R I R
M R F R R R R V R R I R R M R R F R H R H V R H I R H M R H F L L V
L I L M L F L R L R V L R I L R M L R F L H L H V L H I L H M L H F
SEQ ID No 183 F Q K L K L L S L Variant V I M F R R V R I R M R F H
H V H I H M H F R R V R I R M R F R R R R V R R I R R M R R F
R H R H V R H I R H M R H F L L V L I L M L F L R L R V L R I L R M
L R F L H L H V L H I L H M L H F SEQ ID No 229 K E S D G F H R F
Variant W Y L D D W D Y D L Position 1 2 3 4 5 6 7 8 9 10 SEQ ID No
232 A E K D G K L T D Y Variant F W L D F D W D D L
[0152] 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.
[0153] 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-00007 TABLE 7 Combinations of the elongations of peptides
of the invention C-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1
0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0
3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or
4
[0154] 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.
[0155] 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, if they have substantially identical
antigenic activity.
[0156] In an alternative embodiment, the peptide is elongated on
either or both sides by more than 4 amino acids, preferably to a
total length of up to 30 amino acids. This may lead to MHC class II
binding peptides. Binding to MHC class II can be tested by methods
known in the art.
[0157] 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.
[0158] 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.
[0159] 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 pM, and most
preferably no more than about 10 pM. 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.
[0160] 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: 268.
[0161] "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 268 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.
[0162] 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 "i") 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, to be specifically
targeted by said antibody, or, for example, to or into an antibody
that is specific for dendritic cells as described herein.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] Briefly, modification of e.g. arginyl residues in proteins
is often based on the reaction of vicinal dicarbonyl compounds such
as phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form
an adduct. Another example is the reaction of methylglyoxal with
arginine residues. Cysteine can be modified without concomitant
modification of other nucleophilic sites such as lysine and
histidine. As a result, a large number of reagents are available
for the modification of cysteine. The websites of companies such as
Sigma-Aldrich (http://www.sigma-aldrich.com) provide information on
specific reagents.
[0170] 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.
[0171] Tetranitromethane and N-acetyl imidazole can be used for the
modification of tyrosyl residues. Cross-linking via the formation
of dityrosine can be accomplished with hydrogen peroxide/copper
ions.
[0172] 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).
[0173] 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.
[0174] A peptide or variant, wherein the peptide is modified or
includes non-peptide bonds is a preferred embodiment of the
invention.
[0175] 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: 268 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.4Cl, NH.sub.4Br, NH.sub.4NO.sub.3,
NH.sub.4CO.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.2S.sub.04, 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.
[0176] 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-trimethylbenzenesuphonyl 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 dimethyl acrylamide (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% trifluoracetic 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).
[0177] Trifluoracetic 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).
[0178] 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.
[0179] 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.
[0180] 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
colorectal cancer, glioblastoma, gastric cancer, hepatocellular
carcinoma, and renal cell carcinoma samples (N=35 CRC, 12 GBM, 10
GC, 22 HCC, and 79 RCC 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 158 colorectal
cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, and
renal cell carcinoma patients (cf. Example 1).
[0181] 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.
[0182] HLA-peptide complexes from colorectal cancer, glioblastoma,
gastric cancer, hepatocellular carcinoma, and renal cell carcinoma
tissue samples were purified and HLA-associated peptides were
isolated and analyzed by LC-MS (see example 1). All TUMAPs
contained in the present application were identified with this
approach on primary colorectal cancer, glioblastoma, gastric
cancer, hepatocellular carcinoma, and renal cell carcinoma samples
confirming their presentation on primary colorectal cancer,
glioblastoma, gastric cancer, hepatocellular carcinoma, and renal
cell carcinoma.
[0183] Besides presentation of the peptide, mRNA expression of the
underlying gene was tested. mRNA data were obtained via RNASeq
analyses of normal tissues and cancer tissues (cf. Example 2, FIGS.
1A-1P). Peptides which are derived from proteins whose coding mRNA
is highly expressed in cancer tissue, but very low or absent in
vital normal tissues, were preferably included in the present
invention.
[0184] The present invention provides peptides that are useful in
treating cancers/tumors, preferably colorectal cancer,
glioblastoma, gastric cancer, hepatocellular carcinoma, and renal
cell carcinoma 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 colorectal
cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, and
renal cell carcinoma samples.
[0185] 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 brain cells, kidney
cells, liver cells, large intestine cells, stomach cells or other
normal tissue cells, demonstrating a high degree of tumor
association of the source genes (see Example 2). Moreover, the
peptides themselves are presented on tumor tissue--"tumor tissue"
in relation to this invention shall mean a sample from a patient
suffering from colorectal cancer, glioblastoma, gastric cancer,
hepatocellular carcinoma, and renal cell carcinoma.
[0186] 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. colorectal
cancer, glioblastoma, gastric cancer, hepatocellular carcinoma, and
renal cell carcinoma cells presenting the derived peptides.
[0187] The peptides of the present invention have been shown to be
capable of stimulating T cell responses and/or are over-presented
and thus can be used for the production of antibodies and/or TCRs,
such as soluble TCRs, according to the present invention (see
Example 3, Example 4). Furthermore, the peptides when complexed
with the respective MHC can be used for the production of
antibodies and/or TCRs, in particular sTCRs, according to the
present invention, as well. Respective methods are well known to
the person of skill, and can be found in the respective literature
as well (see also below). 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.
[0188] 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 according to the invention
capable of binding to TCRs and antibodies when presented by an MHC
molecule.
[0189] The present description also relates to fragments of the
TCRs according to the invention that are capable of binding to a
peptide antigen according to the present invention when presented
by an HLA molecule. The term particularly relates to soluble TCR
fragments, for example TCRs missing the transmembrane parts and/or
constant regions, single chain TCRs, and fusions thereof to, for
example, with Ig.
[0190] 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.
[0191] 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.
[0192] 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.
[0193] 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.
[0194] 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.
[0195] 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.
[0196] 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.
[0197] 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.
[0198] 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.
[0199] With or without the introduced inter-chain bond mentioned
above, alpha/beta heterodimeric 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.
[0200] 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.
[0201] 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.
[0202] In an embodiment, a TCR comprising at least one mutation in
the TCR alpha chain and/or TCR beta chain has a binding affinity
for, and/or a binding half-life for, a 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 peptides can be enhanced by
methods well known in the art.
[0203] 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 fluorescence activated cell sorting
(FACS)-Calibur analysis.
[0204] The present description further relates to a method of
identifying and isolating a TCR according to the present
description, said method comprising obtaining a transgenic mouse
with the entire human TCR.alpha..beta. gene loci (1.1 and 0.7 Mb),
whose T-cells express a diverse human TCR repertoire that
compensates for mouse TCR deficiency, immunizing the mouse with a
peptide, 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.
[0205] 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.
[0206] In another aspect, to obtain T-cells expressing TCRs of the
present description, TCR RNAs are synthesized by techniques known
in the art, e.g., in vitro transcription systems. The in
vitro-synthesized TCR RNAs are then introduced into primary CD8+
T-cells obtained from healthy donors by electroporation to
re-express tumor specific TCR-alpha and/or TCR-beta chains.
[0207] 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.
[0208] 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).
[0209] 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.
[0210] 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).
[0211] Nucleic acids encoding TCRs of the present description may
be codon optimized to increase expression from a host cell.
Redundancy in the genetic code allows some amino acids to be
encoded by more than one codon, but certain codons are less
"op-timal" than others because of the relative availability of
matching tRNAs as well as other factors (Gustafsson et al., 2004).
Modifying the TCR-alpha and TCR-beta gene sequences such that each
amino acid is encoded by the optimal codon for mammalian gene
expression, as well as eliminating mRNA instability motifs or
cryptic splice sites, has been shown to significantly enhance
TCR-alpha and TCR-beta gene expression (Scholten et al., 2006).
[0212] 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).
[0213] To reduce mispairing, the C-terminus domain of the
introduced TCR chains of the present description may be modified in
order to promote interchain affinity, while de-creasing the ability
of the introduced chains to pair with the endogenous TCR. These
strategies may include replacing the human TCR-alpha and TCR-beta
C-terminus domains with their murine counterparts (murinized
C-terminus domain); generating a second interchain disulfide bond
in the C-terminus domain by introducing a second cysteine residue
into both the TCR-alpha and TCR-beta chains of the introduced TCR
(cysteine modification); swapping interacting residues in the
TCR-alpha and TCR-beta chain C-terminus domains ("knob-in-hole");
and fusing the variable domains of the TCR-alpha and TCR-beta
chains directly to CD3((CD3(fusion) (Schmitt et al., 2009).
[0214] 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.
[0215] 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.
[0216] 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-toluene
sulfonic 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.
[0217] In an especially preferred embodiment, the pharmaceutical
compositions comprise the peptides as salts of acetic acid
(acetates), trifluoro acetates or hydrochloric acid
(chlorides).
[0218] 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.
[0219] 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.
268, 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.
[0220] 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.
[0221] 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.
[0222] 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.
[0223] A desirable method of modifying the DNA encoding the
polypeptide of the invention employs the polymerase chain reaction
as disclosed by Saiki R K, et al. (Saiki et al., 1988). This method
may be used for introducing the DNA into a suitable vector, for
example by engineering in suitable restriction sites, or it may be
used to modify the DNA in other useful ways as is known in the art.
If viral vectors are used, pox- or adenovirus vectors are
preferred.
[0224] 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.
[0225] 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.
[0226] 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.
[0227] Alternatively, the gene for such selectable trait can be on
another vector, which is used to co-transform the desired host
cell.
[0228] 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.
[0229] 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. A typical
mammalian cell vector plasmid for constitutive expression comprises
the CMV or SV40 promoter with a suitable poly A tail and a
resistance marker, such as neomycin. One example is pSVL available
from Pharmacia, Piscataway, N.J., USA. An example of an inducible
mammalian expression vector is pMSG, also available from Pharmacia.
Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are
generally available from Stratagene Cloning Systems, La Jolla,
Calif. 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are
Yeast Integrating plasmids (YIps) and incorporate the yeast
selectable markers HIS3, TRP1, LEU2 and URA3. 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.
[0230] 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.
[0231] 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.
[0232] 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 Balbes 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.
[0233] 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.
[0234] 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.
[0235] 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.
[0236] 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).
[0237] 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.
[0238] 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).
[0239] 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.
[0240] 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, Juvlmmune.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).
[0241] 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.
[0242] 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.
[0243] 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.
[0244] 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.
[0245] 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) and anti-CD40 mAB, or combinations thereof.
[0246] 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.
[0247] 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).
[0248] 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.
[0249] "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 another 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.
[0250] Each scaffold can comprise a labeling 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.
[0251] Each scaffold can be conjugated with a second active
molecule such as for example IL-21, anti-CD3, and anti-CD28.
[0252] For further information on polypeptide scaffolds see for
example the background section of WO 2014/071978A1 and the
references cited therein.
[0253] 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.
[0254] 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.
[0255] 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.
[0256] Further, investigation of cell-binding behavior with flow
cytometry showed that the aptamers revealed very good apparent
affinities that are within the nanomolar range.
[0257] 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.
[0258] 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 268, according to the invention at hand with the MHC
molecule, using the cell-SELEX (Systematic Evolution of Ligands by
Exponential enrichment) technique.
[0259] 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.
[0260] 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 (preferably a
peptide according to the present invention), 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.
[0261] It is thus 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.
[0262] 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.
[0263] 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.
[0264] 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: 268, or a variant thereof which is at least 88%
homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 268
or a variant thereof that induces T cells cross-reacting with said
peptide, wherein said peptide is not the underlying full-length
polypeptide.
[0265] 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: 268 or a variant thereof which is at
least 88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ
ID NO: 268, 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.
[0266] 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.
[0267] 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: 268.
[0268] The present invention further relates to the peptides
according to the invention, wherein the peptide is (chemically)
modified and/or includes non-peptide bonds.
[0269] 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 (li), or wherein the
peptide is fused to (or into) an antibody, such as, for example, an
antibody that is specific for dendritic cells.
[0270] 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.
[0271] The present invention further relates to the nucleic acid
according to the invention that is DNA, cDNA, PNA, RNA or
combinations thereof.
[0272] The present invention further relates to an expression
vector capable of expressing a nucleic acid according to the
present invention.
[0273] 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
colorectal cancer, glioblastoma, gastric cancer, hepatocellular
carcinoma, and renal cell carcinoma.
[0274] 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.
[0275] 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.
[0276] 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.
[0277] 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.
[0278] 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: 268 or said variant amino
acid sequence.
[0279] 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.
[0280] 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.
[0281] 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.
[0282] 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.
[0283] The present invention further relates to a use according to
the invention, wherein said cancer cells are colorectal cancer,
glioblastoma, gastric cancer, hepatocellular carcinoma, and renal
cell carcinoma cells or other solid or hematological tumor cells
such as chronic lymphocytic leukemia, chronic myeloid leukemia and
acute myeloid leukemia, and other lymphoid neoplasms, for example,
Non-Hodgkin lymphoma, post-transplant lymphoproliferative disorders
(PTLD) as well as other myeloid neoplasms, such as primary
myelofibrosis, essential thrombocytopenia, polycythemia vera, as
well as other neoplasms such as esophageal cancer, non-small cell
lung cancer, small cell lung cancer, pancreatic cancer, prostate
cancer, melanoma, breast cancer, gallbladder cancer and
cholangiocarcinoma, urinary bladder cancer, uterine cancer, head
and neck squamous cell carcinoma, mesothelioma.
[0284] 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 colorectal cancer, glioblastoma,
gastric cancer, hepatocellular carcinoma, and renal cell carcinoma.
The present invention also relates to the use of these novel
targets for cancer treatment.
[0285] 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
colorectal cancer, glioblastoma, gastric cancer, hepatocellular
carcinoma, and renal cell carcinoma marker (poly)peptide, delivery
of a toxin to a colorectal cancer, glioblastoma, gastric cancer,
hepatocellular carcinoma, and renal cell carcinoma cell expressing
a cancer marker gene at an increased level, and/or inhibiting the
activity of a colorectal cancer, glioblastoma, gastric cancer,
hepatocellular carcinoma, and renal cell carcinoma marker
polypeptide) according to the invention.
