NZ788577A - Novel peptides, combination of peptides as targets and for use in immunotherapy against gallbladder cancer and cholangiocarcinoma and other cancers - Google Patents
Novel peptides, combination of peptides as targets and for use in immunotherapy against gallbladder cancer and cholangiocarcinoma and other cancersInfo
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- NZ788577A NZ788577A NZ788577A NZ78857717A NZ788577A NZ 788577 A NZ788577 A NZ 788577A NZ 788577 A NZ788577 A NZ 788577A NZ 78857717 A NZ78857717 A NZ 78857717A NZ 788577 A NZ788577 A NZ 788577A
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Abstract
The present invention relates to peptides comprising the amino acid sequence SLSPDLSQV (SEQ ID NO: 18) and proteins, nucleic acids and cells comprising the same 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 comprising SEQ ID NO: 18, 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. rthermore relates to tumor-associated T-cell peptide epitopes comprising SEQ ID NO: 18, 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.
Description
Novel peptides, combination of peptides as targets and for use in immunotherapy
against gallbladder cancer and cholangiocarcinoma and other cancers
The present application is a divisional application from New d patent application
number 749560, the entire disclosure of which is incorporated herein by way of this
reference.
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 s to tumorassociated
T-cell peptide es, alone or in ation with other tumor-associated
peptides that can for e serve as active pharmaceutical ingredients of vaccine
compositions that stimulate anti-tumor immune ses, 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 dies, soluble T-cell
receptors, and other binding les.
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 / logically active nds and cells.
BACKGROUND OF THE INVENTION
The most common form of biliary tract cancer is an adenocarcinoma of the bile duct
epithelium and includes cholangiocarcinoma (CCC) and gallbladder adenocarcinoma
(GBC). Both diseases are characterized by an increasing incidence and poor outcome.
Cholangiocarcinoma is the second most common liver cancer after cellular
adenocarcinoma (HCC). giocarcinoma can develop in any part of the bile duct
system and is therefore classified into intrahepatic, perihilar and distal. The incidence
varies extremely worldwide with the highest rates in Northeast Thailand (>80 per
100,000 population) and low rates in the Western world (0.3-3 per 100,000)
(Bridgewater et al., 2014). Although it is not very common in western countries,
incidence rates are increasing due to aging populations. In Germany CCC mortality
more than tripled between 1998 and 2008 due to demographic changes (von Hahn et
al., 2011). The average age of people diagnosed with CCC is around 70 years
(American Cancer y, 2015).
Risk s for cho|angiocarcinoma e chronic liver and bile tract diseases such as
primary sing cholangitis, hepatolithiasis, bile duct stones, gallbladder polyps, liver
fluke infections, cirrhosis, but also ions with hepatitis B or C, inflammatory bowel
diseases, older age, obesity, exposure to the radioactive substance Thorotrast, family
history, diabetes and alcohol consumption (World Cancer Report, 2014).
Cholangiocarcinoma is much more common in South-East Asia where parasitic
infections with Clonorchis and Opisthorchis species are endemic. Beyond these regions
characterized by a high incidence of rne liver flukes causing chronic inflammation
of the biliary tree, cho|angiocarcinoma is sporadic and still rather uncommon to rare.
giocarcinoma is mostly identified in advanced stages e it is difficult to
diagnose. Symptoms are unspecific and diagnosis of biliary origin remains challenging
since there is no specific nic marker. ore, diagnosis of CCC requires clinical
and radiological exclusion of metastasis from other sites. Rising levels of serum
markers such as CA19—9 and CEA may be helpful in patients with underlying hepatic
diseases (World Cancer Report, 2014).
Molecular carcinogenesis of CCC includes many known nes and signaling
ys. Activating KRAS mutations, loss-of-function mutations of TP53, FGFR2
fusion genes, lDH1/2 mutations, hypermethylation of p16INK4A and SOCS3, JAK-STAT
activation, over-expression of EGFR/HER2, aberrant MAPK/ERK activation and c-Met
over-expression are commonly found in CCC. The link between chronic biliary infection
and CCC carcinogenesis is thought to be the activation of the lL-6/STAT3 pathway. lL-6
is not only secreted by tumor cells enhancing cell growth through autocrine mechanisms
but also regulates the expression of other genes, such as EGFR (World Cancer Report,
WO 02806
_ 3 _
2014). However, molecular stratification based on these genetic abnormalities is not
ready for clinical use (Bridgewater et al., 2014).
Cholangiocarcinoma is difficult to treat and is usually lethal. The only curative treatment
option is complete resection (R0). Unfortunately, only around 30% of tumors are
able. Most stage 0, l and II, and some stage III tumors are resectable depending
on their exact location, while other stage III and most stage IV tumors are unresectable
can Cancer Society, 2015). The 5-year survival after curative resection (R0) is
40%. There is no ce that adjuvant chemotherapy prolongs 5-year survival after
tumor resection. Lymph node involvement is t in one third of patients eligible for
surgical treatment and is associated with poor surgical outcome. 5-year survival after
non-curative resection (R1) is 20%. Given its prognostic value, denectomy of
regional lymph nodes is ended. While N1 mes still is considered suitable
for surgical management, for N2 and M1 disease surgery is contraindicated
(Bridgewater et al., 2014).
If resection of the tumor is not feasible, treatment options are very limited. Different
palliative chemotherapeutic drugs such as 5-fluorouracil, gemcitabine, cisplatin,
capecitabine, latin are in use (American Cancer Society, 2015). Standard of care
for palliative chemotherapy is combination of gemcitabine and cisplatin. The median
sun/ival after chemotherapy is only 12 months.
Liver transplantation can be indicated for patients with early stage unresectable tumors
but is discussed controversially.
The efficacy of biological therapies in biliary tract cancers has been mixed. Drugs
targeting blood vessel growth such as nib, bevacizumab, pazopanib and
regorafenib are now studied for the treatment of CCC. Additionally, drugs that target
EGFR such as cetuximab and panitumumab are used in clinical studies in combination
with chemotherapy (American Cancer Society, 2015). For most drugs tested so far
disease control and overall survival were not improved significantly but there are further
clinical trials ongoing.
Gallbladder cancer is the most common and sive malignancy of the biliary tract
ide. Unspecific clinical presentation also delays diagnosis and leads to the fact
that only 10% of all ts are candidates for surgery. Due to the anatomical
complexity of the biliary system and the high recurrence rate surgery is only curative in
the minority of cases. Risk factors are similar to CCC but GBC is three times more
common in females. Additionally, to gallbladder pathologies, infections with Salmonella
or Helicobacter are common risk factors. GBC is common in South Americans, lndians,
Pakistani, se and Koreans, while it is rare in the western world. c s
in GBC are poorly understood. Molecular changes such as p53 mutation, COX2
overexpression, CDKN inactivation, KRAS mutations but also microsatellite instability
are thought to be involved in GBC carcinogenesis (Kanthan et al., 2015).
As for GBC only 10% of tumors are resectable and even with surgery most progress to
metastatic disease, prognosis is even worse than for CCC with a 5-year survival of less
than 5%. gh most tumors are unresectable there is still no effective adjuvant
therapy (Rakic et al., 2014). Some studies showed that combination of
chemotherapeutic drugs or combination of targeted y (anti-VEGFR/EGFR) with
chemotherapy led to an increased overall survival and might be promising ent
options for the future (Kanthan et al., 2015).
Due to the rarity of carcinomas of the biliary tract in general there are only a few GBC or
CCC specific studies, while most of them include all biliary tract cancers. This is the
reason why treatment did not improve during the last decades and R0 resection still is
the only curative treatment option.
These unsatisfactory treatment options and low survival rates display the need for
innovative treatment. There are some clinical studies using therapy for the
treatment of CCC and GBC. Success was reported when CCC with lymph node
_ 5 _
metastasis was treated by surgery and post-operative immunotherapy consisting of
CD3-activated T cells and tumor lysate (Higuchi et al., 2006).
For CCC and GBC peptide-based vaccines ing WT1, NUF2, CDH3, KIF20A,
LY6K, TTK, IGFZBP3, or DEPDC1, either as triple/quadruple y or erapy
combined with gemcitabine, increased overall survival about 9-12 months in phase I
al trials. Peptide-based es seem to be well tolerated, but only show a
modest anti-tumor effect when administered as monotherapy. Dendritic cell-based
vaccines targeting MUC1 or WT1 showed even more promising results. Therapies using
cytokine d killer cell monotherapy or tumor infiltrating lymphocytes are in phase
l/ll al trials (Marks and Yee, 2015).
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
adder cancer and cholangiocarcinoma in particular. There is also a need to identify
factors representing biomarkers for cancer in general and gallbladder cancer and
cholangiocarcinoma in particular, leading to better diagnosis of cancer, assessment of
prognosis, and prediction of treatment success.
lmmunotherapy 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.
The current classification of tumor associated antigens (TAAs) ses the following
major groups:
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, onally,
in placenta. Since the cells of testis do not express class I and II HLA molecules, these
antigens cannot be ized by T cells in normal tissues and can therefore be
_ 6 _
considered as logically tumor-specific. Well-known examples for CT antigens
are the MAGE family members and NY-ESO-1.
b) entiation 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.
c) xpressed TAAs: Genes encoding widely expressed TAAs have been detected
in histologically different types of tumors as well as in many normal s, generally
with lower expression levels. It is possible that many of the epitopes processed and
potentially presented by normal s 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. ent examples for this
class of TAAs are Her-2/neu, survivin, telomerase, or WT1.
d) Tumor-specific antigens: These unique TAAs arise from mutations of normal genes
(such as B-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 g the risk for autoimmune
reactions against normal tissues. On the other hand, these TAAs are in most cases only
nt 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.
e) TAAs arising from abnormal post-translational modifications: Such TAAs may arise
from proteins which are r ic nor overexpressed in tumors but nevertheless
become tumor associated by posttranslational processes primarily active in tumors.
Examples for this class arise from altered glycosylation patterns leading to novel
epitopes in tumors as for MUC1 or events like protein splicing during degradation which
may or may not be tumor specific.
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 sed in al oma.
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 ized 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.
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 betamicroglobulin, MHC class
II molecules of an alpha and a beta chain. Their three-dimensional conformation s
in a binding groove, which is used for non-covalent interaction with peptides.
MHC class I molecules can be found on most nucleated cells. They present peptides
that result from proteolytic cleavage of predominantly endogenous proteins, defective
mal products (DRlPs) and larger peptides. However, peptides derived from
endosomal compartments or exogenous s 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 ns that
are taken up by APCs e.g. during tosis, and are subsequently processed.
Complexes of peptide and MHC class I are recognized by CD8—positive T cells bearing
the riate T-cell receptor (TCR), whereas complexes of peptide and MHC class II
les are recognized by CD4-positive-helper—T cells bearing the appropriate TCR.
_ 8 _
It is well known that the TCR, the peptide and the MHC are thereby present in a
stoichiometric amount of 1:1 :1.
CD4-positive helper T cells play an ant 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 ceutical products for triggering anti-tumor immune
responses (anatic 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).
In the absence of inflammation, sion of MHC class II molecules is mainly
restricted to cells of the immune system, ally 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).
Elongated (longer) peptides of the invention can act as MHC class II active epitopes.
T-helper cells, activated by MHC class II epitopes, play an important role in
trating the effector function of CTLs in umor ty. 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 es. In this way
associated T-helper cell peptide epitopes, alone or in combination with other
tumor-associated peptides, can serve as active pharmaceutical ingredients of e
compositions that stimulate anti-tumor immune responses.
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
_ g _
(lFNy) (Beatty and Paterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T
cells as direct anti-tumor effectors uller et al., 2013; Tran et al., 2014).
Since the constitutive expression of HLA class II molecules is usually limited to immune
cells, the possibility of isolating class II es directly from primary tumors was
usly not considered possible. However, Dengjel et al. were successful in
identifying a number of MHC Class II es directly from tumors (
EP 1 760 088 B1).
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 d: MHC class II molecule +
peptide epitope) is important in the development of tumor vaccines.
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 ce of the peptide. MHC-class-l-
binding es 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.
In the MHC class I dependent immune reaction, es not only have to be able to
bind to n MHC class I molecules expressed by tumor cells, they subsequently also
have to be recognized by T cells g specific T cell receptors (TCR).
For proteins to be recognized by T-lymphocytes as tumor-specific or -associated
antigens, and to be used in a therapy, particular uisites 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 rmore
desirable that the tive 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 associated antigens are often derived from proteins directly involved
in ormation 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 ct tumor-associated antigens may also be targets of a
vaccination ch (Singh-Jasuja et al., 2004). It is essential that epitopes are
present in the amino acid ce 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 -response.
lly, 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.
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 e only an individual subpopulation of
epitopes of these antigens are le for such an application since a T cell with a
corresponding TCR has to be t 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 ation with a specific antigen can
be clonally expanded and is able to execute effector functions (“effector T cell”).
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 ary. In these cases, the presentation is the
determining factor.
SUMMARY OF THE INVENTION
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: 32 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: 32, wherein said variant binds to MHC and/or s T cells
cross-reacting with said peptide, or a pharmaceutical acceptable salt thereof, wherein
said peptide is not the underlying full-length polypeptide.
The present invention further relates to a peptide of the present ion comprising a
ce that is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 32
or a variant thereof, which is at least 77%, preferably at least 88%, homologous
rably at least 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 32,
wherein said peptide or t f has an overall length of between 8 and 100,
preferably between 8 and 30, and most preferred of between 8 and 14 amino acids.
The following tables show the peptides according to the t invention, their
respective SEQ ID NOs, and the prospective source (underlying) genes for these
peptides. All peptides in Table 1 and Table 2 bind to HLA-A*O2. The es in Table 2
have been disclosed before in large listings as results of high-throughput screenings
with high error rates or calculated using algorithms, but have not been associated with
cancer at all before. The peptides in Table 3 are additional peptides that may be useful
in combination with the other peptides of the ion. The peptides in Tables 4A and B
are furthermore useful in the diagnosis and/or treatment of various other malignancies
that e an xpression or over-presentation of the respective underlying
polypeptide.
Table 1: Peptides according to the present ion.
SEQ ID No. Sequence GenelD(s) Official Gene Symbol(s)
1 YAAEIASAL figggégi’g?’ SGK1, SGK3, C8orf44-SGK3
2 AAYPEIVAV 348654 GEN1
3 EMDSTVITV 26137 ZBTB20
4 FLLEAQNYL 149281 METTL11B
GLIDEVMVLL 54905 CYP2W1
6 LLLPLLPPLSPS 347252 IGFBPL1
7 LLLSDPDKVTI 3700, 375346 lTlH4, TMEM110
8 RIL 55655 NLRP2
9 RLAKLTAAV 283209 PGM2L1
VTVSL 79939 SLC35E1
11 SIIDFTVTM 1767 DNAH5
12 TILPGNLQSW 80317, 387032 ZKSCAN3, ZKSCAN4
13 VLPRAFTYV 5314 PKHD1
14 YGIEFVVGV 56670 SUCNR1
SVIDSLPEI 79830 ZMYM1
16 AVMTDLPVI 23041 MON2
17 VLYDNTQLQL 389072 PLEKHM3
18 SLSPDLSQV 2153 F5
19 TAYPQWVV 57494 RIMKLB
VLQDELPQL 1953 MEGF6
21 IAFPTSISV 5036 PA2G4
22 SAFGFPVIL 54741 LEPROT
23 SLLSELLGV 11135 CDC42EP1
Table 2: Additional peptides according to the present invention with no prior known
cancer association.
SEQ ID No. Sequence GenelD(s) Official Gene Symbol(s)
24 ISAPLVKTL 994 CDC25B
NLSETASTMAL 25897 RNF19A
26 TAQTLVRIL 3608 lLF2
27 ALAEQVQKA 79078 C1 orf50
28 YASGSSASL 5339 PLEC
SEQ ID No. ce GenelD(s) Official Gene (s)
29 FASEVSNVL 8027 STAM
FASGLIHRV 200185 KRTCAP2
31 IKL 26090 ABHD12
32 FEI 10447, 51384 FAM3C, WNT16
Table 3: Peptides of the invention useful for e.g. personalized cancer therapies.
SEQ ID No. Sequence GenelD(s) Official Gene Symbol(s)
33 ILGTEDLIVEV 79719 AAGAB
34 LLWGNLPEI 729533, 653820 FAM72A, FAM72B
GLIDEVMVL 54905 CYP2W1
36 lLVDWLVQV 9133 CCNB2
37 KIQEMQHFL 4321 MMP12
38 KIQEILTQV 10643 IGF2BP3
The present invention furthermore generally relates to the peptides according to the
present invention for use in the treatment of proliferative diseases, such as, for
e, acute myeloid leukemia, melanoma, small cell lung cancer, non-small cell lung
cancer, non-Hodgkin lymphoma, c lymphocytic leukemia, pancreatic cancer, liver
cancer, ovarian cancer, head and neck cancer, urinary bladder cancer, breast cancer,
and kidney cancer.
Particularly red 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:
32. More preferred are the peptides — alone or in combination - selected from the group
consisting of SEQ ID NO: 1 to SEQ ID NO: 16 (see Table 1), and their uses in the
immunotherapy of gallbladder cancer and cholangiocarcinoma, acute myeloid leukemia,
melanoma, small cell lung cancer, all cell lung cancer, dgkin lymphoma,
chronic lymphocytic leukemia, pancreatic , liver cancer, ovarian cancer, head and
neck cancer, urinary bladder cancer, breast cancer, and kidney cancer, and preferably
adder cancer and cholangiocarcinoma. As shown in the following Tables 4A and
B, many of the peptides according to the present invention are also found on other
tumor types and can, thus, also be used in the immunotherapy of other indications. Also
refer to Figure 1 and Example 1.
WO 02806
Table 4A: Peptides ing to the present invention and their specific uses in other
proliferative diseases, especially in other cancerous diseases. The table shows for
selected peptides on which additional tumor types they were found and either over-
presented on more than 5% of the measured tumor samples, or ted on more
than 5% of the measured tumor s with a ratio of geometric means tumor vs
normal tissues being larger than 3. Over-presentation is defined as higher presentation
on the tumor sample as ed to the normal sample with highest presentation.
