U.S. patent application number 17/665117 was filed with the patent office on 2022-07-21 for novel peptides and combination of peptides for use in immunotherapy against cll and other cancers.
The applicant listed for this patent is Immatics Biotechnologies GmbH. Invention is credited to Jens FRITSCHE, Andrea MAHR, Oliver SCHOOR, Harpreet SINGH, Toni WEINSCHENK, Anita WIEBE.
Application Number | 20220226450 17/665117 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-21 |
United States Patent
Application |
20220226450 |
Kind Code |
A1 |
MAHR; Andrea ; et
al. |
July 21, 2022 |
NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY
AGAINST CLL AND OTHER CANCERS
Abstract
The present invention relates to peptides, proteins, nucleic
acids and cells for use in immunotherapeutic methods. In
particular, the present invention relates to the immunotherapy of
cancer. The present invention furthermore relates to
tumor-associated T-cell peptide epitopes, alone or in combination
with other tumor-associated peptides that can for example serve as
active pharmaceutical ingredients of vaccine compositions that
stimulate anti-tumor immune responses, or to stimulate T cells ex
vivo and transfer into patients. Peptides bound to molecules of the
major histocompatibility complex (MHC), or peptides as such, can
also be targets of antibodies, soluble T-cell receptors, and other
binding molecules.
Inventors: |
MAHR; Andrea; (Tuebingen,
DE) ; WEINSCHENK; Toni; (Aichwald, DE) ;
WIEBE; Anita; (Ruebgarten, DE) ; SCHOOR; Oliver;
(Tuebingen, DE) ; FRITSCHE; Jens; (Dusslingen,
DE) ; SINGH; Harpreet; (Muenchen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Immatics Biotechnologies GmbH |
Tuebingen |
|
DE |
|
|
Appl. No.: |
17/665117 |
Filed: |
February 4, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17320854 |
May 14, 2021 |
11260115 |
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17665117 |
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16054503 |
Aug 3, 2018 |
11083784 |
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17320854 |
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15375070 |
Dec 9, 2016 |
10398766 |
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16054503 |
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62265615 |
Dec 10, 2015 |
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International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 14/47 20060101 C07K014/47; A61K 39/39 20060101
A61K039/39; C07K 7/06 20060101 C07K007/06; C07K 7/08 20060101
C07K007/08; C07K 14/725 20060101 C07K014/725; C07K 16/30 20060101
C07K016/30; C12N 5/0783 20060101 C12N005/0783; C12N 15/115 20060101
C12N015/115; C07K 14/74 20060101 C07K014/74; C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 10, 2015 |
GB |
1521746.6 |
Claims
1. A peptide consisting of the amino acid sequence GIIDGSPRL (SEQ
ID NO: 62) in the form of a pharmaceutically acceptable salt.
2. The peptide of claim 1, wherein said peptide has the ability to
bind to an MHC class-I molecule, and wherein said peptide, when
bound to said MHC, is capable of being recognized by CD8 T
cells.
3. The peptide of claim 1, wherein the pharmaceutically acceptable
is chloride salt.
4. The peptide of claim 1, wherein the pharmaceutically acceptable
is acetate salt.
5. A composition comprising the peptide of claim 1, wherein the
composition comprises an adjuvant and a pharmaceutically acceptable
carrier.
6. The composition of claim 5, wherein the peptide is in the form
of a chloride salt.
7. The composition of claim 5, wherein the peptide is in the form
of an acetate salt.
8. The composition of claim 5 wherein the adjuvant is selected from
the group consisting of anti-CD40 antibody, imiquimod, resiquimod,
GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha,
interferon-beta, CpG oligonucleotides and derivatives, poly-(I:C)
and derivatives, RNA, sildenafil, particulate formulations with
poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1,
IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.
9. The composition of claim 8, wherein the adjuvant is IL-2.
10. The composition of claim 8, wherein the adjuvant is IL-7.
11. The composition of claim 8, wherein the adjuvant is IL-12.
12. The composition of claim 8, wherein the adjuvant is IL-15.
13. The composition of claim 8, wherein the adjuvant is IL-21.
14. A pegylated peptide consisting of the amino acid sequence of
GIIDGSPRL (SEQ ID NO: 62) or a pharmaceutically acceptable salt
thereof.
15. The peptide of claim 14, wherein the pharmaceutically
acceptable is chloride salt.
16. The peptide of claim 14, wherein the pharmaceutically
acceptable is acetate salt.
17. A composition comprising the pegylated peptide of claim 14 or
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier.
18. A peptide consisting of the amino acid sequence of GIIDGSPRL
(SEQ ID NO: 62), wherein at least one amino acid of the peptide is
a D-amino acid.
19. The peptide in the form of a pharmaceutically acceptable salt
of claim 1, wherein said peptide is produced by solid phase peptide
synthesis or produced by a yeast cell or bacterial cell expression
system.
20. A composition comprising the peptide of claim 1, wherein the
composition is a pharmaceutical composition and comprises water and
a buffer.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 17/320,854, filed May 14, 2021, which is a Continuation of U.S.
application Ser. No. 16/054,503, filed Aug. 3, 2018, now U.S. Pat.
No. 11,083,784, issued Aug. 10, 2021, which is a Continuation of
U.S. application Ser. No. 15/375,070, filed Dec. 9, 2016, now U.S.
Pat. No. 10,398,766, issued Sep. 3, 2019, which claims the benefit
of U.S. Provisional Application Ser. No. 62/265,615, filed 10 Dec.
2015, and Great Britain Application No. 1521746.6, filed 10 Dec.
2015, the content of each of these applications is herein
incorporated by reference in their entirety.
[0002] This application also is related to PCT/EP2016/078718 filed
24 Nov. 2016, the content of which is incorporated herein by
reference in its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT
FILE (.TXT)
[0003] Pursuant to the EFS-Web legal framework and 37 CFR
.sctn..sctn. 1.821-825 (see MPEP .sctn. 2442.03(a)), a Sequence
Listing in the form of an ASCII-compliant text file (entitled
"Sequence_Listing_2912919-058013_ST25.txt" created on 4 Feb. 2022,
and 82,532 bytes in size) is submitted concurrently with the
instant application, and the entire contents of the Sequence
Listing are incorporated herein by reference.
FIELD
[0004] The present invention relates to peptides, proteins, nucleic
acids and cells for use in immunotherapeutic methods. In
particular, the present invention relates to the immunotherapy of
cancer. The present invention furthermore relates to
tumor-associated T-cell peptide epitopes, alone or in combination
with other tumor-associated peptides that can for example serve as
active pharmaceutical ingredients of vaccine compositions that
stimulate anti-tumor immune responses, or to stimulate T cells ex
vivo and transfer into patients. Peptides bound to molecules of the
major histocompatibility complex (MHC), or peptides as such, can
also be targets of antibodies, soluble T-cell receptors, and other
binding molecules.
[0005] The present invention relates to several novel peptide
sequences and their variants derived from HLA class I molecules of
human tumor cells that can be used in vaccine compositions for
eliciting anti-tumor immune responses, or as targets for the
development of pharmaceutically/immunologically active compounds
and cells.
BACKGROUND OF THE INVENTION
[0006] Chronic lymphocytic leukemia (CLL) is a B-cell neoplasm with
morphologically mature, immunologically not completely matured B
lymphocytes. CLL is diagnosed when at least 5000 B
lymphocytes/.mu.l are present in peripheral blood, whereof up to
55% are immature. Abnormal B cells show a characteristic phenotype
expressing CD19, dim CD20, dim CD5, CD23, CD79a and dim IgM or dim
IgD (Gribben, 2010).
[0007] Two different staging systems are used to classify CLL: The
Rai system and the Binet system. The Rai staging system comprises
five stages (0-IV) that classify the disease according to the
progressive accumulation of abnormal cells. The Binet system uses
three stages (A, B, C) to rank the disease according to the number
of involved sites
(http://www.cancer.gov/cancertopics/pdq/treatment/CLL/Patient).
[0008] CLL is primarily a disease of the elderly. The mean age at
diagnosis is 72 years for sporadic cases and 58 years for familial
cases. The incidence of CLL is higher in males than in females,
with a male:female ratio of about 2:1 (Cartwright et al.,
2002).
[0009] CLL is the most common leukemia in the Western world where
it comprises about 1/3rd of all leukemias. Incidence rates are
similar in the US and Europe, and estimated new cases are about
16,000 per year. CLL is more common in Caucasians than in Africans,
rarer in Hispanics and Native Americans and seldom in Asians. In
people of Asian origin, CLL incidence rates are 3-fold lower than
in Caucasians (Gunawardana et al., 2008).
[0010] The five-year overall survival for patients with CLL is
about 79%
(http://www.cancernet/cancer-types/leukemia-chronic-lymphocytic-cll/stati-
stics). The prognosis for individual patients depends on the stage
at the time of diagnosis and the occurrence of several prognostic
factors. Following the Rai system, patients with stage 0 show a
median survival of 10 years or more, while patients at stage I/II
have a median survival of 7 years and patients at stage III/IV
0.75-4 years (Gribben, 2010). Several factors are associated with
poor prognosis in CLL. These include high expression levels of
ZAP-70 or CD38, an IgVH unmutated status and the cytogenetic
aberrations del 17p or del 11q (Gribben, 2010).
[0011] While CLL is not curable at present, many patients show only
slow progression of the disease or worsening of symptoms. As
patients do not benefit from an early onset of treatment, the
initial approach is "watch and wait" (Richards et al., 1999). For
patients with symptomatic or rapidly progressing disease, several
treatment options are available. These include chemotherapy,
targeted therapy, immune-based therapies like monoclonal
antibodies, CARs and active immunotherapy, and stem cell
transplants.
[0012] Chemotherapeutic drugs used for CLL treatment are mostly
alkylating agents like chlorambucil and cyclophosphamide or purine
analogues like fludarabine. The German CLL Study Group (GCLLSG)
CCL4 demonstrated that fludarabine/cyclophosphamide combinational
therapy is superior to sole fludarabine treatment (complete
remission (CR) of 24% vs. 7%) (Eichhorst et al., 2006).
[0013] Ibrutinib and idelalisib are kinase inhibitors that target
molecules in the B-cell receptor signaling cascade. Ibrutinib
inhibits Bruton's tyrosine kinase (BTK), a src-related cytoplasmic
tyrosine kinase important for B-cell maturation, and is used for
initial or second-line therapy (Byrd et al., 2013; O'Brien et al.,
2014). Idelalisib is a PI3K-delta inhibitor used in combination
with rituximab in refractory CLL (Furman et al., 2014).
[0014] Hematopoietic stem cell transplants (HSCTs) can be
considered for patients with poor prognosis, e.g. patients with del
17p or p53 mutations. HSCTs can either be allogeneic, where the
transplanted cells are donated from an HLA-matched person, or
autologous, where the patients' own stem cells are re-infused after
chemotherapy (Schetelig et al., 2008).
[0015] Monoclonal antibodies are widely used in hematologic
malignancies. This is due to the knowledge of suitable antigens
based on the good characterization of immune cell surface molecules
and the accessibility of tumor cells in blood or bone marrow.
Common monoclonal antibodies used in CLL therapy target either CD20
or CD52. Rituximab, the first monoclonal anti-CD20 antibody
originally approved by the FDA for treatment of NHLs, is now widely
used in CLL therapy. Combinational treatment with
rituximab/fludarabine/cyclophosphamide leads to higher CR rates and
improved overall survival (OS) than the combination
fludarabine/cyclophosphamide and has become the preferred treatment
option (GCLLSG CLLR) (Hallek et al., 2008). Ofatumomab targets CD20
and is used for therapy of refractory CLL patients (Wierda et al.,
2011). Obinutuzumab is another monoclonal anti-CD20 antibody used
in first-line treatment in combination with chlorambucil (Goede et
al., 2014).
[0016] Alemtuzumab is an anti-CD52 antibody used for treatment of
patients with chemotherapy-resistant disease or patients with poor
prognostic factors as del 17p or p53 mutations (Parikh et al.,
2011). Novel monoclonal antibodies target CD37 (otlertuzumab, BI
836826, IMGN529 and (177)Lu-tetulomab) or CD40 (dacetuzumab and
lucatumumab) and are tested in pre-clinical settings (Robak and
Robak, 2014).
[0017] Several completed and ongoing trials are based on engineered
autologous chimeric antigen receptor (CAR)-modified T cells with
CD19 specificity (Maus et al., 2014). So far, only the minority of
patients showed detectable or persistent CARs. One partial response
(PR) and two complete responses (CR) have been detected in the CAR
T-cell trials by Porter et al. and Kalos et al. (Kalos et al.,
2011; Porter et al., 2011).
[0018] Active immunotherapy includes the following strategies: gene
therapy, whole modified tumor cell vaccines, DC-based vaccines and
TAA-derived peptide vaccines.
[0019] Approaches in gene therapy make use of autologous
genetically modified tumor cells. These B-CLL cells are transfected
with immuno-(co-)stimulatory genes like IL-2, IL-12, TNF-alpha,
GM-CSF, CD80, CD40L, LFA-3 and ICAM-1 to improve antigen
presentation and T cell activation (Carballido et al., 2012). While
specific T-cell responses and reduction in tumor cells are readily
observed, immune responses are only transient.
[0020] Several studies have used autologous DCs as antigen
presenting cells to elicit anti-tumor responses. DCs have been
loaded ex vivo with tumor associated peptides, whole tumor cell
lysate, tumor-derived RNA or DNA. Another strategy uses whole tumor
cells for fusion with DCs and generation of DC-B-CLL-cell hybrids.
Transfected DCs initiated both CD4+ and CD8+ T-cell responses
(Muller et al., 2004). Fusion hybrids and DCs loaded with tumor
cell lysate or apoptotic bodies increased tumor-specific CD8+
T-cell responses. Patients that showed a clinical response had
increased IL-12 serum levels and reduced numbers of Tregs (Palma et
al., 2008).
[0021] Different approaches use altered tumor cells to initiate or
increase CLL-specific immune responses. An example for this
strategy is the generation of trioma cells: B-CLL cells are fused
to anti-Fc receptor expressing hybridoma cells that have anti-APC
specificity. Trioma cells induced CLL-specific T-cell responses in
vitro (Kronenberger et al., 2008). Another strategy makes use of
irradiated autologous CLL cells with Bacillus Calmette-Guerin as an
adjuvant as a vaccine. Several patients showed a reduction in
leukocyte levels or stable disease (Hus et al., 2008). Besides
isolated CLL cells, whole blood from CLL patients has been used as
a vaccine after preparation in a blood treatment unit. The vaccine
elicited CLL-specific T-cell responses and led to partial clinical
responses or stable disease in several patients (Spaner et al.,
2005).
[0022] Several TAAs are overexpressed in CLL and are suitable for
vaccinations. These include fibromodulin (Mayr et al., 2005),
RHAMM/CD168 (Giannopoulos et al., 2006), MDM2 (Mayr et al., 2006),
hTERT (Counter et al., 1995), the oncofetal antigen-immature
laminin receptor protein (OFAiLRP) (Siegel et al., 2003),
adipophilin (Schmidt et al., 2004), survivin (Granziero et al.,
2001), KW1 to KW14 (Krackhardt et al., 2002) and the tumor-derived
IgVHCDR3 region (Harig et al., 2001; Carballido et al., 2012).
[0023] A phase I clinical trial was conducted using the
RHAMM-derived R3 peptide as a vaccine. 5 of 6 patients had
detectable R3-specific CD8+ T-cell responses (Giannopoulos et al.,
2010).
[0024] 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 CLL in
particular. There is also a need to identify factors representing
biomarkers for cancer in general and CLL in particular, leading to
better diagnosis of cancer, assessment of prognosis, and prediction
of treatment success.
[0025] Immunotherapy of cancer represents an option of specific
targeting of cancer cells while minimizing side effects. Cancer
immunotherapy makes use of the existence of tumor associated
antigens.
[0026] The current classification of tumor associated antigens
(TAAs) comprises 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,
occasionally, in placenta. Since the cells of testis do not express
class I and II HLA molecules, these antigens cannot be recognized
by T cells in normal tissues and can therefore be considered as
immunologically tumor-specific. Well-known examples for CT antigens
are the MAGE family members and NY-ESO-1. b) Differentiation
antigens: These TAAs are shared between tumors and the normal
tissue from which the tumor arose. Most of the known
differentiation antigens are found in melanomas and normal
melanocytes. Many of these melanocyte lineage-related proteins are
involved in biosynthesis of melanin and are therefore not tumor
specific but nevertheless are widely used for cancer immunotherapy.
Examples include, but are not limited to, tyrosinase and
Melan-A/MART-1 for melanoma or PSA for prostate cancer. c)
Over-expressed TAAs: Genes encoding widely expressed TAAs have been
detected in histologically different types of tumors as well as in
many normal tissues, generally with lower expression levels. It is
possible that many of the epitopes processed and potentially
presented by normal tissues are below the threshold level for
T-cell recognition, while their over-expression in tumor cells can
trigger an anticancer response by breaking previously established
tolerance. Prominent examples for this class of TAAs are Her-2/neu,
survivin, telomerase, or WT1. d) Tumor-specific antigens: These
unique TAAs arise from mutations of normal genes (such as
.beta.-catenin, CDK4, etc.). Some of these molecular changes are
associated with neoplastic transformation and/or progression.
Tumor-specific antigens are generally able to induce strong immune
responses without bearing the risk for autoimmune reactions against
normal tissues. On the other hand, these TAAs are in most cases
only relevant to the exact tumor on which they were identified and
are usually not shared between many individual tumors.
Tumor-specificity (or -association) of a peptide may also arise if
the peptide originates from a tumor- (-associated) exon in case of
proteins with tumor-specific (-associated) isoforms. e) TAAs
arising from abnormal post-translational modifications: Such TAAs
may arise from proteins which are neither specific nor
overexpressed in tumors but nevertheless become tumor associated by
posttranslational processes primarily active in tumors. Examples
for this class arise from altered glycosylation patterns leading to
novel epitopes in tumors as for MUC1 or events like protein
splicing during degradation which may or may not be tumor specific.
f) Oncoviral proteins: These TAAs are viral proteins that may play
a critical role in the oncogenic process and, because they are
foreign (not of human origin), they can evoke a T-cell response.
Examples of such proteins are the human papilloma type 16 virus
proteins, E6 and E7, which are expressed in cervical carcinoma.
[0027] T-cell based immunotherapy targets peptide epitopes derived
from tumor-associated or tumor-specific proteins, which are
presented by molecules of the major histocompatibility complex
(MHC). The antigens that are recognized by the tumor specific T
lymphocytes, that is, the epitopes thereof, can be molecules
derived from all protein classes, such as enzymes, receptors,
transcription factors, etc. which are expressed and, as compared to
unaltered cells of the same origin, usually up-regulated in cells
of the respective tumor.
[0028] There are two classes of MHC-molecules, MHC class I and MHC
class II. MHC class I molecules are composed of an alpha heavy
chain and beta-2-microglobulin, MHC class II molecules of an alpha
and a beta chain. Their three-dimensional conformation results in a
binding groove, which is used for non-covalent interaction with
peptides.
[0029] MHC class I molecules can be found on most nucleated cells.
They present peptides that result from proteolytic cleavage of
predominantly endogenous proteins, defective ribosomal products
(DRIPs) and larger peptides. However, peptides derived from
endosomal compartments or exogenous sources are also frequently
found on MHC class I molecules. This non-classical way of class I
presentation is referred to as cross-presentation in the literature
(Brossart and Bevan, 1997; Rock et al., 1990). MHC class II
molecules can be found predominantly on professional antigen
presenting cells (APCs), and primarily present peptides of
exogenous or transmembrane proteins that are taken up by APCs e.g.
during endocytosis, and are subsequently processed.
[0030] Complexes of peptide and MHC class I are recognized by
CD8-positive T cells bearing the appropriate T-cell receptor (TCR),
whereas complexes of peptide and MHC class II molecules are
recognized by CD4-positive-helper-T cells bearing the appropriate
TCR. It is well known that the TCR, the peptide and the MHC are
thereby present in a stoichiometric amount of 1:1:1.
[0031] CD4-positive helper T cells play an important role in
inducing and sustaining effective responses by CD8-positive
cytotoxic T cells. The identification of CD4-positive T-cell
epitopes derived from tumor associated antigens (TAA) is of great
importance for the development of pharmaceutical products for
triggering anti-tumor immune responses (Gnjatic et al., 2003). At
the tumor site, T helper cells, support a cytotoxic T cell- (CTL-)
friendly cytokine milieu (Mortara et al., 2006) and attract
effector cells, e.g. CTLs, natural killer (NK) cells, macrophages,
and granulocytes (Hwang et al., 2007).
[0032] In the absence of inflammation, expression of MHC class II
molecules is mainly restricted to cells of the immune system,
especially professional antigen-presenting cells (APC), e.g.,
monocytes, monocyte-derived cells, macrophages, dendritic cells. In
cancer patients, cells of the tumor have been found to express MHC
class II molecules (Dengjel et al., 2006).
[0033] Elongated (longer) peptides of the invention can act as MHC
class II active epitopes.
[0034] T-helper cells, activated by MHC class II epitopes, play an
important role in orchestrating the effector function of CTLs in
anti-tumor immunity. T-helper cell epitopes that trigger a T-helper
cell response of the TH1 type support effector functions of
CD8-positive killer T cells, which include cytotoxic functions
directed against tumor cells displaying tumor-associated
peptide/MHC complexes on their cell surfaces. In this way
tumor-associated T-helper cell peptide epitopes, alone or in
combination with other tumor-associated peptides, can serve as
active pharmaceutical ingredients of vaccine compositions that
stimulate anti-tumor immune responses.
[0035] It was shown in mammalian animal models, e.g., mice, that
even in the absence of CD8-positive T lymphocytes, CD4-positive T
cells are sufficient for inhibiting manifestation of tumors via
inhibition of angiogenesis by secretion of interferon-gamma
(IFN.gamma.) (Beatty and Paterson, 2001; Mumberg et al., 1999).
There is evidence for CD4 T cells as direct anti-tumor effectors
(Braumuller et al., 2013; Tran et al., 2014).
[0036] Since the constitutive expression of HLA class II molecules
is usually limited to immune cells, the possibility of isolating
class II peptides directly from primary tumors was previously not
considered possible. However, Dengjel et al. were successful in
identifying a number of MHC Class II epitopes directly from tumors
(WO 2007/028574, EP 1 760 088 B1).
[0037] Since both types of response, CD8 and CD4 dependent,
contribute jointly and synergistically to the anti-tumor effect,
the identification and characterization of tumor-associated
antigens recognized by either CD8+ T cells (ligand: MHC class I
molecule+peptide epitope) or by CD4-positive T-helper cells
(ligand: MHC class II molecule+peptide epitope) is important in the
development of tumor vaccines.
[0038] For an MHC class I peptide to trigger (elicit) a cellular
immune response, it also must bind to an MHC-molecule. This process
is dependent on the allele of the MHC-molecule and specific
polymorphisms of the amino acid sequence of the peptide.
MHC-class-1-binding peptides are usually 8-12 amino acid residues
in length and usually contain two conserved residues ("anchors") in
their sequence that interact with the corresponding binding groove
of the MHC-molecule. In this way, each MHC allele has a "binding
motif" determining which peptides can bind specifically to the
binding groove.
[0039] In the MHC class I dependent immune reaction, peptides not
only have to be able to bind to certain MHC class I molecules
expressed by tumor cells, they subsequently also have to be
recognized by T cells bearing specific T cell receptors (TCR).
[0040] For proteins to be recognized by T-lymphocytes as
tumor-specific or -associated antigens, and to be used in a
therapy, particular prerequisites must be fulfilled. The antigen
should be expressed mainly by tumor cells and not, or in comparably
small amounts, by normal healthy tissues. In a preferred
embodiment, the peptide should be over-presented by tumor cells as
compared to normal healthy tissues. It is furthermore desirable
that the respective antigen is not only present in a type of tumor,
but also in high concentrations (i.e. copy numbers of the
respective peptide per cell). Tumor-specific and tumor-associated
antigens are often derived from proteins directly involved in
transformation of a normal cell to a tumor cell due to their
function, e.g. in cell cycle control or suppression of apoptosis.
Additionally, downstream targets of the proteins directly causative
for a transformation may be up-regulated and thus may be indirectly
tumor-associated. Such indirect tumor-associated antigens may also
be targets of a vaccination approach (Singh-Jasuja et al., 2004).
It is essential that epitopes are present in the amino acid
sequence of the antigen, in order to ensure that such a peptide
("immunogenic peptide"), being derived from a tumor associated
antigen, leads to an in vitro or in vivo T-cell-response.
[0041] Basically, any peptide able to bind an MHC molecule may
function as a T-cell epitope. A prerequisite for the induction of
an in vitro or in vivo T-cell-response is the presence of a T cell
having a corresponding TCR and the absence of immunological
tolerance for this particular epitope.
[0042] Therefore, TAAs are a starting point for the development of
a T cell based therapy including but not limited to tumor vaccines.
The methods for identifying and characterizing the TAAs are usually
based on the use of T-cells that can be isolated from patients or
healthy subjects, or they are based on the generation of
differential transcription profiles or differential peptide
expression patterns between tumors and normal tissues. However, the
identification of genes over-expressed in tumor tissues or human
tumor cell lines, or selectively expressed in such tissues or cell
lines, does not provide precise information as to the use of the
antigens being transcribed from these genes in an immune therapy.
This is because only an individual subpopulation of epitopes of
these antigens are suitable for such an application since a T cell
with a corresponding TCR has to be present and the immunological
tolerance for this particular epitope needs to be absent or
minimal. In a very preferred embodiment of the invention it is
therefore important to select only those over- or selectively
presented peptides against which a functional and/or a
proliferating T cell can be found. Such a functional T cell is
defined as a T cell, which upon stimulation with a specific antigen
can be clonally expanded and is able to execute effector functions
("effector T cell").
[0043] In case of targeting peptide-MHC by specific TCRs (e.g.
soluble TCRs) and antibodies or other binding molecules (scaffolds)
according to the invention, the immunogenicity of the underlying
peptides is secondary. In these cases, the presentation is the
determining factor.
SUMMARY
[0044] 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:
385 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: 385, wherein said variant
binds to MHC and/or induces T cells cross-reacting with said
peptide, or a pharmaceutical acceptable salt thereof, wherein said
peptide is not the underlying full-length polypeptide.
[0045] The present invention further relates to a peptide of the
present invention comprising a sequence that is selected from the
group consisting of SEQ ID NO: 1 to SEQ ID NO: 385 or a variant
thereof, which is at least 77%, preferably at least 88%, homologous
(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1
to SEQ ID NO: 385, wherein said peptide or variant thereof has an
overall length of between 8 and 100, preferably between 8 and 30,
and most preferred of between 8 and 14 amino acids.
[0046] The following tables show the peptides according to the
present invention, their respective SEQ ID NOs, and the prospective
source (underlying) genes for these peptides. All peptides in Table
1 and Table 2 bind to HLA-A*02. The peptides in Table 2 have been
disclosed before in large listings as results of high-throughput
screenings with high error rates or calculated using algorithms,
but have not been associated with cancer at all before. The
peptides in Table 3 are additional peptides that may be useful in
combination with the other peptides of the invention. The peptides
in Table 4 are furthermore useful in the diagnosis and/or treatment
of various other malignancies that involve an over-expression or
over-presentation of the respective underlying polypeptide.
TABLE-US-00001 TABLE 1 Peptides according to the present invention.
SEQ ID No. Sequence GeneID(s) Official Gene Symbol(s) 1 AIPPSFASIFL
3507 IGHM 2 ALHRPDVYL 3507 IGHM 3 VIAELPPKV 3507 IGHM 4 VIAELPPKVSV
3507 IGHM 5 ALIFKIASA 4214 MAP3K1 6 ALDTLEDDMTI 2222 FDFT1 7
ALLERTGYTL 10236, 10492 HNRNPR, SYNCRIP 8 ALAASALPALV 6124 RPL4 9
ALCDTLITV 27033 ZBTB32 10 FVYGESVEL 27033 ZBTB32 11 ALFTFJPLTV
54665 RSBN1 12 ALGEDEITL 25909, 285116 AHCTF1, AHCTF1P1 13
VVDGMPPGV 2969, 2970 GTF2I, GTF2IP1 14 ALLRLLPGL 25920 COBRA1 15
ALPEVSVEA 79649 MAP7D3 16 ALPGGAAVAAV 79697 C14orf169 17 ALTKTNLQL
9750 FAM65B 18 LLGEFSIKM 9750 FAM65B 19 QVMEKLAAV 9750 FAM65B 20
ALVDPGPDFVV 546 ATRX 21 ALWAGLLTL 2208 FCER2 22 ALWDPVIEL 27340
UTP20 23 ALYLTEVFL 55024 BANK1 24 AMAGDVVYA 160365 CLECL1 25
ATYSGLESQSV 3185 HNRNPF 26 AVLLVLPLV 11322 TMC6 27 AVLGLVWLL 11322
TMC6 28 AVLHLLLSV 1535 CYBA 29 AVLQAVTAV 58155 PTBP2 30 ELLEGSEIYL
147007, 23098 TMEM199, SARM1 31 ELMHGVAGV 165631 PARP15 32
FIDKFTPPV 3117, 3122 HLA-DQA1, HLA-DRA 33 FIINSSNIFL 253959
RALGAPA1 34 YLPYIFPNI 253959 RALGAPA1 35 FILPSSLYL 81539 SLC38A1 36
FILTHVDQL 257106 ARHGAP30 37 FIMEGGAMVL 4174 MCMS 38 FIMEGGAMV 4174
MCMS 39 FLDALLTDV 79915 ATAD5 40 FLDEDDMSL 23141 ANKLE2 41
FLDPSLDPLL 58508, 85318 MLL3, BAGE3 42 FLEEGGVVTV 5810 RAD1 43
FLLGPEALSFA 55183 RIF1 44 FLLSINDFL 157680 VPS13B 45 FLPELPADLEA
11064 CNTRL 46 YIIDSAQAV 11064 CNTRL 47 FLSDQPEPYL 160518, 23258
DENND5B, DENND5A 48 FLSPQQPPLL 8621 CDK13 49 FLTDLFAQL 55671 SMEK1
50 FLFEPVVKAFL 55671 SMEK1 51 FLVEAPHDWDL 9790 BMS1 52 FLVETGFLHV
100268168, 114987, WDR31, MYB 4602 53 FLWQHVELVL 55181 SMG8 54
FLYPFPLALF 440026 TMEM41B 55 FMEPTLLML 23 ABCF1 56 FVFEAPYTL 139818
DOCK11 57 GLSEISLRL 139818 DOCK11 58 YIQQGIFSV 139818 DOCK11 59
FVFGDENGTVSL 79084 WDR77 60 FVLDHEDGLNL 4926 NUMA1 61 FVYFIVREV
285527 FRYL 62 GIIDGSPRL 80183 KIAA0226L 63 SLAHVAGCEL 80183
KIAA0226L 64 KLLESVASA 80183 KIAA0226L 65 GLDDMKANL 5079 PAX5 66
SLAGGLDDMKA 5079 PAX5 67 GLDDVTVEV 51019 CCDC53 68 GLDQQFAGLDL 1654
DDX3X 69 GLHQREIFL 9922 IQSEC1 70 FVPDTPVGV 9922 IQSEC1 71
GLKHDIARV 388512 CLEC17A 72 GLLDAGKMYV 911 CD1C 73 GLLEVISALQL 9816
URB2 74 GLLRASFLL 11344, 54106 TWF2, TLR9 75 GLSIFAQDLRL 11344,
54106 TWF2, TLR9 76 GLLRIIPYL 23352 UBR4 77 GLLRLTWFL 11214 AKAP13
78 GLPSFLTEV 100532732, 4439 MSH5-SAPCD1, MSH5 79 GLQAKIQEA 23224
SYNE2 80 VLIEDELEEL 23224 SYNE2 81 WLVGQEFEL 23224 SYNE2 82
GLQSGVDIGV 1265 CNN2 83 GQGEVLVYV 2316 FLNA 84 GVMDVNTAL 391370,
6206 RPS12P4, RPS12 85 HLMLHTAAL 80012 PHC3 86 HLYPGAVTI 55103
RALGPS2 87 HQIEAVDGEEL 10567 RABAC1 88 ILDEIGADVQA 29920 PYCR2 89
ILDFGTFQL 54832 VPS13C 90 VIADLGLIRV 54832 VPS13C 91 ILDLNTYNV 5336
PLCG2 92 ILEPLNPLL 9330 GTF3C3 93 ILFNTQINI 64801 ARV1 94 ILFPLRFTL
9675 TTI1 95 ILGYMAHEHKV 6530 SLC6A2 96 ILIDKTSFV 951 CD37 97
LLFATQITL 951 CD37 98 SLIKYFLFV 951 CD37 99 ILIFHSVAL 84636 GPR174
100 ILNNEVFAI 26999 CYFIP2 101 ILVVIEPLL 23451 SF3B1 102 IQDRAVPSL
3930 LBR 103 KLGGTPAPA 5218 CDK14 104 KLILLDTPLFL 253190, 94009
SERHL2, SERHL 105 KLMNDIADI 472 ATM 106 FMASHLDYL 472 ATM 107
ILYNLYDLL 472 ATM 108 VIYTLIHYI 472 ATM 109 KLWEGLTELV 10235
RASGRP2 110 LLFDHLEPMEL 10235 RASGRP2 111 KLWNVAAPLYL 92105 INTS4
112 KTLDVDATYEI 3978 LIG1 113 KVPAEEVLVAV 91782 CHMP7 114 LIPEGPPQV
64224 HERPUD2 115 LLFDKLYLL 25885 POLR1A 116 LLIGATMQV 23225 NUP210
117 LLILENILL 23225 NUP210 118 RLLILENILL 23225 NUP210 119
VLPAEFFEV 23225 NUP210 120 AIDAALTSV 23225 NUP210 121 RLYEITIEV
23225 NUP210 122 LLIPVVPGV 55178 RNMTL1
123 LLLAEAELLTL 57724 EPG5 124 LLLEETEKQAV 92154, 9788 MTSS1L,
MTSS1 125 LLLEIGEVGKLFV 23158 TBC1D9 126 LLPEGGITAI 9904 RBM19 127
LLPTAPTTV 55729 ATF7IP 128 LLSEEEYHL 9793 CKAP5 129 LLVGTLDVV
23061, 23158 TBC1D9B, TBC1D9 130 LLVLIPVYL 23344 ESYT1 131
LQALEVLKI 27304 MOCS3 132 LVYEAIIMV 27250 PDCD4 133 YLLSGDISEA
27250 PDCD4 134 MLLEHGITLV 200576 PIKFYVE 135 MTAGFSTIAGSV 64078
SLC28A3 136 NLDKLWTLV 389435, 6157 RPL27AP6, RPL27A 137 NLIKTVIKL
25816 TNFAIP8 138 NLLDIDAPVTV 5530 PPP3CA 139 NLTDVVEKL 120892
LRRK2 140 QIAELPATSV 401145 CCSER1 141 QILSEIVEA 10724 MGEA5 142
QLDEPAPQV 387032, 80317 ZKSCAN4, ZKSCAN3 143 QLLDTYFTL 7405 UVRAG
144 QLPPFPREL 8498 RANBP3 145 SALDTITTV 3832 KIF11 146 SIIEGPIIKL
23347 SMCHD1 147 SILETVATL 253714 MMS22L 148 SIVASLITV 221188
GPR114 149 SLDNGGYYI 4067 LYN 150 SLFDQPLSII 117289 TAGAP 151
SLFDSAYGA 2313 FLI1 152 SLIRILQTI 160518 DENND5B 153 SLLAELHVL
115352 FCRL3 154 SLLAELHVLTV 115352 FCRL3 155 SLMLEVPAL 1756 DMD
156 SLNIGDVQL 973 CD79A 157 SLNIRDFTM 147694, 7565 ZNF548, ZNF17
158 SLPEAPLDV 29123 ANKRD11 159 SLQEEKLIYV 84668 FAM126A 160
SLSFLVPSL 9631 NUP155 161 SMDDGMINV 7011 TEP1 162 SMKDDLENV 3688
ITGB1 163 SQLDISEPYKV 3394 IRF8 164 SVHKGFAFV 101060301, 3183,
HNRNPC, HNRNPCL1 343069, 440563, 649330 165 TLDDDLDTV 51092 SIDT2
166 TLDPNQVSL 85464 SSH2 167 TLDTSKLYV 5923 RASGRF1 168 YLLDQSFVM
5923 RASGRF1 169 TLLLGLTEV 55423 SIRPG 170 TLTFRVETV 51379 CRLF3
171 TLVPPAALISI 6907 TBL1X 172 TLYDMLASI 10973 ASCC3 173 TVIENIHTI
23358 USP24 174 VLAELPIIVV 8295 TRRAP 175 FTVPRVVAV 8295 TRRAP 176
VLAEQNIIPSA 54855 FAM46C 177 VLDDRELLL 23178 PASK 178 VLFFNVQEV
57536 KIAA1328 179 VLLGLEMTL 1606 DGKA 180 LLKDGPEIGL 1606 DGKA 181
VLLSIPFVSV 94103 ORMDL3 182 VLLSVPGPPV 2264 FGFR4 183 VLMPTVYQQGV
54472 TOLLIP 184 VLSHNLYTV 50619 DEF6 185 VMDDQRDLI 972 CD74 186
VMDPTKILI 255967 PAN3 187 VMDTHLVNI 9779 TBC1D5 188 VMGDIPAAV 4678
NASP 189 VMLEMTPEL 3112, 6891 HLA-DOB, TAP2 190 VVMGTVPRL 1729
DIAPH1 191 YIFDGSDGGV 5287 PIK3C2B 192 YIQEYLTLL 57674 RNF213 193
YLDLSNNRL 81793 TLR10 194 YLDNVLAEL 1840, 23220 DTX1, DTX4 195
YLGGFALSV 6850 SYK 196 YLLLQTYVL 51259 TMEM216 197 YLQEVPILTL 1633
DCK 198 YLTFLPAEV 64135 IFIH1 199 YLVELSSLL 80279 CDK5RAP3 200
YMFEEVPIVI 8667 EIF3H 201 YQLELHGIEL 5698 PSMB9 202 YVDDVFLRV 10347
ABCA7 203 ALLSSQLAL 8925 HERC1 204 GLLQINDKIAL 5000 ORC4 205
GLSQANFTL 10288, 10859, LILRB2, LILRB1, LILRB5, 10990, 11024,
LILRA1, LILRB3, LILRA3, 11025, 11026, 11027, LILRA2, LILRA4,
LILRP2, 23547, 79166, 79168 LILRA6 206 HMQDVRVLL 84824 FCRLA 207
IIADLDTTIMFA 7094, 83660 TLN1, TLN2 208 ILLKTEGINL 166379 BBS12 209
ILQAELPSL 57508 INTS2 210 KLLVQDFFL 8564 KMO 211 LIDVKPLGV 134510
UBLCP1 212 NIIEAINELLV 8892 EIF2B2 213 RLLYQLVFL 3566 IL4R 214
RLQELTEKL 255394 TCP11L2 215 VMQDIVYKL 84759 PCGF1 216 WLAGDVPAA
24148 PRPF6 217 ALDEPPYLTV 51742 ARID4B 218 ALGEEWKGYVV 6194,
728131 RPS6 219 ALLNLLESA 5788 PTPRC 220 ALPEILFAKV 643 CXCR5 221
ALVSTIIMV 80228 ORAI2 222 ALWELSLKI 2889 RAPGEF1 223 ALWVSQPPEI
259197 NCR3 224 AMEALVVEV 1660 DHX9 225 ILQERELLPV 1660 DHX9 226
AMNISVPQV 23387 SIK3 227 FLAEASVMTQL 1445 CSK 228 FLGGLSPGV 2968
GTF2H4 229 FLLNLQNCHL 55608 ANKRD10 230 FLQDSKVIFV 4772 NFATC1 231
FLYIRQLAI 26155, 401010 NOC2L 232 FMHQIIDQV 79789 CLMN 233
GIIDINVRL 51163 DBR1 234 GLDDAEYAL 7376 NR1H2 235 GLDDLLLFL 8914
TIMELESS 236 GLLESGRHYL 9744 ACAP1 237 GLQENLDVVV 8881 CDC16 238
GLVETELQL 55690 PACS1 239 ILAGEMLSV 7203 CCT3 240 ILARDILEI 5714
PSMD8 241 ILGDILLKV 79831 KDM8 242 ILLGIQELL 7329 UBE2I 243
ILPTLEKELFL 79886 CAAP1 244 ILQALAVHL 54936 ADPRHL2 245 KIMDYSLLLGV
23396 PIP5K1C
246 KLDETGVAL 7155 TOP2B 247 KLKDRLPSI 81606 LBH 248 KTVEPPISQV
57466 SCAF4 249 LLPTGVFQV 51131 PHF11 250 LLVQEPDGLMV 404734, 54882
ANKHD1-EIF4EBP3, ANKHD1 251 LLYDNVPGA 11262 SP140 252 NLLDPGSSYLL
5074 PAWR 253 NLWSVDGEVTV 92017 SNX29 254 QLIPKLIFL 57705 WDFY4 255
YLFEEAISM 57705 WDFY4 256 QLLPVSNVVSV 55374 TMCO6 257 RIINGIIISV
84329 HVCN1 258 RLDYITAEI 10471 PFDN6 259 RLLDEQFAVL 9026 HIP1R 260
SLDDVEGMSV 80196 RNF34 261 SLVEAQGWLV 161176 SYNE3 262 SLWNAGTSV
3495 IGHD 263 SQWEDIHVV 55843 ARHGAP15 264 TILDYINVV 89790, 89858
SIGLEC10, SIGLEC12 265 TLLADDLEIKL 2124 EVI2B 266 TLLDQLDTQL 8678
BECN1 267 TLLDWQDSL 8289 ARID1A 268 TLLQVFHLL 63892 THADA 269
TLTDEQFLV 738 VPS51 270 TVLPVPPLSV 5430 POLR2A 271 VIRNIVEAA
100128168, RPS26P39, RPS26P32, 100996747, 389472, RPS26P11, RPS26,
441502, 6231, 643003, RPS26P28, RPS26P25, 728937, 729188 RPS26P58
272 VLDELPPLI 55832, 91689 CAND1, C22orf32 273 VLGEYSYLL 23431
AP4E1 274 VLLEYHIAYL 8087 FXR1 275 VLLFIEHSV 134265, 7805 AFAP1L1,
LAPTM5 276 VLNDGAPNV 117246 FTSJ3 277 VMILKLPFL 25929 GEMIN5 278
YLDDLLPKL 89910 UBE3B 279 YMAPEVVEA 2872 MKNK2 280 YTLDSLYWSV 81887
LAS1L J = phospho-serine
[0047] KIAA0226L (also known as C13orf18) encodes KIAA0226-like and
is located on chromosome 13q14.13 (RefSeq, 2002). KIAA0226L is
thought to be a tumor suppressor gene and is hyper-methylated in
cervical cancer. Re-activation of KIAA0226L leads to decreased cell
growth, viability, and colony formation (Huisman et al., 2015;
Eijsink et al., 2012; Huisman et al., 2013). The methylation
pattern of KIAA0226L can be used to differ between precursor
lesions and normal cervix cancer (Milutin et al., 2015). The
methylation pattern of KIAA0226L cannot be used as specific
biomarker for cervical cancer (Sohrabi et al., 2014). Re-activation
of KIAA0226L partially de-methylates its promotor region and also
decreases repressive histone methylations (Huisman et al.,
2013).