[0286] Whenever possible, the antibodies of the invention may be
purchased from commercial sources. The antibodies of the invention
may also be generated using well-known methods. The skilled artisan
will understand that either full length colorectal cancer,
glioblastoma, gastric cancer, hepatocellular carcinoma, and renal
cell carcinoma 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.
[0287] 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: 268 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 colorectal cancer, glioblastoma, gastric cancer,
hepatocellular carcinoma, and renal cell carcinoma marker
polypeptide used to generate the antibody according to the
invention.
[0288] 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.
[0289] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a substantially homogeneous population of
antibodies, i.e.; the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. The monoclonal
antibodies herein specifically include "chimeric" antibodies in
which a portion of the heavy and/or light chain is identical with
or homologous to corresponding sequences in antibodies derived from
a particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is identical with or
homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, so long as they exhibit
the desired antagonistic activity (U.S. Pat. No. 4,816,567, which
is hereby incorporated in its entirety).
[0290] 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.
[0291] 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).
[0292] 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.
[0293] 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.
[0294] 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.
[0295] 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.
[0296] 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.
[0297] 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.
[0298] 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.
[0299] 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
colorectal cancer, glioblastoma, gastric cancer, hepatocellular
carcinoma, and renal cell carcinoma, 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.
[0300] It is a further aspect of the invention to provide a method
for producing a soluble T-cell receptor (sTCR) recognizing a
specific peptide-MHC complex. Such soluble T-cell receptors can be
generated from specific T-cell clones, and their affinity can be
increased by mutagenesis targeting the complementarity-determining
regions. For the purpose of T-cell receptor selection, phage
display can be used (US 2010/0113300, (Liddy et al., 2012)). For
the purpose of stabilization of T-cell receptors during phage
display and in case of practical use as drug, alpha and beta chain
can be linked e.g. by non-native disulfide bonds, other covalent
bonds (single-chain T-cell receptor), or by dimerization domains
(Boulter et al., 2003; Card et al., 2004; Willcox et al., 1999).
The T-cell receptor can be linked to toxins, drugs, cytokines (see,
for example, US 2013/0115191), and domains recruiting effector
cells such as an anti-CD3 domain, etc., in order to execute
particular functions on target cells. Moreover, it could be
expressed in T cells used for adoptive transfer. Further
information can be found in WO 2004/033685A1 and WO 2004/074322A1.
A combination of sTCRs is described in WO 2012/056407A1. Further
methods for the production are disclosed in WO 2013/057586A1.
[0301] 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.
[0302] 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.
[0303] Antibodies for diagnostic use may be labeled with probes
suitable for detection by various imaging methods. Methods for
detection of probes include, but are not limited to, fluorescence,
light, confocal and electron microscopy; magnetic resonance imaging
and spectroscopy; fluoroscopy, computed tomography and positron
emission tomography. Suitable probes include, but are not limited
to, fluorescein, rhodamine, eosin and other fluorophores,
radioisotopes, gold, gadolinium and other lanthanides, paramagnetic
iron, fluorine-18 and other positron-emitting radionuclides.
Additionally, probes may be bi- or multi-functional and be
detectable by more than one of the methods listed. These antibodies
may be directly or indirectly labeled with said probes. Attachment
of probes to the antibodies includes covalent attachment of the
probe, incorporation of the probe into the antibody, and the
covalent attachment of a chelating compound for binding of probe,
amongst others well recognized in the art. For
immunohistochemistry, the disease tissue sample may be fresh or
frozen or may be embedded in paraffin and fixed with a preservative
such as formalin. The fixed or embedded section contains the sample
are contacted with a labeled primary antibody and secondary
antibody, wherein the antibody is used to detect the expression of
the proteins in situ.
[0304] 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.
[0305] 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.
[0306] 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).
[0307] 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.
[0308] In case of a MHC class I epitope being used as an antigen,
the T cells are CD8-positive T cells.
[0309] 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: 268, or a variant amino acid sequence thereof.
[0310] 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.
[0311] Allogeneic cells may also be used in the preparation of T
cells and a method is described in detail in WO 97/26328,
incorporated herein by reference. For example, in addition to
Drosophila cells and T2 cells, other cells may be used to present
antigens such as CHO cells, baculovirus-infected insect cells,
bacteria, yeast, and vaccinia-infected target cells. In addition,
plant viruses may be used (see, for example, Porta et al. (Porta et
al., 1994) which describes the development of cowpea mosaic virus
as a high-yielding system for the presentation of foreign
peptides.
[0312] 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.
[0313] 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 268.
[0314] 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.
[0315] 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)).
[0316] 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.
[0317] 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.
[0318] T cells may be obtained by methods known in the art, e.g.
those described above.
[0319] 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).
[0320] 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.
[0321] 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).
[0322] The present invention is further directed at a kit
comprising:
[0323] (a) a container containing a pharmaceutical composition as
described above, in solution or in lyophilized form;
[0324] (b) optionally a second container containing a diluent or
reconstituting solution for the lyophilized formulation; and
[0325] (c) optionally, instructions for (i) use of the solution or
(ii) reconstitution and/or use of the lyophilized formulation.
[0326] 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.
[0327] 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.
[0328] 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).
[0329] 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.
[0330] 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.
[0331] 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.
[0332] 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.
[0333] 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.
[0334] Since the peptides of the invention were isolated from
colorectal cancer, glioblastoma, gastric cancer, hepatocellular
carcinoma, and renal cell carcinoma, the medicament of the
invention is preferably used to treat colorectal cancer,
glioblastoma, gastric cancer, hepatocellular carcinoma, and renal
cell carcinoma.
[0335] 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.
[0336] 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.
[0337] 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 colorectal cancer,
glioblastoma, gastric cancer, hepatocellular carcinoma, and renal
cell carcinoma 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 colorectal cancer, glioblastoma, gastric
cancer, hepatocellular carcinoma, and renal cell carcinoma tissues,
the warehouse may contain HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*24,
HLA-B*07, HLA-B*08 and HLA-B*44 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.
[0338] 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.
[0339] 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, colorectal cancer, glioblastoma, gastric
cancer, hepatocellular carcinoma, and renal cell carcinoma samples
from patients and blood from healthy donors were analyzed in a
stepwise approach:
[0340] 1. HLA ligands from the malignant material were identified
by mass spectrometry
[0341] 2. Genome-wide messenger ribonucleic acid (mRNA) expression
analysis was used to identify genes over-expressed in the malignant
tissue (colorectal cancer, glioblastoma, gastric cancer,
hepatocellular carcinoma, and renal cell carcinoma) compared with a
range of normal organs and tissues
[0342] 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.
[0343] 4. Literature research was performed in order to identify
additional evidence supporting the relevance of the identified
peptides as TUMAPs
[0344] 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.
[0345] 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 colorectal cancer, glioblastoma,
gastric cancer, hepatocellular carcinoma, and renal cell carcinoma
patients.
[0346] 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.
[0347] 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 multi-target 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.
[0348] 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.
[0349] 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.
[0350] Preferably, the peptides included in the vaccine are
identified by a method comprising: (a) identifying tumor-associated
peptides (TUMAPs) presented by a tumor sample from the individual
patient; (b) comparing the peptides identified in (a) with a
warehouse (database) of peptides as described above; and (c)
selecting at least one peptide from the warehouse (database) that
correlates with a tumor-associated peptide identified in the
patient. For example, the TUMAPs presented by the tumor sample are
identified by: (a1) comparing expression data from the tumor sample
to expression data from a sample of normal tissue corresponding to
the tissue type of the tumor sample to identify proteins that are
over-expressed or aberrantly expressed in the tumor sample; and
(a2) correlating the expression data with sequences of MHC ligands
bound to MHC class I and/or class II molecules in the tumor sample
to identify MHC ligands derived from proteins over-expressed or
aberrantly expressed by the tumor. Preferably, the sequences of MHC
ligands are identified by eluting bound peptides from MHC molecules
isolated from the tumor sample, and sequencing the eluted ligands.
Preferably, the tumor sample and the normal tissue are obtained
from the same patient.
[0351] In addition to, or as an alternative to, selecting peptides
using a warehousing (database) model, TUMAPs may be identified in
the patient de novo, and then included in the vaccine. As one
example, candidate TUMAPs may be identified in the patient by (a1)
comparing expression data from the tumor sample to expression data
from a sample of normal tissue corresponding to the tissue type of
the tumor sample to identify proteins that are over-expressed or
aberrantly expressed in the tumor sample; and (a2) correlating the
expression data with sequences of MHC ligands bound to MHC class I
and/or class II molecules in the tumor sample to identify MHC
ligands derived from proteins over-expressed or aberrantly
expressed by the tumor. As another example, proteins may be
identified containing mutations that are unique to the tumor sample
relative to normal corresponding tissue from the individual
patient, and TUMAPs can be identified that specifically target the
mutation. For example, the genome of the tumor and of corresponding
normal tissue can be sequenced by whole genome sequencing: For
discovery of non-synonymous mutations in the protein-coding regions
of genes, genomic DNA and RNA are extracted from tumor tissues and
normal non-mutated genomic germline DNA is extracted from
peripheral blood mononuclear cells (PBMCs). The applied NGS
approach is confined to the re-sequencing of protein coding regions
(exome re-sequencing). For this purpose, exonic DNA from human
samples is captured using vendor-supplied target enrichment kits,
followed by sequencing with e.g. a HiSeq2000 (Illumina).
Additionally, tumor mRNA is sequenced for direct quantification of
gene expression and validation that mutated genes are expressed in
the patients' tumors. The resultant millions of sequence reads are
processed through software algorithms. The output list contains
mutations and gene expression. Tumor-specific somatic mutations are
determined by comparison with the PBMC-derived germline variations
and prioritized. The de novo identified peptides can then be tested
for immunogenicity as described above for the warehouse, and
candidate TUMAPs possessing suitable immunogenicity are selected
for inclusion in the vaccine.
[0352] 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 over presentation 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.
[0353] 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.
[0354] 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.
[0355] 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.
[0356] 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.
[0357] 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 colorectal cancer,
glioblastoma, gastric cancer, hepatocellular carcinoma, and renal
cell carcinoma cells 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.
[0358] 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 colorectal cancer, glioblastoma,
gastric cancer, hepatocellular carcinoma, and renal cell carcinoma.
Presence of groups of peptides can enable classification or
sub-classification of diseased tissues.
[0359] 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.
[0360] 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.
[0361] 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.
EXAMPLES
Example 1
[0362] Identification of Tumor Associated Peptides Presented on the
Cell Surface
[0363] Tissue Samples
[0364] Patients' tumor tissues and normal tissues were obtained
from the University Hospital Tubingen (Tubingen, Germany). 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. Sample
numbers for TUMAP selection were: for renal cancer N=79, for
colorectal cancer N=35, for hepatocellular carcinoma N=22, for
gastric cancer N=10, and for glioblastoma N=12.
[0365] Isolation of HLA Peptides from Tissue Samples
[0366] HLA peptide pools from shock-frozen tissue samples were
obtained by immune precipitation from solid tissues according to a
slightly modified protocol (Falk et al., 1991; Seeger et al., 1999)
using the HLA-A*02-specific antibody BB7.2, the HLA-A, --B,
C-specific antibody W6/32, the HLA-DR specific antibody L243 and
the pan-HLA class II specific antibody T039, CNBr-activated
sepharose, acid treatment, and ultrafiltration.
[0367] Mass Spectrometry Analyses
[0368] The HLA peptide pools as obtained were separated according
to their hydrophobicity by reversed-phase chromatography (Ultimate
3000 RSLC Nano UHPLC System, Dionex) and the eluting peptides were
analyzed in LTQ-Orbitrap and Fusion Lumos hybrid mass spectrometers
(ThermoElectron) equipped with an ESI source. Peptide samples were
loaded with 3% of solvent B (20% H.sub.2O, 80% acetonitrile and
0.04% formic acid) on a 2 cm PepMap 100 C18 Nanotrap column
(Dionex) at a flowrate of 4 .mu.l/min for 10 min. Separation was
performed on either 25 cm or 50 cm PepMap C18 columns with a
particle size of 2 .mu.m (Dionex) mounted in a column oven running
at 50.degree. C. The applied gradient ranged from 3 to 32% solvent
B within 90 min at a flow rate of 300 nl/min (for 25 cm columns) or
140 min at a flow rate of 175 nl/min (for 50 cm columns). (Solvent
A: 99% H.sub.2O, 1% ACN and 0.1% formic acid; Solvent B: 20%
H.sub.2O, 80% ACN and 0.1% formic acid).
[0369] Mass spectrometry analysis was performed in data dependent
acquisition mode employing a top five method (i.e. during each
survey scan the five most abundant precursor ions were selected for
fragmentation). Alternatively, a TopSpeed method was employed for
analysis on Fusion Lumos instruments.
[0370] Survey scans were recorded in the Orbitrap at a resolution
of 60,000 (for Orbitrap XL) or 120,000 (for Orbitrap Fusion Lumos).
MS/MS analysis was performed by collision induced dissociation
(CID, normalized collision energy 35%, activation time 30 ms,
isolation width 1.3 m/z) with subsequent analysis in the linear
trap quadrupole (LTQ). Mass range for HLA class I ligands was
limited to 400-650 m/z with possible charge states 2+ and 3+
selected for fragmentation. For HLA class II mass range was set to
300-1500 m/z allowing for fragmentation with all positive charge
states .gtoreq.2.
[0371] Tandem mass spectra were interpreted by MASCOT or SEQUEST at
a fixed false discovery rate (q.ltoreq.0.05) and additional manual
control. In cases where the identified peptide sequence was
uncertain it was additionally validated by comparison of the
generated natural peptide fragmentation pattern with the
fragmentation pattern of a synthetic sequence-identical reference
peptide.
[0372] Table 8a and 8b 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 colorectal
cancer (Table 8a) and for GBC and GC (Table 8b), peptide SEQ ID No.
50 for colorectal cancer and hepatocellular carcinoma (Table 8a)
and for CCC, GBM, HNSCC, NHL, NSCLCsquam, PACA, SCLC and UBC (Table
8b)).