Normal tissues against which over-presentation was tested were: adipose tissue,
adrenal gland, artery, bone marrow, brain, central nerve, colon, duodenum, esophagus,
eye, adder, heart, kidney, liver, lung, lymph node, mononuclear white blood cells,
pancreas, parathyroid gland, peripheral nerve, peritoneum, pituitary, , rectum,
salivary gland, skeletal , skin, small intestine, spleen, stomach, thyroid gland,
trachea, ureter, urinary bladder, vein.
SEQ ID No. Sequence Other relevant organs / diseases
1 YAAEIASAL AML, Melanoma
6 LLLPLLPPLSPS SCLC, PC
7 LLLSDPDKVTI HCC
16 AVMTDLPVI CLL, NHL, AML
17 VLYDNTQLQL NHL, AML
18 SLSPDLSQV HCC, NHL, AML
VLQDELPQL NSCLC, NHL, AML, HNSCC, CC
21 IAFPTSISV BRCA
NLSETASTMAL SCLC, NHL, Urinary bladder cancer
26 TAQTLVRIL CLL, BRCA
28 YASGSSASL RCC, AML, Melanoma
29 FASEVSNVL SCLC, RCC, CLL, AML, Melanoma
HRV CLL, AML
31 IAIPFLIKL SCLC, NHL
32 YVISQVFEI CLL, Melanoma
NSCLC= non-small cell lung cancer, SCLC= small cell lung cancer, RCC= kidney
cancer, HCC= liver , PC= pancreatic cancer, BRCA=breast cancer, CLL=chronic
lymphocytic leukemia, AML=acute myeloid leukemia, NHL=non-Hodgkin lymphoma,
OC=ovarian cancer, HNSCC=head and neck cancer.
_ 15 _
Table 4B: Peptides according to the present invention and their ic uses in other
proliferative es, ally in other cancerous diseases. The table shows for
selected peptides on which additional tumor types they were found and either over-
presented on more than 5% of the measured tumor samples, or presented on more
than 5% of the measured tumor samples with a ratio of geometric means tumor vs
normal tissues being larger than three. Over-presentation is defined as higher
presentation on the tumor sample as compared to the normal sample with highest
presentation. Normal tissues t which over-presentation was tested were: adipose
tissue, adrenal gland, artery, bone marrow, brain, central nerve, colon, esophagus, eye,
gallbladder, heart, kidney, liver, lung, lymph node, white blood cells, pancreas,
yroid gland, eral nerve, peritoneum, pituitary, pleura, rectum, salivary
gland, skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid gland,
trachea, ureter, urinary bladder, vein.
SEQ ID
No Sequence Additional Entities
6 LLLPLLPPLSPS Brain Cancer
17 VLYDNTQLQL CLL, OC, Urinary bladder cancer
24 ISAPLVKTL CLL
31 IAIPFLIKL HCC, BRCA
32 YVISQVFEI NSCLC
NSCLC: non-small cell lung cancer, HCC= liver cancer, BRCA=breast cancer,
CLL=chronic lymphocytic leukemia, OC=ovarian cancer.
Thus, another aspect of the present invention s to the use of at least one peptide
according to the present ion according to any one of SEQ ID No. 1, 16, 17, 18, 20,
28, 29, and 30 for the — in one red embodiment combined - treatment of acute
myeloid leukemia.
Thus, another aspect of the present invention relates to the use of at least one peptide
according to the present invention according to any one of SEQ ID No. 1, 28, 29, and 32
for the — in one preferred embodiment combined - treatment of melanoma.
_ 16 _
Thus, another aspect of the present ion relates to the use of at least one peptide
according to the present invention ing to any one of SEQ ID No. 6, 25, 29, and 31
for the — in one preferred embodiment combined - treatment of small cell lung cancer.
Thus, another aspect of the present invention relates to the use of at least one peptide
according to the present invention according to any one of SEQ ID No. 32, and 20 for
the — in one preferred embodiment combined - treatment of non-small cell lung cancer.
Thus, r aspect of the present invention relates to the use of at least one peptide
according to the t invention according to any one of SEQ ID No. 16, 17, 18, 20,
, and 31 for the — in one preferred embodiment combined - treatment of non-Hodgkin
lymphoma.
Thus, another aspect of the present invention relates to the use of at least one peptide
according to the present invention according to any one of SEQ ID No. 16, 26, 29, 30,
and 32 for the — in one preferred embodiment combined - treatment of chronic
lymphocytic leukemia.
Thus, another aspect of the present invention relates to the use of at least one e
according to the present invention according to any one of SEQ ID No. 6 for the — in one
preferred embodiment ed - treatment of pancreatic cancer.
Thus, another aspect of the present invention relates to the use of at least one peptide
according to the present invention according to any one of SEQ ID No. 7, 31, and 18 for
the — in one preferred embodiment combined - treatment of liver cancer.
Thus, another aspect of the t invention relates to the use of at least one peptide
according to the present invention ing to any one of SEQ ID No. 17, and 20 for
the — in one red embodiment combined - treatment of ovarian cancer.
Thus, another aspect of the present invention relates to the use of at least one peptide
according to the present invention according to any one of SEQ ID No. 20 for the — in
one preferred embodiment ed - ent of head and neck cancer.
Thus, another aspect of the present ion relates to the use of at least one peptide
according to the present invention according to any one of SEQ ID No. 17, and 25 for
the — in one preferred embodiment combined - treatment of urinary bladder cancer.
Thus, another aspect of the present invention relates to the use of at least one peptide
according to the present invention according to any one of SEQ ID No. 21, 31, and 26
for the — in one preferred embodiment combined - treatment of breast cancer.
Thus, another aspect of the present invention relates to the use of at least one peptide
ing to the present invention according to any one of SEQ ID No. 28, and 29 for
the — in one preferred embodiment combined - treatment of kidney cancer.
Thus, another aspect of the present invention relates to the use of at least one peptide
ing to the t invention according to any one of SEQ ID No. 17, and 24 for
the — in one red embodiment combined - treatment of CLL.
Thus, r aspect of the present invention relates to the use of the peptides
according to the present invention for the - preferably combined - treatment of a
erative e selected from the group of gallbladder cancer and
cholangiocarcinoma, acute myeloid leukemia, melanoma, small cell lung cancer, non-
small cell lung cancer, non-Hodgkin lymphoma, chronic lymphocytic leukemia,
pancreatic cancer, liver cancer, ovarian cancer, head and neck cancer, urinary bladder
cancer, breast cancer, and kidney cancer.
The present invention furthermore s to es according to the present invention
that have the ability to bind to a molecule of the human major histocompatibility complex
(MHC) class-l or - in an elongated form, such as a length-variant - MHC class -II.
The present invention further s to the peptides ing 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: 32.
The present invention further relates to the peptides according to the present invention,
n said peptide is modified and/or includes non-peptide bonds.
The t invention further relates to the peptides according to the present invention,
wherein said peptide is part of a fusion protein, in particular fused to the N-terminal
amino acids of the HLA-DR antigen-associated invariant chain (Ii), or fused to (or into
the sequence of) an antibody, such as, for example, an antibody that is specific for
dendritic cells.
The present invention further relates to a nucleic acid, ng the peptides according
to the present invention. The present ion further relates to the nucleic acid
according to the present invention that is DNA, cDNA, PNA, RNA or ations
thereof.
The present invention further relates to an expression vector capable of expressing
and/or expressing a nucleic acid according to the present invention.
The present invention r s 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 es and in medicine, in particular in
the treatment of cancer.
The present invention further relates to antibodies that are specific against the peptides
ing to the present ion or complexes of said peptides according to the
present invention with MHC, and methods of making these.
WO 02806
_ 1g _
The present invention further relates to T-cell receptors (TCRs), in particular e
TCR (sTCRs) and cloned TCRs engineered into autologous or allogeneic T cells, and
s of making these, as well as NK cells or other cells bearing said TCR or cross-
reacting with said TCRs.
The antibodies and TCRs are additional embodiments of the therapeutic use of
the peptides according to the invention at hand.
The t 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 ion that is an
n presenting cell, and preferably is a dendritic cell.
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.
The present ion 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 n-presenting cell or artificial antigen-presenting cell by
contacting a sufficient amount of the antigen with an antigen-presenting cell.
The present invention further relates to the method according to the present invention,
wherein the antigen-presenting cell comprises an expression vector capable of
sing or expressing said peptide containing SEQ ID No. 1 to SEQ ID No.: 32,
preferably containing SEQ ID No. 1 to SEQ ID No. 16, or a variant amino acid
sequence.
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.
The t invention further relates to a method of killing target cells in a t which
target cells aberrantly express a polypeptide comprising any amino acid sequence
according to the present invention, the method sing administering to the patient
an effective number of T cells as produced according to the t invention.
The present invention further s 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 ing to the present invention, the activated T lymphocyte, the T
cell receptor or the antibody or other peptide- and/or e-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.
Preferably, said medicament is a cellular therapy, a vaccine or a protein based on a
soluble TCR or antibody.
The present ion further relates to a use according to the present invention,
wherein said cancer cells are adder cancer and cholangiocarcinoma, acute
myeloid leukemia, ma, small cell lung cancer, non-small cell lung cancer, non-
Hodgkin lymphoma, chronic lymphocytic leukemia, pancreatic cancer, liver cancer,
ovarian cancer, head and neck cancer, urinary bladder cancer, breast cancer, and
kidney cancer, and preferably gallbladder cancer and cholangiocarcinoma cells.
The present invention further relates to biomarkers based on the peptides according to
the present invention, herein called ts” that can be used in the diagnosis of
cancer, preferably gallbladder cancer and cholangiocarcinoma. The marker can be
over-presentation of the peptide(s) themselves, or over-expression of the corresponding
gene(s). The s 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.
Optionally the antibody carries a r effector function such as an immune stimulating
domain or toxin.
The present invention also s to the use of these novel targets in the context of
cancer treatment.
Both therapeutic and diagnostic uses against additional cancerous diseases are
disclosed in the following more detailed description of the underlying sion
ts eptides) of the peptides according to the invention.
CDC2SB is known to be a downstream target of the oncogenic transcription factor
FoxM1. FoxM1 and its downstream target ors are down-regulated in gastric
cancer, gliomas, cholangiocarcinoma, and acute myeloid leukemia (Zhang et al., 2014a;
Chan-On et al., 2015; Niu et al., 2015; Li et al., 2016). MicroRNA-211 was shown to be
a direct negative regulator of CDC2SB in triple-negative breast cancer cells. The loss of
miRNA-211 and the resulting increase of CDC2SB expression lead to increased
genomic instability (Song and Zhao, 2015). CDC2SB was shown to be up-regulated in
gastric cancer cells by YWHAE silencing inducing cell proliferation, invasion and
migration (Leal et al., 2016). CDC2SB was shown to be down-regulated by the
bromodomain inhibitor JQ1 which suppresses growth of pancreatic ductal
adenocarcinoma in patient-derived xenograft models (Garcia et al., 2016). CDC2SB was
shown to be down-regulated in small intestinal neuroendocrine tumors (Kim et al.,
2016b).
CYP2W1 is over-expressed in a variety of human cancers including hepatocellular,
colorectal and gastric cancer. CYP2W1 over-expression is associated with tumor
ssion and poor survival (Aung et al., 2006; Gomez et al., 2010; Zhang et al.,
2014b). Due to tumor-specific expression, CYP2W1 is an interesting drug target or
enzymatic tor of pro-drugs during cancer therapy (Karlgren and lngelman-
Sundberg, 2007; Nishida et al., 2010).
The DNAH5 gene was ed to be recurrently mutated in myeloma and its
expression was shown to be commonly dysregulated in colorectal cancer (Walker et al.,
2012; Xiao et al., 2015).
It was shown that venous thromboembolism (VTE) occurs frequently in cancer patients.
A combination of F5 variants that are associated with VTE and cancer synergistically
increases the risk of VTE (Gran et al., 2016). The gulant state in cancer
increases the thrombotic risk, but also supports tumor progression. Four SNPs in F5
were shown to be associated with breast cancer. Therefore, targeting the ation
processes in cancer is of high importance (Tinholt et al., 2014). It was shown that F5
mutation is a risk factor for thromboemboli occurrence in children with acute
lymphoblastic leukemia (Sivaslioglu et al., 2014). F5 was shown to be a ate
serum biomarker for prostate adenocarcinoma (Klee et al., 2012).
A change in expression of FAMC3 has been noted in pancreatic cancer-derived cells
(RefSeq, 2002). In melanoma, FAMC3 has been identified as a candidate biomarker for
agy, an important tumor cell survival mechanism (Kraya et al., 2015). FAMC3
plays an essential role in the lial-mesenchymal transition which correlates with
aggressiveness, metastatic progression of tumors and poor survival especially in
cellular cancer, colorectal , lung and breast cancers (Csiszar et al., 2014;
Gao et al., 2014; Song et al., 2014; Chaudhury et al., 2010; Lahsnig et al., 2009).
Mutations in GEN1 have been reported to be associated with breast cancer risk, but
other studies could not confirm the role of GEN1 as a breast cancer predisposition gene
(Kuligina et al., 2013; Sun et al., 2014; Turnbull et al., 2010).
IGFBPL1 is a regulator of insulin-growth factors and is down-regulated in breast cancer
cell lines by aberrant ethylation. ation in |GFBPL1 was clearly associated
with worse overall survival and disease-free survival (Smith et al., 2007).
lLF2, also known as NF45, encodes a transcription factor required for T-cell expression
of the interleukin 2 gene (RefSeq, 2002). lLF2 was shown to be up-regulated in
hepatocellular carcinoma, pancreatic ductal adenocarcinoma and non-small cell lung
cancer (Ni et al., 2015; Wan et al., 2015; Cheng et al., 2016). Expression of lLF2 in liver
cancer cells was described as being associated with the tion of cell growth and
apoptosis via regulation of Bcl-2, Bok, BAX, and clAP1 (Cheng et al., 2016). Expression
of lLF2 was shown to correlate with tumor size, histological differentiation and TNM
stage, while xpression of lLF2 was shown to be associated with poor prognosis of
pancreatic ductal adenocarcinoma. The differentiated expression of lLF2 in pancreatic
ductal adenocarcinoma cell cultures showed effects on cell cycle progression (Wan et
al., 2015). Up-regulated sion of lLF2 in all cell lung cancer was shown to
be associated with tumor cell proliferation and poor prognosis (Ni et al., 2015).
|T|H4 is a member of the lTl family of plasma protease inhibitors that contribute to
extracellular matrix stability by covalent linkage to hyaluronan. |T|H4 was down-
regulated in l tumor s including colon, stomach, ovary, lung, kidney, rectum
and prostate (Hamm et al., 2008). Serum lT|H4 levels are reduced in HCC patients
compared to that in chronic hepatitis B and cirrhosis patients, and low serum |T|H4
levels are associated with shorter survival in HBV-associated HCC patients (Noh et al.,
2014).
KRTCAP2 encodes keratinocyte associated protein 2 and is localized on chromosome
1q22 (RefSeq, 2002). Studies uncovered a cancer-enriched chimeric RNA as the result
of splicing between MUC1, TRIM46, and KRTCAP2 in high-grade serous ovarian
cancer (HGSC) cells, which might be used as a al biomarker and therapeutic target
(Kannan et al., 2015).
MEGF6, also known as EGFL3, encodes the multiple EGF like domains 6 protein and is
located on chromosome 1p36.3 (RefSeq, 2002). MEGF6 was described as a
hepatocellular carcinoma-related gene which shows several polymorphisms in the
tissues of hepatocellular carcinomas (Wang et al., 2005).
NLRP2 (also known as NALP2) encodes the NLR family, pyrin domain containing 2
protein and is involved in the activation of caspase-1 and may also form protein
complexes activating proinflammatory caspases. NLRP7 is a paralog of NLRP2
(RefSeq, 2002; Wu et al., 2010; Slim et al., 2012). The PYRIN domain of NLRP2 inhibits
cell proliferation and tumor growth of glioblastoma (Wu et al., 2010). An
ATM/NLRP2/MDC1-dependent pathway may shut down ribosomal gene ription in
response to chromosome breaks (Kruhlak et al., 2007). ons in NLRP2 can cause
rare human imprinting disorders such as familial hydatidiform mole, th-
ann syndrome and familial transient neonatal es mellitus (Aghajanova et
al., 2015; Dias and Maher, 2013; Ulker et al., 2013). NLRP2 inhibits paB
activation (Kinoshita et al., 2005; ita et al., 2006; Fontalba et al., 2007; Bruey et
aL,2004)
PA2G4 s proliferation-associated 2G, an RNA-binding protein that is ed in
growth regulation and might be involved in ribosome assembly. It has been implicated in
induction of differentiation of human cancer cells (RefSeq, 2002). PA2G4 was identified
to be down-regulated in esophageal squamous cell carcinoma. Over-expression of
PA2G4 inhibited the tumorigenesis and growth of the cells and induced apoptosis.
These results indicate that PA2G4 may suppress the growth of esophageal carcinoma
cells (Jiang et al., 2016). PA2G4 was shown to be up-regulated in cervical cancer
tissues and might be ed with p53 expression levels an effective predictor of
metastatic potential and patient prognosis (Liu et al., 2015c; Liu et al., 2015b). PA2G4
was shown to be up-regulated in pancreatic ductal adenocarcinoma and could serve as
a prognostic indicator and potential target (Gong et al., 2015). Forced PA2G4
expression was shown to suppress growth, ion and invasion in thyroid cancer
cells by up-regulating a major tumor-suppressor RASAL (Liu et al., 2015a). PA2G4 has
been reported to be down-regulated in hepatocellular oma (HCC) and might serve
as a prognostic marker and promising therapeutic target of HCC (Hu et al., 2014).
PKHD1 encodes polycystic kidney and hepatic disease 1. Mutations in this gene cause
autosomal recessive polycystic kidney disease (RefSeq, 2002). PKHD1 was seen to
have loss of function mutations in anap|astic d carcinoma (Jeon et al., 2016).