[0048] FCRL3, also known as FCRH3 or IRTA3, encodes Fc receptor
like 3, one of several FC receptor-like glycoproteins of the
immunoglobulin receptor superfamily which may play a role in
regulation of the immune system (RefSeq, 2002). FCRL3 is
up-regulated on CD4+CD26- T cells in Sezary syndrome patients with
a high tumor burden, suggesting that FCRL3 expression correlates
with a high circulating tumor burden (Wysocka et al., 2014). FCRL3
is up-regulated in chronic lymphocytic leukemia (Poison et al.,
2006). FCRL3 is a potential marker with prognostic relevance in
chronic lymphocytic leukemia (Zucchetto et al., 2011)
TABLE-US-00002 TABLE 2 Additional peptides according to the present
invention with no prior known cancer association. SEQ ID No.
Sequence GeneID(s) Official Gene Symbol(s) 281 NLLDDRGMTAL 146206
RLTPR 282 LLRDGIELV 152926 PPM1K 283 ILQPMDIHV 54832 VPS13C 284
LLSAAEPVPA 974 CD79B 285 GVATAGCVNEV 3394 IRF8 286 FLLEDLSQKL 2177
FANCD2 287 FLWEEKFNSL 10750 GRAP 288 GLAESTGLLAV 50855, 84552
PARD6A, PARD6G 289 ILEEQPMDMLL 84301 DDI2 290 LANPHELSL 84301 DDI2
291 ILLNEDDLVTI 57724 EPG5 292 AAALIIHHV 25942 SIN3A 293 ALDIMIPMV
4217 MAP3K5 294 ALLDQLHTLL 8202 NCOA3 295 ALLQKLQQL 122618 PLD4 296
FIAPTGHSL 23157, 55752 SEPT6, SEPT11 297 FLVEPQEDTRL 8237 USP11 298
IILPVEVEV 23505 TMEM131 299 ILEENIPVL 55653 BCAS4 300 ILLNPAYDVYL
23225 NUP210 301 AASPIITLV 23225 NUP210 302 ILQDLTFVHL 2889 RAPGEF1
303 ILSQPTPSL 29028 ATAD2 304 LAIVPVNTL 23132 RAD54L2 305
LLFPQIEGIKI 26263 FBXO22 306 VVAEELENV 26263 FBXO22 307 LLLTKPTEA
5336 PLCG2 308 SLYDVSRMYV 5336 PLCG2 309 ILYGTQFVL 5336 PLCG2 310
LLSTLHLLV 64098 PARVG 311 LLVDVEPKV 100129492, 6632 SNRPD1 312
LLYNSTDPTL 64005 MYO1G 313 LMADLEGLHL 8546 AP3B1 314 LMKDCEAEV 5518
PPP2R1A 315 LVYEAPETV 57448 BIRC6 316 MLLEHGITL 200576 PIKFYVE 317
NLLAHIWAL 58513 EPS15L1 318 NLQVTQPTV 100505503, RPS17L, RPS17P16,
402057, 442216, RPS17P5, RPS17 6218 319 QVIPQLQTV 6671 SP4 320
TIAPVTVAV 6671 SP4 321 RLLEFELAQL 6093 ROCK1 322 SLASIHVPL 23163
GGA3 323 SLDLFNCEV 10541, 23520, ANP32B, ANP32C, 646791, 647020,
ANP32BP1, ANP32AP1, 723972, 8125 ANP32A 324 SLYSALQQA 11190 CEP250
325 TLENGVPCV 23240 KIAA0922 326 VLAFLVHEL 11176 BAZ2A 327
VLIKWFPEV 54509 RHOF 328 VLLPQETAEIHL 5292 PIM1 329 VLMDGSVKL 11184
MAP4K1 330 VLMWEIYSL 695 BTK 331 VLWELAHLPTL 23358 USP24 332
VMIQHVENL 4033 LRMP 333 VTLEFPQLIRV 126382 NR2C2AP 334 YLLEEKIASL
51199 NIN 335 YLYQEQYFI 10668 CGRRF1 336 YMAVTTQEV 79840 NHEJ1 337
YMYEKESEL 22806 IKZF3 338 FLDMTNWNL 64771 C6orf106 339 GLWGTVVNI
25914 RTTN 340 KLLEEICNL 10075 HUWE1 341 LLAELPASVHA 2319 FLOT2 342
SLITPLQAV 5591 PRKDC 343 TLLEALDCI 158078, 1915 EEF1A1P5, EEF1A1
344 VLAFENPQV 25852 ARMC8 345 YLIEPDVELQRI 25852 ARMC8 346
VLVQVSPSL 23404 EXOSC2 347 YLGPVSPSL 4154 MBNL1 348 ALAKPPVVSV
57634 EP400 349 ALATHILSL 11215 AKAP11 350 ALEDRVWEL 285025 CCDC141
351 ALSEKLARL 115106 HAUS1 352 ALVFELHYV 57705 WDFY4 353 ATPMPTPSV
55206 SBNO1 354 FIMDDPAGNSYL 8882 ZNF259 355 FIWPMLIHI 11325 DDX42
356 FLHDHQAEL 259197 NCR3 357 FLIQEIKTL 23352 UBR4 358 FLTDYLNDL
54880 BCOR 359 FMQDPMEVFV 10212 DDX39A 360 HLIDTNKIQL 55619 DOCK10
361 ILQEFESKL 2074, 267004 ERCC6, PGBD3 362 ILTELGGFEV 51366 UBR5
363 ITTEVVNELYV 57222 ERGIC1 364 KMDWIFHTI 5290 PIK3CA 365
LISPLLLPV 144132 DNHD1 366 LLSETCVTI 23469 PHF3 367 NLWSLVAKV 9889
ZBED4 368 QLQPTDALLCV 125950 RAVER1 369 RLLDLENSLLGL 54899 PXK 370
SIFASPESV 10285 SMNDC1 371 SLADDSVLERL 23195 MDN1 372
SLFGPLPGPGPALV 9595 CYTIP 373 TLLADQGEIRV 9219 MTA2 374 VLSVITEEL
55621 TRMT1 375 VLWFKPVEL 55720 TSR1 376 VLYNQRVEEI 6730 SRP68 377
VVDGTCVAV 57589 KIAA1432 378 YILGKFFAL 8914 TIMELESS 379 YLAELVTPIL
1981 EIF4G1 380 YLDRKLLTL 6850 SYK 381 YLLEENKIKL 139818 DOCK11 382
YLLPLLQRL 55794 DDX28 383 YLLREWVNL 23019 CNOT1 384 YMIGSEVGNYL
6598 SMARCB1 385 YTIPLAIKL 128869 PIGU
TABLE-US-00003 TABLE 3 Peptides useful for e.g. personalized cancer
therapies. SEQ Official Gene ID No. Sequence GeneID(s) Symbol(s)
386 FLGDYVENL 54832 VPS13C 387 YLILSSHQL 1269 CNR2 388 GLLSALENV
1269 CNR2 389 GLAALAVHL 2175 FANCA 390 GLEERLYTA 29933 GPR132 391
FLLEREQLL 165055 CCDC138 392 FLWERPTLLV 79922 MRM1 393 FVMEGEPPKL
348654 GEN1 394 ILSTEIFGV 79703 C11orf80 395 ALYGKLLKL 157680
VPS13B 396 TLLGKQVTL 157680 VPS13B 397 FLPPEHTIVYI 9896 FIG4 398
FLAELPGSLSL 5326 PLAGL2 399 ALAAPDIVPAL 79886 CAAP1 400 ALFQPHLINV
10097 ACTR2 401 AMADKMDMSL 10189 ALYREF 402 LLVSNLDFGV 10189 ALYREF
403 FIMPATVADATAV 23352 UBR4 404 FLQPDLDSL 10514 MYBBP1A 405
FLVEKQPPQV 6778 STAT6 406 FLWPKEVEL 146206 RLTPR 407 FMIDASVHPTL
221960, 51622 CCZ1B, CCZ1 408 FMVDNEAIYDI 10376, 112714, TUBA1B,
TUBA3E, 113457, 51807, TUBA3D, TUBA8, 7277, 7278, TUBA4A, TUBA3C,
7846, 84790 TUBA1A, TUBA1C 409 GLDAATATV 9869 SETDB1 410 GLFDGVPTTA
122618 PLD4 411 GLFEDVTQPGILL 23140 ZZEF1 412 ILGTEDLIVEV 79719
AAGAB 413 ILLEHGADPNL 22852 ANKRD26 414 ILSVVNSQL 80183 KIAA0226L
415 ILVTSIFFL 643 CXCR5 416 IMEDIILTL 1656 DDX6 417 IQIGEETVITV
2316 FLNA 418 IVTEIISEI 64151 NCAPG 419 LLNEILEQV 64151 NCAPG 420
KLIDDQDISISL 80208 SPG11 421 KLWTGGLDNTV 7088, 7090, 7091 TLE1,
TLE3, TLE4 422 KQFEGTVEI 675 BRCA2 423 LLIGTDVSL 9730 VPRBP 424
LLPPLESLATV 5518 PPP2R1A 425 LLSDVRFVL 53339 BTBD1 426 LLWGNLPEI
653820, 729533 FAM72B, FAM72A 427 LLYDAVHIV 2899 GRIK3 428
LMYPYIYHV 54954 FAM120C 429 NLLETKLQL 219988 PATL1 430 QLIEKNWLL
56992 KIF15 431 RMVAEIQNV 11262 SP140 432 SAVDFIRTL 9263 STK17A 433
SILDRDDIFV 8237 USP11 434 SIQQSIERLLV 4809 NHP2L1 435 SLFNQEVQI
100528032, KLRK1, KLRC4 22914, 8302 436 SLFSSLEPQIQPV 23029 RBM34
437 SLEPQIQPV 23029 RBM34 438 SLFIGEKAVLL 23029 RBM34 439 SLLDLHTKV
27340 UTP20 440 SLLEQGKEPWMV 147949, 163087, ZNF583, ZNF383,
342892, 374899, ZNF850, ZNF829, 84503 ZNF527 441 SLLEVNEASSV 149628
PYHIN1 442 SLNDLEKDVMLL 6597 SMARCA4 443 SLTIDGIYYV 1659 DHX8 444
SMSGYDQVL 3187, 3188 HNRNPH1, HNRNPH2 445 VIIKGLEEITV 3832 KIF11
446 VILTSSPFL 10800 CYSLTR1 447 VLDDSIYLV 57565 KLHL14 448
VMNDRLYAI 57565 KLHL14 449 LLDAMNYHL 57565 KLHL14 450 VLGPGPPPL
254359 ZDHHC24 451 VLIEYNFSI 55215 FANCI 452 VLLSLLEKV 1130 LYST
453 SLLPLVWKI 1130 LYST 454 VLMDEGAVLTL 54596 L1TD1 455 VLPDPEVLEAV
57326 PBXIP1 456 YAAPGGLIGV 1968, 255308 EIF253 457 YIMEPSIFNTL
51497 TH1L 458 YIQEHLLQI 10625 IVNS1ABP 459 YLDFSNNRL 51284 TLR7
460 YLEDGFAYV 5558 PRIM2 461 YLLNLNHLGL 23471 TRAM1 462 YVLPKLYVKL
100128168, RP526P39, RPS26P11, 100996747, RP526, RP526P28, 441502,
6231, RP526P20, RP526P15, 643003, 644166, RP526P50, RP526P2,
644928, 644934, RP526P25, RP526P58 646753, 728937, 729188 463
ALMAVVSGL 55103 RALGPS2 464 ALSDETKNNWEV 5591 PRKDC 465
FLYDEIEAEVNL 23191, 26999 CYFIP1, CYFIP2 466 FLYDEIEAEV 23191,
26999 CYFIP1, CYFIP2 467 IMQDFPAEIFL 25914 RTTN 468 KIQEILTQV 10643
IGF2BP3 469 SLDGTELQL 284114 TMEM102 470 TLTNIIHNL 94101 ORMDL1 471
VLLAAGPSAA 23225 NUP210 472 YLFESEGLVL 283431 GAS2L3 473 ALADDDFLTV
4173 MCM4 474 ALADLIEKELSV 4850 CNOT4 475 ALDDMISTL 7203 CCT3 476
ALITEVVRL 26005 C2CD3 477 ALLDQTKTLAESAL 7094 TLN1 478 AMFESSQNVLL
64328 XPO4 479 YLAHFIEGL 64328 XPO4 480 FLAEDPKVTL 60489 APOBEC3G
481 FLIDTSASM 203522, 26512 DDX26B, INT56 482 FLTDLEDLTL 26151 NAT9
483 FLTEMVHFI 93517 SDR42E1 484 FLVDGPRVQL 90204 ZSWIM1 485
GLLDCPIFL 2177 FANCD2 486 GLLDLPFRVGV 23347 SMCHD1 487 GLSDGNPSL
79684 MSANTD2 488 GVYDGEEHSV 4113 MAGEB2 489 HLANIVERL 117854,
445372, TRIM6, TRIM6-TRIM34, 53840 TRIM34 490 ILDEKPVII 23460 ABCA6
491 ILFSEDSTKLFV 84916 CIRH1A 492 ILLDDNMQIRL 5261 PHKG2 493
LIIDQADIYL 27042 DIEXF 494 LLGDSSFFL 283254 HARBI1 495 LLLDEEGTFSL
27013 CNPPD1 496 LLVEQPPLAGV 4773 NFATC2 497 LLWDVPAPSL 1388, 7148
ATF6B, TNXB 498 LTAPPEALLMV 79050 NOC4L 499 NLMEMVAQL 55636 CHD7
500 QIITSVVSV 5378 PMS1 501 QLALKVEGV 25896 INTS7
502 QLLDETSAITL 10915 TCERG1 503 QLYEEPDTKL 10270 AKAP8 504
RLYSGISGLEL 84172 POLR1B 505 RVLEVGALQAV 25885 POLR1A 506
SLLPLDDIVRV 3708, 3709 ITPR1, ITPR2 507 SLLTEQDLWTV 90806 ANGEL2
508 SVDSAPAAV 10635 RAD51AP1 509 TLQEVVTGV 55750 AGK 510 TVDVATPSV
8924 HERC2 511 VLAYFLPEA 4171 MCM2 512 VLISVLQAI 26999 CYFIP2 513
YLIPFTGIVGL 26001 RNF167 514 YLLDDGTLVV 8872 CDC123 515 YLMPGFIHL
168400, 55510 DDX53, DDX43 516 YLPDIIKDQKA 5496 PPM1G 517 YLQLTQSEL
283237 TTC9C 518 YLTEVFLHVV 55024 BANK1 519 YLVEGRFSV 55125 CEP192
520 YLVYILNEL 51202 DDX47 521 YLYGQTTTYL 7153 TOP2A 522 YVLTQPPSV
28796, 28815, IGLV3-21, IGLV2-14, 28831, 3537, IGLJ3, IGLC1, IGLC2
3538
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] FIGS. 1A-1V describe relative presentation of various
peptides of the present disclosure.
[0050] FIGS. 1A to 1V show the overpresentation of various peptides
in normal tissues (white bars) and CLL (black bars). FIG. 1A) Gene:
IGHM, Peptide: ALHRPDVYL (SEQ ID NO.: 2). Tissues from left to
right: 2 adipose tissues, 3 adrenal glands, 4 blood cells, 10 blood
vessels, 6 bone marrows, 7 brains, 6 breasts, 2 cartilages, 2 eyes,
3 gallbladders, 6 hearts, 14 kidneys, 19 large intestines, 20
livers, 45 lungs, 6 lymph nodes, 7 nerves, 3 ovaries, 10
pancreases, 3 parathyroid glands, 1 peritoneum, 5 pituitary glands,
6 placentas, 3 pleuras, 3 prostates, 7 salivary glands, 5 skeletal
muscles, 6 skins, 4 small intestines, 9 spleens, 5 stomachs, 6
testes, 2 thymi, 3 thyroid glands, 9 tracheas, 3 ureters, 6 urinary
bladders, 2 uteri, 6 esophagi, 17 chronic lymphocytic leukemia
samples. The peptide has additionally been detected on 2/21
non-Hodgkin lymphoma samples. FIG. 1B) Gene: IGHM, Peptide:
VIAELPPKV (SEQ ID NO.: 3). Tissues from left to right: 2 adipose
tissues, 3 adrenal glands, 4 blood cells, 10 blood vessels, 6 bone
marrows, 7 brains, 6 breasts, 2 cartilages, 2 eyes, 3 gallbladders,
6 hearts, 14 kidneys, 19 large intestines, 20 livers, 45 lungs, 6
lymph nodes, 7 nerves, 3 ovaries, 10 pancreases, 3 parathyroid
glands, 1 peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3
prostates, 7 salivary glands, 5 skeletal muscles, 6 skins, 4 small
intestines, 9 spleens, 5 stomachs, 6 testes, 2 thymi, 3 thyroid
glands, 9 tracheas, 3 ureters, 6 urinary bladders, 2 uteri, 6
esophagi, 17 chronic lymphocytic leukemia samples. The peptide has
additionally been detected on 5/21 non-Hodgkin lymphoma samples and
1/90 lung cancers. FIG. 1C), Gene: IGHM, Peptide: VIAELPPKVSV (SEQ
ID No.: 4), Tissues from left to right: 2 adipose tissues, 3
adrenal glands, 4 blood cells, 10 blood vessels, 6 bone marrows, 7
brains, 6 breasts, 2 cartilages, 2 eyes, 3 gallbladders, 6 hearts,
14 kidneys, 19 large intestines, 20 livers, 45 lungs, 6 lymph
nodes, 7 nerves, 3 ovaries, 10 pancreases, 3 parathyroid glands, 1
peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3
prostates, 7 salivary glands, 5 skeletal muscles, 6 skins, 4 small
intestines, 9 spleens, 5 stomachs, 6 testes, 2 thymi, 3 thyroid
glands, 9 tracheas, 3 ureters, 6 urinary bladders, 2 uteri, 6
esophagi, 17 chronic lymphocytic leukemia samples. FIG. 1D), Gene:
NUP210, Peptide: RLYEITIEV (SEQ ID No.: 121). Samples from left to
right: 1 PBMC culture, 1 benign prostate, 8 normal tissues (3
lungs, 4 spleens, 1 trachea), 49 cancer tissues (2 brain cancers, 2
breast cancers, 1 colon cancer, 13 leukocytic leukemia cancers, 13
lung cancers, 8 lymph node cancers, 1 myeloid cells cancer, 4
ovarian cancers, 2 skin cancers, 1 urinary bladder cancer, 2 uterus
cancers). Discrepancies regarding the list of tumor types between
FIG. 1D and table 4 may be due to the more stringent selection
criteria applied in table 4 (for details please refer to table 4).
The normal tissue panel and the cancer cell lines and xenografts
tested were the same as in FIGS. 1A-1C. FIG. 1E), Gene: COBRA1,
Peptide: ALLRLLPGL (SEQ ID No.: 14). Samples from left to right: 1
cell line, 11 cancer tissues (1 breast cancer, 1 colon cancer, 1
head and neck cancer, 2 leukocytic leukemia cancers, 1 liver
cancer, 1 lung cancer, 2 lymph node cancers, 1 myeloid cell cancer,
1 skin cancer). FIG. 1F), Gene: KIAA0226L, Peptide: GIIDGSPRL (SEQ
ID No.: 62). Samples from left to right: 2 normal tissues (1 lymph
node, 1 spleen), 18 cancer tissues (2 colon cancers, 9 leukocytic
leukemia cancers, 6 lymph node cancers, 1 rectum cancer). FIG. 1G),
Gene: DDX3X, Peptide: GLDQQFAGLDL (SEQ ID No.: 68). Samples from
left to right: 1 cell line, 2 primary cultures, 21 cancer tissues
(1 bile duct cancer, 1 brain cancer, 1 breast cancer, 1 head and
neck cancer, 2 kidney cancers, 3 leukocytic leukemia cancers, 2
liver cancers, 2 lung cancers, 2 lymph node cancers, 3 ovarian
cancers, 1 rectum cancer, 1 skin cancer, 1 uterus cancer). FIG.
1H), Gene: LIG1, Peptide: KTLDVDATYEI (SEQ ID No.: 112). Samples
from left to right: 2 cell lines, 1 primary culture, 13 cancer
tissues (1 esophageal cancer, 2 leukocytic leukemia cancers, 1
liver cancer, 3 lung cancers, 2 lymph node cancers, 2 ovarian
cancers, 1 skin cancer, 1 uterus cancer). FIG. 1I), Gene: SMCHD1,
Peptide: SIIEGPIIKL (SEQ ID No.: 146). Samples from left to right:
14 cancer tissues (1 head and neck cancer, 3 leukocytic leukemia
cancers, 6 lung cancers, 1 lymph node cancer, 1 ovarian cancer, 2
urinary bladder cancers). FIG. 1J), Gene: MMS22L, Peptide:
SILETVATL (SEQ ID No.: 147). Samples from left to right: 1 primary
culture, 11 cancer tissues (1 colon cancer, 4 leukocytic leukemia
cancers, 1 lung cancer, 3 lymph node cancers, 1 myeloid cell
cancer, 1 skin cancer). FIG. 1K), Gene: FCRL3, Peptide: SLLAELHVL
(SEQ ID No.: 153). Samples from left to right: 1 cell line, 9
normal tissues (1 lymph node, 1 rectum, 7 spleens), 27 cancer
tissues (1 breast cancer, 1 kidney cancer, 10 leukocytic leukemia
cancers, 14 lymph node cancers, 1 skin cancer). FIG. 1L), Gene:
FAM126A, Peptide: SLQEEKLIYV (SEQ ID No.: 159). Samples from left
to right: 2 cell lines, 23 cancer tissues (5 brain cancers, 1
breast cancer, 1 esophageal cancer, 3 head and neck cancers, 2
leukocytic leukemia cancers, 5 lung cancers, 3 lymph node cancers,
1 skin cancer, 2 urinary bladder cancers). FIG. 1M), Gene: HNRNPC,
Peptide: SVHKGFAFV (SEQ ID No.: 164). Samples from left to right:
10 cell lines, 1 primary culture, 1 normal tissue (1 adrenal
gland), 36 cancer tissues (12 brain cancers, 1 colon cancer, 2 head
and neck cancers, 2 kidney cancers, 3 leukocytic leukemia cancers,
1 liver cancer, 5 lung cancers, 1 myeloid cell cancer, 1 ovarian
cancer, 1 rectum cancer, 1 skin cancer, 2 stomach cancers, 2
urinary bladder cancers, 2 uterus cancers). FIG. 1N), Gene:
RASGRF1, Peptide: TLDTSKLYV (SEQ ID No.: 167). Samples from left to
right: 13 cancer tissues (1 brain cancer, 8 leukocytic leukemia
cancers, 4 lymph node cancers). FIG. 1O), Gene: RASGRF1, Peptide:
YLLDQSFVM (SEQ ID No.: 168). Samples from left to right: 16 cancer
tissues (10 leukocytic leukemia cancers, 6 lymph node cancers).
FIG. 1P), Gene: RNF213, Peptide: YIQEYLTLL (SEQ ID No.: 192).
Samples from left to right: 2 cell lines, 18 cancer tissues (1
colon cancer, 1 head and neck cancer, 1 kidney cancer, 4 leukocytic
leukemia cancers, 5 lung cancers, 3 lymph node cancers, 1 myeloid
cell cancer, 1 stomach cancer, 1 urinary bladder cancer). FIG. 1Q),
Gene: DCK, Peptide: YLQEVPILTL (SEQ ID No.: 197). Samples from left
to right: 11 cancer tissues (5 leukocytic leukemia cancers, 4 lymph
node cancers, 1 skin cancer, 1 uterus cancer). FIG. 1R), Gene:
EIF3H, Peptide: YMFEEVPIVI (SEQ ID No.: 200). Samples from left to
right: 4 cell lines, 2 primary cultures, 34 cancer tissues (1 bile
duct cancer, 2 breast cancers, 1 colon cancer, 1 esophageal cancer,
1 gallbladder cancer, 5 head and neck cancers, 2 leukocytic
leukemia cancers, 2 liver cancers, 9 lung cancers, 4 lymph node
cancers, 2 ovarian cancers, 1 rectum cancer, 2 skin cancers, 1
urinary bladder cancer). FIG. 1S), Gene: UBLCP1, Peptide: LIDVKPLGV
(SEQ ID No.: 211). Samples from left to right: 3 cell lines, 23
cancer tissues (5 brain cancers, 1 breast cancer, 1 esophageal
cancer, 1 head and neck cancer, 1 kidney cancer, 3 leukocytic
leukemia cancers, 2 liver cancers, 2 lung cancers, 4 lymph node
cancers, 1 ovarian cancer, 1 prostate cancer, 1 skin cancer). FIG.
1T), Gene: TMCO6, Peptide: QLLPVSNVVSV (SEQ ID No.: 256). Samples
from left to right: 2 primary cultures, 13 cancer tissues (2 head
and neck cancers, 3 leukocytic leukemia cancers, 1 liver cancer, 2
lung cancers, 4 lymph node cancers, 1 ovarian cancer). FIG. 1U),
Gene: MY1OG, Peptide: LLYNSTDPTL (SEQ ID No.: 312). Samples from
left to right: 1 cell line, 2 primary cultures, 14 cancer tissues
(1 colon cancer, 4 leukocytic leukemia cancers, 2 lung cancers, 5
lymph node cancers, 1 ovarian cancer, 1 urinary bladder cancer).
FIG. 1V), Gene: PIM1, Peptide: VLLPQETAEIHL (SEQ ID No.: 328).
Samples from left to right: 3 primary cultures, 27 cancer tissues
(1 breast cancer, 1 colon cancer, 5 leukocytic leukemia cancers, 5
lung cancers, 7 lymph node cancers, 2 ovarian cancers, 2 skin
cancers, 1 stomach cancer, 3 urinary bladder cancers).
[0051] FIGS. 2A-2D describe relative expression of various peptides
of the present disclosure. FIGS. 2A to 2D show exemplary expression
profiles of source genes of the present invention that are highly
over-expressed or exclusively expressed in CLL in a panel of normal
tissues (white bars) and 17 CLL samples (black bars). Tissues from
left to right: 6 arteries, 1 blood cells, 1 brain, 1 heart, 2
livers, 2 lungs, 2 veins, 1 adipose tissue, 1 adrenal gland, 6 bone
marrows, 1 cartilage, 1 colon, 1 esophagus, 2 eyes, 2 gallbladders,
1 kidney, 6 lymph nodes, 5 pancreases, 2 pituitary glands, 1
rectum, 1 salivary gland, 1 skeletal muscle, 1 skin, 1 small
intestine, 1 spleen, 1 stomach, 1 thyroid gland, 7 tracheas, 1
urinary bladder, 1 breast, 5 ovaries, 3 placentas, 1 prostate, 1
testis, 1 thymus, 1 uterus, 10 chronic lymphocytic leukemia
samples. FIG. 2A) Gene symbol: FCER2; FIG. 2B) Gene symbol:
KIAA0226L; FIG. 2C) Gene symbol: PAX5; FIG. 2D) Gene symbol:
CLEC17A.
[0052] FIGS. 3A-3F describe simulation graphs of various peptides
of the present disclosure. FIGS. 3A-3F show exemplary
immunogenicity data: flow cytometry results after peptide-specific
multimer staining. CD8+ T cells were primed using artificial APCs
coated with anti-CD28 mAb and HLA-A*02 in complex with SeqID No 1
peptide (FIG. 3C, left panel), SeqID No 5 peptide (FIG. 3D left
panel), SeqID No 32 peptide (FIG. 3E, left panel) and SeqID No 220
peptide (FIG. 3F, left panel), respectively. After three cycles of
stimulation, the detection of peptide-reactive cells was performed
by 2D multimer staining with A*02/SeqID No 1 (FIG. 3C), A*02/SeqID
No 5 (D), A*02/SeqID No 32 (FIG. 3E) or A*02/SeqID No 220 (FIG.
3F). Right panels (FIGS. 3C, 3D, 3E and 3F) show control staining
of cells stimulated with irrelevant A*02/peptide complexes. Viable
singlet cells were gated for CD8+ lymphocytes. Boolean gates helped
excluding false-positive events detected with multimers specific
for different peptides. Frequencies of specific multimer+ cells
among CD8+ lymphocytes are indicated.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
[0053] The present invention furthermore generally relates to the
peptides according to the present invention for use in the
treatment of proliferative diseases, such as, for example, acute
myelogenous leukemia, bile duct cancer, brain cancer, breast
cancer, colorectal carcinoma, esophageal cancer, gallbladder
cancer, gastric cancer, hepatocellular cancer, Merkel cell
carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung
cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal
cell cancer, small cell lung cancer, urinary bladder cancer and
uterine cancer.
[0054] Particularly preferred are the peptides--alone or in
combination--according to the present invention selected from the
group consisting of SEQ ID NO: 1 to SEQ ID NO: 385. More preferred
are the peptides--alone or in combination--selected from the group
consisting of SEQ ID NO: 1 to SEQ ID NO: 202 (see Table 1), and
their uses in the immunotherapy of CLL, acute myelogenous leukemia,
bile duct cancer, brain cancer, breast cancer, colorectal
carcinoma, esophageal cancer, gallbladder cancer, gastric cancer,
hepatocellular cancer, Merkel cell carcinoma, melanoma, non-Hodgkin
lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic
cancer, prostate cancer, renal cell cancer, small cell lung cancer,
urinary bladder cancer and uterine cancer, glioblastoma, renal cell
carcinoma, hepatocellular carcinoma, and testis cancer, and
preferably CLL.
[0055] 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 FIGS. 1A-1V and Example 1.