TABLE-US-00008 TABLE 8a Overview of presentation of selected tumor-
associated peptides of the present invention across exemplary and
preferred entities (diseases). Peptide Presentation SEQ ID on
cancer No. Sequence entities 1 RSDPVTLDV CRC 2 LPSPTDSNFY RCC 3
ASSTDSASYY HCC 4 NSDLKYNAL HCC 5 SILGSDVRVPSY HCC 6 VLDLSTNVY HCC 7
LITGDPKAAYDY CRC 8 TPVTEFSLNTY CRC 9 FITAQNHGY HCC 10 ITAQNHGY HCC
11 LSAGSGPGQY HCC 12 ITFGERFEY RCC 13 GSTMVEHNY RCC 14 YTERDGSAMVY
GBM 15 LTDYLKNTY HCC 16 LSLIDRLVLY RCC 17 YTDKLQHY CRC 18
EVSNGKWLLY RCC 19 VSNGKWLLY RCC 20 STDEITTRY HCC 21 STDIGALMY CRC
22 TLEQVQLYY CRC 23 TASEDVFQY CRC 24 YTHHLFIFY CRC 25 LMKEVMEHY RCC
26 EVLDSHIHAY GBM 27 LDSHIHAY GBM 28 LTDYINANY GBM 29 SVTDLEMPHY
GBM 30 VLDSHIHAY GBM 31 VTDLEMPHY GBM 32 ATVGYFIFY RCC 33 FADKIHLAY
RCC 34 ITDFNNIRY CRC 35 FASDLLHLY HCC 36 YAAYIIHAY RCC 37 LTDSFPLKV
HCC 38 VMLNSNVLL GBM 39 YLLPSVVLL GBM 40 KIDDIWNLEV HCC 41
SLQDTKITL HCC 42 KMMALVAEL RCC 43 GLMTIVTSL CRC 44 SQTGFVVLV GBM 45
KLLDEVTYL RCC 46 VLITGLPLI RCC 47 YQDSWFQQL RCC 48 NLTFIIILI HCC 49
NLASRPYSL HCC 50 ELMPRVYTL CRC, HCC 51 ALAAELNQL GBM 52 YVSSGEMMV
GBM 53 LLMTSLTES GBM 54 YLPPTDPRMSV GBM 55 RLWQIQHHL RCC 56
FLNQIYTQL GC 57 GLTGVIMTI CRC 58 MLCLLLTL RCC 59 KLHEIYIQA GBM 60
GLPDFVKEL GC 61 RLFGLFLNNV RCC 62 GSYSALLAKK HCC 63 KVLGPNGLLK HCC
64 STTKLYLAK HCC 65 VLGPNGLLK HCC 66 ATYEGIQKK HCC 67 ATALSLSNK HCC
68 ATAYGSTVSK HCC 69 ATAYGSTVSKR HCC 70 ATWSASLKNK HCC 71 KLGNNPVSK
HCC 72 KQVFPGLNY HCC 73 KSFDRHFEK HCC 74 QLYSKFLLK HCC 75 QVPTFTIPK
HCC 76 SAFGYVFPK HCC 77 SSASLAHMK HCC 78 STKSTSPPK HCC 79 STNNEGNLK
HCC 80 STSHHLVSR HCC 81 SVKLQGTSK HCC 82 TAYGSTVSK HCC 83
TAYGSTVSKR HCC 84 TVASLHTEK HCC 85 KMAAWPFSR HCC 86 KTPSGALHRK HCC
87 SSYSRSSAVK GBM 88 MLLQQPLIY RCC 89 KITDFGLAK RCC 90 GSRLGKYYVK
RCC 91 SLIDRLVLY RCC 92 AVLDLGSLLAK RCC 93 ALDKPGKSK GBM 94
KTYVGHPVKM RCC 95 RLFESSFHY RCC 96 FSLAGALNAGFK GBM 97 RMPPPLPTR
GBM 98 KLYPTYSTK HCC 99 ATMQSKLIQK RCC 100 ALLGVIIAK RCC 101
GVIIAKKYFFK RCC 102 IIAKKYFFK RCC 103 KSWTASSSY RCC 104 STQDTLLIK
CRC 105 GSAALYLLR RCC 106 RLSPNDQYK RCC 107 EIYGGHHAGF GBM 108
LLKSSVGNFY GBM 109 KIIAPLVTR RCC 110 GTESGTILK RCC 111 KIKEHVRSK
HCC 112 KMMADYGIRK HCC 113 VWAKILSAF HCC 114 KFLDSNIKF HCC 115
YFEEAANFL HCC 116 LVLDYSKDYNHW HCC 117 NFLPPIIARF RCC 118 TYISKTIAL
RCC 119 YMKALGVGF RCC 120 MYAKEFDLL HCC
121 SYIEKVRFL GBM 122 KLYGMPTDFGF GC 123 RQYLAINQI HCC 124
EVYSPEADQW GBM 125 IYGPKYIHPSF GC 126 TFQDKTLNF CRC 127 IFINLSPEF
GC 128 SYTKVEARL GC 129 VFLNQIYTQL GC 130 VYGDGHYLTF GC 131
KQLDHNLTF CRC 132 VYNPVIYVF HCC 133 SFDSNLLSF CRC 134 TYLTGRQF CRC
135 VIAPIISNF CRC 136 EYNNIQHLF RCC 137 KYLSLSNSF RCC 138 KYLSIPTVF
HCC 139 PYASLASELF HCC 140 KYLSIPAVF HCC 141 KYLSIPAVFF HCC 142
SSFPGAGNTW GBM 143 FELPTGAGLQL HCC 144 IPEPSAQQL HCC 145 RVPSYTLIL
HCC 146 SPGDKRLAA HCC 147 SPIKVPLLL HCC 148 VPDGVSKVL HCC 149
YPLTGDTRL HCC 150 KPSSKALGTSL CRC 151 VVHPRTLLL RCC 152 IPSRLLAIL
RCC 153 APAAVPSAPA GBM 154 GPGTRLSL GBM 155 FPYPYAERL CRC 156
HPQVVILSL RCC 157 SPSPGKDPTL RCC 158 VPERGEPEL RCC 159 FPAHPSLLL
RCC 160 RPAPADSAL RCC 161 NPYEGRVEV RCC 162 MPMISIPRV RCC 163
RPASSLRP CRC, HCC 164 ISTPSEVSTPL GC 165 TPIAKVSEL CRC 166
HDPDVGSNSL GBM 167 YPSEVEHMF RCC 168 IPTDKLLVI RCC 169 FPTEVTPHAF
GBM 170 SPMWHVQQL RCC 171 APKLFAVAF RCC 172 KPAHYPLIAL RCC 173
MVPSAGQLALF RCC 174 VPSLQRLML RCC 175 HPIETLVDIF RCC 176 AAMSRYEL
HCC 177 DLKYNALDL HCC 178 HAKEKLTAL HCC 179 IQIYKKLRTSSF HCC 180
LLKAEPLAF HCC 181 YKKLRTSSF HCC 182 LPFLRENDL GBM 183 FQKLKLLSL CRC
184 EPVKKSRL RCC 185 NPNLKTLL GBM 186 SLIDRLVL RCC 187 YVKERSKAM
RCC 188 SALDHVTRL RCC 189 HIFLRTTL HCC 190 SRSMRLLLL GC 191
LINLKYLSL RCC 192 LPMLKVLNL RCC 193 LSYNKYLQL RCC 194 EAKRHLLQV HCC
195 AEAVLKTLQEL HCC 196 AEQTGTWKL HCC 197 EEAKQVLFL HCC 198
FELPTGAGL HCC 199 GEATLQRIY HCC 200 GEELGFASL HCC 201 GEHTSKATL HCC
202 KEFNLQNMGL HCC 203 KENFAGEATL HCC 204 KESQLPTVM HCC 205
QEVLLQTFL HCC 206 SEPINIIDAL HCC 207 TEATMTFKY HCC 208 AEHDAVRNAL
CRC 209 YEVDTVLRY GBM 210 SENIVIQVY HCC 211 TEKEMIQKL RCC 212
AEETCAPSV RCC 213 TTMDQKSLW GBM 214 AEQPDGLIL HCC 215 AFITAQNHGY
HCC 216 LQEEKVPAIY HCC 217 NEINEKIAPSF HCC 218 AEGGKVPIKW GBM, RCC
219 AENAEYLRV GBM 220 KEITGFLLI GBM 221 AEERAEAKKKF RCC 222
NEISTFHNL HCC 223 SEVPVARVW HCC 224 SESAVFHGF RCC 225 SEAFPSRAL RCC
226 EELLHGQLF GC 227 TEHTQSQAAW RCC 228 AEKQTPDGRKY GBM 229
KESDGFHRF RCC 230 AENLFRAFL CRC 231 AEIHTAEI RCC 232 AEKDGKLTDY GBM
233 DESEKTTKSF GBM 234 EEESLLTSF GBM 235 EEFETLKEF GBM 236
EEKLIIQDF GBM 237 LEMPHYSTF GBM 238 SENPETITY GBM 239 TEVLDSHIHAY
GBM 240 HELENHSMY GBM 241 REAEPIPKM GBM 242 FSDKELAAY HCC 243
RSPNNFLSY RCC 244 RSDPVTLNV CRC 245 ITEKNSGLY CRC
246 YSDLHAFYY GBM 247 RSDPGGGGLAY GBM 248 YSHAAGQGTGLY CRC 249
ALFPERITV GBM 250 KMILKMVQL RCC 251 RLASRPLLL GBM 252 RIYNGIGVSR
RCC 253 KLFGTSGQK GBM 254 AVATKFVNK GBM 255 LPDGSRVEL CRC 256
LPALPQQLI CRC 257 SPLRGGSSL CRC 258 APSGTRVVQVL GBM 259 RPAVGHSGL
HCC 260 EEAPLVTKAF HCC 261 IEALLESSL RCC 262 MELLLVNKL RCC 263
QQATPGPAY CRC 264 DEYLIPQQGF RCC 265 EEVDVPIKLY CRC 266 ARLTPIPFGL
HCC GBM = glioblastoma, CRC = colorectal cancer, RCC = renal cell
carcinoma, HCC = hepatocellular carcinoma, GC = gastric cancer.