PLEC encodes the plakin family member plectin, a protein involved in the cross-linking
and organization of the cytoskeleton and adhesion complexes (Bouameur et al., 2014).
PLEC is over-expressed in colorectal adenocarcinoma, head and neck squamous cell
carcinoma and pancreatic cancer (Lee et al., 2004; Katada et al., 2012; Bausch et al.,
2011).
RNF19A encodes ring finger n 19A, RBR E3 ubiquitin n ligase. The encoded
protein may be involved in amyotrophic lateral sis and Parkinson’s disease
(RefSeq, 2002). RNF19A mRNA levels were shown to be 2-fold higher in the blood of
patients with prostate cancer than in healthy controls. Therefore, RNF19A might be a
relevant biomarker for prostate cancer detection (Bai et al., 2012). RNF19A was
identified being differentially expressed in cancer-associated fibroblasts that are
important for cancer development and progression (Bozoky et al., 2013).
SGK1 encodes a serine/threonine protein kinase that plays an important role in cellular
stress se. It activates certain ium, sodium, and chloride channels,
suggesting an involvement in the regulation of processes such as cell survival, neuronal
excitability, and renal sodium excretion. High levels of expression may contribute to
conditions such as hypertension and diabetic pathy. SGK1 can be activated by
insulin and growth factors via PI3K and PDK1 (RefSeq, 2002). SGK1 expression is
rapidly up-regulated by glucocorticoid stration which may decrease
chemotherapy effectiveness in n cancer. In turn, the isoflavinoid Genistein has
been found to have an inhibitory effect on colorectal cancer by reducing SGK1
_ 26 _
expression (Melhem et al., 2009; Qin et al., 2015). Increased SGK1 expression has
been found in several human tumors, including te carcinoma, non-small cell lung
cancer and hepatocellular carcinoma. SGK1 has anti-apoptotic properties and regulates
cell survival, proliferation and differentiation via phosphorylation of MDM2, which leads
to the ubiquitination and proteasomal degradation of p53. Direct SGK1 inhibition can be
effective in hepatic cancer therapy, either alone or in combination with radiotherapy
(Lang et al., 2010; Abbruzzese et al., 2012; lsikbay et al., 2014; Talarico et al., 2015).
SGK3 encodes a phosphoprotein of the Ser/Thr protein kinase family which
phosphorylates several target proteins and has a role in neutral amino acid transport
and activation of potassium and chloride channels (RefSeq, 2002). SGK3 function was
shown to be associated with the oncogenic driver INPP4B in colon cancer and in breast
cancer (Gasser et al., 2014; Guo et al., 2015). SGK3 was bed as a down-stream
mediator of atidylinositol 3-kinase oncogenic signaling which mediates pivotal
roles in oncogenic progress in various cancers, including breast cancer, n cancer
and hepatocellular carcinoma (Hou et al., 2015). SGK3 was described to serve as a
hallmark cting with numerous molecules in cell proliferation, growth, ion and
tumor angiogenesis (Hou et al., 2015). SGK3 was shown to promote hepatocellular
carcinoma growth and survival through inactivating glycogen synthase kinase 3 beta
and Bclassociated death promoter, respectively (Liu et al., 2012). SGK3 was shown
to be associated with poor e in hepatocellular carcinoma patients (Liu et al.,
2012). Thus, SGK3 may provide a stic biomarker for hepatocellular carcinoma
outcome prediction and a novel therapeutic target (Liu et al., 2012). SGK3 was
described as an important mediator of PDK1 activities in melanoma cells which
contributes to the growth of BRAF-mutant melanomas and may be a potential
therapeutic target (Scortegagna et al., 2015). SGK3 was described as an androgen
receptor riptional target that promotes prostate cell proliferation through activation
of p70 86 kinase and up-regulation of cyclin D1 (Wang et al., 2014). Knock-down of
SGK3 was shown to se LNCaP prostate cancer cell eration by inhibiting G1
to S phase cell cycle progression (Wang et al., 2014). SGK3 was shown to be
associated with estrogen receptor expression in breast cancer and its expression was
shown to be positively ated with tumor prognosis (Xu et al., 2012).
SLC35E1 s the n solute carrier family 35, member E1 and is d on
some 19p13.11 (RefSeq, 2002). SLC35E1 was shown to be associated with
rectal carcinoma se to neoadjuvant radiochemotherapy (Rimkus et al., 2008).
STAM encodes a member of the signal-transducing adaptor molecule family that
mediates down-stream signaling of cytokine receptors and also plays a role in ER to
Golgi trafficking. STAM associates with hepatocyte growth factor-regulated substrates
to form the endosomal sorting complex required for transport-0 (ESCRT-O), which sorts
ubiquitinated membrane proteins to the ESCRT-1 x for lysosomal degradation
(RefSeq, 2002). STAM has been found to be xpressed in locally ed
cen/ical cancer and in tumors in young patients with spinal ependymomas (Korshunov
et al., 2003; Campos-Parra et al., 2016). STAM is a ream target of ZNF331, a
gene down-regulated in gastric cancer, which then leads to down-regulation of STAM as
well (Yu et al., 2013). STAM has been associated with the unfavorable 11q deletion in
chronic lymphocytic leukemia (Aalto et al., 2001).
WNT16, wingless-type MMTV integration site family, member 16 encodes a secreted
signaling protein which is implicated in oncogenesis and in several developmental
processes, including regulation of cell fate and patterning during embryogenesis
(RefSeq, 2002). The expression of WNT16 was shown to be up-regulated in t(1;19)
chromosomal translocation-containing acute lymphoblastoid leukemia (ALL) and play an
important role in leukemogenesis (Casagrande et al., 2006; Mazieres et al., 2005). A
study of ALL cell lines and samples from patients with ALL showed that the up-
regulation of WNT16 and few other Wnt target genes was caused by the methylation of
Wnt inhibitors which was further associated with significantly decreased 10-year
disease-free sun/ival and overall survival (Roman-Gomez et al., 2007).
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_ 28 _
ZBTB20 encodes zinc finger and BTB domain containing 20 and is d on
chromosome 3q13.2 (RefSeq, 2002). ZBTB20 promotes cell proliferation in non-small
cell lung cancer through repression of FoxO1 (Zhao et al., 2014). ZBTB20 sion is
increased in hepatocellular carcinoma and associated with poor prognosis (Wang et al.,
2011). Polymorphism in ZBTB20 gene is associated with gastric cancer (Song et al.,
2013).
ZKSCAN3 encodes zinc finger with KRAB and SCAN domains 3 and is located on
chromosome 6p22.1 (RefSeq, 2002). ZKSCAN3 is ulated in invasive colonic
tumor cells and their liver metastases. ZKSCAN3 is sed in a majority of prostate
cancer samples, but not in normal prostate tissues. ZKSCAN3 gene amplification was
observed in metastatic prostate cancers and lymph node metastases but not in primary
prostate s. ZKSCAN3 plays a critical role in promoting prostate cancer cell
migration (Zhang et al., 2012). ZKSCAN3 is a driver of colon cancer progression which
regulates the expression of several genes favoring tumor progression (Yang et al.,
2008). ZKSCAN3 mutation contributes to myelomagenesis as well as ormation
from myeloma to overt extramedullary disease such as secondary plasma cell leukemia
(Egan et al., 2012). ZKSCAN3 suppression reduces cyclin D2 levels and ts
myeloma cell line proliferation. ZKSCAN3 over-expression induces cyclin D2 in
a cell lines and primary samples (Yang et al., 2011).
ZKSCAN4, also known as ZNF307, encodes zinc finger with KRAB and SCAN domains
4 and is located on chromosome 6p21 (RefSeq, 2002). ZKSCAN4 might suppress the
p53-p21 pathway through activating MDM2 and EP300 expression and inducing p53
degradation (Li et al., 2007).
ZMYM1 encodes zinc finger MYM-type containing 1 and is located on chromosome
1p34.3 (RefSeq, 2002). ZMYM1 is a major interactor of ZNF131 which acts in estrogen
signaling and breast cancer eration (Oh and Chung, 2012; Kim et al., 2016a).
DETAILED PTION OF THE INVENTION
Stimulation of an immune response is dependent upon the presence of antigens
recognized as n by the host immune system. The ery 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.
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 ns or defect
ribosomal products (DRIPS) located in the cytosol, play an ant role in this
response. The MHC-molecules of the human are also ated as human leukocyte-
ns (HLA).
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 xic T
cells, effector functions may be lysis of peptide-pulsed, peptide-precursor pulsed or
naturally peptide-presenting target cells, secretion of cytokines, preferably Interferongamma
, TNF-alpha, or lL-2 induced by e, secretion of effector molecules,
preferably granzymes or ins induced by peptide, or degranulation.
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 amino and
yl 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.
Furthermore, the term “peptide” shall include salts of a series of amino acid residues,
connected one to the other typically by e 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 e
(trifluoroacetate) salts. It has to be noted that the salts of the es according to the
present ion differ substantially from the peptides in their state(s) in vivo, as the
peptides are not salts in vivo.
The term “peptide” shall also include “oligopeptide”. The term peptide” is used
herein to designate a series of amino acid es, 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.
The term eptide” 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
eptide, the term polypeptide is meant to refer to les containing more than
about 30 amino acid residues.
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 e
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
_ 31 _
MHC, oligopeptide, and/or protein that is used to raise specific antibodies or TCRs
against it.
A class I T cell “epitope” requires a short e that is bound to a class I MHC
receptor, forming a ternary complex (MHC class I alpha chain, betamicroglobulin,
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.
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 ns (HLA)):
HLA-A, HLA-B, and HLA-C. 01, 02, and HLA-B*07 are examples of
different MHC class I alleles that can be expressed from these loci.
Table 5: Expression frequencies F of HLA-A*02 and HLA-A*24 and the most frequent
HLA-DR serotypes. ncies are deduced from haplotype frequencies Gf within the
American population adapted from Mori et al. (Mori et al., 1997) ing the Hardy-
Weinberg formula F = 1 — )2. Combinations of A*02 or A*24 with certain HLA-DR
alleles might be ed or less frequent than expected from their single frequencies
due to linkage disequilibrium. For details refer to Chanock et al. (Chanock et al., 2004).
Allele Population Calculated phenotype from
allele frequency
A*02 Caucasian (North America) 49.1%
A*02 African American (North America) 34.1%
A*02 Asian American (North America) 43.2%
A*02 Latin American (North American) 48.3%
DR1 ian (North America) 19.4%
DR2 ian ( North America ) 28.2%
DR3 Caucasian ( North America ) 20.6%
DR4 Caucasian ( North America ) 30.7%
DR5 Caucasian (North America) 23.3%
DR6 Caucasian ( North America ) 26.7%
DR7 Caucasian ( North America ) 24.8%
_ 32 _
Allele Population Calculated phenotype from
allele frequency
DR8 Caucasian (North America) 5.7%
DR9 Caucasian (North America) 2.1%
DR1 African (North) American 13.20%
DR2 African (North) American 29.80%
DR3 African (North) an 24.80%
DR4 African (North) American 11.10%
DR5 African (North) American 31.10%
DR6 African (North) an 33.70%
DR7 African (North) American 19.20%
DR8 African (North) American 12.10%
DR9 African (North) American 5.80%
DR1 Asian (North) an 6.80%
DR2 Asian (North) American 33.80%
DR3 Asian (North) American 9.20%
DR4 Asian (North) American 28.60%
DR5 Asian (North) American 30.00%
DR6 Asian (North) American 25.10%
DR7 Asian (North) American 13.40%
DR8 Asian (North) American 12.70%
DR9 Asian (North) an 18.60%
DR1 Latin (North) American 15.30%
DR2 Latin (North) American 21.20%
DR3 Latin (North )American 15.20%
DR4 Latin (North can 36.80%
DR5 Latin (North) American 20.00%
DR6 Latin ) American 31.10%
DR7 Latin (North) American 20.20%
DR8 Latin (North) American 18.60%
DR9 Latin (North) American 2.10%
A*24 Philippines 65%
A*24 Russia Nenets 61%
A*24:02 Japan 59%
A*24 Malaysia 58%
2 Philippines 54%
A*24 India 47%
A*24 South Korea 40%
A*24 Sri Lanka 37%
A*24 China 32%
_ 33 _
Allele Population Calculated phenotype from
allele frequency
A*24:02 India 29%
A*24 lia West 22%
A*24 USA 22%
A*24 Russia Samara 20%
A*24 South America 20%
A*24 Europe 18%
The peptides of the invention, preferably when included into a e of the invention
as described herein bind to A*02. A vaccine may also include pan-binding MHC class II
peptides. ore, the vaccine of the invention can be used to treat cancer in patients
that are A*02 positive, whereas no selection for MHC class II allotypes is necessary due
to the pan-binding nature of these peptides.
|f A*02 peptides of the invention are combined with peptides binding to another allele,
for example A*24, a higher tage of any patient tion 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 tion. 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).
In a preferred embodiment, the term “nucleotide sequence” refers to a heteropolymer of
deoxyribonucleotides.
The nucleotide sequence coding for a particular e, 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 e a synthetic gene that is capable of being expressed in a
_ 34 _
recombinant transcriptional unit comprising regulatory elements derived from a
microbial or viral operon.
As used herein the term “a tide 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 sed by,
for example, a tic cell or r cell system useful for the production of TCRs.
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 e stranded DNA) and the
ment of such sequence.
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.
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 sized in the
laboratory using methods well known to those of skill in the art of DNA synthesis.
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 c code degeneracy and thus coding for the same amino acid(s).
The term “fragment”, when referring to a coding sequence, means a portion of DNA
comprising less than the complete coding region, whose expression product s
essentially the same biological function or activity as the sion product of the
complete coding region.
The term “DNA segment” refers to a DNA polymer, in the form of a te 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 tration enabling identification, manipulation, and recovery of
the segment and its component nucleotide ces by rd biochemical
methods, for example, by using a cloning vector. Such segments are provided in the
form of an open g frame uninterrupted by internal non-translated ces, or
introns, which are typically present in eukaryotic genes. ces 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.
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.
The term “promoter” means a region of DNA involved in binding of RNA polymerase to
initiate transcription.
The term “isolated” means that the material is removed from its original environment
(e.g., the natural environment, if it is lly 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 ed in that such vector or composition is not part of its natural environment.
The polynucleotides, and recombinant or genic 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 ve 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 neity. 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.
The nucleic acids and ptide expression products disclosed ing to the
present invention, as well as expression vectors ning such nucleic acids and/or
such polypeptides, may be in hed 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%,
%, and 20% by weight are also contemplated. The sequences, constructs, vectors,
clones, and other materials comprising the t 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 se
(i.e., has immunogenic activity) when administered, alone or optionally with a suitable
adjuvant or in a , to an , such as a , 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 e fragment" may also be used to induce a T-cell response in vitro.
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 eptides resulting from such treatment would represent portions,
segments or nts of the starting polypeptide. When used in relation to
polynucleotides, these terms refer to the products produced by ent of said
polynucleotides with any of the endonucleases.
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 ce to be compared (the
“Compared Sequence”) with the described or claimed sequence (the “Reference
ce”). The percent identity is then determined according to the following formula:
percent identity = 100 [1 -(C/R)]
wherein C is the number of differences n the Reference Sequence and the
ed Sequence over the length of alignment between the Reference Sequence
and the Compared Sequence, wherein
(i) each base or amino acid in the Reference Sequence that does not have a
corresponding aligned base or amino acid in the Compared Sequence and
(ii) each gap in the Reference Sequence and
(iii) each aligned base or amino acid in the Reference Sequence that is different from an
aligned base or amino acid in the Compared Sequence, constitutes a difference and
(iiii) the alignment has to start at position 1 of the aligned sequences;
and R is the number of bases or amino acids in the Reference Sequence over the
length of the alignment with the ed Sequence with any gap created in the
Reference Sequence also being counted as a base or amino acid.
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
ied minimum Percent ldentity then the ed 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
As mentioned above, the present invention thus provides a peptide comprising a
ce that is selected from the group of consisting of SEQ ID NO: 1 to SEQ ID NO:
_ 38 _
32 or a variant thereof which is 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 32, or
a variant f 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-l or elongated versions of said es to class II.
In the present invention, the term “homologous” refers to the degree of ty (see
percent identity above) between sequences of two amino acid sequences, i.e. peptide
or polypeptide sequences. The aforementioned “homology” is ined 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
e, the ClustalW algorithm. Commonly available sequence analysis software,
more ically, Vector NTI, GENETYX or other tools are ed by public
databases.
A person skilled in the art will be able to assess, r 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).
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: 32. For example, a peptide may be modified
so that it at least maintains, if not es, 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.
These T cells can subsequently cross-react with cells and kill cells that express a
polypeptide that contains the natural amino acid ce of the cognate peptide as
defined in the aspects of the ion. As can be derived from the scientific literature
_ 39 _
and databases (Rammensee et a|., 1999; Godkin et al., 1997), certain positions of HLA
g es 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 ties 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 32, 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.
The original (unmodified) peptides as sed 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 ure 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 al nature, such as where leucine is replaced by
isoleucine. In s of sequence variations in es of naturally occurring
homologous proteins, certain amino acid substitutions are more often tolerated than
others, and these are often show correlation with rities in size, charge, polarity,
and hydrophobicity between the original amino acid and its ement, and such is
the basis for defining rvative substitutions.”
Conservative substitutions are herein defined as exchanges within one of the ing
five groups: Group 1-small aliphatic, nonpolar or slightly polar residues (Ala, Ser, Thr,
Pro, Gly); Group 2-polar, negatively d 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, lle, Val, Cys); and Group e, aromatic
residues (Phe, Tyr, Trp).
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 cine residue. Highly non-conservative
replacements might e substituting an acidic amino acid for one that is polar, or
even for one that is basic in character. Such al” 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.