TABLE-US-00004 TABLE 4A Peptides according to the present invention
and their specific uses in other proliferative diseases, especially
in other cancerous diseases. The table shows for selected peptides
on which additional tumor types they were found and either
over-presented on more than 5% of the measured tumor samples, or
presented on more than 5% of the measured tumor samples with a
ratio of geometric means tumor vs normal tissues being larger than
3. Over- presentation is defined as higher presentation on the
tumor sample as compared to the normal sample with highest
presentation. Normal tissues against which over-presentation was
tested were: adipose tissue, adrenal gland, blood cells, blood
vessel, bone marrow, brain, breast, esophagus, eye, gallbladder,
heart, kidney, large intestine, liver, lung, lymph node, nerve,
pancreas, parathyroid gland, peritoneum, pituitary gland, pleura,
salivary gland, skeletal muscle, skin, small intestine, spleen,
stomach, thymus, thyroid gland, trachea, ureter, and urinary
bladder. SEQ ID No. Sequence Other relevant organs/diseases 1
AIPPSFASIFL NHL 2 ALHRPDVYL NHL 3 VIAELPPKV NHL 5 ALIFKIASA AML,
BRCA 7 ALLERTGYTL HCC, NHL 8 ALAASALPALV AML 9 ALCDTLITV NHL 10
FVYGESVEL NHL 12 ALGEDEITL NHL, AML, Melanoma 13 VVDGMPPGV NHL 14
ALLRLLPGL NHL, AML 15 ALPEVSVEA NHL, AML, Melanoma 19 QVMEKLAAV NHL
20 ALVDPGPDFVV NHL 21 ALWAGLLTL NHL 22 ALWDPVIEL NHL 24 AMAGDVVYA
NHL 25 ATYSGLESQSV Melanoma 26 AVLLVLPLV NHL 27 AVLGLVWLL NSCLC,
NHL, Uterine Cancer 28 AVLHLLLSV NHL, BRCA 29 AVLQAVTAV SCLC, NHL,
AML, Melanoma, OC, Gallbladder Cancer, Bile Duct Cancer 30
ELLEGSEIYL NHL 32 FIDKFTPPV NHL 33 FIINSSNIFL NHL 35 FILPSSLYL RCC,
NHL, AML, Gallbladder Cancer, Bile Duct Cancer 37 FIMEGGAMVL AML 38
FIMEGGAMV NHL 39 FLDALLTDV SCLC, NHL, AML, Melanoma 40 FLDEDDMSL
SCLC, NHL, Urinary bladder cancer 42 FLEEGGVVTV NHL, Melanoma 43
FLLGPEALSFA NHL, AML 44 FLLSINDFL Melanoma 45 FLPELPADLEA NHL,
Melanoma 46 YIIDSAQAV NHL 48 FLSPQQPPLL NHL 49 FLTDLFAQL NHL 52
FLVETGFLHV Melanoma 55 FMEPTLLML NHL, Urinary bladder cancer 56
FVFEAPYTL NHL, AML 57 GLSEISLRL NHL, AML, Melanoma 58 YIQQGIFSV NHL
59 FVFGDENGTVSL NHL, Esophageal Cancer, Urinary bladder cancer,
Uterine Cancer 60 FVLDHEDGLNL Melanoma, Esophageal Cancer 62
GIIDGSPRL NHL 63 SLAHVAGCEL NHL 64 KLLESVASA NHL 66 SLAGGLDDMKA NHL
67 GLDDVTVEV HCC, NHL, Melanoma, Esophageal Cancer, OC, Gallbladder
Cancer, Bile Duct Cancer 68 GLDQQFAGLDL RCC, HCC, NHL, Melanoma 69
GLHQREIFL NHL 70 FVPDTPVGV NHL 71 GLKHDIARV NHL 72 GLLDAGKMYV NHL,
Melanoma 74 GLLRASFLL NHL, AML, BRCA 75 GLSIFAQDLRL NHL 76
GLLRIIPYL NHL 78 GLPSFLTEV HCC, NHL, AML, Melanoma 79 GLQAKIQEA
NHL, Esophageal Cancer, Uterine Cancer 80 VLIEDELEEL NHL 81
WLVGQEFEL NHL 82 GLQSGVDIGV NHL 83 GQGEVLVYV SCLC, GC, Esophageal
Cancer 86 HLYPGAVTI HCC, NHL 87 HQIEAVDGEEL GC, NHL, Esophageal
Cancer 88 ILDEIGADVQA PC, NHL 89 ILDFGTFQL NHL 91 ILDLNTYNV NHL 92
ILEPLNPLL NHL 93 ILFNTQINI SCLC, NHL, AML, Melanoma, OC 94
ILFPLRFTL NSCLC, RCC, CRC, NHL, Melanoma, OC 96 ILIDKTSFV NHL 97
LLFATQITL NHL 98 SLIKYFLFV NHL 99 ILIFHSVAL NHL, AML 100 ILNNEVFAI
NHL, AML 101 ILVVIEPLL NHL 102 IQDRAVPSL AML 103 KLGGTPAPA NHL 105
KLMNDIADI NHL 109 KLWEGLTELV AML 110 LLFDHLEPMEL NHL 111
KLWNVAAPLYL OC 112 KTLDVDATYEI HCC, NHL, Melanoma, Esophageal
Cancer, OC 113 KVPAEEVLVAV NHL 116 LLIGATMQV SCLC, NHL, AML 117
LLILENILL NHL, AML, Melanoma 118 RLLILENILL NHL 119 VLPAEFFEV NHL,
AML, Urinary bladder cancer, Uterine Cancer 120 AIDAALTSV NHL, AML
121 RLYEITIEV SCLC, NHL, AML, BRCA, OC 122 LLIPVVPGV NSCLC, NHL 124
LLLEETEKQAV RCC, HCC 125 LLLEIGEVGKLFV NHL 126 LLPEGGITAI NHL 127
LLPTAPTTV NHL 130 LLVLIPVYL NHL, Melanoma 133 YLLSGDISEA NHL 136
NLDKLWTLV GC, Esophageal Cancer 137 NLIKTVIKL NHL 138 NLLDIDAPVTV
SCLC, NHL 139 NLTDVVEKL RCC, NHL 141 QILSEIVEA NHL, Melanoma 145
SALDTITTV NHL 146 SIIEGPIIKL NHL, Urinary bladder cancer 147
SILETVATL NHL, AML 148 SIVASLITV NHL, AML 149 SLDNGGYYI NHL 150
SLFDQPLSII NHL 151 SLFDSAYGA AML 152 SLIRILQTI HCC
153 SLLAELHVL NHL 154 SLLAELHVLTV NHL 155 SLMLEVPAL HCC, NHL 156
SLNIGDVQL NHL 157 SLNIRDFTM AML 158 SLPEAPLDV NHL, AML, Uterine
Cancer 159 SLQEEKLIYV Brain Cancer, NHL, MCC, Melanoma, Esophageal
Cancer, Urinary bladder cancer 160 SLSFLVPSL NHL, AML, Urinary
bladder cancer 161 SMDDGMINV CRC, NHL, Gallbladder Cancer, Bile
Duct Cancer 162 SMKDDLENV NSCLC, SCLC, RCC, HCC, NHL, BRCA,
Melanoma, Esophageal Cancer, Uterine Cancer, Gallbladder Cancer,
Bile Duct Cancer 163 SQLDISEPYKV NHL 164 SVHKGFAFV NSCLC, RCC,
Brain Cancer, GC, AML, Esophageal Cancer, Urinary bladder cancer,
Uterine Cancer 165 TLDDDLDTV NHL 166 TLDPNQVSL AML 167 TLDTSKLYV
NHL 168 YLLDQSFVM NHL 169 TLLLGLTEV NHL, Melanoma 170 TLTFRVETV
NHL, Melanoma 172 TLYDMLASI CRC, NHL, AML, Melanoma 175 FTVPRVVAV
NHL 177 VLDDRELLL NHL 178 VLFFNVQEV SCLC, NHL 179 VLLGLEMTL NHL,
Esophageal Cancer 180 LLKDGPEIGL NHL 181 VLLSIPFVSV HCC, NHL 183
VLMPTVYQQGV AML 185 VMDDQRDLI AML 186 VMDPTKILI AML, Urinary
bladder cancer 187 VMDTHLVNI NHL, Urinary bladder cancer 188
VMGDIPAAV NHL, AML, Gallbladder Cancer, Bile Duct Cancer 189
VMLEMTPEL SCLC, PC, NHL, BRCA, Melanoma, Gallbladder Cancer, Bile
Duct Cancer 190 VVMGTVPRL NHL, AML 192 YIQEYLTLL NHL 193 YLDLSNNRL
NHL 194 YLDNVLAEL NHL 195 YLGGFALSV NHL, AML 196 YLLLQTYVL NHL 197
YLQEVPILTL NHL, Melanoma 199 YLVELSSLL HCC, NHL, AML NSCLC, SCLC,
CRC, HCC, NHL, Melanoma, Esophageal Cancer, OC, Urinary bladder
cancer, Gallbladder Cancer, 200 YMFEEVPIVI Bile Duct Cancer 201
YQLELHGIEL Esophageal Cancer 202 YVDDVFLRV Urinary bladder cancer
203 ALLSSQLAL NHL 204 GLLQINDKIAL SCLC, RCC, HCC, Melanoma, OC,
Urinary bladder cancer 205 GLSQANFTL NHL 207 IIADLDTTIMFA SCLC,
NHL, Melanoma, OC 208 ILLKTEGINL NHL, BRCA, OC, Urinary bladder
cancer 209 ILQAELPSL NHL 210 KLLVQDFFL HCC, NHL 211 LIDVKPLGV NHL,
BRCA, Melanoma 212 NIIEAINELLV NHL, BRCA, Melanoma 213 RLLYQLVFL
NHL, Esophageal Cancer, Urinary bladder cancer, Gallbladder Cancer,
Bile Duct Cancer 214 RLQELTEKL NHL, AML 215 VMQDIVYKL NHL,
Esophageal Cancer, Urinary bladder cancer 216 WLAGDVPAA SCLC, NHL
217 ALDEPPYLTV NHL, Melanoma 218 ALGEEWKGYVV Esophageal Cancer 219
ALLNLLESA NHL, AML, OC 220 ALPEILFAKV NHL 221 ALVSTIIMV NHL, AML
222 ALWELSLKI NHL 223 ALWVSQPPEI NHL 224 AMEALVVEV NHL 225
ILQERELLPV NHL, Melanoma, OC, Urinary bladder cancer 227
FLAEASVMTQL NHL 228 FLGGLSPGV NHL, Esophageal Cancer 229 FLLNLQNCHL
NHL 230 FLQDSKVIFV NHL 231 FLYIRQLAI AML 232 FMHQIIDQV RCC, CRC,
Melanoma 233 GIIDINVRL PC, NHL, Melanoma, Esophageal Cancer,
Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile
Duct Cancer 234 GLDDAEYAL NHL, AML, Melanoma, Urinary bladder
cancer 235 GLDDLLLFL SCLC, PC, NHL, AML, Urinary bladder cancer,
Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 236 GLLESGRHYL
NHL, AML 237 GLQENLDVVV SCLC, NHL, Melanoma, Uterine Cancer 238
GLVETELQL NHL, AML 239 ILAGEMLSV NHL, AML, Melanoma, Gallbladder
Cancer, Bile Duct Cancer 240 ILARDILEI NHL, Urinary bladder cancer
241 ILGDILLKV AML 242 ILLGIQELL NHL, AML 243 ILPTLEKELFL AML 244
ILQALAVHL NHL, AML, OC 245 KIMDYSLLLGV SCLC, RCC, OC 246 KLDETGVAL
HCC, NHL, AML, Melanoma 247 KLKDRLPSI NHL 248 KTVEPPISQV NHL 249
LLPTGVFQV Urinary bladder cancer 250 LLVQEPDGLMV SCLC, HCC,
Melanoma, Urinary bladder cancer 251 LLYDNVPGA NHL 252 NLLDPGSSYLL
Esophageal Cancer 253 NLWSVDGEVTV NHL 254 QLIPKLIFL NHL, Melanoma
255 YLFEEAISM NHL 257 RIINGIIISV NHL 258 RLDYITAEI RCC, NHL, AML,
Melanoma 260 SLDDVEGMSV NHL, Gallbladder Cancer, Bile Duct Cancer
261 SLVEAQGWLV NHL 263 SQWEDIHVV NHL, AML 264 TILDYINVV Brain
Cancer, NHL, AML, BRCA, OC, Uterine Cancer 265 TLLADDLEIKL
Esophageal Cancer 266 TLLDQLDTQL NHL, OC, Urinary bladder cancer
267 TLLDWQDSL NHL, AML, Melanoma 268 TLLQVFHLL NHL 269 TLTDEQFLV
NHL, AML 271 VIRNIVEAA GC, CRC, PC, AML, Esophageal Cancer 272
VLDELPPLI SCLC, NHL 273 VLGEYSYLL NHL 274 VLLEYHIAYL NSCLC, SCLC,
HCC, NHL, Urinary bladder cancer 275 VLLFIEHSV NHL 276 VLNDGAPNV
NSCLC, SCLC, NHL, Melanoma, Uterine Cancer, Gallbladder Cancer,
Bile Duct Cancer 278 YLDDLLPKL PC, NHL 280 YTLDSLYWSV NHL 281
NLLDDRGMTAL NHL, AML 282 LLRDGIELV NHL, Melanoma, OC 283 ILQPMDIHV
NHL 284 LLSAAEPVPA NHL 286 FLLEDLSQKL NHL 288 GLAESTGLLAV NHL 290
LANPHELSL AML
291 ILLNEDDLVTI SCLC, NHL, Melanoma 292 AAALIIHHV Urinary bladder
cancer 294 ALLDQLHTLL NHL 295 ALLQKLQQL NHL 296 FIAPTGHSL SCLC,
NHL, AML, BRCA 297 FLVEPQEDTRL NHL, Melanoma 298 IILPVEVEV SCLC,
NHL, AML, Urinary bladder cancer 299 ILEENIPVL NHL 300 ILLNPAYDVYL
NHL, Melanoma 303 ILSQPTPSL NHL, AML, Melanoma, OC 304 LAIVPVNTL
AML 305 LLFPQIEGIKI NHL, Melanoma 306 VVAEELENV NHL, Melanoma, OC
307 LLLTKPTEA NHL 308 SLYDVSRMYV NHL 309 ILYGTQFVL NHL 310
LLSTLHLLV NHL 311 LLVDVEPKV NSCLC, SCLC, RCC, HCC, PC, NHL, AML,
Melanoma 312 LLYNSTDPTL NHL 313 LMADLEGLHL Melanoma, Urinary
bladder cancer 314 LMKDCEAEV NHL 316 MLLEHGITL NHL 317 NLLAHIWAL
AML 320 TIAPVTVAV NHL 321 RLLEFELAQL SCLC, CRC, NHL, OC 322
SLASIHVPL NHL 323 SLDLFNCEV NHL 324 SLYSALQQA NHL 325 TLENGVPCV NHL
326 VLAFLVHEL Esophageal Cancer 327 VLIKWFPEV NHL 328 VLLPQETAEIHL
NHL, BRCA, Melanoma, OC, Urinary bladder cancer 329 VLMDGSVKL NHL
330 VLMWEIYSL NHL 332 VMIQHVENL NHL 333 VTLEFPQLIRV SCLC, HCC, NHL
334 YLLEEKIASL NHL 335 YLYQEQYFI SCLC, NHL, AML, Urinary bladder
cancer 337 YMYEKESEL NHL 338 FLDMTNWNL HCC, NHL, Melanoma, OC,
Urinary bladder cancer 339 GLWGTVVNI NHL, AML, Melanoma, Uterine
Cancer 341 LLAELPASVHA HCC, NHL, Urinary bladder cancer 342
SLITPLQAV NHL, AML, Urinary bladder cancer, Gallbladder Cancer,
Bile Duct Cancer 343 ILLEALDCI NHL, AML, Melanoma, Esophageal
Cancer 344 VLAFENPQV Esophageal Cancer 346 VLVQVSPSL RCC, NHL, AML,
Gallbladder Cancer, Bile Duct Cancer 347 YLGPVSPSL NHL 348
ALAKPPVVSV SCLC, HCC, Melanoma, Esophageal Cancer 349 ALATHILSL
NHL, Urinary bladder cancer 350 ALEDRVWEL NHL, Esophageal Cancer
351 ALSEKLARL HCC, NHL, AML, Urinary bladder cancer 352 ALVFELHYV
NHL 353 ATPMPTPSV SCLC, NHL 354 FIMDDPAGNSYL CRC, NHL, AML,
Melanoma, Esophageal Cancer, Urinary bladder cancer, Gallbladder
Cancer, Bile Duct Cancer 357 FLIQEIKTL NHL, OC, Urinary bladder
cancer 358 FLTDYLNDL NHL 359 FMQDPMEVFV Melanoma 360 HLIDTNKIQL NHL
362 ILTELGGFEV NHL 363 ITTEVVNELYV NHL, Melanoma 364 KMDWIFHTI
Urinary bladder cancer 365 LISPLLLPV NHL, AML, Melanoma, Urinary
bladder cancer 367 NLWSLVAKV NSCLC, NHL, AML, Melanoma, OC 369
RLLDLENSLLGL AML 370 SIFASPESV Melanoma, Esophageal Cancer, Urinary
bladder cancer 371 SLADDSVLERL RCC, HCC, NHL, AML, Esophageal
Cancer, Urinary bladder cancer 373 TLLADQGEIRV NHL, Melanoma 374
VLSVITEEL NHL, AML 375 VLWFKPVEL NSCLC, HCC, NHL, Melanoma,
Esophageal Cancer 376 VLYNQRVEEI Urinary bladder cancer 377
VVDGTCVAV NHL, Melanoma 378 YILGKFFAL NHL, AML 379 YLAELVTPIL
NSCLC, SCLC, NHL, Melanoma 380 YLDRKLLTL NHL 381 YLLEENKIKL NHL 382
YLLPLLQRL NHL, AML 383 YLLREWVNL HCC, NHL 384 YMIGSEVGNYL NHL,
Urinary bladder cancer 385 YTIPLAIKL NHL, AML, BRCA, Esophageal
Cancer NSCLC = non-small cell lung cancer, SCLC = small cell lung
cancer, RCC = kidney cancer, CRC = colon or rectum cancer, GC =
stomach cancer, HCC = liver cancer, PC = pancreatic cancer, PrC =
prostate cancer, leukemia, BRCA = breast cancer, MCC = Merkel cell
carcinoma, NHL = Non Hodgkin lymphoma
[0056] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 1, 2, 3, 7, 9, 10, 13, 14, 15,
19, 20, 21, 22, 24, 26, 27, 28, 29, 30, 32, 33, 35, 38, 39, 40, 42,
43, 45, 46, 48, 49, 55, 56, 57, 58, 59, 62, 63, 64, 66, 67, 68, 69,
70, 71, 72, 74, 75, 76, 78, 79, 80, 81, 82, 86, 87, 88, 89, 91, 92,
93, 94, 96, 97, 98, 99, 100, 101, 103, 105, 110, 112, 113, 116,
117, 118, 119, 120, 121, 122, 125, 126, 127, 130, 133, 137, 138,
139, 141, 145, 146, 147, 148, 149, 150, 153, 154, 155, 156, 158,
159, 160, 161, 162, 163, 165, 167, 168, 169, 170, 172, 175, 177,
178, 179, 180, 181, 187, 188, 189, 190, 192, 193, 194, 195, 196,
197, 199, 200, 203, 205, 207, 208, 209, 210, 211, 212, 213, 214,
215, 216, 217, 219, 220, 221, 222, 223, 224, 225, 227, 228, 229,
230, 233, 234, 235, 236, 237, 238, 239, 240, 242, 244, 246, 247,
248, 251, 253, 254, 255, 257, 258, 260, 261, 263, 264, 266, 267,
268, 269, 272, 273, 274, 275, 276, 278, 280, 281, 282, 283, 284,
286, 288, 290, 291, 294, 295, 296, 297, 298, 299, 300, 303, 305,
306, 307, 308, 309, 310, 311, 312, 314, 316, 320, 321, 322, 323,
324, 325, 327, 328, 329, 330, 332, 333, 334, 335, 337, 338, 339,
341, 342, 343, 346, 347, 349, 350, 351, 352, 353, 354, 357, 358,
360, 362, 363, 365, 367, 371, 373, 374, 375, 377, 378, 379, 380,
381, 382, 383, 384, and 385 for the--in one preferred embodiment
combined--treatment of NHL.
[0057] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 5, 8, 12, 14, 15, 29, 35, 37,
39, 43, 56, 57, 74, 78, 93, 99, 100, 102, 109, 116, 117, 119, 120,
121, 147, 148, 151, 157, 158, 160, 164, 166, 172, 183, 185, 186,
188, 190, 195, 199, 214, 219, 221, 231, 234, 235, 236, 238, 239,
241, 242, 243, 244, 246, 258, 263, 264, 267, 269, 271, 281, 290,
296, 298, 303, 304, 311, 317, 335, 339, 342, 343, 346, 351, 354,
365, 367, 369, 374, 378, 382, and 385 for the--in one preferred
embodiment combined--treatment of AML.
[0058] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 5, 28, 74, 121, 162, 208, 211,
212, 264, 296, 328, and 385 for the--in one preferred embodiment
combined--treatment of B RCA.
[0059] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 7, 67, 68, 78, 86, 112, 124,
152, 155, 162, 181, 199, 200, 204, 210, 246, 250, 274, 311, 333,
338, 341, 348, 351, 371, 375, and 383 for the--in one preferred
embodiment combined--treatment of HCC.
[0060] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 12, 15, 25, 29, 39, 42, 44, 45,
52, 57, 60, 67, 68, 72, 78, 93, 94, 112, 117, 130, 141, 159, 162,
169, 170, 172, 189, 197, 200, 204, 207, 211, 212, 217, 225, 232,
233, 234, 237, 239, 246, 250, 254, 258, 267, 276, 282, 291, 297,
300, 303, 305, 306, 311, 313, 328, 338, 339, 343, 348, 354, 359,
363, 365, 367, 370, 373, 375, 377, and 379 for the--in one
preferred embodiment combined--treatment of melanoma.
[0061] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 27, 94, 122, 162, 164, 200, 274,
276, 311, 367, 375, and 379 for the--in one preferred embodiment
combined--treatment of NSCLC.
[0062] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 27, 59, 79, 119, 158, 162, 164,
233, 235, 237, 264, 276, and 339 for the--in one preferred
embodiment combined--treatment of uterine cancer.
[0063] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 29, 67, 93, 94, 111, 112, 200,
204, 207, 208, 219, 225, 244, 245, 264, 266, 282, 303, 306, 321,
328, 338, 357, and 367 for the--in one preferred embodiment
combined--treatment of ovarian cancer (OC).
[0064] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 29, 35, 67, 161, 162, 188, 189,
200, 213, 233, 235, 239, 260, 276, 342, 346, and 354 for the--in
one preferred embodiment combined--treatment of gallbladder cancer
and/or bile duct cancer.
[0065] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 35, 68, 94, 124, 139, 162, 164,
197, 204, 232, 245, 258, 311, 346, and 371 for the--in one
preferred embodiment combined--treatment of RCC.
[0066] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 39, 40, 83, 93, 121, 138, 162,
178, 189, 200, 204, 207, 216, 235, 237, 245, 250, 272, 274, 276,
291, 296, 298, 311, 321, 333, 335, 348, 353, and 379 for the--in
one preferred embodiment combined--treatment of SCLC.
[0067] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 40, 55, 59, 119, 146, 159, 160,
164, 186, 187, 200, 202, 204, 208, 213, 215, 225, 233, 234, 235,
240, 249, 250, 266, 274, 292, 298, 313, 328, 335, 338, 341, 342,
349, 351, 354, 357, 364, 365, 370, 371, 376, and 384 for the--in
one preferred embodiment combined--treatment of urinary bladder
cancer.
[0068] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 59, 60, 67, 79, 83, 87, 112,
136, 159, 162, 164, 179, 200, 201, 213, 215, 218, 228, 233, 252,
265, 271, 326, 343, 344, 348, 350, 354, 370, 371, 375, and 385 for
the--in one preferred embodiment combined--treatment of esophageal
cancer.
[0069] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 83, 87, 136, 164, and 271 for
the--in one preferred embodiment combined--treatment of gastric
cancer (GC).
[0070] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 88, 189, 233, 235, 271, 278, and
311 for the--in one preferred embodiment combined--treatment of
pancreatic cancer (PC).
[0071] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 94, 161, 172, 200, 232, 271,
321, and 354 for the--in one preferred embodiment
combined--treatment of colorectal cancer (CRC).
[0072] Thus, another aspect of the present invention relates to the
use of the peptide according to the present invention according to
SEQ ID No. 159 for the treatment of MCC.
[0073] Thus, another aspect of the present invention relates to the
use of the peptides according to the present invention for
the--preferably combined--treatment of a proliferative disease
selected from the group of CLL, acute myelogenous leukemia, bile
duct cancer, brain cancer, breast cancer, colorectal carcinoma,
esophageal cancer, gallbladder cancer, gastric cancer,
hepatocellular cancer, Merkel cell carcinoma, melanoma, non-Hodgkin
lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic
cancer, prostate cancer, renal cell cancer, small cell lung cancer,
urinary bladder cancer and uterine cancer.
TABLE-US-00005 TABLE 4B Peptides according to the present invention
and their specific uses in other proliferative diseases, especially
in other cancerous diseases. The table shows, like TABLE 4A, for
selected peptides on which additional tumour types they were found
showing over-presentation (including specific presentation) on more
than 5% of the measured tumour samples, or presentation on more
than 5% of the measured tumour samples with a ratio of geometric
means tumour vs normal tissues being larger than 3. Over-
presentation is defined as higher presentation on the tumour sample
as compared 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, oesophagus, eye, gallbladder, heart, kidney,
liver, lung, lymph node, mononuclear white blood cells, pancreas,
parathyroid gland, peripheral nerve, peritoneum, pituitary, pleura,
rectum, salivary gland, skeletal muscle, skin, small intestine,
spleen, stomach, thyroid gland, trachea, ureter, urinary bladder,
vein. SEQ ID NO. Sequence Additional Entities 5 ALIFKIASA Uterine
Cancer 7 ALLERTGYTL Uterine Cancer, AML 8 ALAASALPALV AML, HNSCC 11
ALFTFSPLTV AML, HNSCC, NHL, NSCLC, OC, SCLC 13 VVDGMPPGV BRCA,
Uterine Cancer 14 ALLRLLPGL BRCA, Melanoma, HNSCC 15 ALPEVSVEA
Esophageal Cancer 25 ATYSGLESQSV AML 26 AVLLVLPLV NHL, NSCLC 27
AVLGLVWLL HNSCC 29 AVLQAVTAV Uterine Cancer 30 ELLEGSEIYL CRC,
BRCA, Melanoma, Uterine Cancer 35 FILPSSLYL SCLC, Uterine Cancer 37
FIMEGGAMVL NHL 38 FIMEGGAMV HNSCC 40 FLDEDDMSL HNSCC 41 FLDPSLDPLL
Uterine Cancer 42 FLEEGGVVTV SCLC, AML 44 FLLSINDFL RCC, Uterine
Cancer, AML, NHL 45 FLPELPADLEA NSCLC, Uterine Cancer, OC 48
FLSPQQPPLL HNSCC 49 FLTDLFAQL NHL, Melanoma 51 FLVEAPHDWDL Uterine
Cancer 55 FMEPTLLML HNSCC 58 YIQQGIFSV NHL, NSCLC 59 FVFGDENGTVSL
SCLC, HNSCC 67 GLDDVTVEV AML 68 GLDQQFAGLDL BRCA, OC, Uterine
Cancer, Gallbladder Cancer, Bile Duct Cancer, HNSCC 76 GLLRIIPYL
RCC 82 GLQSGVDIGV AML, Esophageal Cancer, PC 83 GQGEVLVYV NHL 84
GVMDVNTAL AML, HCC, HNSCC, NHL, NSCLC, GC, RCC 86 HLYPGAVTI AML 87
HQIEAVDGEEL PC 89 ILDFGTFQL BRCA 90 VIADLGLIRV Melanoma 91
ILDLNTYNV AML 93 ILFNTQINI Uterine Cancer, HNSCC 94 ILFPLRFTL
Gallbladder Cancer, Bile Duct Cancer, AML, HNSCC 101 ILVVIEPLL
Uterine Cancer 107 ILYNLYDLL Melanoma 108 VIYTLIHYI BRCA, Melanoma
111 KLWNVAAPLYL Melanoma 112 KTLDVDATYEI Uterine Cancer 113
KVPAEEVLVAV NHL, NSCLC 114 LIPEGPPQV AML, HNSCC 115 LLFDKLYLL
Uterine Cancer 118 RLLILENILL HNSCC 119 VLPAEFFEV SCLC 124
LLLEETEKQAV HNSCC 125 LLLEIGEVGKLFV BRCA 126 LLPEGGITAI SCLC, HCC,
Melanoma, Uterine Cancer, HNSCC 127 LLPTAPTTV SCLC 128 LLSEEEYHL
Urinary Bladder Cancer, NHL, HNSCC 130 LLVLIPVYL Uterine Cancer 136
NLDKLWTLV AML, CRC, Melanoma, NSCLC, Brain Cancer 138 NLLDIDAPVTV
Uterine Cancer, AML 141 QILSEIVEA Uterine Cancer 145 SALDTITTV
NSCLC, RCC, GC, HCC, AML 146 SIIEGPIIKL HNSCC 147 SILETVATL
Melanoma 152 SLIRILQTI SCLC 156 SLNIGDVQL AML 159 SLQEEKLIYV HNSCC
160 SLSFLVPSL Uterine Cancer 161 SMDDGMINV SCLC, Melanoma, Uterine
Cancer, AML, HNSCC 162 SMKDDLENV PC, OC, HNSCC 164 SVHKGFAFV CRC,
Melanoma, HNSCC 172 TLYDMLASI SCLC, Urinary Bladder Cancer, Uterine
Cancer 173 TVIENIHTI CRC, HCC, Melanoma, NHL, NSCLC, OC, Esophageal
Cancer, GC, RCC 174 VLAELPIIVV NSCLC, OC 176 VLAEQNIIPSA NSCLC,
SCLC, GC, Melanoma, NHL, HNSCC 179 VLLGLEMTL HNSCC 181 VLLSIPFVSV
Melanoma, Gallbladder Cancer, Bile Duct Cancer 184 VLSHNLYTV NHL
187 VMDTHLVNI CRC, Melanoma, Uterine Cancer 188 VMGDIPAAV Urinary
Bladder Cancer, HNSCC 189 VMLEMTPEL HNSCC 190 VVMGTVPRL Melanoma
191 YIFDGSDGGV Uterine Cancer 192 YIQEYLTLL SCLC, Urinary Bladder
Cancer, AML, HNSCC 197 YLQEVPILTL Uterine Cancer 198 YLTFLPAEV
BRCA, Uterine Cancer 199 YLVELSSLL Melanoma 200 YMFEEVPIVI BRCA,
HNSCC 201 YQLELHGIEL NHL 202 YVDDVFLRV AML, HNSCC 203 ALLSSQLAL
NHL, AML, NSCLC 204 GLLQINDKIAL BRCA, Uterine Cancer, AML 208
ILLKTEGINL Melanoma, HNSCC 211 LIDVKPLGV RCC, Brain Cancer, HCC,
Esophageal Cancer, HNSCC 212 NIIEAINELLV Uterine Cancer, AML, HNSCC
215 VMQDIVYKL HNSCC 216 WLAGDVPAA AML, HNSCC 217 ALDEPPYLTV AML 218
ALGEEWKGYVV AML 221 ALVSTIIMV Brain Cancer 224 AMEALVVEV AML 226
AMNISVPQV AML, PrC, Brain Cancer, Gallbladder Cancer, CCC, HNSCC,
NHL, NSCLC, OC, SCLC, Uterine Cancer 228 FLGGLSPGV SCLC, BRCA,
Uterine Cancer, AML, HNSCC 231 FLYIRQLAI Uterine Cancer 232
FMHQIIDQV Uterine Cancer 234 GLDDAEYAL Uterine Cancer, HNSCC 235
GLDDLLLFL BRCA, HNSCC 237 GLQENLDVVV AML 239 ILAGEMLSV SCLC, HCC
240 ILARDILEI Melanoma 242 ILLGIQELL BRCA 247 KLKDRLPSI HCC 257
RIINGIIISV AML 258 RLDYITAEI GC, Uterine Cancer 260 SLDDVEGMSV
SCLC, HNSCC
261 SLVEAQGWLV AML 264 TILDYINVV HNSCC 266 TLLDQLDTQL CRC, BRCA,
Uterine Cancer 268 TLLQVFHLL Uterine Cancer 269 TLTDEQFLV BRCA 272
VLDELPPLI BRCA, Uterine Cancer, HNSCC 273 VLGEYSYLL NHL, HNSCC 274
VLLEYHIAYL Melanoma, HNSCC 275 VLLFIEHSV Melanoma 279 YMAPEVVEA
HNSCC, NHL, NSCLC, SCLC, Uterine Cancer 280 YTLDSLYWSV SCLC, BRCA,
Melanoma, HNSCC 286 FLLEDLSQKL Uterine Cancer 289 ILEEQPMDMLL
Uterine Cancer 293 ALDIMIPMV AML, CRC, Brain Cancer, Gallbladder
Cancer, CCC, HNSCC, Melanoma, NHL, NSCLC, OC, Brain Cancer, RCC,
SCLC, Urinary Bladder Cancer, Uterine Cancer 294 ALLDQLHTLL AML 298
IILPVEVEV CRC, HNSCC 299 ILEENIPVL Uterine Cancer, AML 303
ILSQPTPSL Uterine Cancer 305 LLFPQIEGIKI Urinary Bladder Cancer,
HNSCC 306 VVAEELENV RCC 311 LLVDVEPKV Uterine Cancer, HNSCC 312
LLYNSTDPTL Urinary Bladder Cancer 313 LMADLEGLHL Uterine Cancer,
AML 317 NLLAHIWAL NHL 321 RLLEFELAQL Brain Cancer, PrC, Uterine
Cancer, AML, HNSCC 323 SLDLFNCEV Uterine Cancer, AML 324 SLYSALQQA
SCLC, Uterine Cancer, AML 326 VLAFLVHEL Esophageal Cancer, NSCLC,
RCC 327 VLIKWFPEV Uterine Cancer 333 VTLEFPQLIRV BRCA 334
YLLEEKIASL AML 335 YLYQEQYFI CRC, Brain Cancer, Gallbladder Cancer,
CCC, NSCLC, UterineCancer 336 YMAVTTQEV Uterine Cancer 339
GLWGTVVNI HNSCC 340 KLLEEICNL Melanoma, Uterine Cancer, AML, NHL
342 SLITPLQAV HNSCC 343 TLLEALDCI GC, Uterine Cancer, HNSCC 344
VLAFENPQV HNSCC 346 VLVQVSPSL HNSCC 348 ALAKPPVVSV NSCLC, Urinary
Bladder Cancer, NHL, HNSCC 349 ALATHILSL HCC, Melanoma 351
ALSEKLARL CRC, HNSCC, Melanoma, NSCLC, OC, PC, Brain Cancer, RCC,
Uterine Cancer 353 ATPMPTPSV BRCA, Uterine Cancer, Gallbladder
Cancer, Bile Duct Cancer, AML, HNSCC 354 FIMDDPAGNSYL Brain Cancer,
HNSCC 355 FIWPMLIHI Melanoma, Uterine Cancer 357 FLIQEIKTL HCC 358
FLTDYLNDL AML 359 FMQDPMEVFV Melanoma, NHL, NSCLC, OC 360
HLIDTNKIQL NHL, NSCLC 361 ILQEFESKL AML, NHL 362 ILTELGGFEV AML,
PrC, BRCA, PC, CRC, HCC, HNSCC, Melanoma, NSCLC, OC, Esophageal
Cancer, GC, RCC, SCLC, Urinary Bladder Cancer, Uterine Cancer 363
ITTEVVNELYV BRCA 365 LISPLLLPV SCLC, Uterine Cancer, HNSCC 367
NLWSLVAKV Uterine Cancer 368 QLQPTDALLCV Uterine Cancer 370
SIFASPESV Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer,
AML, HNSCC 374 VLSVITEEL SCLC, HCC, PrC, Urinary Bladder Cancer,
Uterine Cancer 375 VLWFKPVEL BRCA, OC, Uterine Cancer, Gallbladder
Cancer, Bile Duct Cancer, HNSCC 377 VVDGTCVAV BRCA, Uterine Cancer,
HNSCC 378 YILGKFFAL Melanoma, Esophageal Cancer 379 YLAELVTPIL HCC,
BRCA, Uterine Cancer, AML, HNSCC 383 YLLREWVNL Melanoma, Uterine
Cancer 384 YMIGSEVGNYL SCLC, Uterine Cancer 385 YTIPLAIKL CRC NSCLC
= non-small cell lung cancer, SCLC = small cell lung cancer, RCC =
kidney cancer, CRC = colon or rectum cancer, GC = stomach cancer,
HCC = liver cancer, PC = pancreatic cancer, PrC = prostate cancer,
BRCA = breast cancer, NHL = non-Hodgkin lymphoma, AML = acute
myeloid leukemia, OC = ovarian cancer, HNSCC = head and neck
squamous cell carcinoma, CCC = cholangiocarcinoma.
[0074] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 5, 7, 13, 29, 30, 35, 41, 44,
45, 51, 68, 93, 101, 112, 115, 126, 130, 138, 141, 160, 161, 172,
187, 191, 197, 198, 204, 212, 226, 228, 231, 232, 234, 258, 266,
268, 272, 279, 286, 289, 293, 299, 303, 311, 313, 321, 323, 324,
327, 335, 336, 340, 343, 351, 353, 355, 362, 365, 367, 368, 370,
374, 375, 377, 379, 383, and 384 for the--in one preferred
embodiment combined--treatment of uterine cancer.
[0075] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 7, 8, 11, 25, 42, 44, 67, 82,
84, 86, 91, 94, 114, 136, 138, 145, 156, 161, 192, 202, 203, 204,
212, 216, 217, 218, 224, 226, 237, 257, 261, 293, 294, 299, 313,
321, 323, 324, 334, 340, 353, 358, 361, 362, 370, and 379 for
the--in one preferred embodiment combined--treatment of AML.
[0076] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 8, 11, 14, 27, 38, 40, 48, 55,
59, 68, 84, 93, 94, 114, 118, 124, 126, 128, 146, 159, 161, 162,
164, 176, 179, 188, 189, 192, 200, 202, 208, 211, 212, 215, 216,
226, 228, 234, 235, 260, 264, 272, 273, 274, 279, 280, 293, 298,
305, 311, 321, 339, 342, 343, 344, 346, 348, 351, 353, 354, 362,
365, 370, 375, 377, and 379 for the--in one preferred embodiment
combined--treatment of HNSCC.
[0077] 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. 11, 26, 37, 44, 49, 58, 83, 84,
113, 128, 173, 176, 184, 201, 203, 226, 273, 279, 293, 317, 340,
348, 359, 360, and 361 for the--in one preferred embodiment
combined--treatment of NHL.
[0078] 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. 11, 26, 45, 58, 84, 113, 136,
145, 173, 174, 176, 203, 226, 279, 293, 326, 335, 348, 351, 359,
360, and 362 for the--in one preferred embodiment
combined--treatment of NSCLC.
[0079] 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. 11, 45, 68, 162, 173, 174, 226,
293, 351, 359, 362, and 375 for the--in one preferred embodiment
combined--treatment of OC.
[0080] 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. 11, 35, 42, 59, 119, 126, 127,
152, 161, 172, 176, 192, 226, 228, 239, 260, 280, 293, 324, 362,
365, 374, and 384 for the--in one preferred embodiment
combined--treatment of SCLC.
[0081] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 13, 14, 30, 68, 89, 108, 125,
198, 200, 204, 228, 235, 242, 266, 269, 272, 280, 333, 353, 362,
363, 375, 377, and 379 for the--in one preferred embodiment
combined--treatment of BRCA.
[0082] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 14, 30, 49, 90, 107, 108, 111,
126, 136, 147, 161, 164, 173, 176, 181, 187, 190, 199, 208, 240,
274, 275, 280, 293, 340, 349, 351, 355, 359, 362, 378, and 383 for
the--in one preferred embodiment combined--treatment of
melanoma.
[0083] 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. 15, 82, 173, 211, 326, 362, and
378 for the--in one preferred embodiment combined--treatment of
esophageal cancer.
[0084] 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. 30, 136, 164, 173, 187, 266,
293, 298, 335, 351, 362, 363, and 385 for the--in one preferred
embodiment combined--treatment of CRC.
[0085] 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. 44, 76, 84, 145, 173, 211, 293,
306, 326, 351, and 362 for the--in one preferred embodiment
combined--treatment of RCC.
[0086] 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. 68, 94, 181, 226, 293, 335, 353,
370, and 375 for the--in one preferred embodiment
combined--treatment of gallbladder cancer and/or bile duct cancer
and/or cholangiocarcinoma.
[0087] 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. 82, 87, 162, 351, and 362 for
the--in one preferred embodiment combined--treatment of PC.
[0088] 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. 84, 145, 173, 176, 258, 343, and
362 for the--in one preferred embodiment combined--treatment of
GC.
[0089] 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. 84, 126, 145, 173, 211, 239,
247, 349, 357, 362, 374, and 379 for the--in one preferred
embodiment combined--treatment of HCC.
[0090] 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. 136, 211, 221, 226, 293, 321,
335, 351, and 354 for the--in one preferred embodiment
combined--treatment of brain cancer.
[0091] 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. 128, 172, 188, 192, 293, 305,
312, 321, 348, 362, and 374 for the--in one preferred embodiment
combined--treatment of urinary bladder cancer.
[0092] 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. 226, 321, 362, and 374 for
the--in one preferred embodiment combined--treatment of PrC.
[0093] The present invention furthermore relates to peptides
according to the present invention that have the ability to bind to
a molecule of the human major histocompatibility complex (MHC)
class-I or--in an elongated form, such as a length-variant--MHC
class-II.
[0094] The present invention further relates to the peptides
according to the present invention wherein said peptides (each)
consist or consist essentially of an amino acid sequence according
to SEQ ID NO: 1 to SEQ ID NO: 385.
[0095] The present invention further relates to the peptides
according to the present invention, wherein said peptide is
modified and/or includes non-peptide bonds.
[0096] The present invention further relates to the peptides
according to the present invention, wherein said peptide is part of
a fusion protein, in particular fused to the N-terminal amino acids
of the HLA-DR antigen-associated invariant chain (Ii), or fused to
(or into the sequence of) an antibody, such as, for example, an
antibody that is specific for dendritic cells.
[0097] The present invention further relates to a nucleic acid,
encoding the peptides according to the present invention. The
present invention further relates to the nucleic acid according to
the present invention that is DNA, cDNA, PNA, RNA or combinations
thereof.
[0098] The present invention further relates to an expression
vector capable of expressing and/or expressing a nucleic acid
according to the present invention.
[0099] The present invention further relates to a peptide according
to the present invention, a nucleic acid according to the present
invention or an expression vector according to the present
invention for use in the treatment of diseases and in medicine, in
particular in the treatment of cancer.
[0100] The present invention further relates to antibodies that are
specific against the peptides according to the present invention or
complexes of said peptides according to the present invention with
MHC, and methods of making these.
[0101] The present invention further relates to T-cell receptors
(TCRs), in particular soluble TCR (sTCRs) and cloned TCRs
engineered into autologous or allogeneic T cells, and methods of
making these, as well as NK cells or other cells bearing said TCR
or cross-reacting with said TCRs.
[0102] The antibodies and TCRs are additional embodiments of the
immunotherapeutic use of the peptides according to the invention at
hand.
[0103] The present invention further relates to a host cell
comprising a nucleic acid according to the present invention or an
expression vector as described before. The present invention
further relates to the host cell according to the present invention
that is an antigen presenting cell, and preferably is a dendritic
cell.
[0104] 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.
[0105] The present invention further relates to said method
according to the present invention, wherein the antigen is loaded
onto class I or II MHC molecules expressed on the surface of a
suitable antigen-presenting cell or artificial antigen-presenting
cell by contacting a sufficient amount of the antigen with an
antigen-presenting cell.
[0106] The present invention further relates to the method
according to the present invention, wherein the antigen-presenting
cell comprises an expression vector capable of expressing or
expressing said peptide containing SEQ ID No. 1 to SEQ ID No.: 385,
preferably containing SEQ ID No. 1 to SEQ ID No. 202, or a variant
amino acid sequence.
[0107] 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.