TABLE-US-00009 TABLE 8b Overview of presentation of selected
tumor-associated peptides of the present invention across entities
(diseases). SEQ ID No. Sequence Peptide Presentation on tumor types
1 RSDPVTLDV GBC, GC 2 LPSPTDSNFY CCC, OSCAR 5 SILGSDVRVPSY
NSCLCadeno, PACA 6 VLDLSTNVY CCC, GBC 8 TPVTEFSLNTY BRCA, CCC, GBC,
GC, HNSCC, MEL, NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA, RCC, SCLC,
UBC, UEC 9 FITAQNHGY GC 10 ITAQNHGY GC 12 ITFGERFEY CRC, GC, HCC,
HNSCC, MEL, OSCAR, PACA, PRCA, UBC 13 GSTMVEHNY GBC, PRCA 14
YTERDGSAMVY BRCA, SCLC, UEC 15 LTDYLKNTY GBC, GC, NSCLCadeno,
NSCLCsquam, PRCA, RCC, UEC 16 LSLIDRLVLY HCC, NSCLCsquam, UEC 17
YTDKLQHY BRCA, HNSCC, NSCLCadeno, SCLC 21 STDIGALMY BRCA, GBC, GC,
GEJC, HNSCC, MEL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR,
PACA, RCC, SCLC, UBC 22 TLEQVQLYY GC 25 LMKEVMEHY MEL, NSCLCadeno,
NSCLCsquam, OC, SCLC 26 EVLDSHIHAY BRCA, CRC, GC, HNSCC, MEL, NHL,
NSCLCsquam, OSCAR, PACA, PRCA, SCLC, UBC 27 LDSHIHAY MEL 28
LTDYINANY GC, MEL, NSCLCsquam, OSCAR 29 SVTDLEMPHY NSCLCsquam,
OSCAR 30 VLDSHIHAY GC, HNSCC, MEL, NSCLCsquam, OSCAR 31 VTDLEMPHY
GC, HNSCC, MEL, NSCLCsquam, OSCAR 33 FADKIHLAY GBC, GC, OSCAR 34
ITDFNNIRY BRCA, GC, GEJC, HNSCC, MEL, NHL, OSCAR 36 YAAYIIHAY
HNSCC, NSCLCadeno, NSCLCsquam, OC, OSCAR, UEC 37 LTDSFPLKV CCC 39
YLLPSVVLL AML, BRCA, CCC, CLL, CRC, GBC, GC, GEJC, HCC, HNSCC, MEL,
NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA,
RCC, SCLC, UBC, UEC 42 KMMALVAEL OC 44 SQTGFVVLV AML, BRCA, CCC,
CLL, CRC, GBC, GC, GEJC, HCC, HNSCC, MEL, NHL, NSCLCadeno,
NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC
45 KLLDEVTYL BRCA, CCC, CRC, GBC, GBM, GC, GEJC, HCC, HNSCC, MEL,
NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA,
SCLC, UBC, UEC 46 VLITGLPLI BRCA, CCC, CRC, HCC, HNSCC, NSCLCsquam,
UBC 47 YQDSWFQQL HCC, UBC 50 ELMPRVYTL CCC, GBM, HNSCC, NHL,
NSCLCsquam, PACA, SCLC, UBC 51 ALAAELNQL NHL 53 LLMTSLTES AML,
BRCA, CLL, CRC, GBC, GC, HCC, HNSCC, NHL, NSCLCadeno, NSCLCother,
NSCLCsquam, OC, PACA, PRCA, SCLC, UBC, UEC 56 FLNQIYTQL CRC, GEJC,
PACA 60 GLPDFVKEL GBC, MEL, OSCAR 67 ATALSLSNK CCC 68 ATAYGSTVSK
CCC, CRC, GBC, PACA 70 ATWSASLKNK CCC 72 KQVFPGLNY CCC, CRC, RCC,
SCLC 74 QLYSKFLLK CCC, OSCAR, RCC, SCLC 75 QVPTFTIPK AML, CCC,
OSCAR, SCLC 76 SAFGYVFPK AML, BRCA, CCC, CRC, GBC, GC, MEL,
NSCLCother, OSCAR, PACA, PRCA, RCC, SCLC, UBC 82 TAYGSTVSK CCC, NHL
85 KMAAWPFSR BRCA, GBC, NSCLCadeno, NSCLCsquam, OC 89 KITDFGLAK GBM
90 GSRLGKYYVK GBM, HCC, HNSCC, NSCLCadeno, NSCLCother, NSCLCsquam,
OC, PACA, SCLC, UBC, UEC 91 SLIDRLVLY GBC, NSCLCadeno, NSCLCsquam,
PACA, SCLC 92 AVLDLGSLLAK BRCA, CCC, CRC, GBC, GBM, GC, HCC, NHL,
NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, SCLC,
UBC, UEC 94 KTYVGHPVKM CRC, GBC, HNSCC, MEL, NSCLCadeno, PACA 98
KLYPTYSTK OSCAR, SCLC 99 ATMQSKLIQK SCLC 100 ALLGVIIAK CCC, CRC,
NSCLCadeno, OC, UBC, UEC 103 KSWTASSSY CRC 104 STQDTLLIK BRCA, GBC,
GC, HNSCC, NHL, NSCLCsquam, OC, OSCAR, PACA, RCC, SCLC, UBC, UEC
105 GSAALYLLR NSCLCsquam, UBC, UEC 107 EIYGGHHAGF SCLC 109
KIIAPLVTR HCC, NSCLCadeno, OC, UEC 112 KMMADYGIRK BRCA, CCC, CRC,
GBC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,
RCC, UEC 113 VWAKILSAF CLL, GBC, NHL 115 YFEEAANFL CCC, GBC 117
NFLPPIIARF CRC, GBC, GBM, HNSCC, MEL, NSCLCadeno, OC, OSCAR, UEC
118 TYISKTIAL BRCA, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL,
NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, SCLC,
UBC, UEC 119 YMKALGVGF GBM 120 MYAKEFDLL BRCA, CLL, CRC, GBC, GC,
HNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PACA,
PRCA, RCC, UEC 121 SYIEKVRFL BRCA, NHL, SCLC 122 KLYGMPTDFGF BRCA,
CRC, HNSCC, NHL, NSCLCsquam, OC, OSCAR, PACA, PRCA, SCLC, UBC 123
RQYLAINQI BRCA, CCC, CLL, CRC, GBC, GBM, GC, HNSCC, MEL, NHL,
NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC,
SCLC, UBC 124 EVYSPEADQW OSCAR 125 IYGPKYIHPSF GBC, NSCLCadeno 126
TFQDKTLNF GC, MEL, OSCAR, PRCA, SCLC, UBC, UEC 127 IFINLSPEF CCC,
GBC, NSCLCadeno, PACA 128 SYTKVEARL CCC, CRC, GBC, NSCLCadeno, PACA
129 VFLNQIYTQL GBC, PACA 130 VYGDGHYLTF NSCLCadeno, PACA 132
VYNPVIYVF AML, BRCA, CCC, CLL, CRC, GBC, GBM, GC, HNSCC, MEL, NHL,
NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC,
SCLC, UBC, UEC 134 TYLTGRQF GC, OSCAR, PRCA, SCLC, UEC 135
VIAPIISNF AML, BRCA, CCC, CLL, GBC, GBM, GC, HCC, MEL, NHL,
NSCLCadeno, NSCLCother, NSCLCsquam, OC, PACA, PRCA, RCC, SCLC, UEC
136 EYNNIQHLF GBC, GBM, GC, HCC, NSCLCadeno, NSCLCother,
NSCLCsquam, OC, PRCA, UBC, UEC 137 KYLSLSNSF HCC, OC, PACA, PRCA
138 KYLSIPTVF NSCLCadeno 139 PYASLASELF GC 140 KYLSIPAVF CCC, GBC,
GC, HNSCC 141 KYLSIPAVFF GBC, GC, HNSCC, SCLC 142 SSFPGAGNTW OSCAR,
SCLC 144 IPEPSAQQL CCC, CRC, GBC, NHL 145 RVPSYTLIL NSCLCsquam,
PRCA 147 SPIKVPLLL GBC, GC, OSCAR, PACA, RCC 148 VPDGVSKVL CCC 150
KPSSKALGTSL GBC 151 VVHPRTLLL CCC, HCC, UEC 155 FPYPYAERL GC,
HNSCC, NSCLCsquam, OC, OSCAR, PACA 156 HPQVVILSL GBC 157 SPSPGKDPTL
NSCLCadeno, PRCA 159 FPAHPSLLL NSCLCadeno 160 RPAPADSAL GBC,
NSCLCadeno 161 NPYEGRVEV CRC, GBC, GBM, GC, MEL 162 MPMISIPRV GBM,
NHL 163 RPASSLRP OC 165 TPIAKVSEL HCC, OC, UEC 166 HDPDVGSNSL BRCA,
CRC, GBC, GC, HCC, HNSCC, NHL, NSCLCadeno, NSCLCother, NSCLCsquam,
OC, OSCAR, PACA, PRCA, SCLC, UBC, UEC
167 YPSEVEHMF MEL, NSCLCadeno, NSCLCother, NSCLCsquam, PRCA, UEC
168 IPTDKLLVI BRCA, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL,
NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, SCLC, UBC, UEC
169 FPTEVTPHAF HNSCC, MEL, NSCLCsquam, SCLC 171 APKLFAVAF
NSCLCadeno 173 MVPSAGQLALF GC, HNSCC, OSCAR, SCLC 174 VPSLQRLML
CRC, NSCLCadeno 175 HPIETLVDIF CCC, CRC, GBC, GBM, GC, MEL,
NSCLCadeno, NSCLCsquam, OC, SCLC, UBC, UEC 177 DLKYNALDL CCC 178
HAKEKLTAL CCC 180 LLKAEPLAF CCC, GBC, RCC, SCLC 182 LPFLRENDL PRCA,
UEC 183 FQKLKLLSL GBC, PACA 188 SALDHVTRL NHL, PRCA 190 SRSMRLLLL
CRC 192 LPMLKVLNL HNSCC, NSCLCadeno, NSCLCother, NSCLCsquam, OC 193
LSYNKYLQL HCC, UEC 194 EAKRHLLQV CCC, MEL, NHL, NSCLCother,
NSCLCsquam, OC, OSCAR, PACA, SCLC 195 AEAVLKTLQEL CCC 196 AEQTGTWKL
CCC 197 EEAKQVLFL RCC 198 FELPTGAGL CCC, HNSCC, RCC, SCLC 199
GEATLQRIY CCC, CRC 200 GEELGFASL CCC, RCC, SCLC 206 SEPINIIDAL CCC,
RCC, SCLC 207 TEATMTFKY CCC, GBC 208 AEHDAVRNAL GC, HNSCC 209
YEVDTVLRY BRCA, MEL 210 SENIVIQVY CCC, MEL, NSCLCother 212
AEETCAPSV BRCA, MEL, UEC 214 AEQPDGLIL CCC, SCLC 218 AEGGKVPIKW
HNSCC, NSCLCadeno, PRCA, UEC 219 AENAEYLRV RCC 220 KEITGFLLI
NSCLCadeno, UEC 223 SEVPVARVW CCC, CRC, GBC, NSCLCadeno, OSCAR, RCC
224 SESAVFHGF BRCA, CCC, CRC, GBC, GBM, GC, HNSCC, MEL, NHL,
NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA, PRCA, SCLC, UBC, UEC 226
EELLHGQLF CCC, CRC, NSCLCadeno, OSCAR, PACA 227 TEHTQSQAAW CLL,
HCC, HNSCC, NSCLCadeno, UEC 229 KESDGFHRF BRCA, CCC, CRC, GBC, GBM,
GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,
OSCAR, PACA, SCLC, UBC, UEC 230 AENLFRAFL AML, CCC, CLL, GBC, GC,
HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA,
PRCA, RCC, UBC, UEC 232 AEKDGKLTDY MEL 234 EEESLLTSF NSCLCsquam,
OSCAR 235 EEFETLKEF HNSCC, MEL 236 EEKLIIQDF HNSCC, MEL,
NSCLCadeno, NSCLCsquam, OSCAR, PRCA 238 SENPETITY HNSCC 239
TEVLDSHIHAY MEL, NSCLCsquam, OSCAR 240 HELENHSMY OSCAR 242
FSDKELAAY BRCA, CCC, GBC, GBM, GC, MEL, NHL, NSCLCadeno,
NSCLCsquam, OSCAR, PACA 243 RSPNNFLSY GBC 244 RSDPVTLNV BRCA, GBC,
GC, NSCLCadeno, PACA 245 ITEKNSGLY GBC, GC 246 YSDLHAFYY AML, BRCA,
CLL, CRC, GBC, GC, GEJC, HCC, HNSCC, MEL, NHL, NSCLCadeno,
NSCLCother, NSCLCsquam, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 247
RSDPGGGGLAY AML, BRCA, CRC, GBC, GC, GEJC, HCC, HNSCC, MEL, NHL,
NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, SCLC,
UBC, UEC 248 YSHAAGQGTGLY BRCA, GBC, HNSCC, OSCAR, UEC 249
ALFPERITV AML, BRCA, CCC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL,
NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC,
SCLC, UBC, UEC 251 RLASRPLLL NSCLCsquam, OC, OSCAR 252 RIYNGIGVSR
CRC, GBM, MEL, NSCLCadeno 254 AVATKFVNK AML, BRCA, CCC, CRC, GBC,
GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,
OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 255 LPDGSRVEL BRCA 256
LPALPQQLI GBC, GBM, GC, MEL, NHL, NSCLCadeno, NSCLCsquam, OSCAR,
PACA, SCLC, UEC 257 SPLRGGSSL AML, BRCA, GC, HNSCC, MEL, NHL,
NSCLCadeno, NSCLCsquam, OC, OSCAR, SCLC, UBC 258 APSGTRVVQVL BRCA,
CCC, CRC, GBC, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,
NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 259
RPAVGHSGL AML, BRCA, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OC,
OSCAR, RCC 260 EEAPLVTKAF AML, CLL, CRC, MEL, NHL, NSCLCadeno,
NSCLCsquam, PRCA, UEC 261 IEALLESSL BRCA, CCC, CLL, CRC, GBC, GBM,
GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA,
PRCA, SCLC, UBC, UEC 262 MELLLVNKL HCC, HNSCC, MEL, OC, UEC 263
QQATPGPAY AML, BRCA, CCC, GBC, GC, NSCLCadeno, NSCLCother,
NSCLCsquam, OSCAR, PACA, UBC 264 DEYLIPQQGF BRCA, CCC, CRC, GBC,
GBM, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam,
OC, OSCAR, PACA, PRCA, SCLC, UBC, UEC 265 EEVDVPIKLY BRCA, CCC, GC,
HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,
OSCAR, PACA, PRCA, RCC, SCLC, UEC 266 ARLTPIPFGL BRCA, GBM, HNSCC,
MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, RCC, SCLC, UBC,
UEC BRCA = breast cancer, CCC = bile duct cancer, GBM = brain
cancer, CRC = colorectal carcinoma, OSCAR = esophageal cancer, GBC
= gallbladder adenocarcinoma, GC = gastric cancer, HNSCC = head and
neck squamous cell carcinoma, HCC = hepatocellular carcinoma, MEL =
melanoma, NHL = non-Hodgkin lymphoma, NSCLCadeno = non-small cell
lung cancer adenocarcinoma, NSCLCother = NSCLC samples that could
not unambiguously be assigned to NSCLCadeno or NSCLCsquam,
NSCLCsquam = squamous cell non-small cell lung cancer, OC = ovarian
cancer, PACA = pancreatic cancer, PRCA = prostate cancer and benign
prostate hyperplasia, RCC = renal cell carcinoma, SCLC = small cell
lung cancer, UBC = urinary bladder cancer, UEC = uterine
cancer.
Example 2
[0373] Expression Profiling of Genes Encoding the Peptides of the
Invention
[0374] 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.
[0375] RNA sources and preparation 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.
[0376] Total RNA from healthy human tissues for RNASeq experiments
was obtained from: Asterand (Detroit, Mich., USA & Royston,
Herts, UK); Bio-Options Inc. (Brea, Calif., USA); Geneticist Inc.
(Glendale, Calif., USA); ProteoGenex Inc. (Culver City, Calif.,
USA); Tissue Solutions Ltd (Glasgow, UK). Total RNA from tumor
tissues for RNASeq experiments was obtained from: Asterand
(Detroit, Mich., USA & Royston, Herts, UK); BioCat GmbH
(Heidelberg, Germany); BioServe (Beltsville, Md., USA); Geneticist
Inc. (Glendale, Calif., USA); Istituto Nazionale Tumori "Pascale"
(Naples, Italy); ProteoGenex Inc. (Culver City, Calif., USA);
University Hospital Heidelberg (Heidelberg, Germany). 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).
[0377] RNAseq Experiments
[0378] Gene expression analysis of--tumor and normal tissue RNA
samples was performed by next generation sequencing (RNAseq) by
CeGaT (Tabingen, 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.
[0379] Exemplary expression profiles of source genes of the present
invention that are highly over-expressed or exclusively expressed
in colorectal cancer, glioblastoma, gastric cancer, hepatocellular
carcinoma, and renal cell carcinoma are shown in FIGS. 1A-1P.
Expression scores for further exemplary genes are shown in Table
9.