Of course, such substitutions may involve structures other than the common o
acids. Thus, D-amino acids might be substituted for the L-amino acids commonly found
in the antigenic es of the invention and yet still be assed 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
If substitutions at more than one position are found to result in a peptide with
substantially lent or greater antigenic activity as defined below, then
combinations of those substitutions will be tested to ine 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.
A e 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-l or —II is ntially changed or is negatively
affected, when compared to the non-modified peptide. In another embodiment, in a
peptide ting 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-l or —II is substantially changed, or is negatively
affected, when compared to the non-modified peptide.
The amino acid residues that do not substantially contribute to interactions with the T-
cell or can be modified by replacement with other amino acid whose incorporation
does not substantially affect T-cell vity and does not eliminate binding to the
relevant MHC. Thus, apart from the proviso given, the peptide of the invention may be
any e (by which term the inventors include oligopeptide or polypeptide), which
includes the amino acid sequences or a portion or variant thereof as given.
Table 6: Variants and motif of the peptides according to SEQ ID NO: 4, 5, and 7
Position 1234567891011
SEQIDNO.4 NYL
Variants V
—|—|—|—|<<<<>>>>§§§§ V
Position 1 2 3 4 5 6 7 8 9 1O 11
Q V
Q I
Q A
on 1 2 3 4 5 6 7 8 9 1O 11
SEQ ID NO.5 G L | D E V M V L L
Variants V
M V
M |
M A
A V
A |
A A
V V
V |
V A
T V
T |
T A
Q V
Q I
Q A
Position 1 2 3 4 5 6 7 8 9 1O 11
SEQ ID NO? L L L S D P D K V T |
Variants V
M V
M L
M A
WO 02806
PosMon OOOO—l—l—l—l<<<<>>>>l\> 1O
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 es that flank the actual epitope are residues that do not
ntially affect proteolytic cleavage ary to expose the actual epitope during
processing.
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 7: Combinations of the elongations of peptides of the invention
C-terminus N-terminus
4 O
3 0 or 1
2 0 or 1 or 2
1 O or 1 or 2 or 3
0 Oor1or2or3or4
N-terminus C-terminus
C-terminus N-terminus
O—\l\)OO-l> O
Oor1or2
Oor1or20r3or4
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.
Thus, the epitopes of the present invention may be identical to naturally ing
tumor-associated or tumor-specific epitopes or may include epitopes that differ by no
more than four residues from the reference peptide, as long as they have substantially
identical antigenic ty.
In an alternative ment, the peptide is ted 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.
ingly, the present invention provides peptides and variants of MHC class I
epitopes, n the peptide or variant has an overall length of between 8 and 100,
preferably between 8 and 30, and most preferred n 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.
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.
Preferably, when the T cells specific for a peptide ing to the present invention are
tested against the substituted es, the peptide tration 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 pM, more ably 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.
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: 32.
sting 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 32 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.
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 “li”) as derived from the NCBI, GenBank Accession number X00497. In
other s, the peptides of the present invention can be fused to an antibody as
described herein, or a functional part thereof, in particular into a sequence of an
antibody, so as to be specifically targeted by said antibody, or, for example, to or into an
antibody that is specific for dendritic cells as bed .
In addition, the peptide or variant may be modified further to improve ity 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 e bonds or non-peptide bonds.
In a reverse e bond amino acid residues are not joined by peptide (-CO-NH-)
es but the e bond is reversed. Such retro-inverso omimetics may be
made using methods known in the art, for example such as those described in Meziere
et al (1997) re et al., 1997), incorporated herein by reference. This approach
involves making pseudopeptides containing s ing 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.
A non-peptide bond is, for example, -CH2-NH, -CH28-, -CH20H2-, -CH=CH-, -COCH2-, -
CH(OH)CH2—, and -CH280-. US 4,897,445 provides a method for the solid phase
synthesis of non-peptide bonds (-CH2-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
NaCNBHg.
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 loxycarbonyl 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 hobic
group, t-butyloxycarbonyl, or an amido group may be added to the peptides' carboxy
termini.
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
sen/e to increase the stability, bioavailability and/or g action of the peptides of the
invenfion.
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 ized e.g. in R. ad,
al Reagents for Protein Modification, 3rd ed. CRC Press, 2004 lad,
2004), which is incorporated herein by reference. Chemical modification of amino acids
includes but is not d to, modification by acylation, amidination, pyridoxylation of
lysine, ive 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 etamide 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 ln Protein
Science, Eds. Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)
for more ive methodology relating to chemical modification of proteins.
Briefly, modification of e.g. arginyl residues in ns is often based on the reaction of
vicinal dicarbonyl compounds such as phenylglyoxal, 2,3-butanedione, and 1,2-
cyclohexanedione to form an . Another example is the reaction of methylglyoxal
with arginine residues. ne can be modified without concomitant modification of
other nucleophilic sites such as lysine and ine. 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.
Selective reduction of disulfide bonds in ns 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 ic acid residues. N-(3-
(dimethylamino)propyl)-N’-ethylcarbodiimide can be used to form intra-molecular
cross|inks 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 ed using 4-hydroxynonenal. The reaction of lysine
residues and other d-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.
nine es in proteins can be modified with e.g. iodoacetamide,
bromoethylamine, and chloramine T.
Tetranitromethane and N-acetylimidazole can be used for the modification of tyrosyl
residues. Cross-linking via the ion of dityrosine can be accomplished with
hydrogen peroxide/copper ions.
Recent studies on the modification of tryptophan have used N-bromosuccinimide, 2-
hydroxynitrobenzyl bromide or 3-bromomethyl(2-nitrophenylmercapto)-3H-
indole (BPNS-skatole).
sful modification of therapeutic proteins and es 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.
A peptide or variant, wherein the peptide is modified or includes non-peptide bonds is a
preferred embodiment of the ion. Another embodiment of the present invention
s to a non-naturally occurring peptide wherein said e consists or ts
essentially of an amino acid sequence according to SEQ ID No: 1 to SEQ ID No: 32 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 ntially from the es in
their state(s) in vivo, as the es 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
sed 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
ing to the invention include alkaline and earth alkaline salts such as salts of the
Hofmeister series sing as anions P043] 8042', ', Cl', Br', NOg‘, CIO4', l',
SCN' and as cations NH4+, Rb”, K“, Na“, 03*, Li+, Zn2+, Mg”, Ca2+, Mn”, Cu” and
Ba”. Particularly salts are selected from (NH4)3PO4, (NH4)2HPO4, (NH4)H2PO4,
(NH4)2SO4, NH4CchOO, NH4CI, NH4Br, NH4N03, NH4CIO4, NH4I, NH4SCN, Rb3PO4,
Rb2HPO4, RbH2PO4, szSO4, Rb4CHgCOO, Rb4Cl, Rb4Br, , Rb4ClO4, Rb4l,
, K3PO4, K2HPO4, KH2PO4, K2804, KCchOO, KCI, KBr, KNOg, KCIO4, Kl,
KSCN, Na3PO4, Na2HPO4, NaH2PO4, Na2804, NaCHgCOO, NaCl, NaBr, NaN03,
NaClO4, Nal, NaSCN, ZnCI2 Cs3PO4, Cs2HPO4, CsH2PO4, Cs2804, CSCH3COO, CsCl,
CsBr, CsN03, CsClO4, Csl, CsSCN, Li3PO4, Li2HPO4, LiH2PO4, Li2804, LiCchOO,
LiCl, LiBr, LiNO3, LiClO4, Lil, LiSCN, Cu2804, Mg3(PO4)2, MngPO4, Mg(H2PO4)2,
, Mg(CchOO)2, MgClg, MgBrg, Mg(N03)2, Mg(ClO4)2, Mglg, Mg(SCN)2, MnClg,
Ca3(PO4),, Ca2HPO4, Ca(H2PO4)2, CaSO4, Ca(CHgCOO)2, CaClz, CaBr2, Ca(N03)2,
Ca(ClO4)2, Calz, Ca(SCN)2, Ba3(PO4)2, Ba2HPO4, Ba(H2PO4)2, BaSO4, COO)2,
BaClg, BaBrg, Ba(N03)2, Ba(ClO4)2, Balg, and Ba(SCN)2. Particularly preferred are NH
acetate, MgClz, KH2PO4, Na2804, KCI, NaCl, and CaClz, such as, for example, the
chloride or acetate (trifluoroacetate) salts.
Generally, peptides and ts (at least those containing peptide linkages between
amino acid residues) may be synthesized by the olyamide mode of solid-phase
peptide synthesis as disclosed by Lukas et al. (Lukas et al., 1981) and by references as
cited therein. Temporary o group protection is afforded by the 9-
_ 50 _
fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile
protecting group is done using 20% piperidine in N, N-dimethylformamide. Side-chain
onalities 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 tive (in the case of lysine and histidine), trityl derivative (in the
case of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the
case of ne). Where glutamine or asparagine are C-terminal residues, use is made
of the 4,4'-dimethoxybenzhydryl group for protection of the side chain amido
functionalities. The solid-phase support is based on a polydimethyl-acrylamide polymer
constituted from the three monomers dimethylacrylamide (backbone-monomer),
bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester
(functionalizing . The peptide-to-resin cleavable linked agent used is the acidlabile
4-hydroxymethyl-phenoxyacetic acid tive. 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 nic acid or
isotin test procedures. Upon completion of synthesis, peptides are cleaved from the
resin t with concomitant removal of hain ting groups by treatment
with 95% trifluoracetic acid containing a 50 % scavenger mix. Scavengers ly
used include ethanedithiol, , e 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 es is possible
(see, for example, (Bruckdorfer et al., 2004), and the references as cited therein).
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).
Purification may be performed by any one, or a combination of, ques such as re-
crystallization, size ion chromatography, change chromatography,
hydrophobic interaction chromatography and (usually) reverse-phase high mance
liquid chromatography using e.g. acetonitrile/water gradient separation.
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 is after acid
hydrolysis and by fast atom bombardment (FAB) mass ometric analysis, as well
as MALDI and ESl-Q-TOF mass spectrometric analysis.
In order to select over-presented peptides, a presentation e is calculated showing
the median sample presentation as well as replicate variation. The profile juxtaposes
s of the tumor entity of interest to a baseline of normal tissue samples. Each of
these profiles can then be consolidated into an over-presentation score by calculating
the p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015) adjusting for multiple
testing by False Discovery Rate (Benjamini and Hochberg, 1995) (cf. Example 1,
Figures 1).
For the identification and relative quantitation of HLA ligands by mass spectrometry,
HLA molecules from 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 (nanoESl) liquid chromatography-mass
ometry (LC-MS) experiments. The resulting peptide ces were verified by
comparison of the fragmentation pattern of natural tumor-associated peptides
(TUMAPs) recorded from gallbladder cancer and cholangiocarcinoma samples (N = 17
A*02—positive samples) with the fragmentation ns of ponding 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 ed from 17 gallbladder cancer and cholangiocarcinoma patients.
The discovery pipeline XPRESIDENT® v2.1 (see, for example, US 2013-0096016,
which is hereby incorporated by reference in its ty) 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 ent, charge state deconvolution
and normalization.
Presentation levels ing error estimates for each peptide and sample were
established. Peptides ively presented on tumor tissue and peptides over-
ted in tumor versus non-cancerous s and organs have been fied.
HLA-peptide complexes from gallbladder cancer and cholangiocarcinoma tissue
samples were purified and HLA-associated peptides were isolated and analyzed by LC-
MS (see examples). All TUMAPs contained in the present application were identified
with this approach on primary gallbladder cancer and cholangiocarcinoma samples
ming their presentation on primary gallbladder cancer and cholangiocarcinoma.
TUMAPs identified on multiple gallbladder cancer and cholangiocarcinoma and normal
tissues were quantified using unting of label-free LC-MS data. The method
assumes that LC-MS signal areas of a peptide correlate with its abundance in the
sample. All quantitative signals of a peptide in various LC-MS experiments were
normalized based on central tendency, averaged per sample and merged into a bar
plot, called presentation profile. The presentation profile consolidates ent analysis
s like protein database search, spectral clustering, charge state deconvolution
(decharging) and retention time ent and normalization.
WO 02806
_ 53 _
Besides over-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, Figures 2). An additional source of normal tissue data was a
database of publicly available RNA expression data from around 3000 normal tissue
samples (Lonsdale, 2013). Peptides which are derived from proteins whose coding
mRNA is highly expressed in cancer , but very low or absent in vital normal
tissues, were ably included in the t invention.
The t invention provides peptides that are useful in treating s/tumors,
preferably gallbladder cancer and cholangiocarcinoma that over- or exclusively present
the peptides of the invention. These peptides were shown by mass spectrometry to be
lly presented by HLA molecules on primary human gallbladder cancer and
cholangiocarcinoma samples.
Many of the source roteins (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 s — “normal tissues” in relation to this invention
shall mean either healthy gallbladder or bile duct cells or other normal tissue cells,
demonstrating a high degree of tumor association of the source genes (see Example 2).
Moreover, the peptides themselves are strongly over-presented on tumor tissue —
“tumor tissue” in relation to this invention shall mean a sample from a patient suffering
from gallbladder cancer and cholangiocarcinoma, but not on normal tissues (see
Example 1).
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. gallbladder cancer and cholangiocarcinoma cells presenting the derived
peptides.
The es 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 so|ub|e TCRs, according to the present invention (see
Example 3, Example 4). Furthermore, the peptides when complexed with the tive
MHC can be used for the production of antibodies and/or TCRs, in particular sTCRs,
according to the present invention, as well. Respective methods are well known to the
person of skill, and can be found in the respective literature as well. Thus, the peptides
of the present invention are useful for generating an immune response in a patient by
which tumor cells can be destroyed. An immune response in a patient can be induced
by direct administration of the described peptides or suitable precursor substances (e.g.
elongated peptides, proteins, or nucleic acids encoding these peptides) to the patient,
ideally in combination with an agent enhancing the immunogenicity (i.e. an adjuvant).
The immune response originating from such a therapeutic vaccination can be ed
to be highly specific against tumor cells because the target peptides of the present
invention are not presented on normal s in comparable copy numbers, preventing
the risk of red mune reactions against normal cells in the patient.
The t description further relates to T-ce|| receptors (TCRs) comprising an alpha
chain and a beta chain a/beta TCRs”). Also provided are peptides capable of
binding to TCRs and antibodies when presented by an MHC le. The t
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.
The term “T-cell receptor” (abbreviated TCR) refers to a heterodimeric molecule
comprising an alpha ptide 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-ca||ed delta TCRs.
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.
_ 55 _
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 cial antigen-presenting cell by
contacting a sufficient amount of the antigen with an antigen-presenting cell or the
n is loaded onto class I or II MHC tetramers by tetramerizing the antigen/class | or
II MHC complex monomers.
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 e 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 embrane (TM)
domains that anchor the alpha and beta chains to the cell membrane.
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 ellular 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
TRDJ) region, and the term “TCR delta constant domain” refers to the
ellular TRDC region, or to a C-terminal truncated TRDC sequence.
TCRs of the present description ably bind to a peptide-HLA molecule complex
with a g affinity (KD) of about 100 uM or less, about 50 uM or less, about 25 uM
or less, or about 10 uM or less. More preferred are high affinity TCRs having binding
affinities of about 1 uM 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;
_ 56 _
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.
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 uM or less.
Alpha/beta heterodimeric TCRs of the present description may have an introduced
disulfide bond between their constant s. Preferred TCRs of this type include
those which have a TRAC constant domain sequence and a TRBC1 or TRBCZ nt
domain sequence except that Thr 48 of TRAC and Ser 57 of TRBCl or TRBCZ are
replaced by cysteine residues, the said cysteines forming a disulfide bond between the
TRAC constant domain sequence and the TRBC1 or TRBCZ constant domain
sequence of the TCR.
With or t the introduced inter-chain bond mentioned above, beta hetero-
dimeric TCRs of the t description may have a TRAC nt domain sequence
and a TRBCl or TRBCZ constant domain sequence, and the TRAC nt domain
sequence and the TRBC1 or TRBCZ 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 TRBCl or TRBCZ.
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.
In an ment, 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.
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 sing 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 lly about 10-
fold lower when compared to TCRs ic 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 d
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 arily induce a functional immune response against the tumor:
s expressing TCRs that are highly reactive to these antigens will have been
negatively selected within the thymus in a process known as central tolerance, meaning
that only T-cells with low-affinity TCRs for self-antigens remain. Therefore, affinity of
TCRs or variants of the present description to the es according to the invention
can be enhanced by methods well known in the art.
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, ting the PBMCs
with er-phycoerythrin (PE) and ing the high avidity T-cells by scence
activated cell sorting (FACS)—Calibur analysis.
The present description further relates to a method of identifying and isolating a TCR
according to the present ption, said method comprising obtaining a transgenic
mouse with the entire human TCRdB gene loci (1.1 and 0.7 Mb), whose T-cells express
a diverse human TCR repertoire that compensates for mouse TCR deficiency,
immunizing the mouse with peptide of interest, incubating PBMCs obtained from the
transgenic mice with tetramer—phycoerythrin (PE), and isolating the high avidity T-cells
by fluorescence activated cell sorting (FACS)—Calibur is.
In one aspect, to obtain T-cells expressing TCRs of the present description, nucleic
acids encoding pha and/or TCR-beta chains of the present description are
cloned into expression vectors, such as gamma retrovirus or lentivirus. The recombinant
viruses are generated and then tested for functionality, such as antigen specificity and
functional avidity. An aliquot of the final product is then used to transduce the target T-
cell population (generally purified from patient PBMCs), which is expanded before
infusion into the patient. In another , to obtain T-cells expressing TCRs of the
t ption, TCR RNAs are synthesized by techniques known in the art, e.g., in
vitro transcription systems. The in vitro-synthesized TCR RNAs are then uced into
primary CD8+ T-cells obtained from healthy donors by electroporation to re-express
tumor specific TCR-alpha and/or TCR-beta chains.
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), B-actin, tin, and a simian virus 40 (SV40)/CD43 composite
promoter, elongation factor (EF)—1a and the spleen focus-forming virus (SFFV)
promoter. In a preferred embodiment, the promoter is heterologous to the nucleic acid
being expressed. In addition to strong ers, TCR expression tes 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 uck hepatitis
virus anscriptional regulatory element (wPRE), which increases the level of
transgene expression by increasing RNA stability (Zufferey et al., 1999).