[0108] The present invention further relates to a method of killing
target cells in a patient which target cells aberrantly express a
polypeptide comprising any amino acid sequence according to the
present invention, the method comprising administering to the
patient an effective number of T cells as produced according to the
present invention.
[0109] The present invention further relates to the use of any
peptide as described, the nucleic acid according to the present
invention, the expression vector according to the present
invention, the cell according to the present invention, the
activated T lymphocyte, the T cell receptor or the antibody or
other peptide- and/or peptide-MHC-binding molecules according to
the present invention as a medicament or in the manufacture of a
medicament. Preferably, said medicament is active against
cancer.
[0110] Preferably, said medicament is a cellular therapy, a vaccine
or a protein based on a soluble TCR or antibody.
[0111] The present invention further relates to a use according to
the present invention, wherein said cancer cells are CLL, acute
myelogenous leukemia, bile duct cancer, brain cancer, breast
cancer, colorectal carcinoma, esophageal cancer, gallbladder
cancer, gastric cancer, hepatocellular cancer, Merkel cell
carcinoma, melanoma, non-Hodgkin lymphoma, non-small cell lung
cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal
cell cancer, small cell lung cancer, urinary bladder cancer and
uterine cancer, glioblastoma, renal cell carcinoma, hepatocellular
carcinoma, and testis cancer, and preferably CLL cells.
[0112] The present invention further relates to biomarkers based on
the peptides according to the present invention, herein called
"targets", that can be used in the diagnosis of cancer, preferably
CLL. The marker can be over-presentation of the peptide(s)
themselves, or over-expression of the corresponding gene(s). The
markers may also be used to predict the probability of success of a
treatment, preferably an immunotherapy, and most preferred an
immunotherapy targeting the same target that is identified by the
biomarker. For example, an antibody or soluble TCR can be used to
stain sections of the tumor to detect the presence of a peptide of
interest in complex with MHC.
[0113] Optionally the antibody carries a further effector function
such as an immune stimulating domain or toxin. The present
invention also relates to the use of these novel targets in the
context of cancer treatment.
[0114] Stimulation of an immune response is dependent upon the
presence of antigens recognized as foreign by the host immune
system. The discovery of the existence of tumor associated antigens
has raised the possibility of using a host's immune system to
intervene in tumor growth. Various mechanisms of harnessing both
the humoral and cellular arms of the immune system are currently
being explored for cancer immunotherapy.
[0115] Specific elements of the cellular immune response are
capable of specifically recognizing and destroying tumor cells. The
isolation of T-cells from tumor-infiltrating cell populations or
from peripheral blood suggests that such cells play an important
role in natural immune defense against cancer. CD8-positive T-cells
in particular, which recognize class I molecules of the major
histocompatibility complex (MHC)-bearing peptides of usually 8 to
10 amino acid residues derived from proteins or defect ribosomal
products (DRIPS) located in the cytosol, play an important role in
this response. The MHC-molecules of the human are also designated
as human leukocyte-antigens (HLA).
[0116] As used herein and except as noted otherwise all terms are
defined as given below.
[0117] The term "T-cell response" means the specific proliferation
and activation of effector functions induced by a peptide in vitro
or in vivo. For MHC class I restricted cytotoxic T cells, effector
functions may be lysis of peptide-pulsed, peptide-precursor pulsed
or naturally peptide-presenting target cells, secretion of
cytokines, preferably Interferon-gamma, TNF-alpha, or IL-2 induced
by peptide, secretion of effector molecules, preferably granzymes
or perforins induced by peptide, or degranulation.
[0118] The term "peptide" is used herein to designate a series of
amino acid residues, connected one to the other typically by
peptide bonds between the alpha-amino and carbonyl groups of the
adjacent amino acids. The peptides are preferably 9 amino acids in
length, but can be as short as 8 amino acids in length, and as long
as 10, 11, 12, 13, or 14 or longer, and in case of MHC class II
peptides (elongated variants of the peptides of the invention) they
can be as long as 15, 16, 17, 18, 19 or 20 or more amino acids in
length.
[0119] Furthermore, the term "peptide" shall include salts of a
series of amino acid residues, connected one to the other typically
by peptide bonds between the alpha-amino and carbonyl groups of the
adjacent amino acids. Preferably, the salts are pharmaceutical
acceptable salts of the peptides, such as, for example, the
chloride or acetate (trifluoroacetate) salts. It has to be noted
that the salts of the peptides according to the present invention
differ substantially from the peptides in their state(s) in vivo,
as the peptides are not salts in vivo.
[0120] The term "peptide" shall also include "oligopeptide". The
term "oligopeptide" is used herein to designate a series of amino
acid residues, connected one to the other typically by peptide
bonds between the alpha-amino and carbonyl groups of the adjacent
amino acids. The length of the oligopeptide is not critical to the
invention, as long as the correct epitope or epitopes are
maintained therein. The oligopeptides are typically less than about
30 amino acid residues in length, and greater than about 15 amino
acids in length.
[0121] The term "polypeptide" designates a series of amino acid
residues, connected one to the other typically by peptide bonds
between the alpha-amino and carbonyl groups of the adjacent amino
acids. The length of the polypeptide is not critical to the
invention as long as the correct epitopes are maintained. In
contrast to the terms peptide or oligopeptide, the term polypeptide
is meant to refer to molecules containing more than about 30 amino
acid residues.
[0122] The term "full length polypeptide" means a complete protein
(amino acid chain) or complete subunit (amino acid chain) of a
multimeric protein.
[0123] A peptide, oligopeptide, protein or polynucleotide coding
for such a molecule is "immunogenic" (and thus is an "immunogen"
within the present invention), if it is capable of inducing an
immune response. In the case of the present invention,
immunogenicity is more specifically defined as the ability to
induce a T-cell response. Thus, an "immunogen" would be a molecule
that is capable of inducing an immune response, and in the case of
the present invention, a molecule capable of inducing a T-cell
response. In another aspect, the immunogen can be the peptide, the
complex of the peptide with MHC, oligopeptide, and/or protein that
is used to raise specific antibodies or TCRs against it.
[0124] A class I T cell "epitope" requires a short peptide that is
bound to a class I MHC receptor, forming a ternary complex (MHC
class I alpha chain, beta-2-microglobulin, and peptide) that can be
recognized by a T cell bearing a matching T-cell receptor binding
to the MHC/peptide complex with appropriate affinity. Peptides
binding to MHC class I molecules are typically 8-14 amino acids in
length, and most typically 9 amino acids in length.
[0125] In humans, there are three different genetic loci that
encode MHC class I molecules (the MHC-molecules of the human are
also designated human leukocyte antigens (HLA)): HLA-A, HLA-B, and
HLA-C. HLA-A*01, HLA-A*02, and HLA-B*07 are examples of different
MHC class I alleles that can be expressed from these loci.
TABLE-US-00006 TABLE 5 Expression frequencies F of HLA-A*02 and
HLA-A*24 and the most frequent HLA-DR serotypes. Frequencies are
deduced from haplotype frequencies Gf within the American
population adapted from Mori et al. (Mori et al., 1997) employing
the Hardy- Weinberg formula F = 1 - (1 - Gf).sup.2. Combinations of
A*02 or A*24 with certain HLA-DR alleles might be enriched or less
frequent than expected from their single frequencies due to linkage
disequilibrium. For details refer to Chanock et al. (Chanock et
al., 2004). Calculated phenotype from Allele Population allele
frequency A*02 Caucasian (North America) 49.1% A*02 African
American (North America) 34.1% A*02 Asian American (North America)
43.2% A*02 Latin American (North American) 48.3% DR1 Caucasian
(North America) 19.4% DR2 Caucasian (North America) 28.2% DR3
Caucasian (North America) 20.6% DR4 Caucasian (North America) 30.7%
DR5 Caucasian (North America) 23.3% DR6 Caucasian (North America)
26.7% DR7 Caucasian (North America) 24.8% DR8 Caucasian (North
America) 5.7% DR9 Caucasian (North America) 2.1% DR1 African
(North) American 13.20% DR2 African (North) American 29.80% DR3
African (North) American 24.80% DR4 African (North) American 11.10%
DR5 African (North) American 31.10% DR6 African (North) American
33.70% DR7 African (North) American 19.20% DR8 African (North)
American 12.10% DR9 African (North) American 5.80% DR1 Asian
(North) American 6.80% DR2 Asian (North) American 33.80% DR3 Asian
(North) American 9.20% DR4 Asian (North) American 28.60% DR5 Asian
(North) American 30.00% DR6 Asian (North) American 25.10% DR7 Asian
(North) American 13.40% DR8 Asian (North) American 12.70% DR9 Asian
(North) American 18.60% DR1 Latin (North) American 15.30% DR2 Latin
(North) American 21.20% DR3 Latin (North) American 15.20% DR4 Latin
(North) American 36.80% DR5 Latin (North) American 20.00% DR6 Latin
(North) American 31.10% DR7 Latin (North) American 20.20% DR8 Latin
(North) American 18.60% DR9 Latin (North) American 2.10% A*24
Philippines 65% A*24 Russia Nenets 61% A*24:02 Japan 59% A*24
Malaysia 58% A*24:02 Philippines 54% A*24 India 47% A*24 South
Korea 40% A*24 Sri Lanka 37% A*24 China 32% A*24:02 India 29% A*24
Australia West 22% A*24 USA 22% A*24 Russia Samara 20% A*24 South
America 20% A*24 Europe 18%
[0126] The peptides of the invention, preferably when included into
a vaccine of the invention as described herein bind to A*02. A
vaccine may also include pan-binding MHC class II peptides.
Therefore, the vaccine of the invention can be used to treat cancer
in patients that are A*02 positive, whereas no selection for MHC
class II allotypes is necessary due to the pan-binding nature of
these peptides.
[0127] If A*02 peptides of the invention are combined with peptides
binding to another allele, for example A*24, a higher percentage of
any patient population can be treated compared with addressing
either MHC class I allele alone. While in most populations less
than 50% of patients could be addressed by either allele alone, a
vaccine comprising HLA-A*24 and HLA-A*02 epitopes can treat at
least 60% of patients in any relevant population. Specifically, the
following percentages of patients will be positive for at least one
of these alleles in various regions: USA 61%, Western Europe 62%,
China 75%, South Korea 77%, Japan 86% (calculated from
www.allelefrequencies.net).
[0128] In a preferred embodiment, the term "nucleotide sequence"
refers to a heteropolymer of deoxyribonucleotides.
[0129] The nucleotide sequence coding for a particular peptide,
oligopeptide, or polypeptide may be naturally occurring or they may
be synthetically constructed. Generally, DNA segments encoding the
peptides, polypeptides, and proteins of this invention are
assembled from cDNA fragments and short oligonucleotide linkers, or
from a series of oligonucleotides, to provide a synthetic gene that
is capable of being expressed in a recombinant transcriptional unit
comprising regulatory elements derived from a microbial or viral
operon.
[0130] As used herein the term "a nucleotide coding for (or
encoding) a peptide" refers to a nucleotide sequence coding for the
peptide including artificial (man-made) start and stop codons
compatible for the biological system the sequence is to be
expressed by, for example, a dendritic cell or another cell system
useful for the production of TCRs.
[0131] As used herein, reference to a nucleic acid sequence
includes both single stranded and double stranded nucleic acid.
Thus, for example for DNA, the specific sequence, unless the
context indicates otherwise, refers to the single strand DNA of
such sequence, the duplex of such sequence with its complement
(double stranded DNA) and the complement of such sequence.
[0132] 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.
[0133] The coding region can be derived from a non-mutated
("normal"), mutated or altered gene, or can even be derived from a
DNA sequence, or gene, wholly synthesized in the laboratory using
methods well known to those of skill in the art of DNA
synthesis.
[0134] The term "expression product" means the polypeptide or
protein that is the natural translation product of the gene and any
nucleic acid sequence coding equivalents resulting from genetic
code degeneracy and thus coding for the same amino acid(s).
[0135] The term "fragment", when referring to a coding sequence,
means a portion of DNA comprising less than the complete coding
region, whose expression product retains essentially the same
biological function or activity as the expression product of the
complete coding region.
[0136] The term "DNA segment" refers to a DNA polymer, in the form
of a separate fragment or as a component of a larger DNA construct,
which has been derived from DNA isolated at least once in
substantially pure form, i.e., free of contaminating endogenous
materials and in a quantity or concentration enabling
identification, manipulation, and recovery of the segment and its
component nucleotide sequences by standard biochemical methods, for
example, by using a cloning vector. Such segments are provided in
the form of an open reading frame uninterrupted by internal
non-translated sequences, or introns, which are typically present
in eukaryotic genes. Sequences of non-translated DNA may be present
downstream from the open reading frame, where the same do not
interfere with manipulation or expression of the coding
regions.
[0137] 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.
[0138] The term "promoter" means a region of DNA involved in
binding of RNA polymerase to initiate transcription.
[0139] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment, if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or polypeptide, separated
from some or all of the coexisting materials in the natural system,
is isolated. Such polynucleotides could be part of a vector and/or
such polynucleotides or polypeptides could be part of a
composition, and still be isolated in that such vector or
composition is not part of its natural environment.
[0140] The polynucleotides, and recombinant or immunogenic
polypeptides, disclosed in accordance with the present invention
may also be in "purified" form. The term "purified" does not
require absolute purity; rather, it is intended as a relative
definition, and can include preparations that are highly purified
or preparations that are only partially purified, as those terms
are understood by those of skill in the relevant art. For example,
individual clones isolated from a cDNA library have been
conventionally purified to electrophoretic homogeneity.
Purification of starting material or natural material to at least
one order of magnitude, preferably two or three orders, and more
preferably four or five orders of magnitude is expressly
contemplated. Furthermore, a claimed polypeptide which has a purity
of preferably 99.999%, or at least 99.99% or 99.9%; and even
desirably 99% by weight or greater is expressly encompassed.
[0141] The nucleic acids and polypeptide expression products
disclosed according to the present invention, as well as expression
vectors containing such nucleic acids and/or such polypeptides, may
be in "enriched form". As used herein, the term "enriched" means
that the concentration of the material is at least about 2, 5, 10,
100, or 1000 times its natural concentration (for example),
advantageously 0.01%, by weight, preferably at least about 0.1% by
weight. Enriched preparations of about 0.5%, 1%, 5%, 10%, and 20%
by weight are also contemplated. The sequences, constructs,
vectors, clones, and other materials comprising the present
invention can advantageously be in enriched or isolated form. The
term "active fragment" means a fragment, usually of a peptide,
polypeptide or nucleic acid sequence, that generates an immune
response (i.e., has immunogenic activity) when administered, alone
or optionally with a suitable adjuvant or in a vector, to an
animal, such as a mammal, for example, a rabbit or a mouse, and
also including a human, such immune response taking the form of
stimulating a T-cell response within the recipient animal, such as
a human. Alternatively, the "active fragment" may also be used to
induce a T-cell response in vitro.
[0142] As used herein, the terms "portion", "segment" and
"fragment", when used in relation to polypeptides, refer to a
continuous sequence of residues, such as amino acid residues, which
sequence forms a subset of a larger sequence. For example, if a
polypeptide were subjected to treatment with any of the common
endopeptidases, such as trypsin or chymotrypsin, the oligopeptides
resulting from such treatment would represent portions, segments or
fragments of the starting polypeptide. When used in relation to
polynucleotides, these terms refer to the products produced by
treatment of said polynucleotides with any of the
endonucleases.
[0143] In accordance with the present invention, the term "percent
identity" or "percent identical", when referring to a sequence,
means that a sequence is compared to a claimed or described
sequence after alignment of the sequence to be compared (the
"Compared Sequence") with the described or claimed sequence (the
"Reference Sequence"). The percent identity is then determined
according to the following formula:
percent identity=100[1-(C/R)]
wherein C is the number of differences between the Reference
Sequence and the Compared Sequence over the length of alignment
between the Reference Sequence and the Compared Sequence, wherein
(i) each base or amino acid in the Reference Sequence that does not
have a corresponding aligned base or amino acid in the Compared
Sequence and (ii) each gap in the Reference Sequence and (iii) each
aligned base or amino acid in the Reference Sequence that is
different from an aligned base or amino acid in the Compared
Sequence, constitutes a difference and (iiii) the alignment has to
start at position 1 of the aligned sequences; and R is the number
of bases or amino acids in the Reference Sequence over the length
of the alignment with the Compared Sequence with any gap created in
the Reference Sequence also being counted as a base or amino
acid.
[0144] If an alignment exists between the Compared Sequence and the
Reference Sequence for which the percent identity as calculated
above is about equal to or greater than a specified minimum Percent
Identity then the Compared Sequence has the specified minimum
percent identity to the Reference Sequence even though alignments
may exist in which the herein above calculated percent identity is
less than the specified percent identity.
[0145] As mentioned above, the present invention thus provides a
peptide comprising a sequence that is selected from the group of
consisting of SEQ ID NO: 1 to SEQ ID NO: 385 or a variant thereof
which is 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 385, or a
variant thereof that will induce T cells cross-reacting with said
peptide. The peptides of the invention have the ability to bind to
a molecule of the human major histocompatibility complex (MHC)
class-I or elongated versions of said peptides to class II.
[0146] In the present invention, the term "homologous" refers to
the degree of identity (see percent identity above) between
sequences of two amino acid sequences, i.e. peptide or polypeptide
sequences. The aforementioned "homology" is determined by comparing
two sequences aligned under optimal conditions over the sequences
to be compared. Such a sequence homology can be calculated by
creating an alignment using, for example, the ClustalW algorithm.
Commonly available sequence analysis software, more specifically,
Vector NTI, GENETYX or other tools are provided by public
databases.
[0147] A person skilled in the art will be able to assess, whether
T cells induced by a variant of a specific peptide will be able to
cross-react with the peptide itself (Appay et al., 2006; Colombetti
et al., 2006; Fong et al., 2001; Zaremba et al., 1997).
[0148] 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: 385. For example, a peptide may be
modified so that it at least maintains, if not improves, the
ability to interact with and bind to the binding groove of a
suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it
at least maintains, if not improves, the ability to bind to the TCR
of activated T cells.
[0149] These T cells can subsequently cross-react with cells and
kill cells that express a polypeptide that contains the natural
amino acid sequence of the cognate peptide as defined in the
aspects of the invention. As can be derived from the scientific
literature and databases (Rammensee et al., 1999; Godkin et al.,
1997), certain positions of HLA binding peptides are typically
anchor residues forming a core sequence fitting to the binding
motif of the HLA receptor, which is defined by polar,
electrophysical, hydrophobic and spatial properties of the
polypeptide chains constituting the binding groove. Thus, one
skilled in the art would be able to modify the amino acid sequences
set forth in SEQ ID NO: 1 to SEQ ID NO 385, 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.
[0150] The original (unmodified) peptides as disclosed herein can
be modified by the substitution of one or more residues at
different, possibly selective, sites within the peptide chain, if
not otherwise stated. Preferably those substitutions are located at
the end of the amino acid chain. Such substitutions may be of a
conservative nature, for example, where one amino acid is replaced
by an amino acid of similar structure and characteristics, such as
where a hydrophobic amino acid is replaced by another hydrophobic
amino acid. Even more conservative would be replacement of amino
acids of the same or similar size and chemical nature, such as
where leucine is replaced by isoleucine. In studies of sequence
variations in families of naturally occurring homologous proteins,
certain amino acid substitutions are more often tolerated than
others, and these are often show correlation with similarities in
size, charge, polarity, and hydrophobicity between the original
amino acid and its replacement, and such is the basis for defining
"conservative substitutions."
[0151] Conservative substitutions are herein defined as exchanges
within one of the following five groups: Group 1-small aliphatic,
nonpolar or slightly polar residues (Ala, Ser, Thr, Pro, Gly);
Group 2-polar, negatively charged residues and their amides (Asp,
Asn, Glu, Gln); Group 3-polar, positively charged residues (His,
Arg, Lys); Group 4-large, aliphatic, nonpolar residues (Met, Leu,
Ile, Val, Cys); and Group 5-large, aromatic residues (Phe, Tyr,
Trp).
[0152] Less conservative substitutions might involve the
replacement of one amino acid by another that has similar
characteristics but is somewhat different in size, such as
replacement of an alanine by an isoleucine residue. Highly
non-conservative replacements might involve substituting an acidic
amino acid for one that is polar, or even for one that is basic in
character. Such "radical" substitutions cannot, however, be
dismissed as potentially ineffective since chemical effects are not
totally predictable and radical substitutions might well give rise
to serendipitous effects not otherwise predictable from simple
chemical principles.
[0153] Of course, such substitutions may involve structures other
than the common L-amino acids. Thus, D-amino acids might be
substituted for the L-amino acids commonly found in the antigenic
peptides of the invention and yet still be encompassed by the
disclosure herein. In addition, non-standard amino acids (i.e.,
other than the common naturally occurring proteinogenic amino
acids) may also be used for substitution purposes to produce
immunogens and immunogenic polypeptides according to the present
invention.
[0154] If substitutions at more than one position are found to
result in a peptide with substantially equivalent or greater
antigenic activity as defined below, then combinations of those
substitutions will be tested to determine if the combined
substitutions result in additive or synergistic effects on the
antigenicity of the peptide. At most, no more than 4 positions
within the peptide would be simultaneously substituted.
[0155] A peptide consisting essentially of the amino acid sequence
as indicated herein can have one or two non-anchor amino acids (see
below regarding the anchor motif) exchanged without that the
ability to bind to a molecule of the human major histocompatibility
complex (MHC) class-I or -II is substantially changed or is
negatively affected, when compared to the non-modified peptide. In
another embodiment, in a peptide consisting essentially of the
amino acid sequence as indicated herein, one or two amino acids can
be exchanged with their conservative exchange partners (see herein
below) without that the ability to bind to a molecule of the human
major histocompatibility complex (MHC) class-I or -II is
substantially changed, or is negatively affected, when compared to
the non-modified peptide.
[0156] The amino acid residues that do not substantially contribute
to interactions with the T-cell receptor can be modified by
replacement with other amino acids whose incorporation does not
substantially affect T-cell reactivity and does not eliminate
binding to the relevant MHC. Thus, apart from the proviso given,
the peptide of the invention may be any peptide (by which term the
inventors include oligopeptide or polypeptide), which includes the
amino acid sequences or a portion or variant thereof as given.
TABLE-US-00007 TABLE 6 Variants and motif of the peptides according
to SEQ ID NO: 2, 5, 8, 62, and 53 Position 1 2 3 4 5 6 7 8 9 10 11
SEQ ID NO. 2 A L H R P D V Y L Variants V I A M M V M I M A A A V A
I A A V V V V I V A T T V T I T A Q Q V Q I Q A Position 1 2 3 4 5
6 7 8 9 10 11 SEQ ID NO 5 A L I F K I A S A Variants L I V M M L M
I M V A A L A I A V V V L V I V V T T L T I T V Q Q L Q I Q V
Position 1 2 3 4 5 6 7 8 9 10 11 SEQ ID NO. 8 A L A A S A L P A L V
Variants I L A M M I M L M A A A I A L A A V V I V L V A T T I T L
T A Q Q I Q L Q A Position 1 2 3 4 5 6 7 8 9 SEQ ID 62 G I I D G S
P R L Variants L V L I L L A M V M I M M A A V A I A A A V V V I V
V A T V T I T T A Q V Q I Q Q A Position 1 2 3 4 5 6 7 8 9 SEQ ID
153 S L L A E L H V L Variants V I A M V M I M M A A V A I A A A V
V V I V V A T V T I T T A Q V Q I Q Q A
Longer (elongated) peptides may also be suitable. It is possible
that MHC class I epitopes, although usually between 8 and 11 amino
acids long, are generated by peptide processing from longer
peptides or proteins that include the actual epitope. It is
preferred that the residues that flank the actual epitope are
residues that do not substantially affect proteolytic cleavage
necessary to expose the actual epitope during processing.
[0157] The peptides of the invention can be elongated by up to four
amino acids, that is 1, 2, 3 or 4 amino acids can be added to
either end in any combination between 4:0 and 0:4. Combinations of
the elongations according to the invention can be found in Table
7.
TABLE-US-00008 TABLE 7 Combinations of the elongations of peptides
of the invention C-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1
0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0
3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or
4
[0158] 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.
[0159] Thus, the epitopes of the present invention may be identical
to naturally occurring tumor-associated or tumor-specific epitopes
or may include epitopes that differ by no more than four residues
from the reference peptide, as long as they have substantially
identical antigenic activity.
[0160] In an alternative embodiment, the peptide is elongated on
either or both sides by more than 4 amino acids, preferably to a
total length of up to 30 amino acids. This may lead to MHC class II
binding peptides. Binding to MHC class II can be tested by methods
known in the art.
[0161] Accordingly, the present invention provides peptides and
variants of MHC class I epitopes, wherein the peptide or variant
has an overall length of between 8 and 100, preferably between 8
and 30, and most preferred between 8 and 14, namely 8, 9, 10, 11,
12, 13, 14 amino acids, in case of the elongated class II binding
peptides the length can also be 15, 16, 17, 18, 19, 20, 21 or 22
amino acids.
[0162] 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.
[0163] Preferably, when the T cells specific for a peptide
according to the present invention are tested against the
substituted peptides, the peptide concentration at which the
substituted peptides achieve half the maximal increase in lysis
relative to background is no more than about 1 mM, preferably no
more than about 1 .mu.M, more preferably no more than about 1 nM,
and still more preferably no more than about 100 pM, and most
preferably no more than about 10 pM. It is also preferred that the
substituted peptide be recognized by T cells from more than one
individual, at least two, and more preferably three
individuals.
[0164] 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: 385.
[0165] "Consisting essentially of" shall mean that a peptide
according to the present invention, in addition to the sequence
according to any of SEQ ID NO: 1 to SEQ ID NO 385 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.
[0166] Nevertheless, these stretches can be important to provide an
efficient introduction of the peptide according to the present
invention into the cells. In one embodiment of the present
invention, the peptide is part of a fusion protein which comprises,
for example, the 80 N-terminal amino acids of the HLA-DR
antigen-associated invariant chain (p33, in the following "Ii") as
derived from the NCBI, GenBank Accession number X00497. In other
fusions, the peptides of the present invention can be fused to an
antibody as described herein, or a functional part thereof, in
particular into a sequence of an antibody, so as to be specifically
targeted by said antibody, or, for example, to or into an antibody
that is specific for dendritic cells as described herein.
[0167] In addition, the peptide or variant may be modified further
to improve stability and/or binding to MHC molecules in order to
elicit a stronger immune response. Methods for such an optimization
of a peptide sequence are well known in the art and include, for
example, the introduction of reverse peptide bonds or non-peptide
bonds.
[0168] In a reverse peptide bond amino acid residues are not joined
by peptide (--CO--NH--) linkages but the peptide bond is reversed.
Such retro-inverso peptidomimetics may be made using methods known
in the art, for example such as those described in Meziere et al
(1997) (Meziere et al., 1997), incorporated herein by reference.
This approach involves making pseudopeptides containing changes
involving the backbone, and not the orientation of side chains.
Meziere et al. (Meziere et al., 1997) show that for MHC binding and
T helper cell responses, these pseudopeptides are useful.
Retro-inverse peptides, which contain NH--CO bonds instead of
CO--NH peptide bonds, are much more resistant to proteolysis.
[0169] A non-peptide bond is, for example, --CH.sub.2--NH,
--CH.sub.2S--, --CH.sub.2CH.sub.2--, --CH.dbd.CH--, --COCH.sub.2--,
--CH(OH)CH.sub.2--, and --CH.sub.2SO--. U.S. Pat. No. 4,897,445
provides a method for the solid phase synthesis of non-peptide
bonds (--CH.sub.2--NH) in polypeptide chains which involves
polypeptides synthesized by standard procedures and the non-peptide
bond synthesized by reacting an amino aldehyde and an amino acid in
the presence of NaCNBH.sub.3.
[0170] Peptides comprising the sequences described above may be
synthesized with additional chemical groups present at their amino
and/or carboxy termini, to enhance the stability, bioavailability,
and/or affinity of the peptides. For example, hydrophobic groups
such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl groups may be
added to the peptides' amino termini. Likewise, an acetyl group or
a 9-fluorenylmethoxy-carbonyl group may be placed at the peptides'
amino termini. Additionally, the hydrophobic group,
t-butyloxycarbonyl, or an amido group may be added to the peptides'
carboxy termini.
[0171] Further, the peptides of the invention may be synthesized to
alter their steric configuration. For example, the D-isomer of one
or more of the amino acid residues of the peptide may be used,
rather than the usual L-isomer. Still further, at least one of the
amino acid residues of the peptides of the invention may be
substituted by one of the well-known non-naturally occurring amino
acid residues. Alterations such as these may serve to increase the
stability, bioavailability and/or binding action of the peptides of
the invention.
[0172] Similarly, a peptide or variant of the invention may be
modified chemically by reacting specific amino acids either before
or after synthesis of the peptide. Examples for such modifications
are well known in the art and are summarized e.g. in R. Lundblad,
Chemical Reagents for Protein Modification, 3rd ed. CRC Press, 2004
(Lundblad, 2004), which is incorporated herein by reference.
Chemical modification of amino acids includes but is not limited
to, modification by acylation, amidination, pyridoxylation of
lysine, reductive alkylation, trinitrobenzylation of amino groups
with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amide
modification of carboxyl groups and sulphydryl modification by
performic acid oxidation of cysteine to cysteic acid, formation of
mercurial derivatives, formation of mixed disulphides with other
thiol compounds, reaction with maleimide, carboxymethylation with
iodoacetic acid or iodoacetamide and carbamoylation with cyanate at
alkaline pH, although without limitation thereto. In this regard,
the skilled person is referred to Chapter 15 of Current Protocols
In Protein Science, Eds. Coligan et al. (John Wiley and Sons NY
1995-2000) (Coligan et al., 1995) for more extensive methodology
relating to chemical modification of proteins.
[0173] Briefly, modification of e.g. arginyl residues in proteins
is often based on the reaction of vicinal dicarbonyl compounds such
as phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form
an adduct. Another example is the reaction of methylglyoxal with
arginine residues. Cysteine can be modified without concomitant
modification of other nucleophilic sites such as lysine and
histidine. As a result, a large number of reagents are available
for the modification of cysteine. The websites of companies such as
Sigma-Aldrich (http://www.sigma-aldrich.com) provide information on
specific reagents.
[0174] Selective reduction of disulfide bonds in proteins is also
common. Disulfide bonds can be formed and oxidized during the heat
treatment of biopharmaceuticals. Woodward's Reagent K may be used
to modify specific glutamic acid residues.
N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide can be used to
form intra-molecular crosslinks between a lysine residue and a
glutamic acid residue. For example, diethylpyrocarbonate is a
reagent for the modification of histidyl residues in proteins.
Histidine can also be modified using 4-hydroxy-2-nonenal. The
reaction of lysine residues and other .alpha.-amino groups is, for
example, useful in binding of peptides to surfaces or the
cross-linking of proteins/peptides. Lysine is the site of
attachment of poly(ethylene)glycol and the major site of
modification in the glycosylation of proteins. Methionine residues
in proteins can be modified with e.g. iodoacetamide,
bromoethylamine, and chloramine T.
[0175] Tetranitromethane and N-acetylimidazole can be used for the
modification of tyrosyl residues. Cross-linking via the formation
of dityrosine can be accomplished with hydrogen peroxide/copper
ions.
[0176] Recent studies on the modification of tryptophan have used
N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or
3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole
(BPNS-skatole).
[0177] Successful modification of therapeutic proteins and peptides
with PEG is often associated with an extension of circulatory
half-life while cross-linking of proteins with glutaraldehyde,
polyethylene glycol diacrylate and formaldehyde is used for the
preparation of hydrogels. Chemical modification of allergens for
immunotherapy is often achieved by carbamylation with potassium
cyanate.
[0178] A peptide or variant, wherein the peptide is modified or
includes non-peptide bonds is a preferred embodiment of the
invention.
[0179] Another embodiment of the present invention relates to a
non-naturally occurring peptide wherein said peptide consists or
consists essentially of an amino acid sequence according to SEQ ID
No: 1 to SEQ ID No: 385 and has been synthetically produced (e.g.
synthesized) as a pharmaceutically acceptable salt. Methods to
synthetically produce peptides are well known in the art. The salts
of the peptides according to the present invention differ
substantially from the peptides in their state(s) in vivo, as the
peptides as generated in vivo are no salts. The non-natural salt
form of the peptide mediates the solubility of the peptide, in
particular in the context of pharmaceutical compositions comprising
the peptides, e.g. the peptide vaccines as disclosed herein. A
sufficient and at least substantial solubility of the peptide(s) is
required in order to efficiently provide the peptides to the
subject to be treated. Preferably, the salts are pharmaceutically
acceptable salts of the peptides. These salts according to the
invention include alkaline and earth alkaline salts such as salts
of the Hofmeister series comprising as anions PO.sub.4.sup.3-,
SO.sub.4.sup.2-, CH.sub.3COO.sup.-, Cl.sup.-, Br, NO.sub.3.sup.-,
ClO.sub.4.sup.-, I.sup.-, SCN.sup.- and as cations NH.sub.4.sup.+,
Rb.sup.+, K.sup.+, Na.sup.+, Cs.sup.+, Li.sup.+, Zn.sup.2+,
Mg.sup.2+, Ca.sup.2+, Mn.sup.2+, Cu.sup.2+ and Ba.sup.2+.
Particularly salts are selected from (NH.sub.4).sub.3PO.sub.4,
(NH.sub.4).sub.2HPO.sub.4, (NH.sub.4)H.sub.2PO.sub.4,
(NH.sub.4).sub.2SO.sub.4, NH.sub.4CH.sub.3COO, NH.sub.4Cl,
NH.sub.4Br, NH.sub.4NO.sub.3, NH.sub.4ClO.sub.4, NH.sub.4I,
NH.sub.4SCN, Rb.sub.3PO.sub.4, Rb.sub.2HPO.sub.4,
RbH.sub.2PO.sub.4, Rb.sub.2SO.sub.4, Rb.sub.4CH.sub.3COO,
Rb.sub.4Cl, Rb.sub.4Br, Rb.sub.4NO.sub.3, Rb.sub.4ClO.sub.4,
Rb.sub.4I, Rb.sub.4SCN, K.sub.3PO.sub.4, K.sub.2HPO.sub.4,
KH.sub.2PO.sub.4, K.sub.2SO.sub.4, KCH.sub.3COO, KCl, KBr, KNOB,
KClO.sub.4, KI, KSCN, Na.sub.3PO.sub.4, Na.sub.2HPO.sub.4,
NaH.sub.2PO.sub.4, Na.sub.2SO.sub.4, NaCH.sub.3COO, NaCl, NaBr,
NaNO.sub.3, NaClO.sub.4, Nal, NaSCN, ZnCl.sub.2 Cs.sub.3PO.sub.4,
Cs.sub.2HPO.sub.4, CsH.sub.2PO.sub.4, Cs.sub.2SO.sub.4,
CsCH.sub.3COO, CsCl, CsBr, CsNO.sub.3, CsClO.sub.4, Csl, CsSCN,
Li.sub.3PO.sub.4, Li.sub.2HPO.sub.4, LiH.sub.2PO.sub.4,
Li.sub.2SO.sub.4, LiCH.sub.3COO, LiCl, LiBr, LiNO.sub.3,
LiClO.sub.4, Lil, LiSCN, Cu.sub.2SO.sub.4,
Mg.sub.3(PO.sub.4).sub.2, Mg.sub.2HPO.sub.4,
Mg(H.sub.2PO.sub.4).sub.2, Mg.sub.2SO.sub.4, Mg(CH.sub.3COO).sub.2,
MgCl.sub.2, MgBr.sub.2, Mg(NO.sub.3).sub.2, Mg(ClO.sub.4).sub.2,
Mgl.sub.2, Mg(SCN).sub.2, MnCl.sub.2, Ca.sub.3(PO.sub.4),
Ca.sub.2HPO.sub.4, Ca(H.sub.2PO.sub.4).sub.2, CaSO.sub.4,
Ca(CH.sub.3COO).sub.2, CaCl.sub.2), CaBr.sub.2, Ca(NO.sub.3).sub.2,
Ca(ClO.sub.4).sub.2, Cale, Ca(SCN).sub.2, Ba.sub.3(PO.sub.4).sub.2,
Ba.sub.2HPO.sub.4, Ba(H.sub.2PO.sub.4).sub.2, BaSO.sub.4,
Ba(CH.sub.3COO).sub.2, BaCl.sub.2, BaBr.sub.2, Ba(NO.sub.3).sub.2,
Ba(ClO.sub.4).sub.2, Bale, and Ba(SCN).sub.2. Particularly
preferred are NH acetate, MgCl.sub.2, KH.sub.2PO.sub.4,
Na.sub.2SO.sub.4, KCl, NaCl, and CaCl.sub.2), such as, for example,
the chloride or acetate (trifluoroacetate) salts.
[0180] Generally, peptides and variants (at least those containing
peptide linkages between amino acid residues) may be synthesized by
the Fmoc-polyamide mode of solid-phase peptide synthesis as
disclosed by Lukas et al. (Lukas et al., 1981) and by references as
cited therein. Temporary N-amino group protection is afforded by
the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage
of this highly base-labile protecting group is done using 20%
piperidine in N, N-dimethylformamide. Side-chain functionalities
may be protected as their butyl ethers (in the case of serine
threonine and tyrosine), butyl esters (in the case of glutamic acid
and aspartic acid), butyloxycarbonyl derivative (in the case of
lysine and histidine), trityl derivative (in the case of cysteine)
and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the
case of arginine). Where glutamine or asparagine are C-terminal
residues, use is made of the 4,4'-dimethoxybenzhydryl group for
protection of the side chain amido functionalities. The solid-phase
support is based on a polydimethyl-acrylamide polymer constituted
from the three monomers dimethylacrylamide (backbone-monomer),
bisacryloylethylene diamine (cross linker) and acryloylsarcosine
methyl ester (functionalizing agent). The peptide-to-resin
cleavable linked agent used is the acid-labile
4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid
derivatives are added as their preformed symmetrical anhydride
derivatives with the exception of asparagine and glutamine, which
are added using a reversed N, N-dicyclohexyl-carbodiimide/1
hydroxybenzotriazole mediated coupling procedure. All coupling and
deprotection reactions are monitored using ninhydrin,
trinitrobenzene sulphonic acid or isotin test procedures. Upon
completion of synthesis, peptides are cleaved from the resin
support with concomitant removal of side-chain protecting groups by
treatment with 95% trifluoroacetic acid containing a 50% scavenger
mix. Scavengers commonly used include ethanedithiol, phenol,
anisole and water, the exact choice depending on the constituent
amino acids of the peptide being synthesized. Also a combination of
solid phase and solution phase methodologies for the synthesis of
peptides is possible (see, for example, (Bruckdorfer et al., 2004),
and the references as cited therein).
[0181] Trifluoroacetic acid is removed by evaporation in vacuo,
with subsequent trituration with diethyl ether affording the crude
peptide. Any scavengers present are removed by a simple extraction
procedure which on lyophilization of the aqueous phase affords the
crude peptide free of scavengers. Reagents for peptide synthesis
are generally available from e.g. Calbiochem-Novabiochem
(Nottingham, UK).