TABLE-US-00010 TABLE 9 Expression scores. SEQ ID Gene Expression No
Sequence CRC GBM GC HCC RCC 1 RSDPVTLDV + 2 LPSPTDSNFY +++ + 3
ASSTDSASYY ++ 4 NSDLKYNAL + 5 SILGSDVRVPSY + 6 VLDLSTNVY ++ 7
LITGDPKAAYDY + + + 8 TPVTEFSLNTY + + 9 FITAQNHGY + 10 ITAQNHGY + 11
LSAGSGPGQY + 12 ITFGERFEY + 13 GSTMVEHNY + + 14 YTERDGSAMVY + 15
LTDYLKNTY + 16 LSLIDRLVLY + 17 YTDKLQHY + 18 EVSNGKWLLY + 19
VSNGKWLLY + 20 STDEITTRY ++ 21 STDIGALMY + ++ 22 TLEQVQLYY + 23
TASEDVFQY + 24 YTHHLFIFY ++ 25 LMKEVMEHY + 26 EVLDSHIHAY ++ 27
LDSHIHAY ++ 28 LTDYINANY +++ 29 SVTDLEMPHY +++ 30 VLDSHIHAY ++ 31
VTDLEMPHY +++ 32 ATVGYFIFY + + 33 FADKIHLAY ++ + 34 ITDFNNIRY + +
35 FASDLLHLY +++ 36 YAAYIIHAY + 37 LTDSFPLKV + 38 VMLNSNVLL + 39
YLLPSVVLL + 40 KIDDIWNLEV + 41 SLQDTKITL + 42 KMMALVAEL ++ 43
GLMTIVTSL ++ 44 SQTGFVVLV + 45 KLLDEVTYL + 46 VLITGLPLI + 47
YQDSWFQQL + 48 NLTFIIILI +++ 49 NLASRPYSL + 50 ELMPRVYTL + + + + +
51 ALAAELNQL + 52 YVSSGEMMV + 53 LLMTSLTES + 54 YLPPTDPRMSV +++ 55
RLWQIQHHL + 56 FLNQIYTQL + 57 GLTGVIMTI ++ 58 MLCLLLTL ++ + 59
KLHEIYIQA + 60 GLPDFVKEL +++ ++ 61 RLFGLFLNNV + 62 GSYSALLAKK + 63
KVLGPNGLLK + 64 STTKLYLAK + 65 VLGPNGLLK + 66 ATYEGIQKK + 67
ATALSLSNK + 68 ATAYGSTVSK ++ 69 ATAYGSTVSKR ++ 70 ATWSASLKNK + 71
KLGNNPVSK + 72 KQVFPGLNY ++ 73 KSFDRHFEK + 74 QLYSKFLLK + 75
QVPTFTIPK ++ 76 SAFGYVFPK + 77 SSASLAHMK + 78 STKSTSPPK ++ 79
STNNEGNLK + 80 STSHHLVSR + 81 SVKLQGTSK + 82 TAYGSTVSK ++ 83
TAYGSTVSKR ++ 84 TVASLHTEK + 85 KMAAWPFSR + 86 KTPSGALHRK + 87
SSYSRSSAVK + 88 MLLQQPLIY + 89 KITDFGLAK +++ + 90 GSRLGKYYVK + 91
SLIDRLVLY + 92 AVLDLGSLLAK + 93 ALDKPGKSK + 94 KTYVGHPVKM + + + + +
95 RLFESSFHY ++ 96 FSLAGALNAGFK + 97 RMPPPLPTR + 98 KLYPTYSTK + 99
ATMQSKLIQK +++ + 100 ALLGVIIAK ++ 101 GVIIAKKYFFK ++ 102 IIAKKYFFK
++ 103 KSWTASSSY + + + 104 STQDTLLIK + + 105 GSAALYLLR ++ 106
RLSPNDQYK ++ 107 EIYGGHHAGF ++ 108 LLKSSVGNFY + 109 KIIAPLVTR + 110
GTESGTILK ++ +++ 111 KIKEHVRSK + 112 KMMADYGIRK + 113 VWAKILSAF +
114 KFLDSNIKF + 115 YFEEAANFL + 116 LVLDYSKDYNHW + 117 NFLPPIIARF +
118 TYISKTIAL + 119 YMKALGVGF + + 120 MYAKEFDLL + 121 SYIEKVRFL +
122 KLYGMPTDFGF +
123 RQYLAINQI + 124 EVYSPEADQW +++ 125 IYGPKYIHPSF ++ + 126
TFQDKTLNF ++ + 127 IFINLSPEF + 128 SYTKVEARL + 129 VFLNQIYTQL + 130
VYGDGHYLTF + 131 KQLDHNLTF ++ 132 VYNPVIYVF + 133 SFDSNLLSF +++ +
134 TYLTGRQF + 135 VIAPIISNF + 136 EYNNIQHLF + 137 KYLSLSNSF + 138
KYLSIPTVF +++ 139 PYASLASELF + 140 KYLSIPAVF + 141 KYLSIPAVFF + 142
SSFPGAGNTW + 143 FELPTGAGLQL ++ 144 IPEPSAQQL ++ 145 RVPSYTLIL +
146 SPGDKRLAA ++ 147 SPIKVPLLL + 148 VPDGVSKVL ++ 149 YPLTGDTRL ++
150 KPSSKALGTSL + 151 VVHPRTLLL + 152 IPSRLLAIL + 153 APAAVPSAPA ++
++ +++ + 154 GPGTRLSL + 155 FPYPYAERL ++ ++ 156 HPQVVILSL ++ 157
SPSPGKDPTL + 158 VPERGEPEL + 159 FPAHPSLLL + 160 RPAPADSAL +++ 161
NPYEGRVEV + 162 MPMISIPRV + 163 RPASSLRP ++ +++ + 164 ISTPSEVSTPL +
165 TPIAKVSEL + +++ 166 HDPDVGSNSL + 167 YPSEVEHMF + 168 IPTDKLLVI
+ 169 FPTEVTPHAF +++ 170 SPMWHVQQL +++ 171 APKLFAVAF + 172
KPAHYPLIAL + ++ 173 MVPSAGQLALF ++ 174 VPSLQRLML + 175 HPIETLVDIF +
176 AAMSRYEL + 177 DLKYNALDL + 178 HAKEKLTAL + 179 IQIYKKLRTSSF +
180 LLKAEPLAF + 181 YKKLRTSSF + 182 LPFLRENDL + 183 FQKLKLLSL + 184
EPVKKSRL + 185 NPNLKTLL + 186 SLIDRLVL + 187 YVKERSKAM + 188
SALDHVTRL + 189 HIFLRTTL + 190 SRSMRLLLL ++ +++ 191 LINLKYLSL + 192
LPMLKVLNL + 193 LSYNKYLQL + 194 EAKRHLLQV + 195 AEAVLKTLQEL ++ 196
AEQTGTWKL + 197 EEAKQVLFL + 198 FELPTGAGL ++ 199 GEATLQRIY + 200
GEELGFASL ++ 201 GEHTSKATL + 202 KEFNLQNMGL ++ 203 KENFAGEATL + 204
KESQLPTVM ++ 205 QEVLLQTFL ++ 206 SEPINIIDAL + 207 TEATMTFKY ++ 208
AEHDAVRNAL ++ 209 YEVDTVLRY +++ 210 SENIVIQVY + 211 TEKEMIQKL ++
212 AEETCAPSV + 213 TTMDQKSLW + 214 AEQPDGLIL + 215 AFITAQNHGY +
216 LQEEKVPAIY + 217 NEINEKIAPSF + 218 AEGGKVPIKW +++ + 219
AENAEYLRV ++ 220 KEITGFLLI +++ 221 AEERAEAKKKF + 222 NEISTFHNL +
223 SEVPVARVW + 224 SESAVFHGF + 225 SEAFPSRAL +++ 226 EELLHGQLF +
227 TEHTQSQAAW + 228 AEKQTPDGRKY ++ 229 KESDGFHRF + 230 AENLFRAFL +
231 AEIHTAEI +++ 232 AEKDGKLTDY +++ 233 DESEKTTKSF +++ 234
EEESLLTSF +++ 235 EEFETLKEF ++ 236 EEKLIIQDF ++ 237 LEMPHYSTF +++
238 SENPETITY +++ 239 TEVLDSHIHAY ++ 240 HELENHSMY + 241 REAEPIPKM
+ 242 FSDKELAAY + 243 RSPNNFLSY + 244 RSDPVTLNV + 245 ITEKNSGLY ++
246 YSDLHAFYY + 247 RSDPGGGGLAY + 248 YSHAAGQGTGLY +
249 ALFPERITV + 250 KMILKMVQL + + +++ 251 RLASRPLLL + 252
RIYNGIGVSR + + 253 KLFGTSGQK +++ 254 AVATKFVNK + 255 LPDGSRVEL ++ +
+++ ++ 256 LPALPQQLI + + 257 SPLRGGSSL + 258 APSGTRVVQVL + 259
RPAVGHSGL + 260 EEAPLVTKAF + 261 IEALLESSL + 262 MELLLVNKL + 263
QQATPGPAY ++ 264 DEYLIPQQGF +++ + 265 EEVDVPIKLY + 266 ARLTPIPFGL +
The table lists peptides from genes that are very highly
over-expressed in tumors compared to a panel of normal tissues
(+++), highly over-expressed in tumors compared to a panel of
normal tissues (++) or over-expressed in tumors compared to a panel
of normal tissues (+). The baseline for this score was calculated
from measurements of the following relevant normal tissues: blood
cells, blood vessels, brain, heart, liver, lung, adipose tissue,
adrenal gland, bile duct, bladder, bone marrow, cartilage,
esophagus, eye, gallbladder, head and neck, kidney, large
intestine, lymph node, nerve, pancreas, parathyroid, peritoneum,
pituitary, pleura, skeletal muscle, skin, small intestine, spleen,
stomach, thyroid gland, trachea, ureter. 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.
Example 3
[0380] In Vitro immunogenicity for MHC Class I Presented
Peptides
[0381] 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, HLA-A*24:02, HLA-A*01:01,
HLA-A*03:01, HLA-B*07:02 and HLA-B*44:02 restricted TUMAPs of the
invention, demonstrating that these peptides are T-cell epitopes
against which CD8+ precursor T cells exist in humans (Table 10a and
Table 10b).
[0382] In Vitro Priming of CD8+ T Cells
[0383] 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, HLA-A*24, HLA-A*01, HLA-A*03, HLA-B*07 or HLA-B*44
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.
[0384] 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.
[0385] 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.
[0386] 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).
[0387] pMHC used for positive and negative control stimulations
were A*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 269) from
modified Melan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5,
SEQ ID NO. 270), respectively.
[0388] 800.000 beads/200 .mu.l were coated in 96-well plates in the
presence of 4.times.12.5 ng different biotin-pMHC, washed and 600
ng biotin anti-CD28 were added subsequently in a volume of 200
.mu.l. Stimulations were initiated in 96-well plates by
co-incubating 1.times.10.sup.6 CD8+ T cells with 2.times.10.sup.5
washed coated beads in 200 .mu.l TCM supplemented with 5 ng/ml
IL-12 (PromoCell) for 3 days at 37.degree. C. Half of the medium
was then exchanged by fresh TCM supplemented with 80 U/ml IL-2 and
incubating was continued for 4 days at 37.degree. C. This
stimulation cycle was performed for a total of three times. For the
pMHC multimer readout using 8 different pMHC molecules per
condition, a two-dimensional combinatorial coding approach was used
as previously described (Andersen et al., 2012) with minor
modifications encompassing coupling to 5 different fluorochromes.
Finally, multimeric analyses were performed by staining the cells
with Live/dead near IR dye (Invitrogen, Karlsruhe, Germany),
CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany) and
fluorescent pMHC multimers. For analysis, a BD LSRII SORP cytometer
equipped with appropriate lasers and filters was used. Peptide
specific cells were calculated as percentage of total CD8+ cells.
Evaluation of multimeric analysis was done using the FlowJo
software (Tree Star, Oregon, USA). In vitro priming of specific
multimer+CD8+ lymphocytes was detected by comparing to negative
control stimulations. Immunogenicity for a given antigen was
detected if at least one evaluable in vitro stimulated well of one
healthy donor was found to contain a specific CD8+ T-cell line
after in vitro stimulation (i.e. this well contained at least 1% of
specific multimer+ among CD8+ T-cells and the percentage of
specific multimer+ cells was at least 10.times. the median of the
negative control stimulations).
[0389] In Vitro Immunogenicity for Colorectal Cancer, Glioblastoma,
Gastric Cancer, Hepatocellular Carcinoma, and Renal Cell Carcinoma
Peptides
[0390] 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 13 peptides of the invention are shown in
FIGS. 2 to 9 together with corresponding negative controls. Results
for 56 peptides from the invention are summarized in Table 10a and
Table 10b.
TABLE-US-00011 TABLE 10 In vitro immunogenicity of HLA class I
peptides of the invention Seq ID No Sequence Wells positive [%] 267
KTLGKLWRL ++++ 268 DYIPYVFKL ++++ Exemplary results of in vitro
immunogenicity experiments conducted by the applicant for the
peptides of the invention. <20% = +; 20%-49% = ++; 50%-69% =
+++; >=70% = ++++
TABLE-US-00012 TABLE 10b in vitro immunogenicity of HLA class I
peptides of the invention Wells Seq ID positive No Sequence [%] HLA
1 RSDPVTLDV "++" A*01 6 VLDLSTNVY "++" A*01 20 STDEITTRY "+" A*01
24 YTHHLFIFY "+" A*01 28 LTDYINANY "+" A*01 31 VTDLEMPHY "+" A*01
33 FADKIHLAY "++" A*01 37 LTDSFPLKV "+++" A*01 245 ITEKNSGLY "+++"
A*01 247 RSDPGGGGLAY "+" A*01 38 VMLNSNVLL "+" A*02 42 KMMALVAEL
"++++" A*02 43 GLMTIVTSL "++" A*02 50 ELMPRVYTL "+" A*02 56
FLNQIYTQL "+++" A*02 57 GLTGVIMTI "++++" A*02 60 GLPDFVKEL "++"
A*02 249 ALFPERITV "++" A*02 250 KMILKMVQL "++" A*02 64 STTKLYLAK
"+" A*03 66 ATYEGIQKK "+" A*03 82 TAYGSTVSK "+" A*03 94 KTYVGHPVKM
"+" A*03 105 GSAALYLLR "+" A*03 110 GTESGTILK "+" A*03 138
KYLSIPTVF "+" A*24 144 IPEPSAQQL "+" B*07 146 SPGDKRLAA "++" B*07
148 VPDGVSKVL "+++" B*07 155 FPYPYAERL "+++" B*07 156 HPQVVILSL
"++" B*07 164 ISTPSEVSTPL "+" B*07 170 SPMWHVQQL "++++" B*07 172
KPAHYPLIAL "+++" B*07 255 LPDGSRVEL "++++" B*07 195 AEAVLKTLQEL "+"
B*44 198 FELPTGAGL "++" B*44 200 GEELGFASL "+" B*44 204 KESQLPTVM
"++" B*44 205 QEVLLQTFL "+" B*44 208 AEHDAVRNAL "+" B*44 209
YEVDTVLRY "+" B*44 218 AEGGKVPIKW "++" B*44 219 AENAEYLRV "+++"
B*44 220 KEITGFLLI "++" B*44 225 SEAFPSRAL "+++" B*44 232
AEKDGKLTDY "+" B*44 233 DESEKTTKSF "++++" B*44 235 EEFETLKEF "+"
B*44 236 EEKLIIQDF "++++" B*44 237 LEMPHYSTF "+++" B*44 238
SENPETITY "++" B*44 239 TEVLDSHIHAY "++++" B*44 264 DEYLIPQQGF "+"
B*44 Exemplary results of in vitro immunogenicity experiments
conducted by the applicant for the peptides of the invention.