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
d in the same vector.
Achieving high-level TCR surface sion requires that both the TCR-alpha and
TCR-beta chains of the introduced TCR be transcribed at high . To do so, the
TCR-alpha and TCR-beta chains of the t description may be cloned into bi-
cistronic constructs in a single , which has been shown to be capable of over-
coming this obstacle. The use of a viral ibosomal entry site (IRES) between the
TCR-alpha and TCR-beta chains results in the coordinated expression of both chains,
because the TCR-alpha and ta 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).
Nucleic acids encoding TCRs of the present description may be codon zed 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
“optimal” 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 l codon for mammalian
gene expression, as well as eliminating mRNA ility motifs or cryptic splice sites,
has been shown to significantly enhance TCR-alpha and TCR-beta gene expression
(Scholten et al., 2006).
Furthermore, mispairing n 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).
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 decreasing
the ability of the introduced chains to pair with the endogenous TCR. These
strategies may e replacing the human TCR-alpha and TCR-beta C-terminus
domains with their murine counterparts (murinized C-terminus domain); generating a
second interchain ide 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); ng 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 CD3C (CD3C fusion). (Schmitt et al. 2009).
In an ment, a host cell is engineered to express a TCR of the present
description. In preferred ments, the host cell is a human T-cell or T-cell
itor. In some embodiments, the T-cell or T-cell progenitor is ed from a
cancer patient. In other embodiments, the T-cell or T-cell progenitor is ed 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.
A “pharmaceutical composition” is a composition suitable for administration to a human
being in a medical setting. ably, a pharmaceutical composition is sterile and
ed according to GMP guidelines.
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 sed 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 n 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 ic 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 ide, ammonium
ide, calcium hydroxide, trimethylamine or the like.
In an especially preferred ment, the pharmaceutical itions comprise the
peptides as salts of acetic acid (acetates), trifluoro acetates or hydrochloric acid
(chlorides).
Preferably, the medicament of the present ion 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 iv, 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 ected so as to co-express immune-stimulating
cytokines, such as interleukin-2. The peptide may be substantially pure, or ed
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 hemocyanin (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
le. 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 | epitopes
that stimulate CD8 T cells the fusion partner or sections of a hybrid le suitably
provide es 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.
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. 32, and at least one additional peptide,
_ 62 _
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.
A further aspect of the ion provides a nucleic acid (for example a polynucleotide)
encoding a peptide or peptide t of the invention. The cleotide 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 ne and it may or may not contain
introns so long as it codes for the e. Of course, only peptides that contain
naturally occurring amino acid es 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.
A variety of methods have been developed to link cleotides, ally DNA, to
vectors for e via complementary cohesive termini. For instance, complementary
lymer 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.
Synthetic linkers containing one or more restriction sites provide an alternative method
of joining the DNA segment to vectors. Synthetic linkers ning a variety of
restriction endonuclease sites are cially available from a number of sources
including International Biotechnologies Inc. New Haven, CN, USA.
A desirable method of modifying the DNA encoding the polypeptide of the invention
employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al.,
1988). This method may be used for introducing the DNA into a suitable vector, for
example by engineering in suitable restriction sites, or it may be used to modify the DNA
in other useful ways as is known in the art. lf viral vectors are used, pox- or adenovirus
vectors are preferred.
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 t of the invention may be used in ance
with known techniques, appropriately modified in view of the teachings ned
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 sed, for example, in US 4,440,859,
4,530,901, 4,582,800, 4,677,063, 4,678,751, 362, 4,710,463, 4,757,006,
4,766,075, and 4,810,648.
The DNA (or in the case of retroviral vectors, RNA) encoding the polypeptide
tuting 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.
Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper
orientation and correct reading frame for sion. If necessary, the DNA may be
linked to the appropriate transcriptional and ational regulatory control nucleotide
sequences ized by the desired host, gh 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 able trait in the transformed cell,
such as antibiotic resistance.
_ 64 _
Alternatively, the gene for such able trait can be on another , which is used
to co-transform the desired host cell.
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 red.
Many expression systems are known, including bacteria (for example E. coli and
Bacillus subtilis), yeasts (for example romyces cerevisiae), filamentous fungi (for
e 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 SV4O promoter with a le poly A tail and a resistance marker, such as neomycin.
One example is pSVL available from Pharmacia, Piscataway, NJ, USA. An example of
an inducible ian 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 s, La Jolla, CA 92037, USA. Plasmids
, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Ylps) and
incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids
pRS413-416 are Yeast Centromere plasmids (chs). 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, 3xFLAG, c-myc or MAT. These fusion proteins allow for detection, purification
and analysis of recombinant protein. Dual-tagged fusions provide flexibility in ion.
The strong human galovirus (CMV) promoter regulatory region drives
constitutive protein expression levels as high as 1 mg/L in COS cells. For less potent
cell lines, protein levels are typically ~0.1 mg/L. The presence of the SV40 replication
_ 65 _
origin will result in high levels of DNA replication in SV4O replication permissive COS
cells. CMV s, for example, can contain the pMB1 (derivative of pBR322) origin for
replication in bacterial cells, the amase gene for llin 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 . Other vectors
and expression systems are well known in the art for use with a variety of host cells.
In another embodiment two or more peptides or peptide ts 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 onal peptide(s) between them. These constructs can also be
used for cancer therapy, and may induce immune ses both involving MHC l and
MHC II.
The t 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,
CA 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 -derived COS-1 cells ble
from the ATCC as CRL 1650 and 293 cells which are human embryonic kidney cells.
red insect cells are Sf9 cells which can be transfected with baculovirus expression
_ 66 _
vectors. An overview regarding the choice of suitable host cells for expression can be
found in, for example, the textbook of a Balbas and Argelia Lorence “Methods in
Molecular Biology Recombinant Gene Expression, Reviews and Protocols,” Part One,
Second Edition, ISBN 588299, and other literature known to the person of
skill.
ormation 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 m phosphate and DEAE-dextran or liposome formulations, are
available from Stratagene Cloning Systems, or Life logies 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.
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.
It will be appreciated that certain host cells of the invention are useful in the preparation
of the es of the invention, for example bacterial, yeast and insect cells. However,
other host cells may be useful in certain therapeutic methods. For example, npresenting
cells, such as dendritic cells, may usefully be used to s the peptides of
the invention such that they may be loaded into appropriate MHC molecules. Thus, the
t invention es a host cell comprising a nucleic acid or an expression vector
according to the invention.
WO 02806
_ 67 _
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 n
containing tic acid phosphatase (PAP) were approved by the U.S. Food and Drug
Administration (FDA) on April 29, 2010, to treat omatic or minimally symptomatic
metastatic HRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).
A further aspect of the invention provides a method of producing a peptide or its variant,
the method sing culturing a host cell and isolating the peptide from the host cell
or its culture medium.
In another embodiment the peptide, the nucleic acid or the sion vector of the
invention are used in medicine. For example, the peptide or its variant may be prepared
for intravenous (iv) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,
intraperitoneal (i.p.) ion, intramuscular (i.m.) injection. Preferred methods of
peptide injection include s.c., i.d., i.p., i.m., and iv Preferred methods of DNA injection
include i.d., i.m., s.c., i.p. and iv Doses of e.g. between 50 pg and 1.5 mg, preferably
125 pg to 500 pg, 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 r
et al., 2012).
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 oven/iew 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 s based on adenovirus, vaccinia
virus, iruses, herpes virus, adeno-associated virus or hybrids containing ts
of more than one virus. Non-viral ry 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 e with an epitope that stimulates T cells for the respective
opposite CDR as noted above.
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 ed by CD8-positive T cells and helper-T (TH) cells to an
n, 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®, AS15, BCG, CP-870,893, 9, CyaA, dSLlM, flagellin or TLR5
ligands derived from flagellin, FLT3 ligand, GM-CSF, I030, I031, lmiquimod
A®), imod, lmuFact IMP321, Interleukins as IL-2, IL-13, IL-21, Interferon-
alpha or -beta, or ted derivatives f, IS Patch, ISS, ISCOMATRIX, lSCOMs,
Juvlmmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312,
Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, in-oil and oil-in-water
emulsions, OK-432, , OMMP-EC, ONTAK, OspA, PepTel® 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 nts such
as Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are
preferred. l 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
tic 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) (US. 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.
lFN-beta) (Gabrilovich et al., 1996).
CpG immunostimulatory oligonucleotides have also been reported to enhance the
effects of adjuvants in a vaccine g. Without being bound by theory, CpG
o|igonuc|eotides act by activating the innate (non-adaptive) immune system via Toll-like
receptors (TLR), mainly TLR9. CpG triggered TLR9 activation enhances antigenspecific
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 es 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 uc|eotides show
even greater adjuvant activity when formulated or inistered with other adjuvants
or in formulations such as microparticles, nanoparticles, lipid emulsions or similar
formulations, which are especially necessary for ng 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). US 6,406,705 Bi describes the combined use of CpG
o|igonuc|eotides, non-nucleic acid adjuvants and an antigen to induce an antigen-
specific immune response. A CpG TLR9 antagonist is dSLlM e Stem Loop
lmmunomodulator) by Mologen (Berlin, y) 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.
Other examples for useful adjuvants e, but are not limited to chemically ed
Cst (e.g. CpR, ldera), dsRNA analogues such as Poly(l:C) and derivates thereof (e.g.
AmpliGen®, Hiltonol®, poly-(ICLC), poly(IC-R), poly(l:C12U), non-CpG bacterial DNA or
RNA as well as immunoactive small molecules and dies such as
cyclophosphamide, Sunitinib, Bevacizumab®, ex, NCX-4016, afil, tadalafil,
vardenafil, Sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, nib,
VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other dies targeting key structures
of the immune system (e.g. anti-CD40, anti-TGFbeta, anti-TNFalpha receptor) and
_ 70 _
8058175, which may act therapeutically and/or as an adjuvant. The amounts and
concentrations of adjuvants and additives useful in the context of the t invention
can readily be determined by the skilled artisan t undue experimentation.
Preferred adjuvants are anti-CD40, imiquimod, imod, GM-CSF,
cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpG oligonucleotides and
derivates, |:C) and derivates, RNA, sildenafil, and particulate ations with
PLG or mes.
In a preferred embodiment, the pharmaceutical composition according to the invention
the adjuvant is selected from the group ting of colony-stimulating factors, such as
Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, mostim),
cyclophosphamide, imiquimod, imod, and interferon-alpha.
In a preferred embodiment, the pharmaceutical composition according to the invention
the adjuvant is selected from the group consisting of -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, ide ISA 206, Montanide ISA 50V, Montanide ISA-51, poly-
ICLC nol®) and anti-CD40 mAB, or combinations thereof.
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 on, the composition can contain excipients, such as
buffers, binding agents, blasting agents, diluents, s, 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 y of
adenomatous or cancerous diseases. Exemplary ations can be found in, for
example, EP2112253.
It is important to realize that the immune response triggered by the vaccine according to
the invention s the cancer in different cell-stages and different stages of
development. Furthermore, different cancer ated signaling pathways are
attacked. This is an advantage over vaccines that s 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 ns and cover l 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 lection 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).
As used herein, the term old" refers to a molecule that specifically binds to an (e.g.
nic) inant. In one embodiment, a scaffold is able to direct the entity to
which it is attached (e.g. a (second) antigen binding ) to a target site, for example
to a specific type of tumor cell or tumor stroma bearing the antigenic determinant (e.g.
the x 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 n repeat motif and single domain antigen
binding (SDAB) molecules, aptamers, (soluble) TCRs and (modified) cells such as
nic or autologous T cells. To assess whether a molecule is a scaffold binding to a
target, binding assays can be performed.
“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
ld armed with an active molecule that is able to kill a cell bearing the specific
target is not able to kill r cell t the specific target but presenting another
peptide-MHC complex(es). Binding to other peptide-MHC xes 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.
Each scaffold can comprise a labelling which provides that the bound scaffold can be
detected by determining the presence or absence of a signal provided by the label. For
example, the scaffold can be ed 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 scence dye, can
provide a visualization of the bound aptamer by fluorescence or laser scanning
microscopy or flow cytometry.
Each scaffold can be conjugated with a second active molecule such as for e IL-
21, anti-CD3, and anti-CD28.
For further information on polypeptide scaffolds see for example the background section
of WO 71978A1 and the references cited therein.
The present invention further relates to aptamers. Aptamers (see for example WO
2014/191359 and the literature as cited n) 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.
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. lndeed aptamers, for
example prostate-specific ne-antigen recognizing aptamers, have been
successfully ed for targeted ies and shown to be functional in xenograft in
vivo models. Furthermore, aptamers recognizing specific tumor cell lines have been
idenfified.
DNA aptamers can be selected to reveal spectrum recognition ties for
various cancer cells, and particularly those derived from solid , while non-
tumorigenic and primary healthy cells are not ized. If the fied aptamers
ize 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.
r, investigation of cell-binding behavior with flow cytometry showed that the
aptamers revealed very good apparent affinities that are within the nanomolar range.
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.
Aptamers can be selected against complex targets such as cells and s and
complexes of the peptides comprising, preferably consisting of, a sequence according
to any of SEQ ID NO 1 to SEQ ID NO 32, according to the invention at hand with the
MHC molecule, using the cell-SELEX (Systematic Evolution of Ligands by Exponential
enrichment) technique.
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.
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 n, the method
sing: immunizing a genetically engineered non-human mammal comprising cells
sing said human major histocompatibility complex (MHC) class I or II with a
soluble form of a MHC class I or II le being complexed with said stricted
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 ying said antibody specifically binding to said human
major histocompatibility complex (MHC) class I or II being complexed with said HLA-
restricted n.
It is a further aspect of the invention to provide an antibody that specifically binds to a
human major histocompatibility complex (MHC) class I or II being complexed with a
HLA-restricted antigen, wherein the antibody preferably is a polyclonal antibody,
onal antibody, bi-specific antibody and/or a chimeric dy.
Respective methods for producing such antibodies and single chain class I major
histocompatibility xes, as well as other tools for the tion of these
antibodies are disclosed in
_ 75 _
03/070752, and in ations (Cohen et al., 2003a; Cohen et al., 2003b; Denkberg et
al., 2003), which for the purposes of the present invention are all itly incorporated
by reference in their entireties.
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.
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: 32, or a variant thereof which is at
least 88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 32 or a
variant thereof that induces T cells cross-reacting with said peptide, wherein said
peptide is not the underlying full-length polypeptide.
The present invention further relates to a peptide sing a ce that is
selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 32 or a variant
f which is at least 88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ
ID NO: 32, wherein said peptide or variant has an overall length of between 8 and 100,
preferably n 8 and 30, and most preferred between 8 and 14 amino acids.
The present invention further relates to the peptides according to the ion that have
the ability to bind to a molecule of the human major histocompatibility complex (MHC)
class-l or -II.
The present invention further s to the peptides according to the invention n
the peptide ts or consists essentially of an amino acid sequence according to
SEQ ID NO: 1 to SEQ ID NO: 32.
The t invention further relates to the peptides according to the invention, wherein
the peptide is (chemically) modified and/or includes non-peptide bonds.
The present invention r relates to the peptides according to the invention, wherein
the peptide is part of a fusion protein, in particular comprising N-terminal amino acids of
the HLA-DR antigen-associated invariant chain (Ii), or wherein the peptide is fused to
(or into) an antibody, such as, for example, an dy that is specific for dendritic
cells.
The present invention further s to a c acid, encoding the peptides according
to the invention, provided that the peptide is not the complete (full) human protein.
The present invention further relates to the nucleic acid ing to the invention that is
DNA, cDNA, PNA, RNA or combinations thereof.
The present invention further relates to an expression vector capable of expressing a
nucleic acid according to the present invention.
The present invention further relates to a e 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 gallbladder cancer
and cholangiocarcinoma.
The t invention r relates to a host cell comprising a nucleic acid according
to the invention or an expression vector according to the invention.
The t invention further relates to the host cell according to the present invention
that is an antigen presenting cell, and preferably a dendritic cell.
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 ing the e from said host cell or its culture medium.
The present invention r relates to the method according to the present invention,
in the antigen is loaded onto class I or II MHC molecules sed on the
surface of a suitable antigen-presenting cell by ting a sufficient amount of the
n with an antigen-presenting cell.
The present invention further s 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: 32 or said variant amino acid
sequence.
The present invention r 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.
The present invention further relates to a method of killing target cells in a patient which
target cells aberrantly express a polypeptide sing any amino acid ce
according to the present invention, the method comprising administering to the patient
an effective number of T cells as according to the present invention.
The present invention further relates to the use of any e 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
cyte 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 ment is active against cancer.
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.
The present invention further relates to a use according to the ion, wherein said
cancer cells are gallbladder cancer and cholangiocarcinoma cells or other solid or
hematological tumor cells such as acute myeloid leukemia, melanoma, small cell lung
cancer, non-small cell lung cancer, non-Hodgkin lymphoma, chronic lymphocytic
leukemia, pancreatic cancer, liver cancer, ovarian cancer, head and neck cancer,
urinary bladder cancer, breast cancer, and kidney cancer.
The present invention further s to particular marker proteins and kers based
on the peptides according to the present invention, herein called “targets” that can be
used in the diagnosis and/or prognosis of adder cancer and giocarcinoma.
The present invention also relates to the use of these novel targets for cancer
treatment.
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 rs 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 gallbladder cancer and cholangiocarcinoma marker (poly)peptide,
delivery of a toxin to a gallbladder cancer and cholangiocarcinoma cell expressing a
cancer marker gene at an sed level, and/or inhibiting the activity of a gallbladder
cancer and cholangiocarcinoma marker polypeptide) ing to the invention.
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 gallbladder cancer
and cholangiocarcinoma marker polypeptides or fragments thereof may be used to
te 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 inant DNA techniques.