[0182] Purification may be performed by any one, or a combination
of, techniques such as re-crystallization, size exclusion
chromatography, ion-exchange chromatography, hydrophobic
interaction chromatography and (usually) reverse-phase high
performance liquid chromatography using e.g. acetonitrile/water
gradient separation.
[0183] Analysis of peptides may be carried out using thin layer
chromatography, electrophoresis, in particular capillary
electrophoresis, solid phase extraction (CSPE), reverse-phase high
performance liquid chromatography, amino-acid analysis after acid
hydrolysis and by fast atom bombardment (FAB) mass spectrometric
analysis, as well as MALDI and ESI-Q-TOF mass spectrometric
analysis.
[0184] In order to select over-presented peptides, a presentation
profile is calculated showing the median sample presentation as
well as replicate variation. The profile juxtaposes samples of the
tumor entity of interest to a baseline of normal tissue samples.
Each of these profiles can then be consolidated into an
over-presentation score by calculating the p-value of a Linear
Mixed-Effects Model (Pinheiro et al., 2015) adjusting for multiple
testing by False Discovery Rate (Benjamini and Hochberg, 1995) (cf.
Example 1, FIGS. 1A-1V).
[0185] For the identification and relative quantitation of HLA
ligands by mass spectrometry, HLA molecules from shock-frozen
tissue samples were purified and HLA-associated peptides were
isolated. The isolated peptides were separated and sequences were
identified by online nano-electrospray-ionization (nanoESl) liquid
chromatography-mass spectrometry (LC-MS) experiments. The resulting
peptide sequences were verified by comparison of the fragmentation
pattern of natural tumor-associated peptides (TUMAPs) recorded from
CLL samples (N=17 A*02-positive samples) with the fragmentation
patterns of corresponding synthetic reference peptides of identical
sequences. Since the peptides were directly identified as ligands
of HLA molecules of primary tumors, these results provide direct
evidence for the natural processing and presentation of the
identified peptides on primary cancer tissue obtained from 16 CLL
patients.
[0186] The discovery pipeline XPRESIDENT.RTM. v2.1 (see, for
example, US 2013-0096016, which is hereby incorporated by reference
in its entirety) allows the identification and selection of
relevant over-presented peptide vaccine candidates based on direct
relative quantitation of HLA-restricted peptide levels on cancer
tissues in comparison to several different non-cancerous tissues
and organs. This was achieved by the development of label-free
differential quantitation using the acquired LC-MS data processed
by a proprietary data analysis pipeline, combining algorithms for
sequence identification, spectral clustering, ion counting,
retention time alignment, charge state deconvolution and
normalization.
[0187] Presentation levels including error estimates for each
peptide and sample were established. Peptides exclusively presented
on tumor tissue and peptides over-presented in tumor versus
non-cancerous tissues and organs have been identified.
[0188] HLA-peptide complexes from CLL 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 CLL samples
confirming their presentation on primary CLL.
[0189] TUMAPs identified on multiple CLL and normal tissues were
quantified using ion-counting of label-free LC-MS data. The method
assumes that LC-MS signal areas of a peptide correlate with its
abundance in the sample. All quantitative signals of a peptide in
various LC-MS experiments were normalized based on central
tendency, averaged per sample and merged into a bar plot, called
presentation profile. The presentation profile consolidates
different analysis methods like protein database search, spectral
clustering, charge state deconvolution (decharging) and retention
time alignment and normalization.
[0190] Furthermore, the discovery platform Technique
XPRESIDENT.RTM. v2.x (see for example PCT/EP2011/056056 and
PCT/EP2015/079873) allows the direct absolute quantitation of MHC-,
preferably HLA-restricted, peptide levels on cancer or other
infected tissues. Briefly, the total cell count was calculated from
the total DNA content of the analyzed tissue sample. The total
peptide amount for a TUMAP in a tissue sample was measured by
nanoLC-MS/MS as the ratio of the natural TUMAP and a known amount
of an isotope-labelled version of the TUMAP, the so-called internal
standard. The efficiency of TUMAP isolation was determined by
spiking peptide:MHC complexes of all selected TUMAPs into the
tissue lysate at the earliest possible point of the TUMAP isolation
procedure and their detection by nanoLC-MS/MS following completion
of the peptide isolation procedure. The total cell count and the
amount of total peptide were calculated from triplicate
measurements per tissue sample. The peptide-specific isolation
efficiencies were calculated as an average from 10 spike
experiments each measured as a triplicate (see Example 6 and Table
12).
[0191] 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, FIGS.
3A-3F). 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 tissue,
but very low or absent in vital normal tissues, were preferably
included in the present invention.
[0192] The present invention provides peptides that are useful in
treating cancers/tumors, preferably CLL that over- or exclusively
present the peptides of the invention. These peptides were shown by
mass spectrometry to be naturally presented by HLA molecules on
primary human CLL samples.
[0193] Many of the source gene/proteins (also designated
"full-length proteins" or "underlying proteins") from which the
peptides are derived were shown to be highly over-expressed in
cancer compared with normal tissues--"normal tissues" in relation
to this invention shall mean either healthy adipose tissue, adrenal
gland, blood cells, blood vessel, bone marrow, brain, breast,
esophagus, eye, gallbladder, heart, kidney, large intestine, liver,
lung, lymph node, nerve, pancreas, parathyroid gland, peritoneum,
pituitary, pleura, salivary gland, skeletal muscle, skin, small
intestine, spleen, stomach, thymus, thyroid gland, trachea, ureter,
and urinary bladder 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
CLL, but not on normal tissues (see Example 1).
[0194] 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. CLL cells
presenting the derived peptides.
[0195] The peptides of the present invention have been shown to be
capable of stimulating T cell responses and/or are over-presented
and thus can be used for the production of antibodies and/or TCRs,
such as soluble TCRs, according to the present invention (see
Example 3, Example 4). Furthermore, the peptides when complexed
with the respective MHC can be used for the production of
antibodies and/or TCRs, in particular sTCRs, according to the
present invention, as well. Respective methods are well known to
the person of skill, and can be found in the respective literature
as well. Thus, the peptides of the present invention are useful for
generating an immune response in a patient by which tumor cells can
be destroyed. An immune response in a patient can be induced by
direct administration of the described peptides or suitable
precursor substances (e.g. elongated peptides, proteins, or nucleic
acids encoding these peptides) to the patient, ideally in
combination with an agent enhancing the immunogenicity (i.e. an
adjuvant). The immune response originating from such a therapeutic
vaccination can be expected to be highly specific against tumor
cells because the target peptides of the present invention are not
presented on normal tissues in comparable copy numbers, preventing
the risk of undesired autoimmune reactions against normal cells in
the patient.
[0196] The present description further relates to T-cell receptors
(TCRs) comprising an alpha chain and a beta chain ("alpha/beta
TCRs"). Also provided are peptides according to the invention
capable of binding to TCRs and antibodies when presented by an MHC
molecule. The present description also relates to nucleic acids,
vectors and host cells for expressing TCRs and peptides of the
present description; and methods of using the same.
[0197] The term "T-cell receptor" (abbreviated TCR) refers to a
heterodimeric molecule comprising an alpha polypeptide chain (alpha
chain) and a beta polypeptide chain (beta chain), wherein the
heterodimeric receptor is capable of binding to a peptide antigen
presented by an HLA molecule. The term also includes so-called
gamma/delta TCRs.
[0198] 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.
[0199] The description in another aspect relates to methods
according to the description, wherein the antigen is loaded onto
class I or II MHC molecules expressed on the surface of a suitable
antigen-presenting cell or artificial antigen-presenting cell by
contacting a sufficient amount of the antigen with an
antigen-presenting cell or the antigen is loaded onto class I or II
MHC tetramers by tetramerizing the antigen/class I or II MHC
complex monomers.
[0200] The alpha and beta chains of alpha/beta TCR's, and the gamma
and delta chains of gamma/delta TCRs, are generally regarded as
each having two "domains", namely variable and constant domains.
The variable domain consists of a concatenation of variable region
(V), and joining region (J). The variable domain may also include a
leader region (L). Beta and delta chains may also include a
diversity region (D). The alpha and beta constant domains may also
include C-terminal transmembrane (TM) domains that anchor the alpha
and beta chains to the cell membrane.
[0201] With respect to gamma/delta TCRs, the term "TCR gamma
variable domain" as used herein refers to the concatenation of the
TCR gamma V (TRGV) region without leader region (L), and the TCR
gamma J (TRGJ) region, and the term TCR gamma constant domain
refers to the extracellular TRGC region, or to a C-terminal
truncated TRGC sequence. Likewise the term "TCR delta variable
domain" refers to the concatenation of the TCR delta V (TRDV)
region without leader region (L) and the TCR delta D/J (TRDD/TRDJ)
region, and the term "TCR delta constant domain" refers to the
extracellular TRDC region, or to a C-terminal truncated TRDC
sequence.
[0202] TCRs of the present description preferably bind to an
peptide-HLA molecule complex with a binding affinity (KD) of about
100 .mu.M or less, about 50 .mu.M or less, about 25 .mu.M or less,
or about 10 .mu.M or less. More preferred are high affinity TCRs
having binding affinities of about 1 .mu.M or less, about 100 nM or
less, about 50 nM or less, about 25 nM or less. Non-limiting
examples of preferred binding affinity ranges for TCRs of the
present invention include about 1 nM to about 10 nM; about 10 nM to
about 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40
nM; about 40 nM to about 50 nM; about 50 nM to about 60 nM; about
60 nM to about 70 nM; about 70 nM to about 80 nM; about 80 nM to
about 90 nM; and about 90 nM to about 100 nM.
[0203] As used herein in connect with TCRs of the present
description, "specific binding" and grammatical variants thereof
are used to mean a TCR having a binding affinity (KD) for a
peptide-HLA molecule complex of 100 .mu.M or less.
[0204] Alpha/beta heterodimeric TCRs of the present description may
have an introduced disulfide bond between their constant domains.
Preferred TCRs of this type include those which have a TRAC
constant domain sequence and a TRBC1 or TRBC2 constant domain
sequence except that Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2
are replaced by cysteine residues, the said cysteines forming a
disulfide bond between the TRAC constant domain sequence and the
TRBC1 or TRBC2 constant domain sequence of the TCR.
[0205] With or without the introduced inter-chain bond mentioned
above, alpha/beta heterodimeric TCRs of the present description may
have a TRAC constant domain sequence and a TRBC1 or TRBC2 constant
domain sequence, and the TRAC constant domain sequence and the
TRBC1 or TRBC2 constant domain sequence of the TCR may be linked by
the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2
of exon 2 of TRBC1 or TRBC2.
[0206] 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.
[0207] In an embodiment, a TCR of the present description having at
least one mutation in the alpha chain and/or having at least one
mutation in the beta chain has modified glycosylation compared to
the unmutated TCR.
[0208] In an embodiment, a TCR comprising at least one mutation in
the TCR alpha chain and/or TCR beta chain has a binding affinity
for, and/or a binding half-life for, a peptide-HLA molecule
complex, which is at least double that of a TCR comprising the
unmutated TCR alpha chain and/or unmutated TCR beta chain.
Affinity-enhancement of tumor-specific TCRs, and its exploitation,
relies on the existence of a window for optimal TCR affinities. The
existence of such a window is based on observations that TCRs
specific for HLA-A2-restricted pathogens have KD values that are
generally about 10-fold lower when compared to TCRs specific for
HLA-A2-restricted tumor-associated self-antigens. It is now known,
although tumor antigens have the potential to be immunogenic,
because tumors arise from the individual's own cells only mutated
proteins or proteins with altered translational processing will be
seen as foreign by the immune system. Antigens that are upregulated
or overexpressed (so called self-antigens) will not necessarily
induce a functional immune response against the tumor: T-cells
expressing TCRs that are highly reactive to these antigens will
have been negatively selected within the thymus in a process known
as central tolerance, meaning that only T-cells with low-affinity
TCRs for self-antigens remain. Therefore, affinity of TCRs or
variants of the present description to pepides can be enhanced by
methods well known in the art.
[0209] The present description further relates to a method of
identifying and isolating a TCR according to the present
description, said method comprising incubating PBMCs from
HLA-A*02-negative healthy donors with A2/peptide monomers,
incubating the PBMCs with tetramer-phycoerythrin (PE) and isolating
the high avidity T-cells by fluorescence activated cell sorting
(FACS)-Calibur analysis.
[0210] The present description further relates to a method of
identifying and isolating a TCR according to the present
description, said method comprising obtaining a transgenic mouse
with the entire human TCRaI3 gene loci (1.1 and 0.7 Mb), whose
T-cells express a diverse human TCR repertoire that compensates for
mouse TCR deficiency, immunizing the mouse with a peptide,
incubating PBMCs obtained from the transgenic mice with
tetramer-phycoerythrin (PE), and isolating the high avidity T-cells
by fluorescence activated cell sorting (FACS)-Calibur analysis.
[0211] In one aspect, to obtain T-cells expressing TCRs of the
present description, nucleic acids encoding TCR-alpha and/or
TCR-beta chains of the present description are cloned into
expression vectors, such as gamma retrovirus or lentivirus. The
recombinant viruses are generated and then tested for
functionality, such as antigen specificity and functional avidity.
An aliquot of the final product is then used to transduce the
target T-cell population (generally purified from patient PBMCs),
which is expanded before infusion into the patient.
[0212] In another aspect, to obtain T-cells expressing TCRs of the
present description, TCR RNAs are synthesized by techniques known
in the art, e.g., in vitro transcription systems. The in
vitro-synthesized TCR RNAs are then introduced into primary CD8+
T-cells obtained from healthy donors by electroporation to
re-express tumor specific TCR-alpha and/or TCR-beta chains.
[0213] To increase the expression, nucleic acids encoding TCRs of
the present description may be operably linked to strong promoters,
such as retroviral long terminal repeats (LTRs), cytomegalovirus
(CMV), murine stem cell virus (MSCV) U3, phosphoglycerate kinase
(PGK), .beta.-actin, ubiquitin, and a simian virus 40 (SV40)/CD43
composite promoter, elongation factor (EF)-1 a and the spleen
focus-forming virus (SFFV) promoter. In a preferred embodiment, the
promoter is heterologous to the nucleic acid being expressed.
[0214] In addition to strong promoters, TCR expression cassettes of
the present description may contain additional elements that can
enhance transgene expression, including a central polypurine tract
(cPPT), which promotes the nuclear translocation of lentiviral
constructs (Follenzi et al., 2000), and the woodchuck hepatitis
virus posttranscriptional regulatory element (wPRE), which
increases the level of transgene expression by increasing RNA
stability (Zufferey et al., 1999).
[0215] The alpha and beta chains of a TCR of the present invention
may be encoded by nucleic acids located in separate vectors, or may
be encoded by polynucleotides located in the same vector.
[0216] Achieving high-level TCR surface expression requires that
both the TCR-alpha and TCR-beta chains of the introduced TCR be
transcribed at high levels. To do so, the TCR-alpha and TCR-beta
chains of the present description may be cloned into bi-cistronic
constructs in a single vector, which has been shown to be capable
of over-coming this obstacle. The use of a viral intraribosomal
entry site (IRES) between the TCR-alpha and TCR-beta chains results
in the coordinated expression of both chains, because the TCR-alpha
and TCR-beta chains are generated from a single transcript that is
broken into two proteins during translation, ensuring that an equal
molar ratio of TCR-alpha and TCR-beta chains are produced. (Schmitt
et al. 2009).
[0217] Nucleic acids encoding TCRs of the present description may
be codon optimized to increase expression from a host cell.
Redundancy in the genetic code allows some amino acids to be
encoded by more than one codon, but certain codons are less
"op-timal" than others because of the relative availability of
matching tRNAs as well as other factors (Gustafsson et al., 2004).
Modifying the TCR-alpha and TCR-beta gene sequences such that each
amino acid is encoded by the optimal codon for mammalian gene
expression, as well as eliminating mRNA instability motifs or
cryptic splice sites, has been shown to significantly enhance
TCR-alpha and TCR-beta gene expression (Scholten et al., 2006).
[0218] Furthermore, mispairing between the introduced and
endogenous TCR chains may result in the acquisition of
specificities that pose a significant risk for autoimmunity. For
example, the formation of mixed TCR dimers may reduce the number of
CD3 molecules available to form properly paired TCR complexes, and
therefore can significantly decrease the functional avidity of the
cells expressing the introduced TCR (Kuball et al., 2007).
[0219] To reduce mispairing, the C-terminus domain of the
introduced TCR chains of the present description may be modified in
order to promote interchain affinity, while de-creasing the ability
of the introduced chains to pair with the endogenous TCR. These
strategies may include replacing the human TCR-alpha and TCR-beta
C-terminus domains with their murine counterparts (murinized
C-terminus domain); generating a second interchain disulfide bond
in the C-terminus domain by introducing a second cysteine residue
into both the TCR-alpha and TCR-beta chains of the introduced TCR
(cysteine modification); swapping interacting residues in the
TCR-alpha and TCR-beta chain C-terminus domains ("knob-in-hole");
and fusing the variable domains of the TCR-alpha and TCR-beta
chains directly to CD3.zeta. (CD3.zeta. fusion). (Schmitt et al.
2009).
[0220] In an embodiment, a host cell is engineered to express a TCR
of the present description. In preferred embodiments, the host cell
is a human T-cell or T-cell progenitor. In some embodiments the
T-cell or T-cell progenitor is obtained from a cancer patient. In
other embodiments the T-cell or T-cell progenitor is obtained from
a healthy donor. Host cells of the present description can be
allogeneic or autologous with respect to a patient to be treated.
In one embodiment, the host is a gamma/delta T-cell transformed to
express an alpha/beta TCR.
[0221] A "pharmaceutical composition" is a composition suitable for
administration to a human being in a medical setting. Preferably, a
pharmaceutical composition is sterile and produced according to GMP
guidelines.
[0222] The pharmaceutical compositions comprise the peptides either
in the free form or in the form of a pharmaceutically acceptable
salt (see also above). As used herein, "a pharmaceutically
acceptable salt" refers to a derivative of the disclosed peptides
wherein the peptide is modified by making acid or base salts of the
agent. For example, acid salts are prepared from the free base
(typically wherein the neutral form of the drug has a neutral
--NH.sub.2 group) involving reaction with a suitable acid. Suitable
acids for preparing acid salts include both organic acids, e.g.,
acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic
acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric
acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,
mandelic acid, methane sulfonic acid, ethane sulfonic acid,
p-toluenesulfonic acid, salicylic acid, and the like, as well as
inorganic acids, e.g., hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid phosphoric acid and the like.
Conversely, preparation of basic salts of acid moieties which may
be present on a peptide are prepared using a pharmaceutically
acceptable base such as sodium hydroxide, potassium hydroxide,
ammonium hydroxide, calcium hydroxide, trimethylamine or the
like.
[0223] In an especially preferred embodiment, the pharmaceutical
compositions comprise the peptides as salts of acetic acid
(acetates), trifluoro acetates or hydrochloric acid
(chlorides).
[0224] Preferably, the medicament of the present invention is an
immunotherapeutic such as a vaccine. It may be administered
directly into the patient, into the affected organ or systemically
i.d., i.m., s.c., i.p. and i.v., or applied ex vivo to cells
derived from the patient or a human cell line which are
subsequently administered to the patient, or used in vitro to
select a subpopulation of immune cells derived from the patient,
which are then re-administered to the patient. If the nucleic acid
is administered to cells in vitro, it may be useful for the cells
to be transfected so as to co-express immune-stimulating cytokines,
such as interleukin-2. The peptide may be substantially pure, or
combined with an immune-stimulating adjuvant (see below) or used in
combination with immune-stimulatory cytokines, or be administered
with a suitable delivery system, for example liposomes. The peptide
may also be conjugated to a suitable carrier such as keyhole limpet
haemocyanin (KLH) or mannan (see WO 95/18145 and (Longenecker et
al., 1993)). The peptide may also be tagged, may be a fusion
protein, or may be a hybrid molecule. The peptides whose sequence
is given in the present invention are expected to stimulate CD4 or
CD8 T cells. However, stimulation of CD8 T cells is more efficient
in the presence of help provided by CD4 T-helper cells. Thus, for
MHC Class I epitopes that stimulate CD8 T cells the fusion partner
or sections of a hybrid molecule suitably provide epitopes which
stimulate CD4-positive T cells. CD4- and CD8-stimulating epitopes
are well known in the art and include those identified in the
present invention.
[0225] 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.
385, and at least one additional peptide, preferably two to 50,
more preferably two to 25, even more preferably two to 20 and most
preferably two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or
eighteen peptides. The peptide(s) may be derived from one or more
specific TAAs and may bind to MHC class I molecules.
[0226] A further aspect of the invention provides a nucleic acid
(for example a polynucleotide) encoding a peptide or peptide
variant of the invention. The polynucleotide may be, for example,
DNA, cDNA, PNA, RNA or combinations thereof, either single- and/or
double-stranded, or native or stabilized forms of polynucleotides,
such as, for example, polynucleotides with a phosphorothioate
backbone and it may or may not contain introns so long as it codes
for the peptide. Of course, only peptides that contain naturally
occurring amino acid residues joined by naturally occurring peptide
bonds are encodable by a polynucleotide. A still further aspect of
the invention provides an expression vector capable of expressing a
polypeptide according to the invention.
[0227] A variety of methods have been developed to link
polynucleotides, especially DNA, to vectors for example via
complementary cohesive termini. For instance, complementary
homopolymer tracts can be added to the DNA segment to be inserted
to the vector DNA. The vector and DNA segment are then joined by
hydrogen bonding between the complementary homopolymeric tails to
form recombinant DNA molecules.
[0228] Synthetic linkers containing one or more restriction sites
provide an alternative method of joining the DNA segment to
vectors. Synthetic linkers containing a variety of restriction
endonuclease sites are commercially available from a number of
sources including International Biotechnologies Inc. New Haven,
Conn., USA.
[0229] A desirable method of modifying the DNA encoding the
polypeptide of the invention employs the polymerase chain reaction
as disclosed by Saiki R K, et al. (Saiki et al., 1988). This method
may be used for introducing the DNA into a suitable vector, for
example by engineering in suitable restriction sites, or it may be
used to modify the DNA in other useful ways as is known in the art.
If viral vectors are used, pox- or adenovirus vectors are
preferred.
[0230] The DNA (or in the case of retroviral vectors, RNA) may then
be expressed in a suitable host to produce a polypeptide comprising
the peptide or variant of the invention. Thus, the DNA encoding the
peptide or variant of the invention may be used in accordance with
known techniques, appropriately modified in view of the teachings
contained herein, to construct an expression vector, which is then
used to transform an appropriate host cell for the expression and
production of the polypeptide of the invention. Such techniques
include those disclosed, for example, in U.S. Pat. Nos. 4,440,859,
4,530,901, 4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463,
4,757,006, 4,766,075, and 4,810,648.
[0231] The DNA (or in the case of retroviral vectors, RNA) encoding
the polypeptide constituting the compound of the invention may be
joined to a wide variety of other DNA sequences for introduction
into an appropriate host. The companion DNA will depend upon the
nature of the host, the manner of the introduction of the DNA into
the host, and whether episomal maintenance or integration is
desired.
[0232] Generally, the DNA is inserted into an expression vector,
such as a plasmid, in proper orientation and correct reading frame
for expression. If necessary, the DNA may be linked to the
appropriate transcriptional and translational regulatory control
nucleotide sequences recognized by the desired host, although such
controls are generally available in the expression vector. The
vector is then introduced into the host through standard
techniques. Generally, not all of the hosts will be transformed by
the vector. Therefore, it will be necessary to select for
transformed host cells. One selection technique involves
incorporating into the expression vector a DNA sequence, with any
necessary control elements, that codes for a selectable trait in
the transformed cell, such as antibiotic resistance.
[0233] Alternatively, the gene for such selectable trait can be on
another vector, which is used to co-transform the desired host
cell.
[0234] Host cells that have been transformed by the recombinant DNA
of the invention are then cultured for a sufficient time and under
appropriate conditions known to those skilled in the art in view of
the teachings disclosed herein to permit the expression of the
polypeptide, which can then be recovered.
[0235] Many expression systems are known, including bacteria (for
example E. coli and Bacillus subtilis), yeasts (for example
Saccharomyces cerevisiae), filamentous fungi (for example
Aspergillus spec.), plant cells, animal cells and insect cells.
Preferably, the system can be mammalian cells such as CHO cells
available from the ATCC Cell Biology Collection.
[0236] A typical mammalian cell vector plasmid for constitutive
expression comprises the CMV or SV40 promoter with a suitable poly
A tail and a resistance marker, such as neomycin. One example is
pSVL available from Pharmacia, Piscataway, N.J., USA. An example of
an inducible mammalian expression vector is pMSG, also available
from Pharmacia. Useful yeast plasmid vectors are pRS403-406 and
pRS413-416 and are generally available from Stratagene Cloning
Systems, La Jolla, Calif. 92037, USA. Plasmids pRS403, pRS404,
pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and
incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3.
Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps). CMV
promoter-based vectors (for example from Sigma-Aldrich) provide
transient or stable expression, cytoplasmic expression or
secretion, and N-terminal or C-terminal tagging in various
combinations of FLAG, 3.times.FLAG, c-myc or MAT. These fusion
proteins allow for detection, purification and analysis of
recombinant protein. Dual-tagged fusions provide flexibility in
detection.
[0237] The strong human cytomegalovirus (CMV) promoter regulatory
region drives constitutive protein expression levels as high as 1
mg/L in COS cells. For less potent cell lines, protein levels are
typically .about.0.1 mg/L. The presence of the SV40 replication
origin will result in high levels of DNA replication in SV40
replication permissive COS cells. CMV vectors, for example, can
contain the pMB1 (derivative of pBR322) origin for replication in
bacterial cells, the b-lactamase gene for ampicillin resistance
selection in bacteria, hGH polyA, and the f1 origin. Vectors
containing the pre-pro-trypsin leader (PPT) sequence can direct the
secretion of FLAG fusion proteins into the culture medium for
purification using ANTI-FLAG antibodies, resins, and plates. Other
vectors and expression systems are well known in the art for use
with a variety of host cells.
[0238] In another embodiment two or more peptides or peptide
variants of the invention are encoded and thus expressed in a
successive order (similar to "beads on a string" constructs). In
doing so, the peptides or peptide variants may be linked or fused
together by stretches of linker amino acids, such as for example
LLLLLL (SEQ ID NO:525), or may be linked without any additional
peptide(s) between them. These constructs can also be used for
cancer therapy, and may induce immune responses both involving MHC
I and MHC II.
[0239] The present invention also relates to a host cell
transformed with a polynucleotide vector construct of the present
invention. The host cell can be either prokaryotic or eukaryotic.
Bacterial cells may be preferred prokaryotic host cells in some
circumstances and typically are a strain of E. coli such as, for
example, the E. coli strains DH5 available from Bethesda Research
Laboratories Inc., Bethesda, Md., USA, and RR1 available from the
American Type Culture Collection (ATCC) of Rockville, Md., USA (No
ATCC 31343). Preferred eukaryotic host cells include yeast, insect
and mammalian cells, preferably vertebrate cells such as those from
a mouse, rat, monkey or human fibroblastic and colon cell lines.
Yeast host cells include YPH499, YPH500 and YPH501, which are
generally available from Stratagene Cloning Systems, La Jolla,
Calif. 92037, USA. Preferred mammalian host cells include Chinese
hamster ovary (CHO) cells available from the ATCC as CCL61, NIH
Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL
1658, monkey kidney-derived COS-1 cells available from the ATCC as
CRL 1650 and 293 cells which are human embryonic kidney cells.
Preferred insect cells are Sf9 cells which can be transfected with
baculovirus expression vectors. An overview regarding the choice of
suitable host cells for expression can be found in, for example,
the textbook of Paulina Balbas and Argelia Lorence "Methods in
Molecular Biology Recombinant Gene Expression, Reviews and
Protocols," Part One, Second Edition, ISBN 978-1-58829-262-9, and
other literature known to the person of skill.
[0240] Transformation of appropriate cell hosts with a DNA
construct of the present invention is accomplished by well-known
methods that typically depend on the type of vector used. With
regard to transformation of prokaryotic host cells, see, for
example, Cohen et al. (Cohen et al., 1972) and (Green and Sambrook,
2012). Transformation of yeast cells is described in Sherman et al.
(Sherman et al., 1986). The method of Beggs (Beggs, 1978) is also
useful. With regard to vertebrate cells, reagents useful in
transfecting such cells, for example calcium phosphate and
DEAE-dextran or liposome formulations, are available from
Stratagene Cloning Systems, or Life Technologies Inc.,
Gaithersburg, Md. 20877, USA. Electroporation is also useful for
transforming and/or transfecting cells and is well known in the art
for transforming yeast cell, bacterial cells, insect cells and
vertebrate cells.
[0241] 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.
[0242] It will be appreciated that certain host cells of the
invention are useful in the preparation of the peptides of the
invention, for example bacterial, yeast and insect cells. However,
other host cells may be useful in certain therapeutic methods. For
example, antigen-presenting cells, such as dendritic cells, may
usefully be used to express the peptides of the invention such that
they may be loaded into appropriate MHC molecules. Thus, the
current invention provides a host cell comprising a nucleic acid or
an expression vector according to the invention.
[0243] In a preferred embodiment, the host cell is an antigen
presenting cell, in particular a dendritic cell or antigen
presenting cell. APCs loaded with a recombinant fusion protein
containing prostatic acid phosphatase (PAP) were approved by the
U.S. Food and Drug Administration (FDA) on Apr. 29, 2010, to treat
asymptomatic or minimally symptomatic metastatic HRPC
(Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).
[0244] A further aspect of the invention provides a method of
producing a peptide or its variant, the method comprising culturing
a host cell and isolating the peptide from the host cell or its
culture medium.
[0245] In another embodiment, the peptide, the nucleic acid or the
expression vector of the invention are used in medicine. For
example, the peptide or its variant may be prepared for intravenous
(i.v.) injection, sub-cutaneous (s.c.) injection, intradermal
(i.d.) injection, intraperitoneal (i.p.) injection, intramuscular
(i.m.) injection. Preferred methods of peptide injection include
s.c., i.d., i.p., i.m., and i.v. Preferred methods of DNA injection
include i.d., i.m., s.c., i.p. and i.v. Doses of e.g. between 50
.mu.g and 1.5 mg, preferably 125 .mu.g to 500 .mu.g, of peptide or
DNA may be given and will depend on the respective peptide or DNA.
Dosages of this range were successfully used in previous trials
(Walter et al., 2012).
[0246] The polynucleotide used for active vaccination may be
substantially pure, or contained in a suitable vector or delivery
system. The nucleic acid may be DNA, cDNA, PNA, RNA or a
combination thereof. Methods for designing and introducing such a
nucleic acid are well known in the art. An overview is provided by
e.g. Teufel et al. (Teufel et al., 2005). Polynucleotide vaccines
are easy to prepare, but the mode of action of these vectors in
inducing an immune response is not fully understood. Suitable
vectors and delivery systems include viral DNA and/or RNA, such as
systems based on adenovirus, vaccinia virus, retroviruses, herpes
virus, adeno-associated virus or hybrids containing elements of
more than one virus. Non-viral delivery systems include cationic
lipids and cationic polymers and are well known in the art of DNA
delivery. Physical delivery, such as via a "gene-gun" may also be
used. The peptide or peptides encoded by the nucleic acid may be a
fusion protein, for example with an epitope that stimulates T cells
for the respective opposite CDR as noted above.
[0247] The medicament of the invention may also include one or more
adjuvants. Adjuvants are substances that non-specifically enhance
or potentiate the immune response (e.g., immune responses mediated
by CD8-positive T cells and helper-T (TH) cells to an antigen, and
would thus be considered useful in the medicament of the present
invention. Suitable adjuvants include, but are not limited to, 1018
ISS, aluminum salts, AMPLIVAX.RTM., AS15, BCG, CP-870,893, CpG7909,
CyaA, dSLIM, flagellin or TLR5 ligands derived from flagellin, FLT3
ligand, GM-CSF, IC30, IC31, Imiquimod (ALDARA.RTM.), resiquimod,
ImuFact IMP321, Interleukins as IL-2, IL-13, IL-21,
Interferon-alpha or -beta, or pegylated derivatives thereof, IS
Patch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune.RTM., LipoVac, MALP2,
MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA
206, Montanide ISA 50V, Montanide ISA-51, water-in-oil and
oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA,
PepTel.RTM. vector system, poly(lactid co-glycolid) [PLG]-based and
dextran microparticles, talactoferrin SRL172, Virosomes and other
Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan,
Pam3Cys, Aquila's QS21 stimulon, which is derived from saponin,
mycobacterial extracts and synthetic bacterial cell wall mimics,
and other proprietary adjuvants such as Ribi's Detox, Quil, or
Superfos. Adjuvants such as Freund's or GM-CSF are preferred.
Several immunological adjuvants (e.g., MF59) specific for dendritic
cells and their preparation have been described previously (Allison
and Krummel, 1995). Also cytokines may be used. Several cytokines
have been directly linked to influencing dendritic cell migration
to lymphoid tissues (e.g., TNF-), accelerating the maturation of
dendritic cells into efficient antigen-presenting cells for
T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No.
5,849,589, specifically incorporated herein by reference in its
entirety) and acting as immunoadjuvants (e.g., IL-12, IL-15, IL-23,
IL-7, IFN-alpha. IFN-beta) (Gabrilovich et al., 1996).
[0248] CpG immunostimulatory oligonucleotides have also been
reported to enhance the effects of adjuvants in a vaccine setting.
Without being bound by theory, CpG oligonucleotides act by
activating the innate (non-adaptive) immune system via Toll-like
receptors (TLR), mainly TLR9. CpG triggered TLR9 activation
enhances antigen-specific humoral and cellular responses to a wide
variety of antigens, including peptide or protein antigens, live or
killed viruses, dendritic cell vaccines, autologous cellular
vaccines and polysaccharide conjugates in both prophylactic and
therapeutic vaccines. More importantly it enhances dendritic cell
maturation and differentiation, resulting in enhanced activation of
TH1 cells and strong cytotoxic T-lymphocyte (CTL) generation, even
in the absence of CD4 T cell help. The TH1 bias induced by TLR9
stimulation is maintained even in the presence of vaccine adjuvants
such as alum or incomplete Freund's adjuvant (IFA) that normally
promote a TH2 bias. CpG oligonucleotides show even greater adjuvant
activity when formulated or co-administered with other adjuvants or
in formulations such as microparticles, nanoparticles, lipid
emulsions or similar formulations, which are especially necessary
for inducing a strong response when the antigen is relatively weak.
They also accelerate the immune response and enable the antigen
doses to be reduced by approximately two orders of magnitude, with
comparable antibody responses to the full-dose vaccine without CpG
in some experiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1
describes the combined use of CpG oligonucleotides, non-nucleic
acid adjuvants and an antigen to induce an antigen-specific immune
response. A CpG TLR9 antagonist is dSLIM (double Stem Loop
Immunomodulator) by Mologen (Berlin, Germany) which is a preferred
component of the pharmaceutical composition of the present
invention. Other TLR binding molecules such as RNA binding TLR 7,
TLR 8 and/or TLR 9 may also be used.
[0249] Other examples for useful adjuvants include, but are not
limited to chemically modified CpGs (e.g. CpR, Idera), dsRNA
analogues such as Poly(I:C) and derivates thereof (e.g.
AmpliGen.RTM., Hiltonal.RTM., poly-(ICLC), poly(IC-R),
poly(I:C12U), non-CpG bacterial DNA or RNA as well as immunoactive
small molecules and antibodies such as cyclophosphamide, sunitinib,
Bevacizumab.RTM., celebrex, NCX-4016, sildenafil, tadalafil,
vardenafil, sorafenib, temozolomide, temsirolimus, XL-999,
CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other
antibodies targeting key structures of the immune system (e.g.
anti-CD40, anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which
may act therapeutically and/or as an adjuvant. The amounts and
concentrations of adjuvants and additives useful in the context of
the present invention can readily be determined by the skilled
artisan without undue experimentation.
[0250] Preferred adjuvants are anti-CD40, imiquimod, resiquimod,
GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha,
CpG oligonucleotides and derivates, poly-(I:C) and derivates, RNA,
sildenafil, and particulate formulations with PLG or virosomes.
[0251] In a preferred embodiment, the pharmaceutical composition
according to the invention the adjuvant is selected from the group
consisting of colony-stimulating factors, such as Granulocyte
Macrophage Colony Stimulating Factor (GM-CSF, sargramostim),
cyclophosphamide, imiquimod, resiquimod, and interferon-alpha.
[0252] In a preferred embodiment, the pharmaceutical composition
according to the invention the adjuvant is selected from the group
consisting of colony-stimulating factors, such as Granulocyte
Macrophage Colony Stimulating Factor (GM-CSF, sargramostim),
cyclophosphamide, imiquimod and resiquimod. In a preferred
embodiment of the pharmaceutical composition according to the
invention, the adjuvant is cyclophosphamide, imiquimod or
resiquimod. Even more preferred adjuvants are Montanide IMS 1312,
Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, poly-ICLC
(Hiltonol.RTM.) and anti-CD40 mAB, or combinations thereof.
[0253] This composition is used for parenteral administration, such
as subcutaneous, intradermal, intramuscular or oral administration.
For this, the peptides and optionally other molecules are dissolved
or suspended in a pharmaceutically acceptable, preferably aqueous
carrier. In addition, the composition can contain excipients, such
as buffers, binding agents, blasting agents, diluents, flavors,
lubricants, etc. The peptides can also be administered together
with immune stimulating substances, such as cytokines. An extensive
listing of excipients that can be used in such a composition, can
be, for example, taken from A. Kibbe, Handbook of Pharmaceutical
Excipients (Kibbe, 2000). The composition can be used for a
prevention, prophylaxis and/or therapy of adenomatous or cancerous
diseases. Exemplary formulations can be found in, for example,
EP2112253.
[0254] It is important to realize that the immune response
triggered by the vaccine according to the invention attacks the
cancer in different cell-stages and different stages of
development. Furthermore, different cancer associated signaling
pathways are attacked. This is an advantage over vaccines that
address only one or few targets, which may cause the tumor to
easily adapt to the attack (tumor escape). Furthermore, not all
individual tumors express the same pattern of antigens. Therefore,
a combination of several tumor-associated peptides ensures that
every single tumor bears at least some of the targets. The
composition is designed in such a way that each tumor is expected
to express several of the antigens and cover several independent
pathways necessary for tumor growth and maintenance. Thus, the
vaccine can easily be used "off-the-shelf" for a larger patient
population. This means that a pre-selection of patients to be
treated with the vaccine can be restricted to HLA typing, does not
require any additional biomarker assessments for antigen
expression, but it is still ensured that several targets are
simultaneously attacked by the induced immune response, which is
important for efficacy (Banchereau et al., 2001; Walter et al.,
2012).
[0255] As used herein, the term "scaffold" refers to a molecule
that specifically binds to an (e.g. antigenic) determinant. In one
embodiment, a scaffold is able to direct the entity to which it is
attached (e.g. a (second) antigen binding moiety) to a target site,
for example to a specific type of tumor cell or tumor stroma
bearing the antigenic determinant (e.g. the complex of a peptide
with MHC, according to the application at hand). In another
embodiment, a scaffold is able to activate signaling through its
target antigen, for example a T cell receptor complex antigen.