<20% = +; 20%-49% = ++; 50%-69% = +++; >=70% = ++++
Example 4
[0391] Synthesis of Peptides
[0392] 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 lyophilizes (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
[0393] MHC Binding Assays
[0394] 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).
[0395] 96 well MAXISorp plates (NUNC) were coated over night with 2
ug/ml streptavidin in PBS at room temperature, washed 4.times. and
blocked for 1 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-00013 TABLE 11 MHC class I binding scores. Seq ID Peptide
No Sequence exchange 1 RSDPVTLDV "+++" 2 LPSPTDSNFY "++" 3
ASSTDSASYY "+++" 4 NSDLKYNAL "++" 5 SILGSDVRVPSY "++" 6 VLDLSTNVY
"+++" 7 LITGDPKAAYDY "++" 8 TPVTEFSLNTY "++" 9 FITAQNHGY "++" 10
ITAQNHGY "++" 11 LSAGSGPGQY "+++" 12 ITFGERFEY "+++" 13 GSTMVEHNY
"++" 14 YTERDGSAMVY "+++" 15 LTDYLKNTY "+++" 16 LSLIDRLVLY "++" 17
YTDKLQHY "+++" 18 EVSNGKWLLY "+++" 19 VSNGKWLLY "+++" 20 STDEITTRY
"+++" 21 STDIGALMY "+++" 22 TLEQVQLYY "+++" 23 TASEDVFQY "+" 24
YTHHLFIFY "+" 25 LMKEVMEHY "++" 26 EVLDSHIHAY "+" 27 LDSHIHAY "+"
28 LTDYINANY "+++" 29 SVTDLEMPHY "+" 30 VLDSHIHAY "+++" 31
VTDLEMPHY "++++" 32 ATVGYFIFY "++" 33 FADKIHLAY "+++" 34 ITDFNNIRY
"+++" 35 FASDLLHLY "++" 36 YAAYIIHAY "+++" 37 LTDSFPLKV "+++" 242
FSDKELAAY "++++" 243 RSPNNFLSY "++" 244 RSDPVTLNV "+++" 245
ITEKNSGLY "+++" 246 YSDLHAFYY "++++" 247 RSDPGGGGLAY "+++" 248
YSHAAGQGTGLY "+++" Binding of HLA-class I restricted peptides to
HLA-A*01:01 was ranged by peptide exchange yield: >10% = +;
>20% = ++; >50 = +++; >75% = ++++
TABLE-US-00014 TABLE 12 MHC class I binding scores. Seq ID Peptide
No Sequence exchange 38 VMLNSNVLL "++++" 39 YLLPSVVLL "++++" 40
KIDDIWNLEV "+++" 41 SLQDTKITL "+++" 42 KMMALVAEL "++++" 43
GLMTIVTSL "++++" 44 SQTGFVVLV "++" 45 KLLDEVTYL "+++" 46 VLITGLPLI
"++++" 47 YQDSWFQQL "+++" 49 NLASRPYSL "++" 50 ELMPRVYTL "+++" 51
ALAAELNQL "+++" 52 YVSSGEMMV "+++" 53 LLMTSLTES "+++" 54
YLPPTDPRMSV "++++" 55 RLWQIQHHL "+++" 56 FLNQIYTQL "++++" 57
GLTGVIMTI "++++" 59 KLHEIYIQA "++++" 60 GLPDFVKEL "++++" 61
RLFGLFLNNV "++++" 249 ALFPERITV "++++" 250 KMILKMVQL "+" 251
RLASRPLLL "+++" Binding of HLA-class I restricted peptides to
HLA-A*02:01 was ranged by peptide exchange yield: >10% = +;
>20% = ++; >50 +++; >75% = ++++
TABLE-US-00015 TABLE 13 MHC class I binding scores. Seq ID Peptide
No Sequence exchange 62 GSYSALLAKK "+++" 63 KVLGPNGLLK "++" 64
STTKLYLAK "++" 65 VLGPNGLLK "++" 66 ATYEGIQKK "++" 67 ATALSLSNK
"+++" 68 ATAYGSTVSK "++" 70 ATWSASLKNK "+++" 71 KLGNNPVSK "++" 73
KSFDRHFEK "+++" 74 QLYSKFLLK "+++" 75 QVPTFTIPK "++" 76 SAFGYVFPK
"+++" 77 SSASLAHMK "++" 78 STKSTSPPK "++" 79 STNNEGNLK "++" 80
STSHHLVSR "++" 81 SVKLQGTSK "++" 82 TAYGSTVSK "+++" 84 TVASLHTEK
"++" 85 KMAAWPFSR "+++" 86 KTPSGALHRK "++" 87 SSYSRSSAVK "+++" 88
MLLQQPLIY "++" 89 KITDFGLAK "++" 90 GSRLGKYYVK "+" 92 AVLDLGSLLAK
"+++" 93 ALDKPGKSK "++" 94 KTYVGHPVKM "+++" 95 RLFESSFHY "+++" 96
FSLAGALNAGFK "++" 97 RMPPPLPTR "++" 98 KLYPTYSTK "++" 99 ATMQSKLIQK
"+++" 100 ALLGVIIAK "++" 101 GVIIAKKYFFK "++" 102 IIAKKYFFK "++"
104 STQDTLLIK "++" 105 GSAALYLLR "++" 106 RLSPNDQYK "++" 109
KIIAPLVTR "++" 110 GTESGTILK "++" 111 KIKEHVRSK "++" 112 KMMADYGIRK
"+++" 252 RIYNGIGVSR "+++" 253 KLFGTSGQK "+++" 254 AVATKFVNK "++"
Binding of HLA-class I restricted peptides to HLA-A*03:01 was
ranged by peptide exchange yield: >10% = +; >20% = ++; >50
= +++; >75% = ++++
TABLE-US-00016 TABLE 14 MHC class I binding scores. Seq ID Peptide
No Sequence exchange 113 VWAKILSAF "+++" 114 KFLDSNIKF "+++" 115
YFEEAANFL "++" 116 LVLDYSKDYNHW "+" 117 NFLPPIIARF "++++" 118
TYISKTIAL "+++" 120 MYAKEFDLL "+++" 121 SYIEKVRFL "+++" 122
KLYGMPTDFGF "++" 123 RQYLAINQI "+" 125 IYGPKYIHPSF "++++" 126
TFQDKTLNF "+++" 127 IFINLSPEF "++++" 128 SYTKVEARL "+++" 129
VFLNQIYTQL "++" 130 VYGDGHYLTF "+++" 133 SFDSNLLSF "+" 134 TYLTGRQF
"++" 135 VIAPIISNF "+++" 136 EYNNIQHLF "+++" 137 KYLSLSNSF "++++"
138 KYLSIPTVF "++++" 139 PYASLASELF "++++" 140 KYLSIPAVF "++++" 141
KYLSIPAVFF "++++" Binding of HLA-class I restricted peptides to
HLA-A*24:02 was ranged by peptide exchange yield: >10% = +;
>20% = ++; >50 = +++; >75% =++++
TABLE-US-00017 TABLE 15 MHC class I binding scores. Seq ID Peptide
No Sequence exchange 143 FELPTGAGLQL "++" 144 IPEPSAQQL "++" 145
RVPSYTLIL "+++" 146 SPGDKRLAA "+++" 147 SPIKVPLLL "+++" 148
VPDGVSKVL "++" 149 YPLTGDTRL "++" 150 KPSSKALGTSL "+++" 151
VVHPRTLLL "++" 152 IPSRLLAIL "+++" 153 APAAVPSAPA "+++" 154
GPGTRLSL "++" 155 FPYPYAERL "++" 156 HPQVVILSL "+++" 157 SPSPGKDPTL
"++" 158 VPERGEPEL "++" 159 FPAHPSLLL "++" 160 RPAPADSAL "+++" 161
NPYEGRVEV "++" 162 MPMISIPRV "++" 163 RPASSLRP "++" 164 ISTPSEVSTPL
"++" 165 TPIAKVSEL "+++" 166 HDPDVGSNSL "++" 167 YPSEVEHMF "++" 168
IPTDKLLVI "++" 169 FPTEVTPHAF "++" 170 SPMWHVQQL "+++" 171
APKLFAVAF "+++" 172 KPAHYPLIAL "+++" 173 MVPSAGQLALF "++" 174
VPSLQRLML "+++" 175 HPIETLVDIF "++" 255 LPDGSRVEL "++" 256
LPALPQQLI "+++" 257 SPLRGGSSL "++" 258 APSGTRVVQVL "++" 259
RPAVGHSGL "++" Binding of HLA-class I restricted peptides to
HLA-B*07:02 was ranged by peptide exchange yield: >10% = +;
>20% = ++; >50 = +++; >75% = ++++
TABLE-US-00018 TABLE 16 MHC class I binding scores. Seq ID Peptide
No Sequence exchange 195 AEAVLKTLQEL "+++" 196 AEQTGTWKL "++" 197
EEAKQVLFL "++" 198 FELPTGAGL "++" 199 GEATLQRIY "+" 200 GEELGFASL
"++" 201 GEHTSKATL "++" 202 KEFNLQNMGL "+++" 203 KENFAGEATL "++"
204 KESQLPTVM "++" 205 QEVLLQTFL "++++" 206 SEPINIIDAL "++" 207
TEATMTFKY "+++" 208 AEHDAVRNAL "+++" 209 YEVDTVLRY "++" 210
SENIVIQVY "++" 211 TEKEMIQKL "++" 212 AEETCAPSV "++" 213 TTMDQKSLW
"+" 214 AEQPDGLIL "++" 216 LQEEKVPAIY "+" 217 NEINEKIAPSF "+++" 218
AEGGKVPIKW "+++" 219 AENAEYLRV "++" 220 KEITGFLLI "+++" 221
AEERAEAKKKF "++" 222 NEISTFHNL "+++" 223 SEVPVARVW "++++" 224
SESAVFHGF "+++" 225 SEAFPSRAL "+++" 226 EELLHGQLF "++" 227
TEHTQSQAAW "+++" 228 AEKQTPDGRKY "++" 229 KESDGFHRF "+++" 230
AENLFRAFL "+++" 231 AEIHTAEI "++" 232 AEKDGKLTDY "++" 233
DESEKTTKSF "++" 234 EEESLLTSF "+++" 235 EEFETLKEF "++" 236
EEKLIIQDF "++" 237 LEMPHYSTF "++++" 238 SENPETITY "+++" 239
TEVLDSHIHAY "++" 240 HELENHSMY "++" 241 REAEPIPKM "++" 260
EEAPLVTKAF "++" 261 IEALLESSL "++" 262 MELLLVNKL "+++" 263
QQATPGPAY "++" 264 DEYLIPQQGF "+++" 265 EEVDVPIKLY "++" 266
ARLTPIPFGL "+" Binding of HLA-class I restricted peptides to
HLA-B*44:02 was ranged by peptide exchange yield: >10% = +;
>20% = ++; >50 = +++; >75% = ++++
Example 6
[0396] Peptide-MHC Class I Stability
[0397] The peptide-MHC stability for HLA-B*08:01 peptides was
performed by ImmunAware (Copenhagen, Denmark). The data were
obtained using a proximity based, homogenous, real-time assay to
measure the dissociation of peptides from HLA class I molecules.
First human recombinant HLA-B*08:01 and b2m were expressed in E.
coli and purified in a series of liquid chromatography based steps
(Ferre et al., 2003; Ostergaard et al., 2001). Afterwards, the
stability of a peptide-MHC complex (pMHC) can be determined by
measuring the amount of b2m associated with the MHC heavy chain
over time at 37.degree. C. (Harndahl et al., 2012). The stability
of each pMHC, expressed as the half life of b2m associated with the
respective heavy chain, was calculated by fitting the data to a
one-phase dissociation equation.
[0398] The pMHC stability were measured in three independent
experiments and the peptides in question, for HLA-B*08:01, were
found to span the range from weak-binders (+) to very stable
binders (++++). The mean half-life (T1/2) is shown in Table 17.