_ 7g _
For example, a cDNA ng a peptide according to the present invention, such as a
peptide according to SEQ ID NO: 1 to SEQ ID NO: 32 polypeptide, or a variant or
fragment thereof, can be expressed in prokaryotic cells (e.g., ia) 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 gallbladder cancer and cholangiocarcinoma marker polypeptide
used to generate the antibody according to the invention.
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 icity 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 ce on the generation and testing of antibodies,
see, e.g., ield, 2014 (Greenfield, 2014)). For example, the dies may be
tested in ELISA assays or, Western blots, immunohistochemical staining of in-
fixed cancers or frozen tissue sections. After their l in vitro characterization,
antibodies intended for therapeutic or in vivo diagnostic use are tested according to
known clinical testing methods.
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 onal 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 d from a
particular species or belonging to a particular antibody class or ss, while the
remainder of the chain(s) is cal with or homologous to corresponding ces in
antibodies derived from another species or belonging to r antibody class or
subclass, as well as fragments of such antibodies, so long as they exhibit the desired
antagonistic activity (US 567, which is hereby incorporated in its entirety).
onal 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. atively, the
lymphocytes may be immunized in vitro.
The monoclonal antibodies may also be made by recombinant DNA methods, such as
those described in US 4,816,567. DNA ng 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
ng the heavy and light chains of murine antibodies).
In vitro methods are also le 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
med using papain. es of papain digestion are described in WO 94/29348
and US 4,342,566. Papain digestion of antibodies typically produces two identical
antigen binding fragments, called Fab nts, each with a single antigen g
site, and a residual Fc fragment. Pepsin treatment yields a 2 nt and a ch'
fragment.
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 g at the binding domain, etc.
onal 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 y apparent to a skilled practitioner in the art and
can include site-specific mutagenesis of the c acid encoding the antibody
fragment.
The antibodies of the invention may further comprise zed 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 n minimal sequence
derived from non-human globulin. 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 ces, 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 s correspond to
those of a non-human immunoglobulin and all or substantially all of the FR s are
those of a human immunoglobulin consensus sequence. The humanized antibody
optimally also will se at least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin.
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 man. These non-human amino acid residues are often referred
to as t" 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 (US 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 dies are typically
human dies in which some CDR residues and possibly some FR es are
substituted by residues from analogous sites in rodent antibodies.
Transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full
repertoire of human antibodies in the absence of endogenous immunoglobulin
tion can be employed. For example, it has been bed 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 tion. 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 ies.
Antibodies of the invention are preferably administered to a subject in a
pharmaceutically acceptable carrier. Typically, an riate amount of a
pharmaceutically-acceptable salt is used in the formulation to render the ation
isotonic. es 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 es 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 ce, the route
of administration and concentration of antibody being administered.
The antibodies can be administered to the subject, patient, or cell by injection (e.g.,
intravenous, intraperitoneal, subcutaneous, uscular), 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 enous ion is preferred.
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 (pg/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, ably for treating gallbladder
cancer and cholangiocarcinoma, 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 g techniques. A therapeutically-administered antibody that
arrests tumor growth, results in tumor shrinkage, and/or prevents the development of
new tumors, ed to the disease course that would occurs in the absence of
antibody administration, is an efficacious antibody for treatment of cancer.
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 x. Such soluble T-cell
receptors can be generated from specific T-cell clones, and their affinity can be
increased by mutagenesis targeting the mentarity-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 e-chain T-cell or), or by
dimerization s (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 , etc.,
in order to execute ular functions on target cells. Moreover, it could be expressed
_ 84 _
in T cells used for adoptive transfer. Further information can be found in WC
2004/033685A1 and
2012/056407A1. Further methods for the production are disclosed in WC
57586A1.
In addition, the peptides and/or the TCRs or antibodies or other binding molecules of
the present invention can be used to verify a ogist’s diagnosis of a cancer based
on a biopsied sample.
The antibodies or TCRs may also be used for in vivo stic assays. Generally, the
antibody is labeled with a radionucleotide (such as 111in, 99To, 14c, 131i, 3H, 32p or 353)
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 s
of a protein selected from the group consisting of the above-mentioned ns, and
the affinity value (Kd) is less than 1 x 10uM.
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 d
to, fluorescence, light, al and on 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
s listed. These antibodies may be directly or indirectly labeled with said probes.
Attachment of probes to the antibodies includes nt attachment of the probe,
incorporation of the probe into the antibody, and the covalent attachment of a ing
compound for binding of probe, t 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
_ 85 _
and secondary antibody, wherein the antibody is used to detect the expression of the
proteins in situ.
Another aspect of the present invention includes an in vitro method for producing
activated T cells, the method sing contacting in vitro T cells with antigen loaded
human MHC molecules expressed on the surface of a suitable n-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 n is used with an antigen-presenting cell.
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 orter associated with antigen processing.
The human peptide loading deficient cell line T2 is available from the American Type
Culture Collection, 12301 Parklawn Drive, Rockville, nd 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).
Preferably, before transfection the host cell expresses substantially no MHC class I
molecules. It is also preferred that the ator cell expresses a molecule important for
providing a mulatory signal for T-cells such as any of 87.1, 87.2, lCAM-1 and LFA
3. The nucleic acid sequences of numerous MHC class I molecules and of the co-
ator molecules are publicly available from the GenBank and EMBL databases.
In case of a MHC class I epitope being used as an n, the T cells are CD8—positive
T cells.
_ 86 _
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: 32, or a t amino acid sequence thereof.
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.
ski et al. (Plebanski et al., 1995) made use of autologous peripheral blood
lymphocytes (PLBs) in the preparation of T cells. Furthermore, the production of
gous T cells by g tic 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.
8. Walter et al. r 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 med MHC: peptide complexes to the surface of polystyrene
les (microbeads) by biotin: streptavidin biochemistry. This system s the
exact control of the MHC density on aAPCs, which allows to selectively eliciting high- or
low-avidity n-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
ased systems often require the addition of appropriate soluble factors, e. g.
cytokines, like interleukin-12.
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.
The activated T cells that are directed against the peptides of the ion are useful in
therapy. Thus, a further aspect of the invention provides activated T cells obtainable by
the foregoing methods of the ion.
Activated T cells, which are produced by the above method, will selectively recognize a
cell that ntly expresses a polypeptide that comprises an amino acid sequence of
SEQ ID NO: 1 to SEQ ID NO 32.
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 ce of the invention wherein the t 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 ors 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 ed.
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)).
The T cells of the t 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.
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 t in normal tissue; ably at least 2—fold, and more preferably at least
-fold or d the level present in normal tissue.
T cells may be obtained by methods known in the art, e.g. those bed above.
Protocols for this so-called adoptive er 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).
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 uced into a
host cell, preferably a T cell. This engineered T cell can then be transferred to a patient
for therapy of cancer.
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 ng 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 ion or (a) known molecule(s).
The t invention further provides a medicament that is useful in treating cancer, in
particular gallbladder cancer and cholangiocarcinoma and other malignancies.
The present invention is further directed at a kit comprising:
(a) a container containing a pharmaceutical composition as bed above, in solution
or in lyophilized form;
(b) optionally a second container containing a diluent or reconstituting solution for the
lyophilized formulation; and
(c) ally, instructions for (i) use of the solution or (ii) reconstitution and/or use of the
lyophilized formulation.
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 ition is ably
lyophmzed.
Kits of the present invention preferably comprise a lyophilized formulation of the present
ion 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 ner may be formed
from a variety of materials such as glass or plastic. Preferably the kit and/or container
n/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 e concentrations as described above. The
label may further indicate that the formulation is useful or intended for subcutaneous
administration.
The container holding the formulation may be a multi-use vial, which allows for repeat
strations (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).
Upon mixing of the diluent and the lyophilized formulation, the final peptide
concentration in the tituted formulation is preferably at least 0.15 mg/mL/peptide
(=75 pg) and preferably not more than 3 mg/mL/peptide (=1500 pg). The kit may further
include other materials desirable from a commercial and user oint, including
other buffers, diluents, filters, needles, syringes, and package inserts with instructions
for use.
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 ner for each component.
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. ), 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 ner prior to stration to a
patient. The components of the kit may be ed 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 , which may be converted into
liquids by addition of suitable solvents, which are preferably provided in another ct
container.
The container of a therapeutic kit may be a vial, test tube, flask, bottle, syringe, or any
other means of enclosing a solid or . Usually, when there is more than one
component, the kit will n a second vial or other container, which allows for
separate dosing. The kit may also n another container for a pharmaceutically
acceptable liquid. Preferably, a therapeutic kit will contain an apparatus (e.g., one or
more needles, syringes, eye droppers, e, etc.), which enables administration of the
agents of the invention that are components of the present kit.
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,
_ g1 _
intramuscular, intravenous or ermal. Preferably, the stration is so, and
most ably i.d. administration may be by infusion pump.
Since the peptides of the ion were isolated from gallbladder cancer and
cholangiocarcinoma, the medicament of the invention is preferably used to treat
adder cancer and cholangiocarcinoma.
The t invention further relates to a method for producing a alized
pharmaceutical for an individual patient comprising manufacturing a pharmaceutical
composition comprising at least one peptide selected from a warehouse of prescreened
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 ations, such as TCR isolations, or soluble
antibodies, and other treatment options.
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
paflentfissue.
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 ed to imply that the particular peptides included
in the e have been pre-manufactured and stored in a physical facility, although
that possibility is contemplated. It is expressly contemplated that the peptides may be
ctured 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
gallbladder cancer and cholangiocarcinoma 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
_ 92 _
class I peptides. In addition to the tumor associated es collected from several
gallbladder cancer and cholangiocarcinoma tissues, the warehouse may n HLA-
A*02 and HLA-A*24 marker peptides. These es allow comparison of the
magnitude of T-cell immunity d by TUMAPS in a quantitative manner and hence
allow important conclusion to be drawn on the capacity of the e to elicit anti-tumor
responses. Secondly, they function as important positive control peptides derived from a
“non-self” antigen in the case that any vaccine-induced T-cell responses to TUMAPs
derived from “self” antigens in a patient are not observed. And thirdly, it may allow
conclusions to be drawn, regarding the status of competence of the patient.
TUMAPs for the use are identified by using an integrated functional genomics
approach combining gene expression analysis, mass ometry, and T-cell
immunology (XPresident ®). 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, gallbladder cancer and
cholangiocarcinoma samples from patients and blood from healthy donors were
analyzed in a stepwise approach:
1. HLA ligands from the malignant material were identified by mass spectrometry
2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis was used to
identify genes over-expressed in the malignant tissue (gallbladder cancer and
cholangiocarcinoma) compared with a range of normal organs and tissues
3. Identified HLA ligands were compared to gene expression data. Peptides over-
presented or selectively presented on tumor tissue, preferably encoded by selectively
sed or over-expressed genes as detected in step 2 were considered le
TUMAP candidates for a multi-peptide vaccine.
4. Literature research was performed in order to fy onal evidence supporting
the relevance of the identified peptides as TUMAPs
. 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.
6. In order to assess, whether an induction of in vivo T-cell responses by the selected
peptides may be le, in vitro immunogenicity assays were med using human
T cells from healthy donors as well as from gallbladder cancer and cholangiocarcinoma
patients.
In an aspect, the es 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 cial
antigen presenting cells loaded with peptide/MHC complexes and anti-CD28 antibody.
This method is red for rare cancers and patients with a rare sion e. In
contrast to multi-peptide cocktails with a fixed composition as currently developed, the
warehouse allows a icantly higher matching of the actual expression of antigens in
the tumor with the vaccine. Selected single or combinations of several “off-the-shelf”
peptides will be used for each patient in a multitarget approach. In theory, an approach
based on selection of e.g. 5 different antigenic peptides from a library of 50 would
already lead to approximately 17 million possible drug product (DP) compositions.
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 , or as below.
The HLA phenotype, transcriptomic and omic data is gathered from the patient’s
tumor material, and blood samples to identify the most le 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.
_ g4 _
Preferably, the peptides included in the vaccine are fied by a method comprising:
(a) identifying tumor-associated peptides (TUMAPs) presented by a tumor sample from
the individual patient; (b) comparing the peptides fied in (a) with a use
(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 ted 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 xpressed or aberrantly expressed in the tumor sample; and (a2)
correlating the expression data with sequences of MHC s bound to MHC class I
and/or class II les 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.
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 sion 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 sion data with ces 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 ponding tissue from the individual patient, and TUMAPs can be
identified that specifically target the on. 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
WO 02806
_ 95 _
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 (lllumina). Additionally, tumor mRNA is sequenced for direct quantification of
gene expression and tion that mutated genes are expressed in the patients’
tumors. The resultant millions of ce reads are processed through software
algorithms. The output list contains mutations and gene expression. Tumor-specific
somatic mutations are ined 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 ed for inclusion in the vaccine.
In one exemplary embodiment, the peptides included in the e are fied by: (a)
identifying tumor-associated peptides (TUMAPs) presented by a tumor sample from the
individual t by the method as described above; (b) ing the peptides
identified in a) with a warehouse of peptides that have been prescreened for
genicity 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) ally, selecting at least
one peptide identified de novo in (a) confirming its immunogenicity.
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.
Once the peptides for a alized 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.
Each e to be included into a product is dissolved in DMSO. The concentration of
the single peptide solutions has to be chosen depending on the number of peptides to
be included into the product. The single peptide-DMSO solutions are mixed in equal
parts to achieve a solution containing all peptides to be included in the product with a
concentration of ~2.5 mg/ml per peptide. The mixed solution is then d 1:3 with
water for injection to achieve a tration of 0.826 mg/ml per peptide in 33% DMSO.
The diluted on is filtered through a 0.22 pm sterile filter. The final bulk solution is
obtained.
Final bulk solution is filled into vials and stored at -20°C until use. One vial contains 700
pL solution, ning 0.578 mg of each e. Of this, 500 pL (approx. 400 pg per
peptide) will be applied for intradermal injection.
In on to being useful for treating cancer, the peptides of the present invention are
also useful as diagnostics. Since the es were generated from gallbladder cancer
and cholangiocarcinoma 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 .
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 gallbladder cancer and cholangiocarcinoma. Presence of groups of
peptides can enable classification or sub-classification of diseased tissues.
The detection of peptides on diseased tissue specimen can enable the on about
the t 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-
llance. Thus, presence of peptides shows that this mechanism is not exploited by
the analyzed cells.
The peptides of the present ion 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, c
acids, autologous materials, adoptive transfer of lymphocytes. In gene therapy settings,
cyte responses against peptides can be considered in the assessment of side
effects. Monitoring of cyte 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.
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 o. For the purposes of the present invention,
all nces as cited herein are incorporated by reference in their entireties.
Figures 1A through 1D show the over-presentation of various peptides in normal tissues
(white bars) and gallbladder cancer and cholangiocarcinoma (black bars). Figure 1A:
Gene symbol: MON2, Peptide: AVMTDLPVI, (SEQ ID NO.: 16), Tissues from left to
right: 6 adipose tissues, 8 adrenal , 24 blood cells, 17 blood vessels, 1O bone
marrows, 15 brains, 8 breasts, 4 cartilages, 7 esophagi, 2 eyes, 16 hearts, 19 kidneys,
21 large intestines, 25 livers, 49 lungs, 8 lymph nodes, 13 nerves, 3 ovaries, 11
pancreases, 6 parathyroid glands, 1 peritoneum, 6 ary glands, 7 placentas, 1
pleura, 3 prostates, 7 salivary glands, 1O skeletal muscles, 12 skins, 6 small intestines,
12 spleens, 5 stomachs, 7 testes, 2 thymi, 2 thyroid glands, 14 tracheas, 7 ureters, 8
urinary bladders, 6 uteri, 3 adders, 17 adder cancer and cholangiocarcinoma
samples. The peptide has additionally been detected on 5/18 acute myeloid leukemias,
7/48 benign prostatic hyperplasias, 8/18 breast cancers, 4/17 chronic lymphocytic
leukemias, 1/29 colorectal cancers, 3/34 brain cancers, 4/21 liver cancers, 4/10 head
and neck cancers, 4/18 melanomas, 12/20 non-Hodgkin lymphomas, 10/90 non-small
cell lung cancers, 6/20 ovarian cancers, 2/18 esophageal cancers, 3/19 pancreatic
cancers, 3/23 kidney cancers, 6/17 small cell lung cancers, 3/15 urinary bladder
cancers, and 6/16 uterus cancers. Figure 1B: Gene symbol: CDC25B, Peptide:
KTL (SEQ ID NO.: 24), Tissues from left to right: 6 adipose tissues, 8 adrenal
, 24 blood cells, 17 blood vessels, 10 bone marrows, 15 brains, 8 breasts, 4
cartilages, 7 esophagi, 2 eyes, 16 hearts, 19 s, 21 large ines, 25 livers, 49
lungs, 8 lymph nodes, 13 nerves, 3 ovaries, 11 pancreases, 6 parathyroid glands, 1
peritoneum, 6 pituitary glands, 7 placentas, 1 pleura, 3 prostates, 7 salivary glands, 10
skeletal muscles, 12 skins, 6 small intestines, 12 spleens, 5 stomachs, 7 testes, 2 thymi,
2 thyroid glands, 14 tracheas, 7 ureters, 8 urinary bladders, 6 uteri, 3 gallbladders, 17
adder cancer and giocarcinoma samples. The peptide has additionally been
ed on 1/20 ovarian s. Figure 10: Gene symbol: RNF19A, Peptide:
NLSETASTMAL (SEQ ID NO.: 25), Tissues from left to right: 6 adipose tissues, 8
adrenal glands, 24 blood cells, 17 blood vessels, 10 bone marrows, 15 brains, 8
breasts, 4 cartilages, 7 esophagi, 2 eyes, 16 hearts, 19 kidneys, 21 large intestines, 25
, 49 lungs, 8 lymph nodes, 13 nerves, 3 ovaries, 11 pancreases, 6 parathyroid
glands, 1 peritoneum, 6 pituitary glands, 7 placentas, 1 pleura, 3 prostates, 7 salivary
, 10 skeletal muscles, 12 skins, 6 small intestines, 12 spleens, 5 stomachs, 7
testes, 2 thymi, 2 thyroid glands, 14 tracheas, 7 ureters, 8 y bladders, 6 uteri, 3
gallbladders, 17 adder cancer and cholangiocarcinoma samples. The e has
additionally been detected on 1/21 liver cancers, 4/90 non-small cell lung cancers, 1/17
small cell lung s, 1/20 dgkin lymphomas, 1/20 ovarian cancers, and 1/15
urinary bladder cancers. Figure 1D: Gene symbol: MEGF6, Peptide: VLQDELPQL
(SEQ ID NO.: 20), Samples from left to right: 12 normal tissues (1 esophagus, 2 livers,
3 lungs, 1 rectum, 1 skin, 1 small intestine, 1 spleen, 2 tracheas), 45 cancer tissues (3
bile duct cancers, 1 breast cancer, 1 esophageal , 4 gallbladder cancers, 5 head
and neck cancers, 3 kidney cancers, 4 leukocytic leukemia cancers, 2 liver cancers, 9
_ 99 _
lung cancers, 2 lymph node cancers, 1 myeloid cell cancer, 2 ovarian cancers, 3
pancreas cancers, 4 prostate cancers, 1 urinary bladder cancer). Figures 1E h G
show the resentation of various peptides in different cancer tissues (black dots).