Scaffolds include but are not limited to antibodies and fragments
thereof, antigen binding domains of an antibody, comprising an
antibody heavy chain variable region and an antibody light chain
variable region, binding proteins comprising at least one ankyrin
repeat motif and single domain antigen binding (SDAB) molecules,
aptamers, (soluble) TCRs and (modified) cells such as allogenic or
autologous T cells. To assess whether a molecule is a scaffold
binding to a target, binding assays can be performed.
[0256] "Specific" binding means that the scaffold binds the
peptide-MHC-complex of interest better than other naturally
occurring peptide-MHC-complexes, to an extent that a scaffold armed
with an active molecule that is able to kill a cell bearing the
specific target is not able to kill another cell without the
specific target but presenting other peptide-MHC complex(es).
Binding to other peptide-MHC complexes is irrelevant if the peptide
of the cross-reactive peptide-MHC is not naturally occurring, i.e.
not derived from the human HLA-peptidome. Tests to assess target
cell killing are well known in the art. They should be performed
using target cells (primary cells or cell lines) with unaltered
peptide-MHC presentation, or cells loaded with peptides such that
naturally occurring peptide-MHC levels are reached.
[0257] Each scaffold can comprise a labelling which provides that
the bound scaffold can be detected by determining the presence or
absence of a signal provided by the label. For example, the
scaffold can be labelled with a fluorescent dye or any other
applicable cellular marker molecule. Such marker molecules are well
known in the art. For example, a fluorescence-labelling, for
example provided by a fluorescence dye, can provide a visualization
of the bound aptamer by fluorescence or laser scanning microscopy
or flow cytometry. Each scaffold can be conjugated with a second
active molecule such as for example IL-21, anti-CD3, anti-CD28. For
further information on polypeptide scaffolds see for example the
background section of WO 2014/071978A1 and the references cited
therein.
[0258] The present invention further relates to aptamers. Aptamers
(see for example WO 2014/191359 and the literature as cited
therein) are short single-stranded nucleic acid molecules, which
can fold into defined three-dimensional structures and recognize
specific target structures. They have appeared to be suitable
alternatives for developing targeted therapies. Aptamers have been
shown to selectively bind to a variety of complex targets with high
affinity and specificity.
[0259] Aptamers recognizing cell surface located molecules have
been identified within the past decade and provide means for
developing diagnostic and therapeutic approaches. Since aptamers
have been shown to possess almost no toxicity and immunogenicity
they are promising candidates for biomedical applications. Indeed,
aptamers, for example prostate-specific membrane-antigen
recognizing aptamers, have been successfully employed for targeted
therapies and shown to be functional in xenograft in vivo models.
Furthermore, aptamers recognizing specific tumor cell lines have
been identified.
[0260] DNA aptamers can be selected to reveal broad-spectrum
recognition properties for various cancer cells, and particularly
those derived from solid tumors, while non-tumorigenic and primary
healthy cells are not recognized. If the identified aptamers
recognize not only a specific tumor sub-type but rather interact
with a series of tumors, this renders the aptamers applicable as
so-called broad-spectrum diagnostics and therapeutics.
[0261] Further, investigation of cell-binding behavior with flow
cytometry showed that the aptamers revealed very good apparent
affinities that are within the nanomolar range.
[0262] 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 si RNA into tumor
cells.
[0263] Aptamers can be selected against complex targets such as
cells and tissues and complexes of the peptides comprising,
preferably consisting of, a sequence according to any of SEQ ID NO
1 to SEQ ID NO 385, according to the invention at hand with the MHC
molecule, using the cell-SELEX (Systematic Evolution of Ligands by
Exponential enrichment) technique.
[0264] 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.
[0265] Therefore, it is a further aspect of the invention to
provide a method for producing a recombinant antibody specifically
binding to a human major histocompatibility complex (MHC) class I
or II being complexed with a HLA-restricted antigen, the method
comprising: immunizing a genetically engineered non-human mammal
comprising cells expressing said human major histocompatibility
complex (MHC) class I or II with a soluble form of a MHC class I or
II molecule being complexed with said HLA-restricted antigen;
isolating mRNA molecules from antibody producing cells of said
non-human mammal; producing a phage display library displaying
protein molecules encoded by said mRNA molecules; and isolating at
least one phage from said phage display library, said at least one
phage displaying said antibody specifically binding to said human
major histocompatibility complex (MHC) class I or II being
complexed with said HLA-restricted antigen.
[0266] It is a further aspect of the invention to provide an
antibody that specifically binds to a human major
histocompatibility complex (MHC) class I or II being complexed with
a HLA-restricted antigen, wherein the antibody preferably is a
polyclonal antibody, monoclonal antibody, bi-specific antibody
and/or a chimeric antibody.
[0267] Respective methods for producing such antibodies and single
chain class I major histocompatibility complexes, as well as other
tools for the production of these antibodies are disclosed in WO
03/068201, WO 2004/084798, WO 01/72768, WO 03/070752, and in
publications (Cohen et al., 2003a; Cohen et al., 2003b; Denkberg et
al., 2003), which for the purposes of the present invention are all
explicitly incorporated by reference in their entireties.
[0268] 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.
[0269] 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: 385, or a variant thereof which is at least 88%
homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 385
or a variant thereof that induces T cells cross-reacting with said
peptide, wherein said peptide is not the underlying full-length
polypeptide.
[0270] The present invention further relates to a peptide
comprising a sequence that is selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 385 or a variant thereof which is at
least 88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ
ID NO: 385, wherein said peptide or variant has an overall length
of between 8 and 100, preferably between 8 and 30, and most
preferred between 8 and 14 amino acids.
[0271] The present invention further relates to the peptides
according to the invention that have the ability to bind to a
molecule of the human major histocompatibility complex (MHC)
class-I or -II.
[0272] The present invention further relates to the peptides
according to the invention wherein the peptide consists or consists
essentially of an amino acid sequence according to SEQ ID NO: 1 to
SEQ ID NO: 385.
[0273] The present invention further relates to the peptides
according to the invention, wherein the peptide is (chemically)
modified and/or includes non-peptide bonds.
[0274] The present invention further relates to the peptides
according to the invention, wherein the peptide is part of a fusion
protein, in particular comprising N-terminal amino acids of the
HLA-DR antigen-associated invariant chain (Ii), or wherein the
peptide is fused to (or into) an antibody, such as, for example, an
antibody that is specific for dendritic cells.
[0275] The present invention further relates to a nucleic acid,
encoding the peptides according to the invention, provided that the
peptide is not the complete (full) human protein.
[0276] The present invention further relates to the nucleic acid
according to the invention that is DNA, cDNA, PNA, RNA or
combinations thereof.
[0277] The present invention further relates to an expression
vector capable of expressing a nucleic acid according to the
present invention.
[0278] The present invention further relates to a peptide according
to the present invention, a nucleic acid according to the present
invention or an expression vector according to the present
invention for use in medicine, in particular in the treatment of
CLL.
[0279] The present invention further relates to a host cell
comprising a nucleic acid according to the invention or an
expression vector according to the invention.
[0280] The present invention further relates to the host cell
according to the present invention that is an antigen presenting
cell, and preferably a dendritic cell.
[0281] The present invention further relates to a method of
producing a peptide according to the present invention, said method
comprising culturing the host cell according to the present
invention, and isolating the peptide from said host cell or its
culture medium.
[0282] The present invention further relates to the method
according to the present invention, where-in the antigen is loaded
onto class I or II MHC molecules expressed on the surface of a
suitable antigen-presenting cell by contacting a sufficient amount
of the antigen with an antigen-presenting cell.
[0283] The present invention further relates to the method
according to the invention, wherein the antigen-presenting cell
comprises an expression vector capable of expressing or expressing
said peptide containing SEQ ID NO: 1 to SEQ ID NO: 385 or said
variant amino acid sequence.
[0284] The present invention further relates to activated T cells,
produced by the method according to the present invention, wherein
said T cells selectively recognizes a cell which aberrantly
expresses a polypeptide comprising an amino acid sequence according
to the present invention.
[0285] The present invention further relates to a method of killing
target cells in a patient which target cells aberrantly express a
polypeptide comprising any amino acid sequence according to the
present invention, the method comprising administering to the
patient an effective number of T cells as according to the present
invention.
[0286] The present invention further relates to the use of any
peptide described, a nucleic acid according to the present
invention, an expression vector according to the present invention,
a cell according to the present invention, or an activated
cytotoxic T lymphocyte according to the present invention as a
medicament or in the manufacture of a medicament. The present
invention further relates to a use according to the present
invention, wherein the medicament is active against cancer.
[0287] 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.
[0288] The present invention further relates to a use according to
the invention, wherein said cancer cells are CLL cells or other
solid or hematological tumor cells such as acute myelogenous
leukemia, bile duct cancer, brain cancer, breast cancer, colorectal
carcinoma, esophageal cancer, gallbladder cancer, gastric cancer,
hepatocellular cancer, Merkel cell carcinoma, melanoma, non-Hodgkin
lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic
cancer, prostate cancer, renal cell cancer, small cell lung cancer,
urinary bladder cancer, uterine cancer, glioblastoma, renal cell
carcinoma, hepatocellular carcinoma, and testis cancer.
[0289] The present invention further relates to particular marker
proteins and biomarkers based on the peptides according to the
present invention, herein called "targets" that can be used in the
diagnosis and/or prognosis of CLL. The present invention also
relates to the use of these novel targets for cancer treatment.
[0290] The term "antibody" or "antibodies" is used herein in a
broad sense and includes both polyclonal and monoclonal antibodies.
In addition to intact or "full" immunoglobulin molecules, also
included in the term "antibodies" are fragments (e.g. CDRs, Fv, Fab
and Fc fragments) or polymers of those immunoglobulin molecules and
humanized versions of immunoglobulin molecules, as long as they
exhibit any of the desired properties (e.g., specific binding of a
CLL marker (poly)peptide, delivery of a toxin to a CLL cell
expressing a cancer marker gene at an increased level, and/or
inhibiting the activity of a CLL marker polypeptide) according to
the invention.
[0291] 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 CLL marker polypeptides or
fragments thereof may be used to generate the antibodies of the
invention. A polypeptide to be used for generating an antibody of
the invention may be partially or fully purified from a natural
source, or may be produced using recombinant DNA techniques.
[0292] For example, a cDNA encoding a peptide according to the
present invention, such as a peptide according to SEQ ID NO: 1 to
SEQ ID NO: 385 polypeptide, or a variant or fragment thereof, can
be expressed in prokaryotic cells (e.g., bacteria) or eukaryotic
cells (e.g., yeast, insect, or mammalian cells), after which the
recombinant protein can be purified and used to generate a
monoclonal or polyclonal antibody preparation that specifically
bind the CLL marker polypeptide used to generate the antibody
according to the invention.
[0293] One of skill in the art will realize that the generation of
two or more different sets of monoclonal or polyclonal antibodies
maximizes the likelihood of obtaining an antibody with the
specificity and affinity required for its intended use (e.g.,
ELISA, immunohistochemistry, in vivo imaging, immunotoxin therapy).
The antibodies are tested for their desired activity by known
methods, in accordance with the purpose for which the antibodies
are to be used (e.g., ELISA, immunohistochemistry, immunotherapy,
etc.; for further guidance on the generation and testing of
antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). For
example, the antibodies may be tested in ELISA assays or, Western
blots, immunohistochemical staining of formalin-fixed cancers or
frozen tissue sections. After their initial in vitro
characterization, antibodies intended for therapeutic or in vivo
diagnostic use are tested according to known clinical testing
methods.
[0294] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a substantially homogeneous population of
antibodies, i.e.; the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. The monoclonal
antibodies herein specifically include "chimeric" antibodies in
which a portion of the heavy and/or light chain is identical with
or homologous to corresponding sequences in antibodies derived from
a particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is identical with or
homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, so long as they exhibit
the desired antagonistic activity (U.S. Pat. No. 4,816,567, which
is hereby incorporated in its entirety).
[0295] Monoclonal antibodies of the invention may be prepared using
hybridoma methods. In a hybridoma method, a mouse or other
appropriate host animal is typically immunized with an immunizing
agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the immunizing
agent. Alternatively, the lymphocytes may be immunized in
vitro.
[0296] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies).
[0297] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly Fab fragments, can be accomplished using routine
techniques known in the art. For instance, digestion can be
performed using papain. Examples of papain digestion are described
in WO 94/29348 and U.S. Pat. No. 4,342,566. Papain digestion of
antibodies typically produces two identical antigen binding
fragments, called Fab fragments, each with a single antigen binding
site, and a residual Fc fragment. Pepsin treatment yields a F(ab')2
fragment and a pFc' fragment.
[0298] The antibody fragments, whether attached to other sequences
or not, can also include insertions, deletions, substitutions, or
other selected modifications of particular regions or specific
amino acids residues, provided the activity of the fragment is not
significantly altered or impaired compared to the non-modified
antibody or antibody fragment. These modifications can provide for
some additional property, such as to remove/add amino acids capable
of disulfide bonding, to increase its bio-longevity, to alter its
secretory characteristics, etc. In any case, the antibody fragment
must possess a bioactive property, such as binding activity,
regulation of binding at the binding domain, etc. Functional or
active regions of the antibody may be identified by mutagenesis of
a specific region of the protein, followed by expression and
testing of the expressed polypeptide. Such methods are readily
apparent to a skilled practitioner in the art and can include
site-specific mutagenesis of the nucleic acid encoding the antibody
fragment.
[0299] The antibodies of the invention may further comprise
humanized antibodies or human antibodies. Humanized forms of
non-human (e.g., murine) antibodies are chimeric immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab'
or other antigen-binding subsequences of antibodies) which contain
minimal sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues from a complementary determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework (FR) residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin.
[0300] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0301] Transgenic animals (e.g., mice) that are capable, upon
immunization, of producing a full repertoire of human antibodies in
the absence of endogenous immunoglobulin production can be
employed. For example, it has been described that the homozygous
deletion of the antibody heavy chain joining region gene in
chimeric and germ-line mutant mice results in complete inhibition
of endogenous antibody production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge. Human
antibodies can also be produced in phage display libraries.
[0302] Antibodies of the invention are preferably administered to a
subject in a pharmaceutically acceptable carrier. Typically, an
appropriate amount of a pharmaceutically-acceptable salt is used in
the formulation to render the formulation isotonic. Examples of the
pharmaceutically-acceptable carrier include saline, Ringer's
solution and dextrose solution. The pH of the solution is
preferably from about 5 to about 8, and more preferably from about
7 to about 7.5. Further carriers include sustained release
preparations such as semipermeable matrices of solid hydrophobic
polymers containing the antibody, which matrices are in the form of
shaped articles, e.g., films, liposomes or microparticles. It will
be apparent to those persons skilled in the art that certain
carriers may be more preferable depending upon, for instance, the
route of administration and concentration of antibody being
administered.
[0303] The antibodies can be administered to the subject, patient,
or cell by injection (e.g., intravenous, intraperitoneal,
subcutaneous, intramuscular), or by other methods such as infusion
that ensure its delivery to the bloodstream in an effective form.
The antibodies may also be administered by intratumoral or
peritumoral routes, to exert local as well as systemic therapeutic
effects. Local or intravenous injection is preferred.
[0304] Effective dosages and schedules for administering the
antibodies may be determined empirically, and making such
determinations is within the skill in the art. Those skilled in the
art will understand that the dosage of antibodies that must be
administered will vary depending on, for example, the subject that
will receive the antibody, the route of administration, the
particular type of antibody used and other drugs being
administered. A typical daily dosage of the antibody used alone
might range from about 1 (.mu.g/kg to up to 100 mg/kg of body
weight or more per day, depending on the factors mentioned above.
Following administration of an antibody, preferably for treating
CLL, the efficacy of the therapeutic antibody can be assessed in
various ways well known to the skilled practitioner. For instance,
the size, number, and/or distribution of cancer in a subject
receiving treatment may be monitored using standard tumor imaging
techniques. A therapeutically-administered antibody that arrests
tumor growth, results in tumor shrinkage, and/or prevents the
development of new tumors, compared to the disease course that
would occurs in the absence of antibody administration, is an
efficacious antibody for treatment of cancer.
[0305] It is a further aspect of the invention to provide a method
for producing a soluble T-cell receptor (sTCR) recognizing a
specific peptide-MHC complex. Such soluble T-cell receptors can be
generated from specific T-cell clones, and their affinity can be
increased by mutagenesis targeting the complementarity-determining
regions. For the purpose of T-cell receptor selection, phage
display can be used (US 2010/0113300, (Liddy et al., 2012)). For
the purpose of stabilization of T-cell receptors during phage
display and in case of practical use as drug, alpha and beta chain
can be linked e.g. by non-native disulfide bonds, other covalent
bonds (single-chain T-cell receptor), or by dimerization domains
(Boulter et al., 2003; Card et al., 2004; Willcox et al., 1999).
The T-cell receptor can be linked to toxins, drugs, cytokines (see,
for example, US 2013/0115191), domains recruiting effector cells
such as an anti-CD3 domain, etc., in order to execute particular
functions on target cells. Moreover, it could be expressed in T
cells used for adoptive transfer. Further information can be found
in WO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs is
described in WO 2012/056407A1. Further methods for the production
are disclosed in WO 2013/057586A1.
[0306] In addition, the peptides and/or the TCRs or antibodies or
other binding molecules of the present invention can be used to
verify a pathologist's diagnosis of a cancer based on a biopsied
sample.
[0307] The antibodies or TCRs may also be used for in vivo
diagnostic assays. Generally, the antibody is labeled with a
radionucleotide (such as .sup.111In, .sup.99Tc, .sup.14C,
.sup.131I, .sup.3H, .sup.32P or .sup.35S) so that the tumor can be
localized using immunoscintiography. In one embodiment, antibodies
or fragments thereof bind to the extracellular domains of two or
more targets of a protein selected from the group consisting of the
above-mentioned proteins, and the affinity value (Kd) is less than
1.times.100 .mu.M.
[0308] Antibodies for diagnostic use may be labeled with probes
suitable for detection by various imaging methods. Methods for
detection of probes include, but are not limited to, fluorescence,
light, confocal and electron microscopy; magnetic resonance imaging
and spectroscopy; fluoroscopy, computed tomography and positron
emission tomography. Suitable probes include, but are not limited
to, fluorescein, rhodamine, eosin and other fluorophores,
radioisotopes, gold, gadolinium and other lanthanides, paramagnetic
iron, fluorine-18 and other positron-emitting radionuclides.
Additionally, probes may be bi- or multi-functional and be
detectable by more than one of the methods listed. These antibodies
may be directly or indirectly labeled with said probes. Attachment
of probes to the antibodies includes covalent attachment of the
probe, incorporation of the probe into the antibody, and the
covalent attachment of a chelating compound for binding of probe,
amongst others well recognized in the art. For
immunohistochemistry, the disease tissue sample may be fresh or
frozen or may be embedded in paraffin and fixed with a preservative
such as formalin. The fixed or embedded section contains the sample
are contacted with a labeled primary antibody and secondary
antibody, wherein the antibody is used to detect the expression of
the proteins in situ.
[0309] Another aspect of the present invention includes an in vitro
method for producing activated T cells, the method comprising
contacting in vitro T cells with antigen loaded human MHC molecules
expressed on the surface of a suitable antigen-presenting cell for
a period of time sufficient to activate the T cell in an antigen
specific manner, wherein the antigen is a peptide according to the
invention. Preferably a sufficient amount of the antigen is used
with an antigen-presenting cell.
[0310] Preferably the mammalian cell lacks or has a reduced level
or function of the TAP peptide transporter. Suitable cells that
lack the TAP peptide transporter include T2, RMA-S and Drosophila
cells. TAP is the transporter associated with antigen
processing.
[0311] The human peptide loading deficient cell line T2 is
available from the American Type Culture Collection, 12301 Parklawn
Drive, Rockville, Md. 20852, USA under Catalogue No CRL 1992; the
Drosophila cell line Schneider line 2 is available from the ATCC
under Catalogue No CRL 19863; the mouse RMA-S cell line is
described in Ljunggren et al. (Ljunggren and Karre, 1985).
[0312] Preferably, before transfection the host cell expresses
substantially no MHC class I molecules. It is also preferred that
the stimulator cell expresses a molecule important for providing a
co-stimulatory signal for T-cells such as any of B7.1, B7.2, ICAM-1
and LFA 3. The nucleic acid sequences of numerous MHC class I
molecules and of the co-stimulator molecules are publicly available
from the GenBank and EMBL databases.
[0313] In case of a MHC class I epitope being used as an antigen,
the T cells are CD8-positive T cells.
[0314] 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: 385, or a variant amino acid sequence thereof.
[0315] A number of other methods may be used for generating T cells
in vitro. For example, autologous tumor-infiltrating lymphocytes
can be used in the generation of CTL. Plebanski et al. (Plebanski
et al., 1995) made use of autologous peripheral blood lymphocytes
(PLBs) in the preparation of T cells. Furthermore, the production
of autologous T cells by pulsing dendritic cells with peptide or
polypeptide, or via infection with recombinant virus is possible.
Also, B cells can be used in the production of autologous T cells.
In addition, macrophages pulsed with peptide or polypeptide, or
infected with recombinant virus, may be used in the preparation of
autologous T cells. S. Walter et al. (Walter et al., 2003) describe
the in vitro priming of T cells by using artificial antigen
presenting cells (aAPCs), which is also a suitable way for
generating T cells against the peptide of choice. In the present
invention, aAPCs were generated by the coupling of preformed
MHC:peptide complexes to the surface of polystyrene particles
(microbeads) by biotin:streptavidin biochemistry. This system
permits the exact control of the MHC density on aAPCs, which allows
to selectively elicit high- or low-avidity antigen-specific T cell
responses with high efficiency from blood samples. Apart from
MHC:peptide complexes, aAPCs should carry other proteins with
co-stimulatory activity like anti-CD28 antibodies coupled to their
surface. Furthermore, such aAPC-based systems often require the
addition of appropriate soluble factors, e. g. cytokines, like
interleukin-12.
[0316] Allogeneic cells may also be used in the preparation of T
cells and a method is described in detail in WO 97/26328,
incorporated herein by reference. For example, in addition to
Drosophila cells and T2 cells, other cells may be used to present
antigens such as CHO cells, baculovirus-infected insect cells,
bacteria, yeast, vaccinia-infected target cells. In addition plant
viruses may be used (see, for example, Porta et al. (Porta et al.,
1994) which describes the development of cowpea mosaic virus as a
high-yielding system for the presentation of foreign peptides.
[0317] The activated T cells that are directed against the peptides
of the invention are useful in therapy. Thus, a further aspect of
the invention provides activated T cells obtainable by the
foregoing methods of the invention.
[0318] Activated T cells, which are produced by the above method,
will selectively recognize a cell that aberrantly expresses a
polypeptide that comprises an amino acid sequence of SEQ ID NO: 1
to SEQ ID NO 385.
[0319] Preferably, the T cell recognizes the cell by interacting
through its TCR with the HLA/peptide-complex (for example,
binding). The T cells are useful in a method of killing target
cells in a patient whose target cells aberrantly express a
polypeptide comprising an amino acid sequence of the invention
wherein the patient is administered an effective number of the
activated T cells. The T cells that are administered to the patient
may be derived from the patient and activated as described above
(i.e. they are autologous T cells). Alternatively, the T cells are
not from the patient but are from another individual. Of course, it
is preferred if the individual is a healthy individual. By "healthy
individual" the inventors mean that the individual is generally in
good health, preferably has a competent immune system and, more
preferably, is not suffering from any disease that can be readily
tested for, and detected.
[0320] 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)).
[0321] The T cells of the present invention may be used as active
ingredients of a therapeutic composition. Thus, the invention also
provides a method of killing target cells in a patient whose target
cells aberrantly express a polypeptide comprising an amino acid
sequence of the invention, the method comprising administering to
the patient an effective number of T cells as defined above.
[0322] By "aberrantly expressed" the inventors also mean that the
polypeptide is over-expressed compared to levels of expression in
normal tissues or that the gene is silent in the tissue from which
the tumor is derived but in the tumor it is expressed. By
"over-expressed" the inventors mean that the polypeptide is present
at a level at least 1.2-fold of that present in normal tissue;
preferably at least 2-fold, and more preferably at least 5-fold or
10-fold the level present in normal tissue.
[0323] T cells may be obtained by methods known in the art, e.g.
those described above.
[0324] Protocols for this so-called adoptive transfer of T cells
are well known in the art. Reviews can be found in: Gattioni et al.
and Morgan et al. (Gattinoni et al., 2006; Morgan et al.,
2006).
[0325] Another aspect of the present invention includes the use of
the peptides complexed with MHC to generate a T-cell receptor whose
nucleic acid is cloned and is introduced into a host cell,
preferably a T cell. This engineered T cell can then be transferred
to a patient for therapy of cancer.
[0326] Any molecule of the invention, i.e. the peptide, nucleic
acid, antibody, expression vector, cell, activated T cell, T-cell
receptor or the nucleic acid encoding it, is useful for the
treatment of disorders, characterized by cells escaping an immune
response. Therefore, any molecule of the present invention may be
used as medicament or in the manufacture of a medicament. The
molecule may be used by itself or combined with other molecule(s)
of the invention or (a) known molecule(s).
[0327] The present invention is further directed at a kit
comprising:
(a) a container containing a pharmaceutical composition as
described above, in solution or in lyophilized form; (b) optionally
a second container containing a diluent or reconstituting solution
for the lyophilized formulation; and (c) optionally, instructions
for (i) use of the solution or (ii) reconstitution and/or use of
the lyophilized formulation.
[0328] The kit may further comprise one or more of (iii) a buffer,
(iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe. The
container is preferably a bottle, a vial, a syringe or test tube;
and it may be a multi-use container. The pharmaceutical composition
is preferably lyophilized.
[0329] Kits of the present invention preferably comprise a
lyophilized formulation of the present invention in a suitable
container and instructions for its reconstitution and/or use.
Suitable containers include, for example, bottles, vials (e.g. dual
chamber vials), syringes (such as dual chamber syringes) and test
tubes. The container may be formed from a variety of materials such
as glass or plastic. Preferably the kit and/or container contain/s
instructions on or associated with the container that indicates
directions for reconstitution and/or use. For example, the label
may indicate that the lyophilized formulation is to be
reconstituted to peptide concentrations as described above. The
label may further indicate that the formulation is useful or
intended for subcutaneous administration.
[0330] The container holding the formulation may be a multi-use
vial, which allows for repeat administrations (e.g., from 2-6
administrations) of the reconstituted formulation. The kit may
further comprise a second container comprising a suitable diluent
(e.g., sodium bicarbonate solution).
[0331] Upon mixing of the diluent and the lyophilized formulation,
the final peptide concentration in the reconstituted formulation is
preferably at least 0.15 mg/mL/peptide (=75 .mu.g) and preferably
not more than 3 mg/mL/peptide (=1500 .mu.g). The kit may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use.
[0332] Kits of the present invention may have a single container
that contains the formulation of the pharmaceutical compositions
according to the present invention with or without other components
(e.g., other compounds or pharmaceutical compositions of these
other compounds) or may have distinct container for each
component.
[0333] Preferably, kits of the invention include a formulation of
the invention packaged for use in combination with the
co-administration of a second compound (such as adjuvants (e.g.
GM-CSF), a chemotherapeutic agent, a natural product, a hormone or
antagonist, an anti-angiogenesis agent or inhibitor, an
apoptosis-inducing agent or a chelator) or a pharmaceutical
composition thereof. The components of the kit may be pre-complexed
or each component may be in a separate distinct container prior to
administration to a patient. The components of the kit may be
provided in one or more liquid solutions, preferably, an aqueous
solution, more preferably, a sterile aqueous solution. The
components of the kit may also be provided as solids, which may be
converted into liquids by addition of suitable solvents, which are
preferably provided in another distinct container.
[0334] The container of a therapeutic kit may be a vial, test tube,
flask, bottle, syringe, or any other means of enclosing a solid or
liquid. Usually, when there is more than one component, the kit
will contain a second vial or other container, which allows for
separate dosing. The kit may also contain another container for a
pharmaceutically acceptable liquid. Preferably, a therapeutic kit
will contain an apparatus (e.g., one or more needles, syringes, eye
droppers, pipette, etc.), which enables administration of the
agents of the invention that are components of the present kit.
[0335] The present formulation is one that is suitable for
administration of the peptides by any acceptable route such as oral
(enteral), nasal, ophthal, subcutaneous, intradermal,
intramuscular, intravenous or transdermal. Preferably, the
administration is s.c., and most preferably i.d. administration may
be by infusion pump.
[0336] Since the peptides of the invention were isolated from CLL,
the medicament of the invention is preferably used to treat
CLL.
[0337] The present invention further relates to a method for
producing a personalized pharmaceutical for an individual patient
comprising manufacturing a pharmaceutical composition comprising at
least one peptide selected from a warehouse of pre-screened TUMAPs,
wherein the at least one peptide used in the pharmaceutical
composition is selected for suitability in the individual patient.
In one embodiment, the pharmaceutical composition is a vaccine. The
method could also be adapted to produce T cell clones for
down-stream applications, such as TCR isolations, or soluble
antibodies, and other treatment options.
[0338] A "personalized pharmaceutical" shall mean specifically
tailored therapies for one individual patient that will only be
used for therapy in such individual patient, including actively
personalized cancer vaccines and adoptive cellular therapies using
autologous patient tissue.
[0339] As used herein, the term "warehouse" shall refer to a group
or set of peptides that have been pre-screened for immunogenicity
and/or over-presentation in a particular tumor type. The term
"warehouse" is not intended to imply that the particular peptides
included in the vaccine have been pre-manufactured and stored in a
physical facility, although that possibility is contemplated. It is
expressly contemplated that the peptides may be manufactured de
novo for each individualized vaccine produced, or may be
pre-manufactured and stored. The warehouse (e.g. in the form of a
database) is composed of tumor-associated peptides which were
highly overexpressed in the tumor tissue of CLL patients with
various HLA-A HLA-B and HLA-C alleles. It may contain MHC class I
and MHC class II peptides or elongated MHC class I peptides. In
addition to the tumor associated peptides collected from several
CLL tissues, the warehouse may contain HLA-A*02 and HLA-A*24 marker
peptides. These peptides allow comparison of the magnitude of
T-cell immunity induced by TUMAPS in a quantitative manner and
hence allow important conclusion to be drawn on the capacity of the
vaccine to elicit anti-tumor responses. Secondly, they function as
important positive control peptides derived from a "non-self"
antigen in the case that any vaccine-induced T-cell responses to
TUMAPs derived from "self" antigens in a patient are not observed.
And thirdly, it may allow conclusions to be drawn, regarding the
status of immunocompetence of the patient.
[0340] TUMAPs for the warehouse are identified by using an
integrated functional genomics approach combining gene expression
analysis, mass spectrometry, and T-cell immunology
(XPresident.RTM.). The approach assures that only TUMAPs truly
present on a high percentage of tumors but not or only minimally
expressed on normal tissue, are chosen for further analysis. For
initial peptide selection, CLL 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 (CLL) 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 expressed or
over-expressed genes as detected in step 2 were considered suitable
TUMAP candidates for a multi-peptide vaccine. 4. Literature
research was performed in order to identify additional evidence
supporting the relevance of the identified peptides as TUMAPs 5.
The relevance of over-expression at the mRNA level was confirmed by
redetection of selected TUMAPs from step 3 on tumor tissue and lack
of (or infrequent) detection on healthy tissues. 6. In order to
assess, whether an induction of in vivo T-cell responses by the
selected peptides may be feasible, in vitro immunogenicity assays
were performed using human T cells from healthy donors as well as
from CLL patients.
[0341] In an aspect, the peptides are pre-screened for
immunogenicity before being included in the warehouse. By way of
example, and not limitation, the immunogenicity of the peptides
included in the warehouse is determined by a method comprising in
vitro T-cell priming through repeated stimulations of CD8+ T cells
from healthy donors with artificial antigen presenting cells loaded
with peptide/MHC complexes and anti-CD28 antibody.
[0342] This method is preferred for rare cancers and patients with
a rare expression profile. In contrast to multi-peptide cocktails
with a fixed composition as currently developed, the warehouse
allows a significantly higher matching of the actual expression of
antigens in the tumor with the vaccine. Selected single or
combinations of several "off-the-shelf" peptides will be used for
each patient in a multitarget approach. In theory, an approach
based on selection of e.g. 5 different antigenic peptides from a
library of 50 would already lead to approximately 17 million
possible drug product (DP) compositions.
[0343] In one aspect, the peptides are selected for inclusion in
the vaccine based on their suitability for the individual patient
based on the method according to the present invention as described
herein, or as below.
[0344] The HLA phenotype, transcriptomic and peptidomic data is
gathered from the patient's tumor material, and blood samples to
identify the most suitable peptides for each patient containing
"warehouse" and patient-unique (i.e. mutated) TUMAPs. Those
peptides will be chosen, which are selectively or over-expressed in
the patients' tumor and, where possible, show strong in vitro
immunogenicity if tested with the patients' individual PBMCs.
[0345] Preferably, the peptides included in the vaccine are
identified by a method comprising: (a) identifying tumor-associated
peptides (TUMAPs) presented by a tumor sample from the individual
patient; (b) comparing the peptides identified in (a) with a
warehouse (database) of peptides as described above; and (c)
selecting at least one peptide from the warehouse (database) that
correlates with a tumor-associated peptide identified in the
patient. For example, the TUMAPs presented by the tumor sample are
identified by: (a1) comparing expression data from the tumor sample
to expression data from a sample of normal tissue corresponding to
the tissue type of the tumor sample to identify proteins that are
over-expressed or aberrantly expressed in the tumor sample; and
(a2) correlating the expression data with sequences of MHC ligands
bound to MHC class I and/or class II molecules in the tumor sample
to identify MHC ligands derived from proteins over-expressed or
aberrantly expressed by the tumor. Preferably, the sequences of MHC
ligands are identified by eluting bound peptides from MHC molecules
isolated from the tumor sample, and sequencing the eluted ligands.
Preferably, the tumor sample and the normal tissue are obtained
from the same patient.
[0346] In addition to, or as an alternative to, selecting peptides
using a warehousing (database) model, TUMAPs may be identified in
the patient de novo, and then included in the vaccine. As one
example, candidate TUMAPs may be identified in the patient by (a1)
comparing expression data from the tumor sample to expression data
from a sample of normal tissue corresponding to the tissue type of
the tumor sample to identify proteins that are over-expressed or
aberrantly expressed in the tumor sample; and (a2) correlating the
expression data with sequences of MHC ligands bound to MHC class I
and/or class II molecules in the tumor sample to identify MHC
ligands derived from proteins over-expressed or aberrantly
expressed by the tumor. As another example, proteins may be
identified containing mutations that are unique to the tumor sample
relative to normal corresponding tissue from the individual
patient, and TUMAPs can be identified that specifically target the
mutation. For example, the genome of the tumor and of corresponding
normal tissue can be sequenced by whole genome sequencing: For
discovery of non-synonymous mutations in the protein-coding regions
of genes, genomic DNA and RNA are extracted from tumor tissues and
normal non-mutated genomic germline DNA is extracted from
peripheral blood mononuclear cells (PBMCs). The applied NGS
approach is confined to the re-sequencing of protein coding regions
(exome re-sequencing). For this purpose, exonic DNA from human
samples is captured using vendor-supplied target enrichment kits,
followed by sequencing with e.g. a HiSeq2000 (Illumina).
Additionally, tumor mRNA is sequenced for direct quantification of
gene expression and validation that mutated genes are expressed in
the patients' tumors. The resultant millions of sequence reads are
processed through software algorithms. The output list contains
mutations and gene expression. Tumor-specific somatic mutations are
determined by comparison with the PBMC-derived germline variations
and prioritized. The de novo identified peptides can then be tested
for immunogenicity as described above for the warehouse, and
candidate TUMAPs possessing suitable immunogenicity are selected
for inclusion in the vaccine.
[0347] In one exemplary embodiment, the peptides included in the
vaccine are identified by: (a) identifying tumor-associated
peptides (TUMAPs) presented by a tumor sample from the individual
patient by the method as described above; (b) comparing the
peptides identified in a) with a warehouse of peptides that have
been prescreened for immunogenicity and overpresentation in tumors
as compared to corresponding normal tissue; (c) selecting at least
one peptide from the warehouse that correlates with a
tumor-associated peptide identified in the patient; and (d)
optionally, selecting at least one peptide identified de novo in
(a) confirming its immunogenicity.
[0348] 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.
[0349] Once the peptides for a personalized peptide based vaccine
are selected, the vaccine is produced. The vaccine preferably is a
liquid formulation consisting of the individual peptides dissolved
in between 20-40% DMSO, preferably about 30-35% DMSO, such as about
33% DMSO.
[0350] Each peptide to be included into a product is dissolved in
DMSO. The concentration of the single peptide solutions has to be
chosen depending on the number of peptides to be included into the
product. The single peptide-DMSO solutions are mixed in equal parts
to achieve a solution containing all peptides to be included in the
product with a concentration of .about.2.5 mg/ml per peptide. The
mixed solution is then diluted 1:3 with water for injection to
achieve a concentration of 0.826 mg/ml per peptide in 33% DMSO. The
diluted solution is filtered through a 0.22 .mu.m sterile filter.
The final bulk solution is obtained.
[0351] Final bulk solution is filled into vials and stored at
-20.degree. C. until use. One vial contains 700 .mu.L solution,
containing 0.578 mg of each peptide. Of this, 500 .mu.L (approx.
400 .mu.g per peptide) will be applied for intradermal
injection.
[0352] In addition to being useful for treating cancer, the
peptides of the present invention are also useful as diagnostics.
Since the peptides were generated from CLL cells and since it was
determined that these peptides are not or at lower levels present
in normal tissues, these peptides can be used to diagnose the
presence of a cancer.
[0353] 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 CLL. Presence of groups of peptides
can enable classification or sub-classification of diseased
tissues.
[0354] The detection of peptides on diseased tissue specimen can
enable the decision about the benefit of therapies involving the
immune system, especially if T-lymphocytes are known or expected to
be involved in the mechanism of action. Loss of MHC expression is a
well described mechanism by which infected of malignant cells
escape immuno-surveillance. Thus, presence of peptides shows that
this mechanism is not exploited by the analyzed cells.
[0355] The peptides of the present invention might be used to
analyze lymphocyte responses against those peptides such as T cell
responses or antibody responses against the peptide or the peptide
complexed to MHC molecules. These lymphocyte responses can be used
as prognostic markers for decision on further therapy steps. These
responses can also be used as surrogate response markers in
immunotherapy approaches aiming to induce lymphocyte responses by
different means, e.g. vaccination of protein, nucleic acids,
autologous materials, adoptive transfer of lymphocytes. In gene
therapy settings, lymphocyte responses against peptides can be
considered in the assessment of side effects. Monitoring of
lymphocyte responses might also be a valuable tool for follow-up
examinations of transplantation therapies, e.g. for the detection
of graft versus host and host versus graft diseases.
[0356] The present invention will now be described in the following
examples which describe preferred embodiments thereof, and with
reference to the accompanying figures, nevertheless, without being
limited thereto. For the purposes of the present invention, all
references as cited herein are incorporated by reference in their
entireties.