TABLE-US-00019 TABLE 17 Mean half-life (T1/2) based on three
individual measurements. Seq ID Mean Half-life No Sequence (T1/2)
176 AAMSRYEL ++ 177 DLKYNALDL +++ 178 HAKEKLTAL +++ 180 LLKAEPLAF
++ 181 YKKLRTSSF ++ 182 LPFLRENDL ++ 183 FQKLKLLSL + 184 EPVKKSRL
+++ 185 NPNLKTLL ++ 187 YVKERSKAM +++ 189 HIFLRTTL ++ 192 LPMLKVLNL
+ 193 LSYNKYLQL ++ 194 EAKRHLLQV ++ T1/2 > 2 h = +; T1/2 > 4
h = ++; T1/2 > 6 h = +++; T1/2 > 10 h = ++++
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Sequence CWU 1
1
27019PRTHomo sapiens 1Arg Ser Asp Pro Val Thr Leu Asp Val1
5210PRTHomo sapiens 2Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr1 5
10310PRTHomo sapiens 3Ala Ser Ser Thr Asp Ser Ala Ser Tyr Tyr1 5
1049PRTHomo sapiens 4Asn Ser Asp Leu Lys Tyr Asn Ala Leu1
5512PRTHomo sapiens 5Ser Ile Leu Gly Ser Asp Val Arg Val Pro Ser
Tyr1 5 1069PRTHomo sapiens 6Val Leu Asp Leu Ser Thr Asn Val Tyr1
5712PRTHomo sapiens 7Leu Ile Thr Gly Asp Pro Lys Ala Ala Tyr Asp
Tyr1 5 10811PRTHomo sapiens 8Thr Pro Val Thr Glu Phe Ser Leu Asn
Thr Tyr1 5 1099PRTHomo sapiens 9Phe Ile Thr Ala Gln Asn His Gly
Tyr1 5108PRTHomo sapiens 10Ile Thr Ala Gln Asn His Gly Tyr1
51110PRTHomo sapiens 11Leu Ser Ala Gly Ser Gly Pro Gly Gln Tyr1 5
10129PRTHomo sapiens 12Ile Thr Phe Gly Glu Arg Phe Glu Tyr1
5139PRTHomo sapiens 13Gly Ser Thr Met Val Glu His Asn Tyr1
51411PRTHomo sapiens 14Tyr Thr Glu Arg Asp Gly Ser Ala Met Val Tyr1
5 10159PRTHomo sapiens 15Leu Thr Asp Tyr Leu Lys Asn Thr Tyr1
51610PRTHomo sapiens 16Leu Ser Leu Ile Asp Arg Leu Val Leu Tyr1 5
10178PRTHomo sapiens 17Tyr Thr Asp Lys Leu Gln His Tyr1
51810PRTHomo sapiens 18Glu Val Ser Asn Gly Lys Trp Leu Leu Tyr1 5
10199PRTHomo sapiens 19Val Ser Asn Gly Lys Trp Leu Leu Tyr1
5209PRTHomo sapiens 20Ser Thr Asp Glu Ile Thr Thr Arg Tyr1
5219PRTHomo sapiens 21Ser Thr Asp Ile Gly Ala Leu Met Tyr1
5229PRTHomo sapiens 22Thr Leu Glu Gln Val Gln Leu Tyr Tyr1
5239PRTHomo sapiens 23Thr Ala Ser Glu Asp Val Phe Gln Tyr1
5249PRTHomo sapiens 24Tyr Thr His His Leu Phe Ile Phe Tyr1
5259PRTHomo sapiens 25Leu Met Lys Glu Val Met Glu His Tyr1
52610PRTHomo sapiens 26Glu Val Leu Asp Ser His Ile His Ala Tyr1 5
10278PRTHomo sapiens 27Leu Asp Ser His Ile His Ala Tyr1 5289PRTHomo
sapiens 28Leu Thr Asp Tyr Ile Asn Ala Asn Tyr1 52910PRTHomo sapiens
29Ser Val Thr Asp Leu Glu Met Pro His Tyr1 5 10309PRTHomo sapiens
30Val Leu Asp Ser His Ile His Ala Tyr1 5319PRTHomo sapiens 31Val
Thr Asp Leu Glu Met Pro His Tyr1 5329PRTHomo sapiens 32Ala Thr Val
Gly Tyr Phe Ile Phe Tyr1 5339PRTHomo sapiens 33Phe Ala Asp Lys Ile
His Leu Ala Tyr1 5349PRTHomo sapiens 34Ile Thr Asp Phe Asn Asn Ile
Arg Tyr1 5359PRTHomo sapiens 35Phe Ala Ser Asp Leu Leu His Leu Tyr1
5369PRTHomo sapiens 36Tyr Ala Ala Tyr Ile Ile His Ala Tyr1
5379PRTHomo sapiens 37Leu Thr Asp Ser Phe Pro Leu Lys Val1
5389PRTHomo sapiens 38Val Met Leu Asn Ser Asn Val Leu Leu1
5399PRTHomo sapiens 39Tyr Leu Leu Pro Ser Val Val Leu Leu1
54010PRTHomo sapiens 40Lys Ile Asp Asp Ile Trp Asn Leu Glu Val1 5
10419PRTHomo sapiens 41Ser Leu Gln Asp Thr Lys Ile Thr Leu1
5429PRTHomo sapiens 42Lys Met Met Ala Leu Val Ala Glu Leu1
5439PRTHomo sapiens 43Gly Leu Met Thr Ile Val Thr Ser Leu1
5449PRTHomo sapiens 44Ser Gln Thr Gly Phe Val Val Leu Val1
5459PRTHomo sapiens 45Lys Leu Leu Asp Glu Val Thr Tyr Leu1
5469PRTHomo sapiens 46Val Leu Ile Thr Gly Leu Pro Leu Ile1
5479PRTHomo sapiens 47Tyr Gln Asp Ser Trp Phe Gln Gln Leu1
5489PRTHomo sapiens 48Asn Leu Thr Phe Ile Ile Ile Leu Ile1
5499PRTHomo sapiens 49Asn Leu Ala Ser Arg Pro Tyr Ser Leu1
5509PRTHomo sapiens 50Glu Leu Met Pro Arg Val Tyr Thr Leu1
5519PRTHomo sapiens 51Ala Leu Ala Ala Glu Leu Asn Gln Leu1
5529PRTHomo sapiens 52Tyr Val Ser Ser Gly Glu Met Met Val1
5539PRTHomo sapiens 53Leu Leu Met Thr Ser Leu Thr Glu Ser1
55411PRTHomo sapiens 54Tyr Leu Pro Pro Thr Asp Pro Arg Met Ser Val1
5 10559PRTHomo sapiens 55Arg Leu Trp Gln Ile Gln His His Leu1
5569PRTHomo sapiens 56Phe Leu Asn Gln Ile Tyr Thr Gln Leu1
5579PRTHomo sapiens 57Gly Leu Thr Gly Val Ile Met Thr Ile1
5588PRTHomo sapiens 58Met Leu Cys Leu Leu Leu Thr Leu1 5599PRTHomo
sapiens 59Lys Leu His Glu Ile Tyr Ile Gln Ala1 5609PRTHomo sapiens
60Gly Leu Pro Asp Phe Val Lys Glu Leu1 56110PRTHomo sapiens 61Arg
Leu Phe Gly Leu Phe Leu Asn Asn Val1 5 106210PRTHomo sapiens 62Gly
Ser Tyr Ser Ala Leu Leu Ala Lys Lys1 5 106310PRTHomo sapiens 63Lys
Val Leu Gly Pro Asn Gly Leu Leu Lys1 5 10649PRTHomo sapiens 64Ser
Thr Thr Lys Leu Tyr Leu Ala Lys1 5659PRTHomo sapiens 65Val Leu Gly
Pro Asn Gly Leu Leu Lys1 5669PRTHomo sapiens 66Ala Thr Tyr Glu Gly
Ile Gln Lys Lys1 5679PRTHomo sapiens 67Ala Thr Ala Leu Ser Leu Ser
Asn Lys1 56810PRTHomo sapiens 68Ala Thr Ala Tyr Gly Ser Thr Val Ser
Lys1 5 106911PRTHomo sapiens 69Ala Thr Ala Tyr Gly Ser Thr Val Ser
Lys Arg1 5 107010PRTHomo sapiens 70Ala Thr Trp Ser Ala Ser Leu Lys
Asn Lys1 5 10719PRTHomo sapiens 71Lys Leu Gly Asn Asn Pro Val Ser
Lys1 5729PRTHomo sapiens 72Lys Gln Val Phe Pro Gly Leu Asn Tyr1
5739PRTHomo sapiens 73Lys Ser Phe Asp Arg His Phe Glu Lys1
5749PRTHomo sapiens 74Gln Leu Tyr Ser Lys Phe Leu Leu Lys1
5759PRTHomo sapiens 75Gln Val Pro Thr Phe Thr Ile Pro Lys1
5769PRTHomo sapiens 76Ser Ala Phe Gly Tyr Val Phe Pro Lys1
5779PRTHomo sapiens 77Ser Ser Ala Ser Leu Ala His Met Lys1
5789PRTHomo sapiens 78Ser Thr Lys Ser Thr Ser Pro Pro Lys1
5799PRTHomo sapiens 79Ser Thr Asn Asn Glu Gly Asn Leu Lys1
5809PRTHomo sapiens 80Ser Thr Ser His His Leu Val Ser Arg1
5819PRTHomo sapiens 81Ser Val Lys Leu Gln Gly Thr Ser Lys1
5829PRTHomo sapiens 82Thr Ala Tyr Gly Ser Thr Val Ser Lys1
58310PRTHomo sapiens 83Thr Ala Tyr Gly Ser Thr Val Ser Lys Arg1 5
10849PRTHomo sapiens 84Thr Val Ala Ser Leu His Thr Glu Lys1
5859PRTHomo sapiens 85Lys Met Ala Ala Trp Pro Phe Ser Arg1
58610PRTHomo sapiens 86Lys Thr Pro Ser Gly Ala Leu His Arg Lys1 5
108710PRTHomo sapiens 87Ser Ser Tyr Ser Arg Ser Ser Ala Val Lys1 5
10889PRTHomo sapiens 88Met Leu Leu Gln Gln Pro Leu Ile Tyr1
5899PRTHomo sapiens 89Lys Ile Thr Asp Phe Gly Leu Ala Lys1
59010PRTHomo sapiens 90Gly Ser Arg Leu Gly Lys Tyr Tyr Val Lys1 5
10919PRTHomo sapiens 91Ser Leu Ile Asp Arg Leu Val Leu Tyr1
59211PRTHomo sapiens 92Ala Val Leu Asp Leu Gly Ser Leu Leu Ala Lys1
5 10939PRTHomo sapiens 93Ala Leu Asp Lys Pro Gly Lys Ser Lys1
59410PRTHomo sapiens 94Lys Thr Tyr Val Gly His Pro Val Lys Met1 5
10959PRTHomo sapiens 95Arg Leu Phe Glu Ser Ser Phe His Tyr1
59612PRTHomo sapiens 96Phe Ser Leu Ala Gly Ala Leu Asn Ala Gly Phe
Lys1 5 10979PRTHomo sapiens 97Arg Met Pro Pro Pro Leu Pro Thr Arg1
5989PRTHomo sapiens 98Lys Leu Tyr Pro Thr Tyr Ser Thr Lys1
59910PRTHomo sapiens 99Ala Thr Met Gln Ser Lys Leu Ile Gln Lys1 5
101009PRTHomo sapiens 100Ala Leu Leu Gly Val Ile Ile Ala Lys1
510111PRTHomo sapiens 101Gly Val Ile Ile Ala Lys Lys Tyr Phe Phe
Lys1 5 101029PRTHomo sapiens 102Ile Ile Ala Lys Lys Tyr Phe Phe
Lys1 51039PRTHomo sapiens 103Lys Ser Trp Thr Ala Ser Ser Ser Tyr1
51049PRTHomo sapiens 104Ser Thr Gln Asp Thr Leu Leu Ile Lys1
51059PRTHomo sapiens 105Gly Ser Ala Ala Leu Tyr Leu Leu Arg1
51069PRTHomo sapiens 106Arg Leu Ser Pro Asn Asp Gln Tyr Lys1
510710PRTHomo sapiens 107Glu Ile Tyr Gly Gly His His Ala Gly Phe1 5
1010810PRTHomo sapiens 108Leu Leu Lys Ser Ser Val Gly Asn Phe Tyr1
5 101099PRTHomo sapiens 109Lys Ile Ile Ala Pro Leu Val Thr Arg1
51109PRTHomo sapiens 110Gly Thr Glu Ser Gly Thr Ile Leu Lys1
51119PRTHomo sapiens 111Lys Ile Lys Glu His Val Arg Ser Lys1
511210PRTHomo sapiens 112Lys Met Met Ala Asp Tyr Gly Ile Arg Lys1 5
101139PRTHomo sapiens 113Val Trp Ala Lys Ile Leu Ser Ala Phe1
51149PRTHomo sapiens 114Lys Phe Leu Asp Ser Asn Ile Lys Phe1
51159PRTHomo sapiens 115Tyr Phe Glu Glu Ala Ala Asn Phe Leu1
511612PRTHomo sapiens 116Leu Val Leu Asp Tyr Ser Lys Asp Tyr Asn
His Trp1 5 1011710PRTHomo sapiens 117Asn Phe Leu Pro Pro Ile Ile
Ala Arg Phe1 5 101189PRTHomo sapiens 118Thr Tyr Ile Ser Lys Thr Ile
Ala Leu1 51199PRTHomo sapiens 119Tyr Met Lys Ala Leu Gly Val Gly
Phe1 51209PRTHomo sapiens 120Met Tyr Ala Lys Glu Phe Asp Leu Leu1
51219PRTHomo sapiens 121Ser Tyr Ile Glu Lys Val Arg Phe Leu1
512211PRTHomo sapiens 122Lys Leu Tyr Gly Met Pro Thr Asp Phe Gly
Phe1 5 101239PRTHomo sapiens 123Arg Gln Tyr Leu Ala Ile Asn Gln
Ile1 512410PRTHomo sapiens 124Glu Val Tyr Ser Pro Glu Ala Asp Gln
Trp1 5 1012511PRTHomo sapiens 125Ile Tyr Gly Pro Lys Tyr Ile His
Pro Ser Phe1 5 101269PRTHomo sapiens 126Thr Phe Gln Asp Lys Thr Leu
Asn Phe1 51279PRTHomo sapiens 127Ile Phe Ile Asn Leu Ser Pro Glu
Phe1 51289PRTHomo sapiens 128Ser Tyr Thr Lys Val Glu Ala Arg Leu1
512910PRTHomo sapiens 129Val Phe Leu Asn Gln Ile Tyr Thr Gln Leu1 5
1013010PRTHomo sapiens 130Val Tyr Gly Asp Gly His Tyr Leu Thr Phe1
5 101319PRTHomo sapiens 131Lys Gln Leu Asp His Asn Leu Thr Phe1
51329PRTHomo sapiens 132Val Tyr Asn Pro Val Ile Tyr Val Phe1
51339PRTHomo sapiens 133Ser Phe Asp Ser Asn Leu Leu Ser Phe1