Upper part: Median MS signal intensities from technical replicate measurements are
d as dots for single HLA-A*O2 positive normal (grey dots) and tumor samples
(black dots) on which the peptide was detected. Tumor and normal samples are
grouped according to organ of origin, and box-and-whisker plots ent median, 25th
and 75th percentile (box), and minimum and maximum (whiskers) of normalized signal
intensities over multiple samples. Normal organs are ordered according to risk
categories (blood cells, blood vessels, brain, liver, lung: high risk, grey dots;
reproductive , breast, prostate: low risk, grey dots; all other organs: medium risk;
grey dots). Lower part: The relative peptide detection frequency in every organ is shown
as spine plot. Numbers below the panel indicate number of samples on which the
e was detected out of the total number of samples analyzed for each organ (N =
526 for normal samples, N = 562 for tumor samples). If the peptide has been detected
on a sample but could not be quantified for technical reasons, the sample is ed in
this entation of detection frequency, but no dot is shown in the upper part of the
figure. Tissues (from left to right): Normal samples: blood cells; bloodvess (blood
vessels); brain; heart; liver; lung; adipose (adipose tissue); adren.gl. al gland);
bile duct; r; BM (bone marrow); cartilage; esoph agus); eye; gallb
(gallbladder); head&neck; kidney; large_int (large intestine); LN (lymph node); nerve;
as; parathyr (parathyroid gland); perit (peritoneum); pituit (pituitary); pleura;
us (skeletal muscle); skin; int (small intestine); spleen; stomach; thyroid;
trachea; ureter; breast; ovary; ta; prostate; testis; thymus; uterus. Tumor
samples: AML: acute myeloid leukemia; BRCA: breast cancer; CCC: cholangiocellular
carcinoma; CLL: chronic cytic leukemia; CRC: colorectal cancer; GBC:
gallbladder cancer; GBM: glioblastoma; GC: gastric cancer; GEJC: stomach cardia
esophagus, cancer; HCC: hepatocellular carcinoma; HNSCC: head-and-neck cancer;
MEL: melanoma; NHL: non-hodgkin lymphoma; NSCLC: non-small cell lung cancer;
00: ovarian cancer; OSCAR: esophageal cancer; PACA: pancreatic cancer; PRCA:
prostate cancer; RCC: renal cell carcinoma; SCLC: small cell lung cancer; UBC: urinary
bladder carcinoma; UEC: uterine and endometrial cancer. Figure 1E) Gene :
PLEKHM3, e: VLYDNTQLQL (SEQ ID NO.: 17), Figure 1F) Gene symbol: ILF2,
Peptide: TAQTLVRIL (SEQ ID NO.: 26), Figure 1G) Gene symbol: STAM, Peptide:
NVL (SEQ ID NO.: 29).
Figures 2A through 2C show exemplary expression profiles of source genes of the
present invention that are highly xpressed or exclusively expressed in gallbladder
cancer and cholangiocarcinoma in a panel of normal tissues (white bars) and 10
gallbladder cancer and cholangiocarcinoma s (black bars). Tissues from left to
right: 6 arteries, 2 blood cells, 5 brains, 3 hearts, 3 livers, 3 lungs, 2 veins, 1 adipose
tissue, 1 adrenal gland, 1 bile duct, 5 bone marrows, 1 cartilage, 1 colon, 1 esophagus,
2 eyes, 2 gallbladders, 2 salivary glands, 1 kidney, 6 lymph nodes, 4 pancreases, 1
yroid gland, 2 peripheral nerves, 2 peritoneums, 2 pituitary , 2 pleuras, 1
rectum, 2 skeletal muscles, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thyroid
gland, 7 tracheas, 2 ureters, 1 urinary bladder, 1 breast, 5 ovaries, 5 tas, 1
prostate, 1 testis, 1 thymus, 1 , 11 gallbladder cancer and cholangiocarcinoma
samples. Figure 2A) Gene symbol: CYP2W1, B) Gene symbol: PKHD1, C) Gene
symbol: SUCNR1.
Figure 3 shows exemplary immunogenicity data: flow cytometry results after peptide-
specific multimer staining.
Figure 4 shows exemplary s 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 x with SquD No 13 peptide (A, left panel),
SeqlD No 16 peptide (B, left panel) and SquD No 25 peptide (C, left panel),
respectively. After three cycles of stimulation, the detection of peptide-reactive cells was
performed by 2D multimer staining with A*O2/SeqlD No 13 (A), A*02/SeqlD No 16 (B) or
A*O2/SeqlD No 25 (C). Right panels (A, B and C) show control staining of cells
ated with irrelevant A*O2/peptide complexes. Viable singlet cells were gated for
CD8+ lymphocytes. Boolean gates helped excluding false-positive events detected with
multimers specific for different peptides. Frequencies of specific multimer+ cells among
CD8+ lymphocytes are indicated.
EXAMPLES
EXAMPLE 1
Identification and guantitation of tumor associated peptides presented on the cell
surface
Tissue samples
Patients’ tumor tissues were obtained from: Conversant Healthcare Systems Inc.,
Huntsville, AL, USA), ProteoGenex Inc. (Culver City, CA, USA), Tissue Solutions Ltd
(Glasgow, UK), University Hospital TUbingen (TUbingen, Germany). Normal s
were obtained from Asterand (Detroit, MI, USA & Royston, Herts, UK), Bio-Options Inc.
(Brea, CA, USA), BioServe (Beltsville, MD, USA), Capital BioScience Inc. (Rockville,
MD, USA), Geneticist Inc. (Glendale, CA, USA), Kyoto Prefectural University of
Medicine (KPUM) (Kyoto, , ProteoGenex Inc. r City, CA, USA), Tissue
Solutions Ltd (Glasgow, UK), University Hospital Geneva (Geneva, Switzerland),
University al Heidelberg (Heidelberg, Germany), University Hospital Munich
(Munich, Germany), 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 ion of
TUMAPs at -70°C or below.
ion of HLA es from tissue samples
HLA peptide pools from frozen tissue samples were obtained by immune
precipitation from solid tissues according to a slightly ed protocol (Falk et al.,
1991; Seeger et al., 1999) using the HLA-A*02—specific antibody 887.2, the HLA-A, -B, -
C-specific antibody WES/32, CNBr—activated sepharose, acid treatment, and
ultrafiltration.
Mass ometry analyses
The HLA e pools as ed were ted according to their hydrophobicity by
reversed-phase chromatography (nanoAcquity UPLC system, Waters) and the eluting
peptides were analyzed in LTQ- velos and fusion hybrid mass spectrometers
(ThermoElectron) equipped with an ESI source. Peptide pools were loaded directly onto
the analytical fused-silica micro-capillary column (75 um id x 250 mm) packed with 1.7
pm C18 reversed-phase al (Waters) applying a flow rate of 400 nL per minute.
Subsequently, the peptides were separated using a two-step 180 minute-binary gradient
from 10% to 33% B at a flow rate of 300 nL per minute. The gradient was composed of
Solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in itrile). A
gold coated glass capillary (PicoTip, New Objective) was used for introduction into the
nanoESl source. The bitrap mass spectrometers were operated in the data-
ent mode using a TOP5 strategy. In brief, a scan cycle was initiated with a full
scan of high mass accuracy in the ap (R = 30 000), which was followed by MS/MS
scans also in the orbitrap (R = 7500) on the 5 most abundant precursor ions with
dynamic exclusion of previously selected ions. Tandem mass spectra were reted
by SEQUEST and additional manual control. The identified peptide sequence was
assured by comparison of the generated natural peptide fragmentation pattern with the
fragmentation pattern of a synthetic sequence-identical reference peptide.
Label-free relative LC-MS quantitation was performed by ion counting i.e. by extraction
and analysis of LC-MS features (Mueller et al., 2007). The method assumes that the
peptide’s LC-MS signal area correlates with its abundance in the sample. Extracted
features were further processed by charge state deconvolution and ion time
alignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MS features were
cross-referenced with the sequence identification results to combine quantitative data of
different samples and tissues to peptide presentation es. The quantitative data
were normalized in a two-tier fashion according to central tendency to account for
variation within technical and biological replicates. Thus, each fied peptide can be
associated with quantitative data allowing relative quantification n samples and
WO 02806
tissues. In addition, all quantitative data ed for e candidates was inspected
manually to assure data consistency and to verify the accuracy of the automated
analysis. For each peptide, a presentation profile was calculated showing the mean
sample presentation as well as replicate variations. The profiles juxtapose adder
cancer and cholangiocarcinoma samples to a baseline of normal tissue samples.
tation profiles of exemplary over-presented peptides are shown in Figure 1.
Presentation scores for exemplary peptides are shown in Table 8.
Table 8: Presentation scores. The table lists peptides that are very highly over-
presented on tumors compared to a panel of normal tissues (+++), highly over-
presented on tumors compared to a panel of normal tissues (++) or over-presented on
tumors compared to a panel of normal tissues (+).The panel of normal tissues
considered relevant for comparison with tumors consisted of: adipose , l
gland, artery, bone , brain, l nerve, colon, duodenum, esophagus, eye,
gallbladder, heart, kidney, liver, lung, lymph node, mononuclear white blood cells,
pancreas, parathyroid gland, peripheral nerve, peritoneum, pituitary, pleura, rectum,
salivary gland, al muscle, skin, small intestine, spleen, stomach, thyroid gland,
trachea, ureter, urinary bladder, vein.
E150 ID sequence Esejs’tfriation
1 YAAEIASAL +++
2 AAYPEIVAV +++
3 EMDSTVITV +++
4 FLLEAQNYL +++
GLIDEVMVLL +++
6 LLLPLLPPLSPS +++
7 LLLSDPDKVTI +++
8 LSASLVRIL +++
9 RLAKLTAAV +++
SAFPFPVTVSL +++
11 SIIDFTVTM +++
12 TILPGNLQSW +++
13 VLPRAFTYV +++
14 YGIEFVVGV +++
SVIDSLPEI +++
E150 ID sequence Eistfriation
17 VLYDNTQLQL +++
19 TAYPQVVVV +
VLQDELPQL ++
21 IAFPTSISV +++
24 ISAPLVKTL +++
NLSETASTMAL +++
26 RIL +++
27 ALAEQVQKA ++
FASGLIHRV +++
31 IAIPFLIKL ++
32 YVISQVFEI +
EXAMPLE 2
Expression profiling of genes encoding the peptides of the invention
Over-presentation or specific presentation of a e 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 e targets for
immunotherapies. Especially for eutic options with high safety risks, such as
affinity-matured TCRs, the ideal target peptide will be derived from a protein that is
unique to the tumor and not found on normal tissues.
RNA sources and preparation
Surgically removed tissue specimens were provided as indicated above (see Example
1) after written informed consent had been obtained from each t. 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 p with RNeasy
(QIAGEN, Hilden, Germany); both methods were performed ing to the
cturer's protocol.
Total RNA from healthy human tissues for RNASeq ments was ed from:
Asterand (Detroit, MI, USA & Royston, Herts, UK), BioCat GmbH (Heidelberg,
Germany), BioServe ville, MD, USA), Capital BioScience Inc. (Rockville, MD,
USA), Geneticist Inc. (Glendale, CA, USA), to Nazionale Tumori "Pascale" (Naples,
Italy), ProteoGenex Inc. (Culver City, CA, USA), University Hospital Heidelberg
(Heidelberg, Germany).
Total RNA from tumor tissues for RNASeq experiments was obtained from:
ProteoGenex Inc. (Culver City, CA, USA), Tissue Solutions Ltd (Glasgow, UK),
University Hospital Ttibingen (Ttibingen, 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).
RNAseg experiments
Gene expression analysis of - tumor and normal tissue RNA samples was performed by
next generation sequencing (RNAseq) by CeGaT (Ttibingen, Germany). Briefly,
sequencing libraries are prepared using the Illumina HiSeq v4 t kit according to
the provider’s protocol (Illumina Inc., San Diego, CA, 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 ript level as RPKM (Reads Per
Kilobase per Million mapped reads, generated by the software Cufflinks) and on exon
level (total reads, generated by the re ls), based on annotations of the
ensembl sequence database (Ensembl77). Exon reads are normalized for exon length
and alignment size to obtain RPKM values.
Exemplary sion es of source genes of the present invention that are highly
over-expressed or exclusively expressed in gallbladder cancer and cholangiocarcinoma
are shown in Figure 2. Expression scores for r exemplary genes are shown in
Table 9.
Table 9: Expression scores. The table lists peptides from genes that are very highly
over-expressed in tumors compared to a panel of normal tissues (+++), highly overexpressed
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 s: adipose tissue,
adrenal gland, artery, bile duct, blood cells, bone marrow, brain, cartilage, colon,
esophagus, eye, gallbladder, ry gland, heart, kidney, liver, lung, lymph node,
pancreas, parathyroid gland, peripheral nerve, peritoneum, pituitary, , rectum,
skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland, trachea, ureter,
urinary bladder, and vein. In case expression data for several samples of the same
tissue type were ble, the arithmetic mean of all respective samples was used for
the calculation.
SEQ ID No Sequence ngizssion
MVLL +++
8 LSASLVRIL +++
13 VLPRAFTYV +++
14 YGIEFVVGV +++
EXAMPLE 3
In vitro immunogenicity for MHC class I presented peptides
In order to obtain information regarding the immunogenicity of the TUMAPs of the
present invention, the ors 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*0201 restricted TUMAPs of the
invention, demonstrating that these peptides are T-cell epitopes against which CD8+
precursor T cells exist in humans (Table 10).
In vitro priming of CD8+ T cells
In order to m in vitro stimulations by artificial antigen presenting cells loaded with
peptide-MHC complex (pMHC) and anti-CD28 antibody, the inventors first isolated
CD8+ T cells from fresh HLA-A*02 leukapheresis products via positive selection using
CD8 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors
obtained from the University clinics Mannheim, Germany, after informed consent.
PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium (TCM) until
use consisting of RPMl-Glutamax (lnvitrogen, Karlsruhe, Germany) supplemented with
% heat inactivated human AB serum (PAN-Biotech, ach, Germany), 100 U/ml
Penicillin/100 ug/ml omycin (Cambrex, Cologne, Germany), 1 mM sodium
pyruvate (CC Pro, Oberdorla, Germany), 20 pg/ml ycin (Cambrex). 2.5 ng/ml IL-
7 (PromoCell, Heidelberg, Germany) and 10 U/ml lL-2 (Novartis Pharma, Niirnberg,
Germany) were also added to the TCM at this step.
Generation of nti-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.
The purified co-stimulatory mouse lgG2a anti human CD28 Ab 9.3 (Jung et al., 1987)
was chemically biotinylated using Sulfo-N-hydroxysuccinimidobiotin as ended
by the manufacturer (Perbio, Bonn, Germany). Beads used were 5.6 pm diameter
avidin coated polystyrene les (Bangs Laboratories, Illinois, USA).
pMHC used for positive and negative control stimulations were A*0201/MLA-001
(peptide ELAGIGILTV (SEQ ID NO. 39) from modified Melan-A/MART-l) and
/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO. 40), respectively.
800.000 200 ul were coated in l plates in the presence of 4 x 12.5 ng
different biotin-pMHC, washed and 600 ng biotin anti-CD28 were added subsequently in
a volume of 200 pl. Stimulations were initiated in 96-well plates by co-incubating ’Ix’l06
CD8+ T cells with 2x105 washed coated beads in 200 pl TCM supplemented with 5
ng/ml lL-12 (PromoCell) for 3 days at 37°C. Half of the medium was then exchanged by
fresh TCM supplemented with 80 U/ml lL-2 and incubating was continued for 4 days at
37°C. This stimulation cycle was performed for a total of three times. For the pMHC
multimer readout using 8 different pMHC les per condition, a two-dimensional
combinatorial coding approach was used as previously bed (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 (lnvitrogen, Karlsruhe, Germany), CD8—FITC antibody clone 8K1 (BD, Heidelberg,
Germany) and fluorescent pMHC multimers. For analysis, a BD LSRII SORP cytometer
ed 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 ic
multimer+ CD8+ lymphocytes was detected by comparing to negative l
stimulations. lmmunogenicity for a given antigen was detected if at least one ble
in vitro ated 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 ic multimer+ cells was at least 10x the
median of the negative l stimulations).
In vitro lmmunogenicity for gallbladder cancer and giocarcinoma peptides
For tested HLA class I peptides, in vitro immunogenicity could be demonstrated by
tion of peptide specific T-cell lines. Exemplary flow cytometry results after
TUMAP-specific multimer staining for 2 peptides of the invention are shown in Figure 3
together with corresponding negative controls. Additional exemplary flow cytometry
results after TUMAP-specific multimer staining for three peptides of the invention are
shown in Figure 4 together with corresponding negative controls. Results for five
peptides from the invention are summarized in Table 10A. Additional results for four
peptides of the invention are summarized in Table 108.