FIGURES
[0357] FIGS. 1A to 1V show the overpresentation of various peptides
in normal tissues (white bars) and CLL (black bars). FIG. 1A) Gene:
IGHM, Peptide: ALHRPDVYL (SEQ ID NO.: 2). Tissues from left to
right: 2 adipose tissues, 3 adrenal glands, 4 blood cells, 10 blood
vessels, 6 bone marrows, 7 brains, 6 breasts, 2 cartilages, 2 eyes,
3 gallbladders, 6 hearts, 14 kidneys, 19 large intestines, 20
livers, 45 lungs, 6 lymph nodes, 7 nerves, 3 ovaries, 10
pancreases, 3 parathyroid glands, 1 peritoneum, 5 pituitary glands,
6 placentas, 3 pleuras, 3 prostates, 7 salivary glands, 5 skeletal
muscles, 6 skins, 4 small intestines, 9 spleens, 5 stomachs, 6
testes, 2 thymi, 3 thyroid glands, 9 tracheas, 3 ureters, 6 urinary
bladders, 2 uteri, 6 esophagi, 17 chronic lymphocytic leukemia
samples. The peptide has additionally been detected on 2/21
non-Hodgkin lymphoma samples. FIG. 1B) Gene: IGHM, Peptide:
VIAELPPKV (SEQ ID NO.: 3). Tissues from left to right: 2 adipose
tissues, 3 adrenal glands, 4 blood cells, 10 blood vessels, 6 bone
marrows, 7 brains, 6 breasts, 2 cartilages, 2 eyes, 3 gallbladders,
6 hearts, 14 kidneys, 19 large intestines, 20 livers, 45 lungs, 6
lymph nodes, 7 nerves, 3 ovaries, 10 pancreases, 3 parathyroid
glands, 1 peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3
prostates, 7 salivary glands, 5 skeletal muscles, 6 skins, 4 small
intestines, 9 spleens, 5 stomachs, 6 testes, 2 thymi, 3 thyroid
glands, 9 tracheas, 3 ureters, 6 urinary bladders, 2 uteri, 6
esophagi, 17 chronic lymphocytic leukemia samples. The peptide has
additionally been detected on 5/21 non-Hodgkin lymphoma samples and
1/90 lung cancers. FIG. 1C), Gene: IGHM, Peptide: VIAELPPKVSV (SEQ
ID No.: 4), Tissues from left to right: 2 adipose tissues, 3
adrenal glands, 4 blood cells, 10 blood vessels, 6 bone marrows, 7
brains, 6 breasts, 2 cartilages, 2 eyes, 3 gallbladders, 6 hearts,
14 kidneys, 19 large intestines, 20 livers, 45 lungs, 6 lymph
nodes, 7 nerves, 3 ovaries, 10 pancreases, 3 parathyroid glands, 1
peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3
prostates, 7 salivary glands, 5 skeletal muscles, 6 skins, 4 small
intestines, 9 spleens, 5 stomachs, 6 testes, 2 thymi, 3 thyroid
glands, 9 tracheas, 3 ureters, 6 urinary bladders, 2 uteri, 6
esophagi, 17 chronic lymphocytic leukemia samples. FIG. 1D), Gene:
NUP210, Peptide: RLYEITIEV (SEQ ID No.: 121). Samples from left to
right: 1 PBMC culture, 1 benign prostate, 8 normal tissues (3
lungs, 4 spleens, 1 trachea), 49 cancer tissues (2 brain cancers, 2
breast cancers, 1 colon cancer, 13 leukocytic leukemia cancers, 13
lung cancers, 8 lymph node cancers, 1 myeloid cells cancer, 4
ovarian cancers, 2 skin cancers, 1 urinary bladder cancer, 2 uterus
cancers). Discrepancies regarding the list of tumor types between
FIG. 1D and table 4 may be due to the more stringent selection
criteria applied in table 4 (for details please refer to table 4).
The normal tissue panel and the cancer cell lines and xenografts
tested were the same as in FIGS. 1A-1C. FIG. 1E), Gene: COBRA1,
Peptide: ALLRLLPGL (SEQ ID No.: 14). Samples from left to right: 1
cell line, 11 cancer tissues (1 breast cancer, 1 colon cancer, 1
head and neck cancer, 2 leukocytic leukemia cancers, 1 liver
cancer, 1 lung cancer, 2 lymph node cancers, 1 myeloid cell cancer,
1 skin cancer). FIG. 1F), Gene: KIAA0226L, Peptide: GIIDGSPRL (SEQ
ID No.: 62). Samples from left to right: 2 normal tissues (1 lymph
node, 1 spleen), 18 cancer tissues (2 colon cancers, 9 leukocytic
leukemia cancers, 6 lymph node cancers, 1 rectum cancer). FIG. 1G),
Gene: DDX3X, Peptide: GLDQQFAGLDL (SEQ ID No.: 68). Samples from
left to right: 1 cell line, 2 primary cultures, 21 cancer tissues
(1 bile duct cancer, 1 brain cancer, 1 breast cancer, 1 head and
neck cancer, 2 kidney cancers, 3 leukocytic leukemia cancers, 2
liver cancers, 2 lung cancers, 2 lymph node cancers, 3 ovarian
cancers, 1 rectum cancer, 1 skin cancer, 1 uterus cancer). FIG.
1H), Gene: LIG1, Peptide: KTLDVDATYEI (SEQ ID No.: 112). Samples
from left to right: 2 cell lines, 1 primary culture, 13 cancer
tissues (1 esophageal cancer, 2 leukocytic leukemia cancers, 1
liver cancer, 3 lung cancers, 2 lymph node cancers, 2 ovarian
cancers, 1 skin cancer, 1 uterus cancer). FIG. 1I), Gene: SMCHD1,
Peptide: SIIEGPIIKL (SEQ ID No.: 146). Samples from left to right:
14 cancer tissues (1 head and neck cancer, 3 leukocytic leukemia
cancers, 6 lung cancers, 1 lymph node cancer, 1 ovarian cancer, 2
urinary bladder cancers). FIG. 1J), Gene: MMS22L, Peptide:
SILETVATL (SEQ ID No.: 147). Samples from left to right: 1 primary
culture, 11 cancer tissues (1 colon cancer, 4 leukocytic leukemia
cancers, 1 lung cancer, 3 lymph node cancers, 1 myeloid cell
cancer, 1 skin cancer). FIG. 1K), Gene: FCRL3, Peptide: SLLAELHVL
(SEQ ID No.: 153). Samples from left to right: 1 cell line, 9
normal tissues (1 lymph node, 1 rectum, 7 spleens), 27 cancer
tissues (1 breast cancer, 1 kidney cancer, 10 leukocytic leukemia
cancers, 14 lymph node cancers, 1 skin cancer). FIG. 1L), Gene:
FAM126A, Peptide: SLQEEKLIYV (SEQ ID No.: 159). Samples from left
to right: 2 cell lines, 23 cancer tissues (5 brain cancers, 1
breast cancer, 1 esophageal cancer, 3 head and neck cancers, 2
leukocytic leukemia cancers, 5 lung cancers, 3 lymph node cancers,
1 skin cancer, 2 urinary bladder cancers). FIG. 1M), Gene: HNRNPC,
Peptide: SVHKGFAFV (SEQ ID No.: 164). Samples from left to right:
10 cell lines, 1 primary culture, 1 normal tissue (1 adrenal
gland), 36 cancer tissues (12 brain cancers, 1 colon cancer, 2 head
and neck cancers, 2 kidney cancers, 3 leukocytic leukemia cancers,
1 liver cancer, 5 lung cancers, 1 myeloid cell cancer, 1 ovarian
cancer, 1 rectum cancer, 1 skin cancer, 2 stomach cancers, 2
urinary bladder cancers, 2 uterus cancers). FIG. 1N), Gene:
RASGRF1, Peptide: TLDTSKLYV (SEQ ID No.: 167). Samples from left to
right: 13 cancer tissues (1 brain cancer, 8 leukocytic leukemia
cancers, 4 lymph node cancers). FIG. 1O), Gene: RASGRF1, Peptide:
YLLDQSFVM (SEQ ID No.: 168). Samples from left to right: 16 cancer
tissues (10 leukocytic leukemia cancers, 6 lymph node cancers).
FIG. 1P), Gene: RNF213, Peptide: YIQEYLTLL (SEQ ID No.: 192).
Samples from left to right: 2 cell lines, 18 cancer tissues (1
colon cancer, 1 head and neck cancer, 1 kidney cancer, 4 leukocytic
leukemia cancers, 5 lung cancers, 3 lymph node cancers, 1 myeloid
cell cancer, 1 stomach cancer, 1 urinary bladder cancer). FIG. 1Q),
Gene: DCK, Peptide: YLQEVPILTL (SEQ ID No.: 197). Samples from left
to right: 11 cancer tissues (5 leukocytic leukemia cancers, 4 lymph
node cancers, 1 skin cancer, 1 uterus cancer). FIG. 1R), Gene:
EIF3H, Peptide: YMFEEVPIVI (SEQ ID No.: 200). Samples from left to
right: 4 cell lines, 2 primary cultures, 34 cancer tissues (1 bile
duct cancer, 2 breast cancers, 1 colon cancer, 1 esophageal cancer,
1 gallbladder cancer, 5 head and neck cancers, 2 leukocytic
leukemia cancers, 2 liver cancers, 9 lung cancers, 4 lymph node
cancers, 2 ovarian cancers, 1 rectum cancer, 2 skin cancers, 1
urinary bladder cancer). FIG. 1S), Gene: UBLCP1, Peptide: LIDVKPLGV
(SEQ ID No.: 211). Samples from left to right: 3 cell lines, 23
cancer tissues (5 brain cancers, 1 breast cancer, 1 esophageal
cancer, 1 head and neck cancer, 1 kidney cancer, 3 leukocytic
leukemia cancers, 2 liver cancers, 2 lung cancers, 4 lymph node
cancers, 1 ovarian cancer, 1 prostate cancer, 1 skin cancer). FIG.
1T), Gene: TMCO6, Peptide: QLLPVSNVVSV (SEQ ID No.: 256). Samples
from left to right: 2 primary cultures, 13 cancer tissues (2 head
and neck cancers, 3 leukocytic leukemia cancers, 1 liver cancer, 2
lung cancers, 4 lymph node cancers, 1 ovarian cancer). FIG. 1U),
Gene: MY1OG, Peptide: LLYNSTDPTL (SEQ ID No.: 312). Samples from
left to right: 1 cell line, 2 primary cultures, 14 cancer tissues
(1 colon cancer, 4 leukocytic leukemia cancers, 2 lung cancers, 5
lymph node cancers, 1 ovarian cancer, 1 urinary bladder cancer).
FIG. 1V), Gene: PIM1, Peptide: VLLPQETAEIHL (SEQ ID No.: 328).
Samples from left to right: 3 primary cultures, 27 cancer tissues
(1 breast cancer, 1 colon cancer, 5 leukocytic leukemia cancers, 5
lung cancers, 7 lymph node cancers, 2 ovarian cancers, 2 skin
cancers, 1 stomach cancer, 3 urinary bladder cancers).
[0358] FIGS. 2A to 2D show exemplary expression profiles of source
genes of the present invention that are highly over-expressed or
exclusively expressed in CLL in a panel of normal tissues (white
bars) and 17 CLL samples (black bars). Tissues from left to right:
6 arteries, 1 blood cells, 1 brain, 1 heart, 2 livers, 2 lungs, 2
veins, 1 adipose tissue, 1 adrenal gland, 6 bone marrows, 1
cartilage, 1 colon, 1 esophagus, 2 eyes, 2 gallbladders, 1 kidney,
6 lymph nodes, 5 pancreases, 2 pituitary glands, 1 rectum, 1
salivary gland, 1 skeletal muscle, 1 skin, 1 small intestine, 1
spleen, 1 stomach, 1 thyroid gland, 7 tracheas, 1 urinary bladder,
1 breast, 5 ovaries, 3 placentas, 1 prostate, 1 testis, 1 thymus, 1
uterus, 10 chronic lymphocytic leukemia samples. FIG. 2A) Gene
symbol: FCER2; FIG. 2B) Gene symbol: KIAA0226L; FIG. 2C) Gene
symbol: PAX5; FIG. 2D) Gene symbol: CLEC17A.
[0359] FIGS. 3A-3F show exemplary immunogenicity data: flow
cytometry results after peptide-specific multimer staining. CD8+ T
cells were primed using artificial APCs coated with anti-CD28 mAb
and HLA-A*02 in complex with SeqID No 1 peptide (FIG. 3C, left
panel), SeqID No 5 peptide (FIG. 3D left panel), SeqID No 32
peptide (FIG. 3E, left panel) and SeqID No 220 peptide (FIG. 3F,
left panel), respectively. After three cycles of stimulation, the
detection of peptide-reactive cells was performed by 2D multimer
staining with A*02/SeqID No 1 (FIG. 3C), A*02/SeqID No 5 (D),
A*02/SeqID No 32 (FIG. 3E) or A*02/SeqID No 220 (FIG. 3F). Right
panels (FIGS. 3C, 3D, 3E and 3F) show control staining of cells
stimulated with irrelevant A*02/peptide complexes. Viable singlet
cells were gated for CD8+ lymphocytes. Boolean gates helped
excluding false-positive events detected with multimers specific
for different peptides. Frequencies of specific multimer+ cells
among CD8+ lymphocytes are indicated.
EXAMPLES
Example 1
Identification and Quantitation of Tumor Associated Peptides
Presented on the Cell Surface
Tissue Samples
[0360] Patients' tumor tissues were obtained from: ProteoGenex Inc.
(Culver City, Calif., USA); University Hospital Bonn (Bonn,
Germany); University Hospital Tubingen (Tubingen, Germany).
[0361] Normal tissues were obtained from Asterand (Detroit, Mich.,
USA & Royston, Herts, UK); Bio-Options Inc. (Brea, Calif.,
USA); BioServe (Beltsville, Md., USA); Capital BioScience Inc.
(Rockville, Md., USA); Geneticist Inc. (Glendale, Calif., USA);
Kyoto Prefectural University of Medicine (KPUM) (Kyoto, Japan);
ProteoGenex Inc. (Culver City, Calif., USA); Tissue Solutions Ltd
(Glasgow, UK); University Hospital Geneva (Geneva, Switzerland);
University Hospital Heidelberg (Heidelberg, Germany); University
Hospital Munich (Munich, Germany); University Hospital Tubingen
(Tubingen, Germany).
[0362] Written informed consents of all patients had been given
before surgery or autopsy. Tissues were shock-frozen immediately
after excision and stored until isolation of TUMAPs at -70.degree.
C. or below.
Isolation of HLA Peptides from Tissue Samples
[0363] HLA peptide pools from shock-frozen tissue samples were
obtained by immune precipitation from solid tissues according to a
slightly modified protocol (Falk et al., 1991; Seeger et al., 1999)
using the HLA-A*02-specific antibody BB7.2, the HLA-A, --B,
C-specific antibody W6/32, CNBr-activated sepharose, acid
treatment, and ultrafiltration.
Mass Spectrometry Analyses
[0364] The HLA peptide pools as obtained were separated according
to their hydrophobicity by reversed-phase chromatography
(nanoAcquity UPLC system, Waters) and the eluting peptides were
analyzed in LTQ-velos and fusion hybrid mass spectrometers
(ThermoElectron) equipped with an ESI source. Peptide pools were
loaded directly onto the analytical fused-silica micro-capillary
column (75 .mu.m i.d..times.250 mm) packed with 1.7 .mu.m C18
reversed-phase material (Waters) applying a flow rate of 400 nL per
minute. Subsequently, the peptides were separated using a two-step
180 minute-binary gradient from 10% to 33% B at a flow rate of 300
nL per minute. The gradient was composed of Solvent A (0.1% formic
acid in water) and solvent B (0.1% formic acid in acetonitrile). A
gold coated glass capillary (PicoTip, New Objective) was used for
introduction into the nanoESl source. The LTQ-Orbitrap mass
spectrometers were operated in the data-dependent mode using a TOP5
strategy. In brief, a scan cycle was initiated with a full scan of
high mass accuracy in the orbitrap (R=30 000), which was followed
by MS/MS scans also in the orbitrap (R=7500) on the 5 most abundant
precursor ions with dynamic exclusion of previously selected ions.
Tandem mass spectra were interpreted by SEQUEST and additional
manual control. The identified peptide sequence was assured by
comparison of the generated natural peptide fragmentation pattern
with the fragmentation pattern of a synthetic sequence-identical
reference peptide.
[0365] Label-free relative LC-MS quantitation was performed by ion
counting i.e. by extraction and analysis of LC-MS features (Mueller
et al., 2007). The method assumes that the peptide's LC-MS signal
area correlates with its abundance in the sample. Extracted
features were further processed by charge state deconvolution and
retention time alignment (Mueller et al., 2008; Sturm et al.,
2008). Finally, all LC-MS features were cross-referenced with the
sequence identification results to combine quantitative data of
different samples and tissues to peptide presentation profiles. The
quantitative data were normalized in a two-tier fashion according
to central tendency to account for variation within technical and
biological replicates. Thus each identified peptide can be
associated with quantitative data allowing relative quantification
between samples and tissues. In addition, all quantitative data
acquired for peptide candidates was inspected manually to assure
data consistency and to verify the accuracy of the automated
analysis. For each peptide, a presentation profile was calculated
showing the mean sample presentation as well as replicate
variations. The profiles juxtapose CLL samples to a baseline of
normal tissue samples. Presentation profiles of exemplary
over-presented peptides are shown in FIGS. 1A-1V. Presentation
scores for exemplary peptides are shown in Table 8.
TABLE-US-00009 TABLE 8 Presentation scores. The table lists
peptides that are very highly over-presented on tumors compared to
a panel of normal tissues (+++), highly over-presented on tumors
compared to a panel of normal tissues (++) or over-presented on
tumors compared to a panel of normal tissues (+). The panel of
normal tissues considered relevant for comparison with tumors
consisted of: adipose tissue, adrenal gland, blood cells, blood
vessel, bone marrow, brain, breast, esophagus, eye, gallbladder,
heart, kidney, large intestine, liver, lung, lymph node, nerve,
pancreas, parathyroid gland, peritoneum, pituitary, pleura,
salivary gland, skeletal muscle, skin, small intestine, spleen,
stomach, thymus, thyroid gland, trachea, ureter, urinary bladder.
SEQ ID No. Sequence Peptide Presentation 1 AIPPSFASIFL +++ 2
ALHRPDVYL +++ 3 VIAELPPKV +++ 4 VIAELPPKVSV +++ 5 ALIFKIASA +++ 6
ALDTLEDDMTI +++ 7 ALLERTGYTL +++ 8 ALAASALPALV +++ 9 ALCDTLITV +++
10 FVYGESVEL +++ 11 ALFTFJPLTV +++ 12 ALGEDEITL +++ 13 VVDGMPPGV ++
14 ALLRLLPGL + 15 ALPEVSVEA + 16 ALPGGAAVAAV ++ 17 ALTKTNLQL +++ 18
LLGEFSIKM +++ 19 QVMEKLAAV + 20 ALVDPGPDFVV + 21 ALWAGLLTL +++ 22
ALWDPVIEL +++ 23 ALYLTEVFL +++ 24 AMAGDVVYA +++ 25 ATYSGLESQSV + 27
AVLGLVWLL ++ 28 AVLHLLLSV +++ 29 AVLQAVTAV +++ 30 ELLEGSEIYL + 31
ELMHGVAGV ++ 32 FIDKFTPPV +++ 33 FIINSSNIFL +++ 35 FILPSSLYL +++ 36
FILTH VDQL +++ 37 FIMEGGAMVL +++ 39 FLDALLTDV +++ 40 FLDEDDMSL + 41
FLDPSLDPLL +++ 43 FLLGPEALSFA +++ 45 FLPELPADLEA ++ 47 FLSDQPEPYL
++ 48 FLSPQQPPLL +++ 49 FLTDLFAQL + 50 FLFEPVVKAFL +++ 51
FLVEAPHDWDL ++ 52 FLVETGFLHV +++ 53 FLWQHVELVL +++ 54 FLYPFPLALF
+++ 56 FVFEAPYTL +++ 57 GLSEISLRL +++ 58 YIQQGIFSV ++ 59
FVFGDENGTVSL +++ 60 FVLDHEDGLNL ++ 61 FVYFIVREV +++ 62 GIIDGSPRL +
63 SLAHVAGCEL +++ 64 KLLESVASA ++ 65 GLDDMKANL +++ 66 SLAGGLDDMKA
+++ 68 GLDQQFAGLDL +++ 69 GLHQREIFL +++ 70 FVPDTPVGV ++ 71
GLKHDIARV +++ 72 GLLDAGKMYV +++ 73 GLLEVISALQL +++ 74 GLLRASFLL +++
76 GLLRIIPYL ++ 77 GLLRLTWFL ++ 78 GLPSFLTEV +++ 79 GLQAKIQEA +++
80 VLIEDELEEL +++ 81 WLVGQEFEL +++ 82 GLQSGVDIGV + 83 GQGEVLVYV +
86 HLYPGAVTI +++ 88 ILDEIGADVQA +++ 89 ILDFGTFQL +++ 90 VIADLGLIRV
+++ 92 ILEPLNPLL +++ 93 ILFNTQINI +++ 94 ILFPLRFTL +++ 95
ILGYMAHEHKV +++ 96 ILIDKTSFV + 97 LLFATQITL + 98 SLIKYFLFV +++ 99
ILIFHSVAL +++ 100 ILNNEVFAI + 101 ILVVIEPLL +++ 102 IQDRAVPSL +++
103 KLGGTPAPA ++ 104 KLILLDTPLFL +++ 105 KLMNDIADI +++ 106
FMASHLDYL +++ 107 ILYNLYDLL ++ 108 VIYTLIHYI + 109 KLWEGLTELV ++
112 KTLDVDATYEI +++ 113 KVPAEEVLVAV + 114 LIPEGPPQV +++ 115
LLFDKLYLL +++ 116 LLIGATMQV +++ 117 LLILENILL ++ 118 RLLILENILL +++
119 VLPAEFFEV +++ 121 RLYEITIEV ++ 122 LLIPVVPGV ++ 123 LLLAEAELLTL
+++ 124 LLLEETEKQAV ++ 125 LLLEIGEVGKLFV ++ 126 LLPEGGITAI +++ 127
LLPTAPTTV +++ 128 LLSEEEYHL +++ 129 LLVGTLDVV + 130 LLVLIPVYL +++
131 LQALEVLKI +++ 132 LVYEAIIMV +++ 133 YLLSGDISEA +++ 134
MLLEHGITLV +++ 135 MTAGFSTIAGSV +++ 137 NLIKTVIKL + 138 NLLDIDAPVTV
++ 139 NLTDVVEKL +++ 140 QIAELPATSV +++ 141 QILSEIVEA ++ 142
QLDEPAPQV +++ 144 QLPPFPREL +++ 145 SALDTITTV +++ 146 SIIEGPIIKL
+++ 147 SILETVATL +++ 148 SIVASLITV +++ 149 SLDNGGYYI +++ 153
SLLAELHVL ++ 154 SLLAELHVLTV ++ 155 SLMLEVPAL +++ 157 SLNIRDFTM +++
160 SLSFLVPSL +++ 162 SMKDDLENV +++ 163 SQLDISEPYKV + 164 SVHKGFAFV
+++ 165 TLDDDLDTV +++ 166 TLDPNQVSL +++ 167 TLDTSKLYV +++ 168
YLLDQSFVM +++ 169 TLLLGLTEV +++ 170 TLTFRVETV +++ 171 TLVPPAALISI
+++ 172 TLYDMLASI +++ 174 VLAELPIIVV +++ 175 FTVPRVVAV ++ 176
VLAEQNIIPSA +++ 177 VLDDRELLL +++ 178 VLFFNVQEV +++ 179 VLLGLEMTL
+++ 181 VLLSIPFVSV + 182 VLLSVPGPPV +++ 185 VMDDQRDLI + 186
VMDPTKILI +++ 187 VMDTHLVNI +++ 189 VMLEMTPEL +++ 190 VVMGTVPRL +++
191 YIFDGSDGGV +++ 192 YIQEYLTLL +++ 193 YLDLSNNRL +++ 194
YLDNVLAEL + 195 YLGGFALSV +++ 197 YLQEVPILTL +++ 198 YLTFLPAEV +++
199 YLVELSSLL +++ 200 YMFEEVPIVI +++ 201 YQLELHGIEL +++ 202
YVDDVFLRV +++ 204 GLLQINDKIAL ++ 205 GLSQANFTL + 206 HMQDVRVLL +++
208 ILLKTEGINL ++ 209 ILQAELPSL ++ 210 KLLVQDFFL +++ 211 LIDVKPLGV
+++ 212 NIIEAINELLV ++ 213 RLLYQLVFL ++ 214 RLQELTEKL + 215
VMQDIVYKL + 217 ALDEPPYLTV + 218 ALGEEWKGYVV + 219 ALLNLLESA ++ 221
ALVSTIIMV + 225 ILQERELLPV + 226 AMNISVPQV + 227 FLAEASVMTQL ++ 228
FLGGLSPGV ++ 229 FLLNLQNCHL ++ 231 FLYIRQLAI + 232 FMHQIIDQV + 234
GLDDAEYAL + 235 GLDDLLLFL + 236 GLLESGRHYL +++ 237 GLQENLDVVV + 238
GLVETELQL + 240 ILARDILEI ++ 241 ILGDILLKV + 242 ILLGIQELL ++ 243
ILPTLEKELFL + 244 ILQALAVHL ++ 245 KIMDYSLLLGV + 248 KTVEPPISQV +
249 LLPTGVFQV + 251 LLYDNVPGA + 254 QLIPKLIFL +++ 255 YLFEEAISM +
258 RLDYITAEI ++ 259 RLLDEQFAVL + 260 SLDDVEGMSV ++ 261 SLVEAQGWLV
++ 263 SQWEDIHVV ++ 264 TILDYINVV +++ 266 TLLDQLDTQL + 267
TLLDWQDSL + 268 TLLQVFHLL ++ 270 TVLPVPPLSV +++ 271 VIRNIVEAA ++
273 VLGEYSYLL + 275 VLLFIEHSV + 276 VLNDGAPNV + 277 VMILKLPFL + 278
YLDDLLPKL ++ 280 YTLDSLYWSV + 281 NLLDDRGMTAL +++ 282 LLRDGIELV +++
283 ILQPMDIHV +++
284 LLSAAEPVPA +++ 286 FLLEDLSQKL +++ 287 FLWEEKFNSL +++ 289
ILEEQPMDMLL +++ 290 LANPHELSL +++ 291 ILLNEDDLVTI +++ 292 AAALIIHHV
+ 294 ALLDQLHTLL + 297 FLVEPQEDTRL ++ 298 IILPVEVEV +++ 299
ILEENIPVL ++ 300 ILLNPAYDVYL ++ 302 ILQDLTFVHL + 304 LAIVPVNTL +++
305 LLFPQIEGIKI + 307 LLLTKPTEA ++ 308 SLYDVSRMYV +++ 309 ILYGTQFVL
+ 310 LLSTLHLLV + 311 LLVDVEPKV + 312 LLYNSTDPTL +++ 314 LMKDCEAEV
++ 316 MLLEHGITL +++ 317 NLLAHIWAL + 318 NLQVTQPTV +++ 320
TIAPVTVAV + 322 SLASIHVPL +++ 323 SLDLFNCEV +++ 327 VLIKWFPEV +++
328 VLLPQETAEIHL ++ 329 VLMDGSVKL + 330 VLMWEIYSL ++ 331
VLWELAHLPTL +++ 332 VMIQHVENL +++ 334 YLLEEKIASL +++ 336 YMAVTTQEV
+++ 337 YMYEKESEL ++ 338 FLDMTNWNL + 339 GLWGTVVNI + 340 KLLEEICNL
++ 341 LLAELPASVHA ++ 342 SLITPLQAV +++ 343 TLLEALDCI ++ 344
VLAFENPQV ++ 345 YLIEPDVELQRI + 346 VLVQVSPSL +++ 347 YLGPVSPSL +
349 ALATHILSL ++ 350 ALEDRVWEL + 351 ALSEKLARL + 352 ALVFELHYV +
353 ATPMPTPSV ++ 354 FIMDDPAGNSYL + 355 FIWPMLIHI +++ 356 FLHDHQAEL
+ 357 FLIQEIKTL ++ 358 FLTDYLNDL + 359 FMQDPMEVFV ++ 360 HLIDTNKIQL
+ 362 ILTELGGFEV + 363 ITTEVVNELYV + 364 KMDWIFHTI + 365 LISPLLLPV
++ 367 NLWSLVAKV ++ 368 QLQPTDALLCV + 371 SLADDSVLERL + 372
SLFGPLPGPGPALV + 374 VLSVITEEL ++ 375 VLWFKPVEL ++ 377 VVDGTCVAV +
378 YILGKFFAL + 379 YLAELVTPIL + 380 YLDRKLLTL + 382 YLLPLLQRL +++
383 YLLREWVNL +++ 384 YMIGSEVGNYL + 385 YTIPLAIKL +++
Example 2
Expression Profiling of Genes Encoding the Peptides of the
Invention
[0366] Over-presentation or specific presentation of a peptide on
tumor cells compared to normal cells is sufficient for its
usefulness in immunotherapy, and some peptides are tumor-specific
despite their source protein occurring also in normal tissues.
Still, mRNA expression profiling adds an additional level of safety
in selection of peptide targets for immunotherapies. Especially for
therapeutic options with high safety risks, such as
affinity-matured TCRs, the ideal target peptide will be derived
from a protein that is unique to the tumor and not found on normal
tissues.
RNA Sources and Preparation
[0367] Surgically removed tissue specimens were provided as
indicated above (see Example 1) after written informed consent had
been obtained from each patient. Tumor tissue specimens were
snap-frozen immediately after surgery and later homogenized with
mortar and pestle under liquid nitrogen. Total RNA was prepared
from these samples using TRI Reagent (Ambion, Darmstadt, Germany)
followed by a cleanup with RNeasy (QIAGEN, Hilden, Germany); both
methods were performed according to the manufacturer's
protocol.
[0368] Total RNA from healthy human tissues for RNASeq experiments
was obtained from: Asterand (Detroit, Mich., USA & Royston,
Herts, UK); BioCat GmbH (Heidelberg, Germany); BioServe
(Beltsville, Md., USA); Geneticist Inc. (Glendale, Calif., USA);
Istituto Nazionale Tumori "Pascale" (Naples, Italy); ProteoGenex
Inc. (Culver City, Calif., USA); University Hospital Heidelberg
(Heidelberg, Germany).
[0369] Total RNA from tumor tissues for RNASeq experiments was
obtained from: Tissue Solutions Ltd (Glasgow, UK); University
Hospital Bonn (Bonn, Germany)
[0370] 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).
RNAseq Experiments
[0371] Gene expression analysis of--tumor and normal tissue RNA
samples was performed by next generation sequencing (RNAseq) by
CeGaT (Tubingen, Germany). Briefly, sequencing libraries are
prepared using the Illumina HiSeq v4 reagent kit according to the
provider's protocol (Illumina Inc, San Diego, Calif., USA), which
includes RNA fragmentation, cDNA conversion and addition of
sequencing adaptors. Libraries derived from multiple samples are
mixed equimolarly and sequenced on the Illumina HiSeq 2500
sequencer according to the manufacturer's instructions, generating
50 bp single end reads. Processed reads are mapped to the human
genome (GRCh38) using the STAR software. Expression data are
provided on transcript level as RPKM (Reads Per Kilobase per
Million mapped reads, generated by the software Cufflinks) and on
exon level (total reads, generated by the software Bedtools), based
on annotations of the ensembl sequence database (Ensembl77). Exon
reads are normalized for exon length and alignment size to obtain
RPKM values. Exemplary expression profiles of source genes of the
present invention that are highly over-expressed or exclusively
expressed in CLL are shown in FIGS. 3A-3F. Expression scores for
further exemplary genes are shown in Table 9.
TABLE-US-00010 TABLE 9 Expression scores. The table lists peptides
from genes that are very highly over- expressed in tumors compared
to a panel of normal tissues (+++), highly over-expressed in tumors
compared to a panel of normal tissues (++) or over-expressed in
tumors compared to a panel of normal tissues (+). The baseline for
this score was calculated from measurements of the following
relevant normal tissues: adipose tissue, adrenal gland, artery,
blood cells, bone marrow, brain, cartilage, colon, esophagus, eye,
gallbladder, heart, kidney, liver, lung, lymph node, pancreas,
pituitary, rectum, salivary gland, skeletal muscle, skin, small
intestine, spleen, stomach, thyroid gland, trachea, urinary
bladder, and vein. In case expression data for several samples of
the same tissue type were available, the arithmetic mean of all
respective samples was used for the calculation. SEQ ID No Sequence
Gene Expression 1 AIPPSFASIFL ++ 2 ALHRPDVYL ++ 3 VIAELPPKV +++ 4
VIAELPPKVSV +++ 5 ALIFKIASA +++ 9 ALCDTLITV +++ 10 FVYGESVEL +++ 17
ALTKTNLQL +++ 18 LLGEFSIKM +++ 19 QVMEKLAAV +++ 21 ALWAGLLTL +++ 24
AMAGDVVYA +++ 31 ELMHGVAGV +++ 46 YIIDSAQAV ++ 62 GIIDGSPRL +++ 63
SLAHVAGCEL +++ 64 KLLESVASA +++ 65 GLDDMKANL +++ 66 SLAGGLDDMKA +++
71 GLKHDIARV +++ 74 GLLRASFLL ++ 75 GLSIFAQDLRL ++ 85 HLMLHTAAL +
86 HLYPGAVTI +++ 91 ILDLNTYNV + 96 ILIDKTSFV +++ 97 LLFATQITL +++
98 SLIKYFLFV +++ 105 KLMNDIADI ++ 106 FMASHLDYL + 107 ILYNLYDLL ++
108 VIYTLIHYI + 113 KVPAEEVLVAV ++ 116 LLIGATMQV + 117 LLILENILL ++
118 RLLILENILL ++ 119 VLPAEFFEV ++ 120 AIDAALTSV ++ 121 RLYEITIEV
++ 127 LLPTAPTTV + 137 NLIKTVIKL + 146 SIIEGPIIKL +++ 149 SLDNGGYYI
+++ 150 SLFDQPLSII +++ 153 SLLAELHVL +++ 154 SLLAELHVLTV +++ 156
SLNIGDVQL +++ 163 SQLDISEPYKV +++ 177 VLDDRELLL ++ 189 VMLEMTPEL
+++ 193 YLDLSNNRL + 195 YLGGFALSV ++ 205 GLSQANFTL +++ 206
HMQDVRVLL +++ 213 RLLYQLVFL +++ 220 ALPEILFAKV ++ 221 ALVSTIIMV +
223 ALWVSQPPEI ++ 235 GLDDLLLFL + 251 LLYDNVPGA +++ 254 QLIPKLIFL
+++ 255 YLFEEAISM +++ 257 RIINGIIISV +++ 262 SLWNAGTSV ++ 275
VLLFIEHSV ++ 284 LLSAAEPVPA +++ 285 GVATAGCVNEV +++ 286 FLLEDLSQKL
++ 294 ALLDQLHTLL + 299 ILEENIPVL ++ 300 ILLNPAYDVYL ++ 301
AASPIITLV + 307 LLLTKPTEA ++ 308 SLYDVSRMYV + 309 ILYGTQFVL ++ 312
LLYNSTDPTL + 319 QVIPQLQTV ++ 320 TIAPVTVAV ++ 330 VLMWEIYSL ++ 332
VMIQHVENL +++ 337 YMYEKESEL +++ 352 ALVFELHYV +++ 356 FLHDHQAEL +
360 HLIDTNKIQL + 378 YILGKFFAL + 380 YLDRKLLTL ++
Example 3
In Vitro Immunogenicity for MHC Class I Presented Peptides
[0372] In order to obtain information regarding the immunogenicity
of the TUMAPs of the present invention, the inventors performed
investigations using an in vitro T-cell priming assay based on
repeated stimulations of CD8+ T cells with artificial antigen
presenting cells (aAPCs) loaded with peptide/MHC complexes and
anti-CD28 antibody. This way the inventors could show
immunogenicity for HLA-A*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
[0373] In order to perform in vitro stimulations by artificial
antigen presenting cells loaded with peptide-MHC complex (pMHC) and
anti-CD28 antibody, the inventors first isolated CD8+ T cells from
fresh HLA-A*02 leukapheresis products via positive selection using
CD8 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) of
healthy donors obtained from the University clinics Mannheim,
Germany, after informed consent.
[0374] PBMCs and isolated CD8+ lymphocytes were incubated in T-cell
medium (TCM) until use consisting of RPMI-Glutamax (Invitrogen,
Karlsruhe, Germany) supplemented with 10% heat inactivated human AB
serum (PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100
.mu.g/ml Streptomycin (Cambrex, Cologne, Germany), 1 mM sodium
pyruvate (CC Pro, Oberdorla, Germany), 20 .mu.g/ml Gentamycin
(Cambrex). 2.5 ng/ml IL-7 (PromoCell, Heidelberg, Germany) and 10
U/ml IL-2 (Novartis Pharma, N rnberg, Germany) were also added to
the TCM at this step.
[0375] Generation of pMHC/anti-CD28 coated beads, T-cell
stimulations and readout was performed in a highly defined in vitro
system using four different pMHC molecules per stimulation
condition and 8 different pMHC molecules per readout condition.
[0376] The purified co-stimulatory mouse IgG2a anti human CD28 Ab
9.3 (Jung et al., 1987) was chemically biotinylated using
Sulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer
(Perbio, Bonn, Germany). Beads used were 5.6 .mu.m diameter
streptavidin coated polystyrene particles (Bangs Laboratories,
Illinois, USA).
[0377] pMHC used for positive and negative control stimulations
were A*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 523) from
modified Melan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5,
SEQ ID NO. 524), respectively.
[0378] 800.000 beads/200 .mu.I were coated in 96-well plates in the
presence of 4.times.12.5 ng different biotin-pMHC, washed and 600
ng biotin anti-CD28 were added subsequently in a volume of 200
.mu.I. Stimulations were initiated in 96-well plates by
co-incubating 1.times.10.sup.6 CD8+ T cells with 2.times.10.sup.6
washed coated beads in 200 .mu.I TCM supplemented with 5 ng/ml
IL-12 (PromoCell) for 3 days at 37.degree. C. Half of the medium
was then exchanged by fresh TCM supplemented with 80 U/ml IL-2 and
incubating was continued for 4 days at 37.degree. C. This
stimulation cycle was performed for a total of three times. For the
pMHC multimer readout using 8 different pMHC molecules per
condition, a two-dimensional combinatorial coding approach was used
as previously described (Andersen et al., 2012) with minor
modifications encompassing coupling to 5 different fluorochromes.
Finally, multimeric analyses were performed by staining the cells
with Live/dead near IR dye (Invitrogen, Karlsruhe, Germany),
CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany) and
fluorescent pMHC multimers. For analysis, a BD LSRII SORP cytometer
equipped with appropriate lasers and filters was used. Peptide
specific cells were calculated as percentage of total CD8+ cells.