51348PRTHomo sapiens 134Thr Tyr Leu Thr Gly Arg Gln Phe1
51359PRTHomo sapiens 135Val Ile Ala Pro Ile Ile Ser Asn Phe1
51369PRTHomo sapiens 136Glu Tyr Asn Asn Ile Gln His Leu Phe1
51379PRTHomo sapiens 137Lys Tyr Leu Ser Leu Ser Asn Ser Phe1
51389PRTHomo sapiens 138Lys Tyr Leu Ser Ile Pro Thr Val Phe1
513910PRTHomo sapiens 139Pro Tyr Ala Ser Leu Ala Ser Glu Leu Phe1 5
101409PRTHomo sapiens 140Lys Tyr Leu Ser Ile Pro Ala Val Phe1
514110PRTHomo sapiens 141Lys Tyr Leu Ser Ile Pro Ala Val Phe Phe1 5
1014210PRTHomo sapiens 142Ser Ser Phe Pro Gly Ala Gly Asn Thr Trp1
5 1014311PRTHomo sapiens 143Phe Glu Leu Pro Thr Gly Ala Gly Leu Gln
Leu1 5 101449PRTHomo sapiens 144Ile Pro Glu Pro Ser Ala Gln Gln
Leu1 51459PRTHomo sapiens 145Arg Val Pro Ser Tyr Thr Leu Ile Leu1
51469PRTHomo sapiens 146Ser Pro Gly Asp Lys Arg Leu Ala Ala1
51479PRTHomo sapiens 147Ser Pro Ile Lys Val Pro Leu Leu Leu1
51489PRTHomo sapiens 148Val Pro Asp Gly Val Ser Lys Val Leu1
51499PRTHomo sapiens 149Tyr Pro Leu Thr Gly Asp Thr Arg Leu1
515011PRTHomo sapiens 150Lys Pro Ser Ser Lys Ala Leu Gly Thr Ser
Leu1 5 101519PRTHomo sapiens 151Val Val His Pro Arg Thr Leu Leu
Leu1 51529PRTHomo sapiens 152Ile Pro Ser Arg Leu Leu Ala Ile Leu1
515310PRTHomo sapiens 153Ala Pro Ala Ala Val Pro Ser Ala Pro Ala1 5
101548PRTHomo sapiens 154Gly Pro Gly Thr Arg Leu Ser Leu1
51559PRTHomo sapiens 155Phe Pro Tyr Pro Tyr Ala Glu Arg Leu1
51569PRTHomo sapiens 156His Pro Gln Val Val Ile Leu Ser Leu1
515710PRTHomo sapiens 157Ser Pro Ser Pro Gly Lys Asp Pro Thr Leu1 5
101589PRTHomo sapiens 158Val Pro Glu Arg Gly Glu Pro Glu Leu1
51599PRTHomo sapiens 159Phe Pro Ala His Pro Ser Leu Leu Leu1
51609PRTHomo sapiens 160Arg Pro Ala Pro Ala Asp Ser Ala Leu1
51619PRTHomo sapiens 161Asn Pro Tyr Glu Gly Arg Val Glu Val1
51629PRTHomo sapiens 162Met Pro Met Ile Ser Ile Pro Arg Val1
51638PRTHomo sapiens 163Arg Pro Ala Ser Ser Leu Arg Pro1
516411PRTHomo sapiens 164Ile Ser Thr Pro Ser Glu Val Ser Thr Pro
Leu1 5 101659PRTHomo sapiens 165Thr Pro Ile Ala Lys Val Ser Glu
Leu1 516610PRTHomo sapiens 166His Asp Pro Asp Val Gly Ser Asn Ser
Leu1 5 101679PRTHomo sapiens 167Tyr Pro Ser Glu Val Glu His Met
Phe1 51689PRTHomo sapiens 168Ile Pro Thr Asp Lys Leu Leu Val Ile1
516910PRTHomo sapiens 169Phe Pro Thr Glu Val Thr Pro His Ala Phe1 5
101709PRTHomo sapiens 170Ser Pro Met Trp His Val Gln Gln Leu1
51719PRTHomo sapiens 171Ala Pro Lys Leu Phe Ala Val Ala Phe1
517210PRTHomo sapiens 172Lys Pro Ala His Tyr Pro Leu Ile Ala Leu1 5
1017311PRTHomo sapiens 173Met Val Pro Ser Ala Gly Gln Leu Ala Leu
Phe1 5 101749PRTHomo sapiens 174Val Pro Ser Leu Gln Arg Leu Met
Leu1 517510PRTHomo sapiens 175His Pro Ile Glu Thr Leu Val Asp Ile
Phe1 5 101768PRTHomo sapiens 176Ala Ala Met Ser Arg Tyr Glu Leu1
51779PRTHomo sapiens 177Asp Leu Lys Tyr Asn Ala Leu Asp Leu1
51789PRTHomo sapiens 178His Ala Lys Glu Lys Leu Thr Ala Leu1
517912PRTHomo sapiens 179Ile Gln Ile Tyr Lys Lys Leu Arg Thr Ser
Ser Phe1 5 101809PRTHomo sapiens 180Leu Leu Lys Ala Glu Pro Leu Ala
Phe1 51819PRTHomo sapiens 181Tyr Lys Lys Leu Arg Thr Ser Ser Phe1
51829PRTHomo sapiens 182Leu Pro Phe Leu Arg Glu Asn Asp Leu1
51839PRTHomo sapiens 183Phe Gln Lys Leu Lys Leu Leu Ser Leu1
51848PRTHomo sapiens 184Glu Pro Val Lys Lys Ser Arg Leu1
51858PRTHomo sapiens 185Asn Pro Asn Leu Lys Thr Leu Leu1
51868PRTHomo sapiens 186Ser Leu Ile Asp Arg Leu Val Leu1
51879PRTHomo sapiens 187Tyr Val Lys Glu Arg Ser Lys Ala Met1
51889PRTHomo sapiens 188Ser Ala Leu Asp His Val Thr Arg Leu1
51898PRTHomo sapiens 189His Ile Phe Leu Arg Thr Thr Leu1
51909PRTHomo sapiens 190Ser Arg Ser Met Arg Leu Leu Leu Leu1
51919PRTHomo sapiens 191Leu Ile Asn Leu Lys Tyr Leu Ser Leu1
51929PRTHomo sapiens 192Leu Pro Met Leu Lys Val Leu Asn Leu1
51939PRTHomo sapiens 193Leu Ser Tyr Asn Lys Tyr Leu Gln Leu1
51949PRTHomo sapiens 194Glu Ala Lys Arg His Leu Leu Gln Val1
519511PRTHomo sapiens 195Ala Glu Ala Val Leu Lys Thr Leu Gln Glu
Leu1 5 101969PRTHomo sapiens 196Ala Glu Gln Thr Gly Thr Trp Lys
Leu1 51979PRTHomo sapiens 197Glu Glu Ala Lys Gln Val Leu Phe Leu1
51989PRTHomo sapiens 198Phe Glu Leu Pro Thr Gly Ala Gly Leu1
51999PRTHomo sapiens 199Gly Glu Ala Thr Leu Gln Arg Ile Tyr1
52009PRTHomo sapiens 200Gly Glu Glu Leu Gly Phe Ala Ser Leu1
52019PRTHomo sapiens 201Gly Glu His Thr Ser Lys Ala Thr Leu1
520210PRTHomo sapiens 202Lys Glu Phe Asn Leu Gln Asn Met Gly Leu1 5
1020310PRTHomo sapiens 203Lys Glu Asn Phe Ala Gly Glu Ala Thr Leu1
5 102049PRTHomo sapiens 204Lys Glu Ser Gln Leu Pro Thr Val Met1
52059PRTHomo sapiens 205Gln Glu Val Leu Leu Gln Thr Phe Leu1
520610PRTHomo sapiens 206Ser Glu Pro Ile Asn Ile Ile Asp Ala Leu1 5
102079PRTHomo sapiens 207Thr Glu Ala Thr Met Thr Phe Lys Tyr1
520810PRTHomo sapiens 208Ala Glu His Asp Ala Val Arg Asn Ala Leu1 5
102099PRTHomo sapiens 209Tyr Glu Val Asp Thr Val Leu Arg Tyr1
52109PRTHomo sapiens 210Ser Glu Asn Ile Val Ile Gln Val Tyr1
52119PRTHomo sapiens 211Thr Glu Lys Glu Met Ile Gln Lys Leu1
52129PRTHomo sapiens 212Ala Glu Glu Thr Cys Ala Pro Ser Val1
52139PRTHomo sapiens 213Thr Thr Met Asp Gln Lys Ser Leu Trp1
52149PRTHomo sapiens 214Ala Glu Gln Pro Asp Gly Leu Ile Leu1
521510PRTHomo sapiens 215Ala Phe Ile Thr Ala Gln Asn His Gly Tyr1 5
1021610PRTHomo sapiens 216Leu Gln Glu Glu Lys Val Pro Ala Ile Tyr1
5 1021711PRTHomo sapiens 217Asn Glu Ile Asn Glu Lys Ile Ala Pro Ser
Phe1 5 1021810PRTHomo sapiens 218Ala Glu Gly Gly Lys Val Pro Ile
Lys Trp1 5 102199PRTHomo sapiens 219Ala Glu Asn Ala Glu Tyr Leu Arg
Val1 52209PRTHomo sapiens 220Lys Glu Ile Thr Gly Phe Leu Leu Ile1
522111PRTHomo sapiens 221Ala Glu Glu Arg Ala Glu Ala Lys Lys Lys
Phe1 5 102229PRTHomo sapiens 222Asn Glu Ile Ser Thr Phe His Asn
Leu1 52239PRTHomo sapiens 223Ser Glu Val Pro Val Ala Arg Val Trp1
52249PRTHomo sapiens 224Ser Glu Ser Ala Val Phe His Gly Phe1
52259PRTHomo sapiens 225Ser Glu Ala Phe Pro Ser Arg Ala Leu1
52269PRTHomo sapiens 226Glu Glu Leu Leu His Gly Gln Leu Phe1
522710PRTHomo sapiens 227Thr Glu His Thr Gln Ser Gln Ala Ala Trp1 5
1022811PRTHomo sapiens 228Ala Glu Lys Gln Thr Pro Asp Gly Arg Lys
Tyr1 5 102299PRTHomo sapiens 229Lys Glu Ser Asp Gly Phe His Arg
Phe1 52309PRTHomo sapiens 230Ala Glu Asn Leu Phe Arg Ala Phe Leu1
52318PRTHomo sapiens 231Ala Glu Ile His Thr Ala Glu Ile1
523210PRTHomo sapiens 232Ala Glu Lys Asp Gly Lys Leu Thr Asp Tyr1 5
1023310PRTHomo sapiens 233Asp Glu Ser Glu Lys Thr Thr Lys Ser Phe1
5 102349PRTHomo sapiens 234Glu Glu Glu Ser Leu Leu Thr Ser Phe1
52359PRTHomo sapiens 235Glu Glu Phe Glu Thr Leu Lys Glu Phe1
52369PRTHomo sapiens 236Glu Glu Lys Leu Ile Ile Gln Asp Phe1
52379PRTHomo sapiens 237Leu Glu Met Pro His Tyr Ser Thr Phe1
52389PRTHomo sapiens 238Ser Glu Asn Pro Glu Thr Ile Thr Tyr1
523911PRTHomo sapiens 239Thr Glu Val Leu Asp Ser His Ile His Ala
Tyr1 5 102409PRTHomo sapiens 240His Glu Leu Glu Asn His Ser Met
Tyr1 52419PRTHomo sapiens 241Arg Glu Ala Glu Pro Ile Pro Lys Met1
52429PRTHomo sapiens 242Phe Ser Asp Lys Glu Leu Ala Ala Tyr1
52439PRTHomo sapiens 243Arg Ser Pro Asn Asn Phe Leu Ser Tyr1
52449PRTHomo sapiens 244Arg Ser Asp Pro Val Thr Leu Asn Val1
52459PRTHomo sapiens 245Ile Thr Glu Lys Asn Ser Gly Leu Tyr1
52469PRTHomo sapiens 246Tyr Ser Asp Leu His Ala Phe Tyr Tyr1
524711PRTHomo sapiens 247Arg Ser Asp Pro Gly Gly Gly Gly Leu Ala
Tyr1 5 1024812PRTHomo sapiens 248Tyr Ser His Ala Ala Gly Gln Gly
Thr Gly Leu Tyr1 5 102499PRTHomo sapiens 249Ala Leu Phe Pro Glu Arg
Ile Thr Val1 52509PRTHomo sapiens 250Lys Met Ile Leu Lys Met Val
Gln Leu1 52519PRTHomo sapiens 251Arg Leu Ala Ser Arg Pro Leu Leu
Leu1 525210PRTHomo sapiens 252Arg Ile Tyr Asn Gly Ile Gly Val Ser
Arg1 5 102539PRTHomo sapiens 253Lys Leu Phe Gly Thr Ser Gly Gln
Lys1 52549PRTHomo sapiens 254Ala Val Ala Thr Lys Phe Val Asn Lys1
52559PRTHomo sapiens 255Leu Pro Asp Gly Ser Arg Val Glu Leu1
52569PRTHomo sapiens 256Leu Pro Ala Leu Pro Gln Gln Leu Ile1
52579PRTHomo sapiens 257Ser Pro Leu Arg Gly Gly Ser Ser Leu1
525811PRTHomo sapiens 258Ala Pro Ser Gly Thr Arg Val Val Gln Val
Leu1 5 102599PRTHomo sapiens 259Arg Pro Ala Val Gly His Ser Gly
Leu1 526010PRTHomo sapiens 260Glu Glu Ala Pro Leu Val Thr Lys Ala
Phe1 5 102619PRTHomo sapiens 261Ile Glu Ala Leu Leu Glu Ser Ser
Leu1 52629PRTHomo sapiens 262Met Glu Leu Leu Leu Val Asn Lys Leu1
52639PRTHomo sapiens 263Gln Gln Ala Thr Pro Gly Pro Ala Tyr1
526410PRTHomo sapiens 264Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe1 5
1026510PRTHomo sapiens 265Glu Glu Val Asp Val Pro Ile Lys Leu Tyr1
5 1026610PRTHomo sapiens 266Ala Arg Leu Thr Pro Ile Pro Phe Gly
Leu1 5 102679PRTHomo sapiens 267Lys Thr Leu Gly Lys Leu Trp Arg
Leu1 52689PRTHomo sapiens 268Asp Tyr Ile Pro Tyr Val Phe Lys Leu1
526910PRTHomo sapiens 269Glu Leu Ala Gly Ile Gly Ile Leu Thr Val1 5
102709PRTHomo sapiens 270Tyr Leu Leu Pro Ala Ile Val His Ile1 5
* * * * *
References