Table 10A: in vitro immunogenicity of HLA class I peptides of the invention
Exemplary results of in vitro immunogenicity experiments performed by the applicant for
the es of the invention. <20 % = +; 20 % - 49 % = ++; 50 % - 69 %= +++; >= 70 %
= ++++
Seq ID
ILGTEDLIVEV
38 KIQEILTQV ++
Table 108: In vitro immunogenicity of HLA class I peptides of the invention
Exemplary results of in vitro immunogenicity experiments conducted by the applicant for
HLA-A*02 restricted peptides of the invention. Results of in vitro genicity
experiments are indicated. Percentage of positive wells and donors (among evaluable)
are summarized as ted <20 % = +; 20 % - 49 % = ++; 50 % - 69 %= +++; >= 70
%= ++++
SEQ ID No Sequence Wells positive [%]
6 LLLPLLPPLSPS +
13 TYV ++++
16 AVMTDLPVI +
NLSETASTMAL ++
EXAMPLE 4
Synthesis of peptides
All peptides were sized using rd 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 uoro acetate salt) in purities of >50%. All
TUMAPs are preferably administered as trifluoro-acetate salts or acetate salts, other
salt-forms are also possible.
EXAMPLE 5
MHC Binding Assays
Candidate peptides for T cell based therapies ing to the present ion were
further tested for their MHC binding capacity (affinity). The individual peptide-MHC
xes were produced by UV-ligand exchange, where a UV-sensitive peptide is
cleaved upon UV-irradiation, and exchanged with the peptide of interest as ed.
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 (02m) of stabilized MHC complexes. The assay was performed as generally
bed in Rodenko et al. (Rodenko et al., 2006).
96 well MAXISorp plates (NUNC) were coated over night with 2ug/ml streptavidin in
PBS at room temperature, washed 4x and blocked for1h at 37°C in 2% BSA containing
ng buffer. Refolded HLA-A*02:01/MLA-001 rs 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 1h at
37°C, washed four times, incubated with 2ug/ml HRP conjugated anti-02m for 1h at
37°C, washed again and detected with TMB solution that is stopped with NH2804.
Absorption was measured at 450nm. Candidate es 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 xes.
Table 11: MHC class I binding scores. Binding of HLA-class l restricted es to
HLA-A*02:01 was ranged by e exchange yield: >10% = +; >20% = ++; >50 = +++;
> 75% = ++++
SEQ ID No Sequence Peptide exchange
1 YAAEIASAL +++
2 AAYPEIVAV +++
3 EMDSTVITV +++
4 FLLEAQNYL ++++
SEQ ID No Sequence Peptide exchange
GLIDEVMVLL ++++
6 LLLPLLPPLSPS ++++
7 LLLSDPDKVTI ++++
8 LSASLVRIL +
9 RLAKLTAAV ++++
SAFPFPVTVSL ++
11 SIIDFTVTM ++++
12 TILPGNLQSW +
13 VLPRAFTW ++++
14 YGIEFVVGV ++++
SVIDSLPEI ++++
16 AVMTDLPVI ++++
17 VLYDNTQLQL ++++
18 SQV +++
19 TAYPQVVW ++
VLQDELPQL ++++
21 IAFPTSISV +++
22 SAFGFPVIL ++
23 SLLSELLGV ++++
24 ISAPLVKTL +
NLSETASTMAL ++++
26 RIL +
27 ALAEQVQKA +++
28 YASGSSASL +
29 FASEVSNVL ++++
FASGLIHRV +++
31 IAIPFLIKL ++++
32 YVISQVFEI ++++
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Claims (29)
1. A peptide comprising an amino acid sequence ing to SEQ ID No. 18; or a pharmaceutical acceptable salt thereof, wherein said peptide has an overall length of up to 16 amino acids.
2. The peptide according to claim 1, wherein said peptide has the ability to bind to an MHC class-I or –II molecule, and wherein said e, when bound to said MHC, is capable of being recognized by CD4 and/or CD8 T cells.
3. The e pharmaceutical acceptable salt thereof according to any one of claims 1 or 2, wherein said peptide consists of an amino acid sequence according to SEQ ID No.
4. The peptide or pharmaceutical acceptable salt thereof according to any one of claims 1 to 3, wherein said peptide includes non-peptide bonds.
5. A fusion protein comprising the peptide or pharmaceutical acceptable salt thereof according to any one of claims 1 to 4, ses the peptide according to any one of claims 1 to 4 and inal amino acids of the HLA-DR antigen-associated invariant chain (Ii).
6. An antibody, in particular a soluble or membrane-bound antibody, preferably a monoclonal antibody or a g fragment thereof, that specifically recognizes the peptide or pharmaceutical acceptable salt thereof ing to any of claims 1 to 4, or specifically recognizes the peptide or pharmaceutical acceptable salt thereof according to any of claims 1 to 4 when bound to an MHC molecule.
7. A T-cell receptor, a soluble T-cell receptor or a membrane-bound T-cell receptor, or a binding fragment thereof, that is reactive with an HLA , wherein said ligand is the peptide or pharmaceutical acceptable salt thereof according to any one of claims 1 to 4, or said ligand is the peptide or pharmaceutical acceptable salt thereof according to any of claims 1 to 4 when bound to an MHC le.
8. The soluble T-cell receptor according to claim 7, or the soluble T-cell receptor according to claim 7 that carries a further effector function, an immune stimulating domain or a toxin.
9. An aptamer that specifically recognizes the e or pharmaceutical acceptable salt f according to any one of claims 1 to 4, or recognizes the peptide or pharmaceutical acceptable salt thereof according to any one of claims 1 to 4 that is bound to an MHC molecule.
10. A n isolated nucleic acid, encoding for a peptide or pharmaceutical acceptable salt thereof according to any one of claims 1 to 4, an antibody or binding fragment thereof ing to claim 6, or a T-cell or or binding fragment thereof according to claim 7 or 8, or an expression vector e of expressing a nucleic acid encoding for a e according to any one of claims 1 to 4, an antibody or binding fragment thereof according to claim 6, or a T-cell receptor or binding nt thereof according to claim 7 or 8.
11. A recombinant host cell , a recombinant antigen presenting cell, a recombinant dendritic cell, a recombinant T cell or a recombinant NK cell, comprising the peptide according to any one of claims 1 to 4, the fusion protein according to claim 5, the antibody or binding fragment thereof according to claim 6, the T-cell receptor or binding fragment thereof according to claim 7 or 8 or the nucleic acid or the expression vector ing to claim 10, wherein said cell is not a cell within a human body.
12. An in vitro method for producing activated T lymphocytes, the method comprising contacting in vitro T cytes with antigen loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or an artificial uct mimicking an antigen-presenting cell for a period of time sufficient to activate said T lymphocytes in an antigen specific manner, wherein said antigen is a peptide according to any one of claims 1 to 3.
13. An activated T lymphocyte, produced by the method according to claim 12, that selectively recognizes a cell which presents a polypeptide comprising an amino acid ce given in any one of claims 1 to 3.
14. A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of the peptide according to any one of claims 1 to 4, the fusion protein according to claim 5, the antibody or binding fragment thereof according to claim 6, the T-cell receptor or binding fragment thereof according to claim 7 or 8, the aptamer according to claim 9, the nucleic acid or the sion vector according to claim 10, a inant host cell according to claim 11, or the activated T lymphocyte according to claim 13, or a conjugated or labelled active ingredient, and one or more of a pharmaceutically acceptable carrier, pharmaceutically acceptable excipients and/or a stabilizer.
15. The ceutical composition according to claim 14, further comprising one or more adjuvants, optionally wherein the one or more adjuvants are ed from interleukin and immunoadjuvant.
16. The eutic composition according to claim 15, wherein a) the interleukin is IL-2; and/or b) the immunoadjuvant is IL-15.
17. A method for producing the peptide or pharmaceutically acceptable salt f according to any one of claims 1 to 4, the fusion protein according to claim 5, the antibody or binding fragment f according to claim 6, or the T-cell receptor or binding fragment thereof according to claim 7 or 8, the method comprising culturing a recombinant host cell according to claim 11, and isolating the peptide or pharmaceutical acceptable salt thereof, the fusion protein, the antibody or binding fragment thereof or the T cell receptor or binding fragment thereof from said host cell and/or its e medium.
18. Use of the peptide according to any one of claims 1 to 4, the fusion protein according to claim 5, the antibody or binding fragment thereof ing to claim 6, the T-cell receptor or binding fragment thereof according to claim 7 or 8, the aptamer according to claim 9, the nucleic acid or the expression vector ing to claim 10, a recombinant host cell according to claim 11, or the activated T lymphocyte ing to claim 13 for use in the manufacture of a medicament against cancer.
19. The use according to claim 18, wherein said cancer is selected from the group of gallbladder cancer and cholangiocarcinoma, acute myeloid leukemia, melanoma, small cell lung cancer, all cell lung , non-Hodgkin lymphoma, chronic lymphocytic leukemia, pancreatic cancer, liver cancer, n cancer, head and neck , urinary r cancer, breast cancer, and kidney cancer and other tumors that show an overexpression of a protein from which a peptide of SEQ ID No. 18 is derived from.
20. A kit comprising: a) a container comprising a ceutical composition ning the peptide(s) or the pharmaceutical acceptable salt thereof according to any one of claims 1 to 4, the fusion protein according to claim 5 the antibody or binding fragment thereof ing to claim 6, the T-cell receptor or binding fragment thereof according to claim 7 or 8, the aptamer according to claim 9, the nucleic acid or the expression vector according to claim 10, a recombinant host cell according to claim 11, or the activated T lymphocyte according to claim 13, in solution or in lyophilized form.
21. The kit according to claim 20, further comprising one or more of: b) a second container containing a diluent or reconstituting solution for the lyophilized formulation; c) at least one more peptide selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 17 and SEQ ID No. 19 to SEQ ID No. 38; d) instructions for (1) use of the solution or (2) reconstitution and/or use of the lyophilized formulation; e) a buffer; f) a diluent; g) a filter; h) a needle; i) a syringe; and/or j) an adjuvant.
22. A method for producing a personalized anti -cancer vaccine or a compound-based and/or cellular therapy for an individual patient, said method comprising: a) identifying a tumor-associated peptides s) presented by a tumor sample from said individual patient; b) comparing the peptides as identified in a) with a warehouse of peptides that have been pre-screened for immunogenicity and/or over-presentation in tumors as compared to normal tissues; c) ing at least one peptide from the warehouse that s a TUMAP identified in the patient tumor sample; and d) manufacturing and/or formulating the personalized vaccine or compound-based or cellular therapy based on step c) wherein said warehouse comprises a peptide according to SEQ ID No. 18.
23. The method according to claim 22, wherein said TUMAP is 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 fy proteins that are over-expressed or aberrantly expressed in the tumor sample; and a2) ating the expression data with ces 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 ntly expressed by the tumor.
24. The method according to claim 22 or 23, wherein the sequences of MHC ligands are identified by eluting bound peptides from MHC molecules isolated from the tumor sample, and sequencing the eluted ligands.
25. The method according to any one of claims 22 to 24, wherein the normal tissue corresponding to the tissue type of the tumor sample is obtained from the same patient.
26. The method according to any one of claims 22 to 25, wherein the peptide included in the warehouse is identified based on the following steps: aa. Performing genome-wide messenger ribonucleic acid (mRNA) expression analysis by highly parallel methods, such as microarrays or sequencing-based expression profiling, sing identify genes that over-expressed in a malignant tissue, compared with a normal tissue or tissues; ab. Selecting peptides encoded by ively expressed or xpressed genes as ed in step aa, and ac. Determining an induction of in vivo T-cell responses by the es as selected comprising in vitro immunogenicity assays using human T cells from healthy donors or said t; or ba. fying HLA ligands from said tumor sample using mass ometry; bb. Performing genome-wide messenger ribonucleic acid (mRNA) expression analysis by highly parallel methods, such as microarrays or sequencing-based expression profiling, comprising identify genes that over-expressed in a malignant tissue, ed with a normal tissue or tissues; bc. Comparing the identified HLA ligands to said gene expression data; bd. Selecting es encoded by selectively expressed or over-expressed genes as detected in step bc; be. Re-detecting of selected TUMAPs from step bd on tumor tissue and lack of or infrequent detection on healthy tissues and confirming the relevance of over-expression at the mRNA level; and bf. Determining an induction of in vivo T-cell responses by the peptides as selected comprising in vitro immunogenicity assays using human T cells from healthy donors or said patient.
27. The method according to any one of claims 22 to 26, wherein the immunogenicity of the peptides ed in the warehouse is determined by a method sing in vitro immunogenicity assays, patient immunomonitoring for individual HLA binding, MHC multimer staining, ELISPOT assays and/or ellular cytokine staining.
28. The method according to any one of claims 22 to 27, further comprising identifying at least one mutation that is unique to the tumor sample relative to normal corresponding tissue from the individual patient, and ing a peptide that correlates with the mutation for inclusion in the vaccine or for the generation of cellular therapies.
29. The method according to claim 28, wherein said at least one mutation is identified by whole genome sequencing. ha, @33an mfiaéfi fifiwfi «336% Kawfifi “33¢ a a“ wrgmgfi gsgww 333qu *wgufimfim mw £33 m, £§§m¢§ a? hmwmufifim “Egg? gmfim mm 8%???” wag» m g, “533“”; 3 a xggmfificm Mg“: xmwfimwa gfifiwwfi w maps» ”53$ g3” “Eswsfifig a ffiwafi hm “UN “Egg gumfim imamg 6&3 “3%:33 3 “$33, ma $¢§£§m ££§3§ 53%; “62$; $va5» m. *wvgfiw w m3“ 3 w MNEfi 3%th $wa 3, m, gfiafiugg “afiiim m 3,, garéw Sfim§§ gmfim B§m§m 5&333 “mmmfigmw «wwmhwg m, m, 333“ E EEEQ geafim «wwwmgfii m gm. “3??” wgfiwmwfia “WEE“, “32 m m m 5 an 8%: SEE O>>h©mmm_ Em 13539;} émmasg} uaafiaaségg aagzegag E .3“ wwwswwm wfififig gwfigfi £33 £33,.“ ‘awfiww «Eggs gag.m “aficwwfifi *msuafigm 3 3ng m fingfitgfi mg, 33:3» ‘wgmmag .mEE my $3”ngQO vfifi m mm gawmfima ma, w ~m§,:§m§m £33 hmmgag $333?” w “3.3% 333 $333fi§ w .fiwngfi m," mm gafim ‘mfigfiw 33mg 5&3» x 3, amumswwé «w “3&3 m.” €333” “gamma m, 3m figufiwfig wgwfimfiw £$§§w m w." .ggufia mm“ J ‘35 $33 fiafifi 3 M wfigfiugg ‘wwgfiu m 3 fiwfigmfi, 53%, 333% ‘ngfiaga 3%33 figgwm m $333 m, 333% Mama»? 33%? ‘wymfitg w 3 m «3 ~33ng “wwwfigfi ”02 “3%” m N a m: ”gig Q 93m: gm 333?: mmfias} fimazamza agawa E fin wuutummw mamaggm gfimgga £§§n $33» baggage w 33ng gag?” m“ ”mgwawgfi .mfigfiafi m“ ”wuflfi m ”mgatag me mwfigmu *éwfiam 63% flw fivg mm 353mg ma 3 a figmfifimm m£3: £333 figmgfifi m ”Exam m figfi wax: ‘Ewwxgfiya m w samfi ha am, ?&:m§ Q§¢Ex svgmw mam 333 £333»: m mm $§§§ «m agwws figgwmmg w *mufiwxm ASE $3”ng a 3H wmgfiuéfl figfiw gag 3 m “$3,333 xfisfia 352532 a a” aggmw 333% «magamw fimwwgagy fiafiwfi m. xwwwhga h 393» figmg m mN 333: m “m2 gm @ ammmamfig ”$33 gyfifiga .Mfififi m} m, UH ”@333 Q 8%: Em {mmmgwfi gaggwfifiagg mwg my m, $333 w“ahwwfimfi. $§§m§ $33,» 3 ggfix giaya figzgh m$333 £333 m x3 $33», ”$3 «3% E3§§ ”gum?“ m 35%» ”mammfifi m ggfig ”Em: gags.» gggu $quan 3V3 a?” w, 53m gmfi Euumfimwu “Egg a w fgag ”£393 w. £0»qu w, 93¢: $53 $33K”, gag: “aungagv m m égagy 3&3»: £33m H “3% .m m gang» kgmm, ”vawgg gaugagm “533 .433:va $333 auda§> .3 w $3,“: :¢ a am i 3&3: gag Efifig 33% 53$?» fififigm ‘figgg ”a: $3an maggg 993% a m, 3 $3 fi $§§§ W 3 Egg 93m: gm {Sag-3:"; ééasafiwa mmaagm mmag my“?WM«mo—«WNWMWWMMMWM «0:22 mmewmwmgfigfiggww ilfigmw _ $3?» fig g'ggig ‘géggfiagggzgé émsagfiiggé figfimmwmz ng' wag htox~>bsvw+wx¢aifia£wfivthlpcy«c»?>»tt,$a ............ fifiggzfifig ggmégéfi mfimwmém ggfigggfigzs Egg‘éggfi :ggfggggfig (gag: "Eggg Wmfigfifi 33221:: Egrggggg V3§5§€§ gééiiffifid E fifigggggw glam: {A*02) ‘ 34% :gggfagsgs: ,fififimg VLYEENTQLQL ggfigw §$¥fi§§3fi§ 5mg > fiéfi mmm fiffié‘figfii 17 33%;} ragga} :fz‘agams n w W2%W‘33; g..ggg.,gg*‘»..v.~».g».4g>g..g»g.g,:g».Hgg;gg~g.g,gggg.g .»,«ggn._.»»r..g.g u it M) mtg ii} jgfiéfimggg 1E Papfidgfi SEQ WW ,gggfigiiwgfi
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