Evaluation of multimeric analysis was done using the FlowJo
software (Tree Star, Oregon, USA). In vitro priming of specific
multimer+CD8+ lymphocytes was detected by comparing to negative
control stimulations. Immunogenicity for a given antigen was
detected if at least one evaluable in vitro stimulated well of one
healthy donor was found to contain a specific CD8+ T-cell line
after in vitro stimulation (i.e. this well contained at least 1% of
specific multimer+ among CD8+ T-cells and the percentage of
specific multimer+ cells was at least 10.times. the median of the
negative control stimulations).
In Vitro Immunogenicity for CLL Peptides
[0379] For tested HLA class I peptides, in vitro immunogenicity
could be demonstrated by generation of peptide specific T-cell
lines. Exemplary flow cytometry results after TUMAP-specific
multimer staining for two peptides of the invention are shown in
FIGS. 3A-3F together with corresponding negative controls. Results
for two peptides from the invention are summarized in Table 10A,
others in Table 10B.
TABLE-US-00011 TABLE 10A in vitro immunogenicity of HLA class I
peptides of the invention Exemplary results of in vitro
immunogenicity experiments conducted by the applicant for the
peptides of the invention. <20% =+; 20% - 49% =++; 50% - 69%
=+++; >= 70% =++++ Seq ID Sequence wells 427 LLYDAVHIV ++ 521
YLYGQTTTYL ++
TABLE-US-00012 TABLE 10B In vitro immunogenicity of additional HLA
class I peptides of the invention Exemplary results of in vitro
immunogenicity experiments conducted by the applicant for HLA-A*02
restricted peptides of the invention. Results of in vitro
immunogenicity experiments are indicated. Percentage of positive
wells and donors (among evaluable) are summarized as indicated
<20% =+; 20% - 49% =++; 50% - 69% =+++; >= 70% =++++ SEQ ID
NO: Sequence Wells positive [%] 1 AIPPSFASIFL "+++" 2 ALHRPDVYL "+"
5 ALIFKIASA "+++" 17 ALTKTNLQL "+" 26 AVLLVLPLV "+" 32 FIDKFTPPV
"++++" 49 FLTDLFAQL "++" 54 FLYPFPLALF "+" 56 FVFEAPYTL "++" 57
GLSEISLRL "+" 69 GLHQREIFL "+" 81 WLVGQEFEL "+" 86 HLYPGAVTI "+" 96
ILIDKTSFV "+" 100 ILNNEVFAI "++" 101 ILVVIEPLL "+" 102 IQDRAVPSL
"+" 112 KTLDVDATYEI "+" 114 LIPEGPPQV "+" 154 SLLAELHVLTV "+" 192
YIQEYLTLL "+" 197 YLQEVPILTL "+" 200 YMFEEVPIVI "+" 201 YQLELHGIEL
"+" 202 YVDDVFLRV "++" 220 ALPEILFAKV "++" 240 ILARDILEI "+" 281
NLLDDRGMTAL "+" 282 LLRDGIELV "++" 307 LLLTKPTEA "+" 320 TIAPVTVAV
"+" 322 SLASIHVPL "+" 323 SLDLFNCEV "+" 329 VLMDGSVKL "+++" 332
VMIQHVENL "+" 380 YLDRKLLTL "++++"
Example 4
Synthesis of Peptides
[0380] All peptides were synthesized using standard and
well-established solid phase peptide synthesis using the
Fmoc-strategy. Identity and purity of each individual peptide have
been determined by mass spectrometry and analytical RP-HPLC. The
peptides were obtained as white to off-white lyophilizates
(trifluoro acetate salt) in purities of >50%. All TUMAPs are
preferably administered as trifluoro-acetate salts or acetate
salts, other salt-forms are also possible.
Example 5
MHC Binding Assays
[0381] Candidate peptides for T cell based therapies according to
the present invention were further tested for their MHC binding
capacity (affinity). The individual peptide-MHC complexes were
produced by UV-ligand exchange, where a UV-sensitive peptide is
cleaved upon UV-irradiation, and exchanged with the peptide of
interest as analyzed. Only peptide candidates that can effectively
bind and stabilize the peptide-receptive MHC molecules prevent
dissociation of the MHC complexes. To determine the yield of the
exchange reaction, an ELISA was performed based on the detection of
the light chain (.mu.2m) of stabilized MHC complexes. The assay was
performed as generally described in Rodenko et al. (Rodenko et al.,
2006).
[0382] 96 well MAXISorp plates (NUNC) were coated over night with 2
ug/ml streptavidin in PBS at room temperature, washed 4.times. and
blocked forl h at 37.degree. C. in 2% BSA containing blocking
buffer. Refolded HLA-A*02:01/MLA-001 monomers served as standards,
covering the range of 15-500 ng/ml. Peptide-MHC monomers of the
UV-exchange reaction were diluted 100 fold in blocking buffer.
Samples were incubated for 1 h at 37.degree. C., washed four times,
incubated with 2 ug/ml HRP conjugated anti-.beta.2m for 1 h at
37.degree. C., washed again and detected with TMB solution that is
stopped with NH2SO4. Absorption was measured at 450 nm. Candidate
peptides that show a high exchange yield (preferably higher than
50%, most preferred higher than 75%) are generally preferred for a
generation and production of antibodies or fragments thereof,
and/or T cell receptors or fragments thereof, as they show
sufficient avidity to the MHC molecules and prevent dissociation of
the MHC complexes.
TABLE-US-00013 TABLE 11 MHC class I binding scores. Binding of
HLA-class I restricted peptides to HLA-A*02:01 was ranged by
peptide exchange yield: .gtoreq.10% =+; .gtoreq.20% =++; .gtoreq.50
=+++; .gtoreq.75% =++++ SEQID Sequence Peptide Exchange 1
AIPPSFASIFL "+++" 2 ALHRPDVYL "+++" 3 VIAELPPKV "++" 4 VIAELPPKVSV
"+++" 5 ALIFKIASA "+++" 6 ALDTLEDDMTI "+++" 7 ALLERTGYTL "+++" 8
ALAASALPALV "+++" 9 ALCDTLITV "+++" 10 FVYGESVEL "+++" 11
ALFTFJPLTV "+++" 12 ALGEDEITL "+++" 13 VVDGMPPGV "+++" 14 ALLRLLPGL
"+++" 15 ALPEVSVEA "+++" 16 ALPGGAAVAAV "++++" 17 ALTKTNLQL "+++"
18 LLGEFSIKM "+++" 19 QVMEKLAAV "+++" 20 ALVDPGPDFVV "+++" 21
ALWAGLLTL "+++" 22 ALWDPVIEL "+++" 23 ALYLTEVFL "++++" 24 AMAGDVVYA
"+++" 25 ATYSGLESQSV "++" 26 AVLLVLPLV "+++" 27 AVLGLVWLL "+++" 28
AVLHLLLSV "++" 29 AVLQAVTAV "+++" 30 ELLEGSEIYL "+++" 31 ELMHGVAGV
"+++" 32 FIDKFTPPV "++++" 34 YLPYIFPNI "+++" 35 FILPSSLYL "++++" 36
FILTHVDQL "++++" 37 FIMEGGAMVL "+++" 38 FIMEGGAMV "+++" 39
FLDALLTDV "+++" 40 FLDEDDMSL "++" 41 FLDPSLDPLL "+++" 42 FLEEGGVVTV
"+++" 43 FLLGPEALSFA "++++" 44 FLLSINDFL "+++" 45 FLPELPADLEA "+++"
46 YIIDSAQAV "+++" 47 FLSDQPEPYL "++" 48 FLSPQQPPLL "+++" 49
FLTDLFAQL "++++" 50 FLFEPVVKAFL "+++" 51 FLVEAPHDWDL "++++" 52
FLVETGFLHV "++++" 53 FLWQHVELVL "+++" 54 FLYPFPLALF "+++" 55
FMEPTLLML "+++" 56 FVFEAPYTL "+++" 57 GLSEISLRL "++++" 58 YIQQGIFSV
"+++" 59 FVFGDENGTVSL "+++" 60 FVLDHEDGLNL "+++" 61 FVYFIVREV "+++"
62 GIIDGSPRL "+++" 63 SLAHVAGCEL "+++" 64 KLLESVASA "+++" 65
GLDDMKANL "++" 66 SLAGGLDDMKA "+++" 67 GLDDVTVEV "++" 68
GLDQQFAGLDL "+" 69 GLHQREIFL "+++" 70 FVPDTPVGV "+++" 71 GLKHDIARV
"+++" 72 GLLDAGKMYV "+++" 73 GLLEVISALQL "+++" 74 GLLRASFLL "++" 75
GLSIFAQDLRL "++" 76 GLLRIIPYL "+++" 78 GLPSFLTEV "+++" 79 GLQAKIQEA
"+++" 80 VLIEDELEEL "++" 81 WLVGQEFEL "+++" 82 GLQSGVDIGV "+++" 83
GQGEVLVYV "+++" 84 GVMDVNTAL "++" 85 HLMLHTAAL "++++" 86 HLYPGAVTI
"+++" 87 HQIEAVDGEEL "++" 88 ILDEIGADVQA "+++" 89 ILDFGTFQL "+++"
90 VIADLGLIRV "+++" 91 ILDLNTYNV "+++" 92 ILEPLNPLL "+++" 93
ILFNTQINI "+++" 94 ILFPLRFTL "+++" 95 ILGYMAHEHKV "++" 96 ILIDKTSFV
"+++" 97 LLFATQITL "++" 98 SLIKYFLFV "+" 99 ILIFHSVAL "+++" 100
ILNNEVFAI "+++" 101 ILVVIEPLL "++++" 102 IQDRAVPSL "++" 103
KLGGTPAPA "++" 104 KLILLDTPLFL "+++" 105 KLMNDIADI "++" 106
FMASHLDYL "+++" 107 ILYNLYDLL "+++" 108 VIYTLIHYI "+++" 109
KLWEGLTELV "+++" 110 LLFDHLEPMEL "+++" 111 KLWNVAAPLYL "+++" 112
KTLDVDATYEI "+++" 113 KVPAEEVLVAV "+++" 114 LIPEGPPQV "+++" 115
LLFDKLYLL "+++" 116 LLIGATMQV "+++" 117 LLILENILL "+++" 118
RLLILENILL "+++" 119 VLPAEFFEV "+++" 120 AIDAALTSV "+++" 121
RLYEITIEV "++++" 122 LLIPVVPGV "+++" 123 LLLAEAELLTL "++" 124
LLLEETEKQAV "+++" 125 LLLEIGEVGKLFV "+++" 126 LLPEGGITAI "+++" 127
LLPTAPTTV "+++" 128 LLSEEEYHL "+++" 130 LLVLIPVYL "+" 131 LQALEVLKI
"+" 132 LVYEAIIMV "+++" 133 YLLSGDISEA "+++" 134 MLLEHGITLV "++++"
135 MTAGFSTIAGSV "+" 136 NLDKLWTLV "++" 137 NLIKTVIKL "+++" 138
NLLDIDAPVTV "+++" 139 NLTDVVEKL "+++" 140 QIAELPATSV "+++" 141
QILSEIVEA "++" 142 QLDEPAPQV "++" 143 QLLDTYFTL "+++" 145 SALDTITTV
"+++" 146 SIIEGPIIKL "+++" 147 SILETVATL "+++" 148 SIVASLITV "+++"
149 SLDNGGYYI "+++" 150 SLFDQPLSII "+++" 151 SLFDSAYGA "+++" 152
SLIRILQTI "+++" 153 SLLAELHVL "+++" 154 SLLAELHVLTV "++++" 155
SLMLEVPAL "+++" 156 SLNIGDVQL "+++" 157 SLNIRDFTM "+++" 158
SLPEAPLDV "+++" 159 SLQEEKLIYV "+++" 160 SLSFLVPSL "+++" 161
SMDDGMINV "+++" 162 SMKDDLENV "++" 163 SQLDISEPYKV "+++" 164
SVHKGFAFV "+++" 165 TLDDDLDTV "++" 166 TLDPNQVSL "++" 167 TLDTSKLYV
"+++" 168 YLLDQSFVM "++++" 169 TLLLGLTEV "+++" 170 TLTFRVETV "+++"
171 TLVPPAALISI "+++" 172 TLYDMLASI "+++" 173 TVIENIHTI "++" 174
VLAELPIIVV "+++" 175 FTVPRVVAV "+++" 176 VLAEQNIIPSA "+++" 177
VLDDRELLL "+++" 178 VLFFNVQEV "+++" 179 VLLGLEMTL "+++" 180
LLKDGPEIGL "++" 181 VLLSIPFVSV "+++" 182 VLLSVPGPPV "+++" 183
VLMPTVYQQGV "+++" 184 VLSHNLYTV "+++" 185 VMDDQRDLI "++" 186
VMDPTKILI "++++" 187 VMDTHLVNI "++++" 188 VMGDIPAAV "++" 189
VMLEMTPEL "+++" 190 VVMGTVPRL "+++" 191 YIFDGSDGGV "+++" 192
YIQEYLTLL "+++" 193 YLDLSNNRL "+++" 194 YLDNVLAEL "++++" 195
YLGGFALSV "++++" 196 YLLLQTYVL "+" 197 YLQEVPILTL "++++" 198
YLTFLPAEV "+++" 199 YLVELSSLL "++++" 200 YMFEEVPIVI "++++" 201
YQLELHGIEL "++++" 202 YVDDVFLRV "++++" 203 ALLSSQLAL "+++" 204
GLLQINDKIAL "+++" 205 GLSQANFTL "+++" 206 HMQDVRVLL "+++" 207
IIADLDTTIMFA "+++" 208 ILLKTEGINL "+++" 209 ILQAELPSL "+++" 210
KLLVQDFFL "+++" 211 LIDVKPLGV "+++" 214 RLQELTEKL "+++" 215
VMQDIVYKL "+++" 216 WLAGDVPAA "+++" 217 ALDEPPYLTV "+++" 218
ALGEEWKGYVV "++" 219 ALLNLLESA "+++" 220 ALPEILFAKV "+++" 221
ALVSTIIMV "+" 222 ALWELSLKI "+++" 223 ALWVSQPPEI "+++" 224
AMEALVVEV "+++" 225 ILQERELLPV "+++" 226 AMNISVPQV "+++" 227
FLAEASVMTQL "+++" 228 FLGGLSPGV "+++" 229 FLLNLQNCHL "++++" 230
FLQDSKVIFV "+++" 231 FLYIRQLAI "+++" 232 FMHQIIDQV "++++" 233
GIIDINVRL "++" 234 GLDDAEYAL "++" 235 GLDDLLLFL "+++" 236
GLLESGRHYL "+++" 237 GLQENLDVVV "++++" 238 GLVETELQL "+++" 239
ILAGEMLSV "+++" 240 ILARDILEI "+++" 241 ILGDILLKV "+++" 242
ILLGIQELL "+++" 243 ILPTLEKELFL "+++" 244 ILQALAVHL "+++" 245
KIMDYSLLLGV "++++" 246 KLDETGVAL "++" 247 KLKDRLPSI "++" 248
KTVEPPISQV "++"
249 LLPTGVFQV "+++" 250 LLVQEPDGLMV "+++" 251 LLYDNVPGA "+++" 252
NLLDPGSSYLL "+++" 253 NLWSVDGEVTV "+++" 254 QLIPKLIFL "+++" 255
YLFEEAISM "+++" 256 QLLPVSNVVSV "+++" 258 RLDYITAEI "+++" 259
RLLDEQFAVL "+++" 260 SLDDVEGMSV "+++" 261 SLVEAQGWLV "+++" 262
SLWNAGTSV "+++" 263 SQWEDIHVV "+++" 264 TILDYINVV "+++" 265
TLLADDLEIKL "+++" 266 TLLDQLDTQL "++" 267 TLLDWQDSL "+++" 268
TLLQVFHLL "+++" 269 TLTDEQFLV "++" 270 TVLPVPPLSV "+++" 271
VIRNIVEAA "++" 272 VLDELPPLI "+++" 273 VLGEYSYLL "+++" 274
VLLEYHIAYL "+++" 275 VLLFIEHSV "+++" 276 VLNDGAPNV "+++" 277
VMILKLPFL "++" 278 YLDDLLPKL "+++" 279 YMAPEVVEA "+++" 280
YTLDSLYWSV "+++" 281 NLLDDRGMTAL "+++" 282 LLRDGIELV "+++" 283
ILQPMDIHV "+++" 284 LLSAAEPVPA "++" 285 GVATAGCVNEV "++++" 286
FLLEDLSQKL "+++" 287 FLWEEKFNSL "++++" 288 GLAESTGLLAV "+++" 289
ILEEQPMDMLL "+++" 291 ILLNEDDLVTI "+++" 292 AAALIIHHV "+++" 293
ALDIMIPMV "+++" 294 ALLDQLHTLL "++++" 295 ALLQKLQQL "+++" 296
FIAPTGHSL "++" 297 FLVEPQEDTRL "+++" 298 IILPVEVEV "+++" 299
ILEENIPVL "+++" 300 ILLNPAYDVYL "+++" 301 AASPIITLV "+++" 302
ILQDLTFVHL "+++" 303 ILSQPTPSL "+++" 304 LAIVPVNTL "+++" 305
LLFPQIEGIKI "+++" 306 VVAEELENV "++" 307 LLLTKPTEA "+++" 308
SLYDVSRMYV "++++" 309 ILYGTQFVL "+++" 310 LLSTLHLLV "+++" 311
LLVDVEPKV "+++" 312 LLYNSTDPTL "+++" 313 LMADLEGLHL "+++" 314
LMKDCEAEV "+++" 315 LVYEAPETV "+++" 316 MLLEHGITL "+++" 317
NLLAHIWAL "+++" 318 NLQVTQPTV "+++" 319 QVIPQLQTV "+++" 320
TIAPVTVAV "++++" 321 RLLEFELAQL "+++" 322 SLASIHVPL "+++" 323
SLDLFNCEV "++++" 324 SLYSALQQA "+++" 325 TLENGVPCV "+++" 326
VLAFLVHEL "++++" 327 VLIKWFPEV "++++" 328 VLLPQETAEIHL "++++" 329
VLMDGSVKL "++++" 330 VLMWEIYSL "+++" 331 VLWELAHLPTL "+++" 332
VMIQHVENL "+++" 333 VTLEFPQLIRV "+++" 334 YLLEEKIASL "+++" 335
YLYQEQYFI "+++" 336 YMAVTTQEV "++++" 337 YMYEKESEL "++" 338
FLDMTNWNL "++++" 339 GLWGTVVNI "+++" 340 KLLEEICNL "++++" 341
LLAELPASVHA "+++" 342 SLITPLQAV "+++" 344 VLAFENPQV "+++" 345
YLIEPDVELQRI "+++" 346 VLVQVSPSL "+++" 347 YLGPVSPSL "+++" 348
ALAKPPVVSV "+++" 349 ALATHILSL "+++" 350 ALEDRVWEL "+++" 351
ALSEKLARL "+++" 352 ALVFELHYV "+++" 353 ATPMPTPSV "++" 354
FIMDDPAGNSYL "+++" 355 FIWPMLIHI "+++" 356 FLHDHQAEL "++" 357
FLIQEIKTL "+++" 358 FLTDYLNDL "+++" 359 FMQDPMEVFV "+++" 360
HLIDTNKIQL "+++" 361 ILQEFESKL "+++" 362 ILTELGGFEV "+++" 363
ITTEVVNELYV "+++" 364 KMDWIFHTI "+++" 365 LISPLLLPV "+++" 367
NLWSLVAKV "+++" 369 RLLDLENSLLGL "+++" 370 SIFASPESV "+++" 371
SLADDSVLERL "+++" 372 SLFGPLPGPGPALV "+++" 373 TLLADQGEIRV "++" 374
VLSVITEEL "+++" 375 VLWFKPVEL "+++" 376 VLYNQRVEEI "+++" 377
VVDGTCVAV "+++" 378 YILGKFFAL "+++" 379 YLAELVTPIL "+++" 380
YLDRKLLTL "+++" 381 YLLEENKIKL "+++" 382 YLLPLLQRL "++++" 383
YLLREWVNL "++++" 384 YMIGSEVGNYL "++++" 385 YTIPLAIKL "+++"
Example 6
Absolute Quantitation of Tumor Associated Peptides Presented on the
Cell Surface.
[0383] The generation of binders, such as antibodies and/or TCRs,
is a laborious process, which may be conducted only for a number of
selected targets. In the case of tumor-associated and -specific
peptides, selection criteria include but are not restricted to
exclusiveness of presentation and the density of peptide presented
on the cell surface. In addition to the isolation and relative
quantitation of peptides as described in EXAMPLE 1, the inventors
did analyze absolute peptide copies per cell as described in patent
x. The quantitation of TUMAP copies per cell in solid tumor samples
requires the absolute quantitation of the isolated TUMAP, the
efficiency of TUMAP isolation, and the cell count of the tissue
sample analyzed. An overview on our experimental approach is
described below.
Peptide Quantitation by nanoLC-MS/MS
[0384] For an accurate quantitation of peptides by mass
spectrometry, a calibration curve was generated for each peptide
using the internal standard method. The internal standard is a
double-isotope-labelled variant of each peptide, i.e. two
isotope-labelled amino acids were included in TUMAP synthesis. It
differs from the tumor-associated peptide only in its mass but
shows no difference in other physicochemical properties (Anderson
et al., 2012). The internal standard was spiked to each MS sample
and all MS signals were normalized to the MS signal of the internal
standard to level out potential technical variances between MS
experiments. The calibration curves were prepared in at least three
different matrices, i.e. HLA peptide eluates from natural samples
similar to the routine MS samples, and each preparation was
measured in duplicate MS runs. For evaluation, MS signals were
normalized to the signal of the internal standard and a calibration
curve was calculated by logistic regression. For the quantitation
of tumor-associated peptides from tissue samples, the respective
samples were also spiked with the internal standard; the MS signals
were normalized to the internal standard and quantified using the
peptide calibration curve.
Efficiency of Peptide/MHC Isolation
[0385] As for any protein purification process, the isolation of
proteins from tissue samples is associated with a certain loss of
the protein of interest. To determine the efficiency of TUMAP
isolation, peptide/MHC complexes were generated for all TUMAPs
selected for absolute quantitation. To be able to discriminate the
spiked from the natural peptide/MHC complexes,
single-isotope-labelled versions of the TUMAPs were used, i.e. one
isotope-labelled amino acid was included in TUMAP synthesis. These
complexes were spiked into the freshly prepared tissue lysates,
i.e. at the earliest possible point of the TUMAP isolation
procedure, and then captured like the natural peptide/MHC complexes
in the following affinity purification. Measuring the recovery of
the single-labelled TUMAPs therefore allows conclusions regarding
the efficiency of isolation of individual natural TUMAPs.
[0386] The efficiency of isolation was analyzed in a low number of
samples and was comparable among these tissue samples. In contrast,
the isolation efficiency differs between individual peptides. This
suggests that the isolation efficiency, although determined in only
a limited number of tissue samples, may be extrapolated to any
other tissue preparation. However, it is necessary to analyze each
TUMAP individually as the isolation efficiency may not be
extrapolated from one peptide to others.
Determination of the Cell Count in Solid, Frozen Tissue
[0387] In order to determine the cell count of the tissue samples
subjected to absolute peptide quantitation, the inventors applied
DNA content analysis. This method is applicable to a wide range of
samples of different origin and, most importantly, frozen samples
(Alcoser et al., 2011; Forsey and Chaudhuri, 2009; Silva et al.,
2013). During the peptide isolation protocol, a tissue sample is
processed to a homogenous lysate, from which a small lysate aliquot
is taken. The aliquot is divided in three parts, from which DNA is
isolated (QiaAmp DNA Mini Kit, Qiagen, Hilden, Germany). The total
DNA content from each DNA isolation is quantified using a
fluorescence-based DNA quantitation assay (Qubit dsDNA HS Assay
Kit, Life Technologies, Darmstadt, Germany) in at least two
replicates.
[0388] In order to calculate the cell number, a DNA standard curve
from aliquots of single healthy blood cells, with a range of
defined cell numbers, has been generated. The standard curve is
used to calculate the total cell content from the total DNA content
from each DNA isolation. The mean total cell count of the tissue
sample used for peptide isolation is extrapolated considering the
known volume of the lysate aliquots and the total lysate
volume.
Peptide Copies Per Cell
[0389] With data of the aforementioned experiments, the inventors
calculated the number of TUMAP copies per cell by dividing the
total peptide amount by the total cell count of the sample,
followed by division through isolation efficiency. Copy cell number
for selected peptides are shown in Table 12.
TABLE-US-00014 TABLE 12 Absolute copy numbers. The table lists the
results of absolute peptide quantitation in NSCLC tumor samples.
The median number of copies per cell are indicated for each
peptide: <100 =+; >= 100 =++; >= 1,000 +++; >= 10,000
=++++. The number of samples, in which evaluable, high quality MS
data are available, is indicated Number of Copy Number quantifiable
Seq ID Sequence Category samples 62 GIIDGSPRL + 17 153 SLLAELHVL ++
17
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Sequence CWU 1
1
525111PRTHomo sapiens 1Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu1
5 1029PRTHomo sapiens 2Ala Leu His Arg Pro Asp Val Tyr Leu1
539PRTHomo sapiens 3Val Ile Ala Glu Leu Pro Pro Lys Val1
5411PRTHomo sapiens 4Val Ile Ala Glu Leu Pro Pro Lys Val Ser Val1 5
1059PRTHomo sapiens 5Ala Leu Ile Phe Lys Ile Ala Ser Ala1
5611PRTHomo sapiens 6Ala Leu Asp Thr Leu Glu Asp Asp Met Thr Ile1 5
10710PRTHomo sapiens 7Ala Leu Leu Glu Arg Thr Gly Tyr Thr Leu1 5
10811PRTHomo sapiens 8Ala Leu Ala Ala Ser Ala Leu Pro Ala Leu Val1
5 1099PRTHomo sapiens 9Ala Leu Cys Asp Thr Leu Ile Thr Val1
5109PRTHomo sapiens 10Phe Val Tyr Gly Glu Ser Val Glu Leu1
51110PRTHomo sapiens 11Ala Leu Phe Thr Phe Ser Pro Leu Thr Val1 5
10129PRTHomo sapiens 12Ala Leu Gly Glu Asp Glu Ile Thr Leu1
5139PRTHomo sapiens 13Val Val Asp Gly Met Pro Pro Gly Val1
5149PRTHomo sapiens 14Ala Leu Leu Arg Leu Leu Pro Gly Leu1
5159PRTHomo sapiens 15Ala Leu Pro Glu Val Ser Val Glu Ala1
51611PRTHomo sapiens 16Ala Leu Pro Gly Gly Ala Ala Val Ala Ala Val1
5 10179PRTHomo sapiens 17Ala Leu Thr Lys Thr Asn Leu Gln Leu1
5189PRTHomo sapiens 18Leu Leu Gly Glu Phe Ser Ile Lys Met1
5199PRTHomo sapiens 19Gln Val Met Glu Lys Leu Ala Ala Val1
52011PRTHomo sapiens 20Ala Leu Val Asp Pro Gly Pro Asp Phe Val Val1
5 10219PRTHomo sapiens 21Ala Leu Trp Ala Gly Leu Leu Thr Leu1
5229PRTHomo sapiens 22Ala Leu Trp Asp Pro Val Ile Glu Leu1
5239PRTHomo sapiens 23Ala Leu Tyr Leu Thr Glu Val Phe Leu1
5249PRTHomo sapiens 24Ala Met Ala Gly Asp Val Val Tyr Ala1
52511PRTHomo sapiens 25Ala Thr Tyr Ser Gly Leu Glu Ser Gln Ser Val1
5 10269PRTHomo sapiens 26Ala Val Leu Leu Val Leu Pro Leu Val1
5279PRTHomo sapiens 27Ala Val Leu Gly Leu Val Trp Leu Leu1
5289PRTHomo sapiens 28Ala Val Leu His Leu Leu Leu Ser Val1
5299PRTHomo sapiens 29Ala Val Leu Gln Ala Val Thr Ala Val1
53010PRTHomo sapiens 30Glu Leu Leu Glu Gly Ser Glu Ile Tyr Leu1 5
10319PRTHomo sapiens 31Glu Leu Met His Gly Val Ala Gly Val1
5329PRTHomo sapiens 32Phe Ile Asp Lys Phe Thr Pro Pro Val1
53310PRTHomo sapiens 33Phe Ile Ile Asn Ser Ser Asn Ile Phe Leu1 5
10349PRTHomo sapiens 34Tyr Leu Pro Tyr Ile Phe Pro Asn Ile1
5359PRTHomo sapiens 35Phe Ile Leu Pro Ser Ser Leu Tyr Leu1
5369PRTHomo sapiens 36Phe Ile Leu Thr His Val Asp Gln Leu1
53710PRTHomo sapiens 37Phe Ile Met Glu Gly Gly Ala Met Val Leu1 5
10389PRTHomo sapiens 38Phe Ile Met Glu Gly Gly Ala Met Val1
5399PRTHomo sapiens 39Phe Leu Asp Ala Leu Leu Thr Asp Val1
5409PRTHomo sapiens 40Phe Leu Asp Glu Asp Asp Met Ser Leu1
54110PRTHomo sapiens 41Phe Leu Asp Pro Ser Leu Asp Pro Leu Leu1 5
104210PRTHomo sapiens 42Phe Leu Glu Glu Gly Gly Val Val Thr Val1 5
104311PRTHomo sapiens 43Phe Leu Leu Gly Pro Glu Ala Leu Ser Phe
Ala1 5 10449PRTHomo sapiens 44Phe Leu Leu Ser Ile Asn Asp Phe Leu1
54511PRTHomo sapiens 45Phe Leu Pro Glu Leu Pro Ala Asp Leu Glu Ala1
5 10469PRTHomo sapiens 46Tyr Ile Ile Asp Ser Ala Gln Ala Val1
54710PRTHomo sapiens 47Phe Leu Ser Asp Gln Pro Glu Pro Tyr Leu1 5
104810PRTHomo sapiens 48Phe Leu Ser Pro Gln Gln Pro Pro Leu Leu1 5
10499PRTHomo sapiens 49Phe Leu Thr Asp Leu Phe Ala Gln Leu1
55011PRTHomo sapiens 50Phe Leu Phe Glu Pro Val Val Lys Ala Phe Leu1
5 105111PRTHomo sapiens 51Phe Leu Val Glu Ala Pro His Asp Trp Asp
Leu1 5 105210PRTHomo sapiens 52Phe Leu Val Glu Thr Gly Phe Leu His
Val1 5 105310PRTHomo sapiens 53Phe Leu Trp Gln His Val Glu Leu Val
Leu1 5 105410PRTHomo sapiens 54Phe Leu Tyr Pro Phe Pro Leu Ala Leu
Phe1 5 10559PRTHomo sapiens 55Phe Met Glu Pro Thr Leu Leu Met Leu1
5569PRTHomo sapiens 56Phe Val Phe Glu Ala Pro Tyr Thr Leu1
5579PRTHomo sapiens 57Gly Leu Ser Glu Ile Ser Leu Arg Leu1
5589PRTHomo sapiens 58Tyr Ile Gln Gln Gly Ile Phe Ser Val1
55912PRTHomo sapiens 59Phe Val Phe Gly Asp Glu Asn Gly Thr Val Ser
Leu1 5 106011PRTHomo sapiens 60Phe Val Leu Asp His Glu Asp Gly Leu
Asn Leu1 5 10619PRTHomo sapiens 61Phe Val Tyr Phe Ile Val Arg Glu
Val1 5629PRTHomo sapiens 62Gly Ile Ile Asp Gly Ser Pro Arg Leu1
56310PRTHomo sapiens 63Ser Leu Ala His Val Ala Gly Cys Glu Leu1 5
10649PRTHomo sapiens 64Lys Leu Leu Glu Ser Val Ala Ser Ala1
5659PRTHomo sapiens 65Gly Leu Asp Asp Met Lys Ala Asn Leu1
56611PRTHomo sapiens 66Ser Leu Ala Gly Gly Leu Asp Asp Met Lys Ala1
5 10679PRTHomo sapiens 67Gly Leu Asp Asp Val Thr Val Glu Val1
56811PRTHomo sapiens 68Gly Leu Asp Gln Gln Phe Ala Gly Leu Asp Leu1
5 10699PRTHomo sapiens 69Gly Leu His Gln Arg Glu Ile Phe Leu1
5709PRTHomo sapiens 70Phe Val Pro Asp Thr Pro Val Gly Val1
5719PRTHomo sapiens 71Gly Leu Lys His Asp Ile Ala Arg Val1
57210PRTHomo sapiens 72Gly Leu Leu Asp Ala Gly Lys Met Tyr Val1 5
107311PRTHomo sapiens 73Gly Leu Leu Glu Val Ile Ser Ala Leu Gln
Leu1 5 10749PRTHomo sapiens 74Gly Leu Leu Arg Ala Ser Phe Leu Leu1
57511PRTHomo sapiens 75Gly Leu Ser Ile Phe Ala Gln Asp Leu Arg Leu1
5 10769PRTHomo sapiens 76Gly Leu Leu Arg Ile Ile Pro Tyr Leu1
5779PRTHomo sapiens 77Gly Leu Leu Arg Leu Thr Trp Phe Leu1
5789PRTHomo sapiens 78Gly Leu Pro Ser Phe Leu Thr Glu Val1
5799PRTHomo sapiens 79Gly Leu Gln Ala Lys Ile Gln Glu Ala1
58010PRTHomo sapiens 80Val Leu Ile Glu Asp Glu Leu Glu Glu Leu1 5
10819PRTHomo sapiens 81Trp Leu Val Gly Gln Glu Phe Glu Leu1
58210PRTHomo sapiens 82Gly Leu Gln Ser Gly Val Asp Ile Gly Val1 5
10839PRTHomo sapiens 83Gly Gln Gly Glu Val Leu Val Tyr Val1
5849PRTHomo sapiens 84Gly Val Met Asp Val Asn Thr Ala Leu1
5859PRTHomo sapiens 85His Leu Met Leu His Thr Ala Ala Leu1
5869PRTHomo sapiens 86His Leu Tyr Pro Gly Ala Val Thr Ile1
58711PRTHomo sapiens 87His Gln Ile Glu Ala Val Asp Gly Glu Glu Leu1
5 108811PRTHomo sapiens 88Ile Leu Asp Glu Ile Gly Ala Asp Val Gln
Ala1 5 10899PRTHomo sapiens 89Ile Leu Asp Phe Gly Thr Phe Gln Leu1
59010PRTHomo sapiens 90Val Ile Ala Asp Leu Gly Leu Ile Arg Val1 5
10919PRTHomo sapiens 91Ile Leu Asp Leu Asn Thr Tyr Asn Val1
5929PRTHomo sapiens 92Ile Leu Glu Pro Leu Asn Pro Leu Leu1
5939PRTHomo sapiens 93Ile Leu Phe Asn Thr Gln Ile Asn Ile1
5949PRTHomo sapiens 94Ile Leu Phe Pro Leu Arg Phe Thr Leu1
59511PRTHomo sapiens 95Ile Leu Gly Tyr Met Ala His Glu His Lys Val1
5 10969PRTHomo sapiens 96Ile Leu Ile Asp Lys Thr Ser Phe Val1
5979PRTHomo sapiens 97Leu Leu Phe Ala Thr Gln Ile Thr Leu1
5989PRTHomo sapiens 98Ser Leu Ile Lys Tyr Phe Leu Phe Val1
5999PRTHomo sapiens 99Ile Leu Ile Phe His Ser Val Ala Leu1
51009PRTHomo sapiens 100Ile Leu Asn Asn Glu Val Phe Ala Ile1
51019PRTHomo sapiens 101Ile Leu Val Val Ile Glu Pro Leu Leu1
51029PRTHomo sapiens 102Ile Gln Asp Arg Ala Val Pro Ser Leu1
51039PRTHomo sapiens 103Lys Leu Gly Gly Thr Pro Ala Pro Ala1
510411PRTHomo sapiens 104Lys Leu Ile Leu Leu Asp Thr Pro Leu Phe
Leu1 5 101059PRTHomo sapiens 105Lys Leu Met Asn Asp Ile Ala Asp
Ile1 51069PRTHomo sapiens 106Phe Met Ala Ser His Leu Asp Tyr Leu1
51079PRTHomo sapiens 107Ile Leu Tyr Asn Leu Tyr Asp Leu Leu1
51089PRTHomo sapiens 108Val Ile Tyr Thr Leu Ile His Tyr Ile1
510910PRTHomo sapiens 109Lys Leu Trp Glu Gly Leu Thr Glu Leu Val1 5
1011011PRTHomo sapiens 110Leu Leu Phe Asp His Leu Glu Pro Met Glu
Leu1 5 1011111PRTHomo sapiens 111Lys Leu Trp Asn Val Ala Ala Pro
Leu Tyr Leu1 5 1011211PRTHomo sapiens 112Lys Thr Leu Asp Val Asp
Ala Thr Tyr Glu Ile1 5 1011311PRTHomo sapiens 113Lys Val Pro Ala
Glu Glu Val Leu Val Ala Val1 5 101149PRTHomo sapiens 114Leu Ile Pro
Glu Gly Pro Pro Gln Val1 51159PRTHomo sapiens 115Leu Leu Phe Asp
Lys Leu Tyr Leu Leu1 51169PRTHomo sapiens 116Leu Leu Ile Gly Ala
Thr Met Gln Val1 51179PRTHomo sapiens 117Leu Leu Ile Leu Glu Asn
Ile Leu Leu1 511810PRTHomo sapiens 118Arg Leu Leu Ile Leu Glu Asn
Ile Leu Leu1 5 101199PRTHomo sapiens 119Val Leu Pro Ala Glu Phe Phe
Glu Val1 51209PRTHomo sapiens 120Ala Ile Asp Ala Ala Leu Thr Ser
Val1 51219PRTHomo sapiens 121Arg Leu Tyr Glu Ile Thr Ile Glu Val1
51229PRTHomo sapiens 122Leu Leu Ile Pro Val Val Pro Gly Val1
512311PRTHomo sapiens 123Leu Leu Leu Ala Glu Ala Glu Leu Leu Thr
Leu1 5 1012411PRTHomo sapiens 124Leu Leu Leu Glu Glu Thr Glu Lys
Gln Ala Val1 5 1012513PRTHomo sapiens 125Leu Leu Leu Glu Ile Gly
Glu Val Gly Lys Leu Phe Val1 5 1012610PRTHomo sapiens 126Leu Leu
Pro Glu Gly Gly Ile Thr Ala Ile1 5 101279PRTHomo sapiens 127Leu Leu
Pro Thr Ala Pro Thr Thr Val1 51289PRTHomo sapiens 128Leu Leu Ser
Glu Glu Glu Tyr His Leu1 51299PRTHomo sapiens 129Leu Leu Val Gly
Thr Leu Asp Val Val1 51309PRTHomo sapiens 130Leu Leu Val Leu Ile
Pro Val Tyr Leu1 51319PRTHomo sapiens 131Leu Gln Ala Leu Glu Val
Leu Lys Ile1 51329PRTHomo sapiens 132Leu Val Tyr Glu Ala Ile Ile
Met Val1 513310PRTHomo sapiens 133Tyr Leu Leu Ser Gly Asp Ile Ser
Glu Ala1 5 1013410PRTHomo sapiens 134Met Leu Leu Glu His Gly Ile
Thr Leu Val1 5 1013512PRTHomo sapiens 135Met Thr Ala Gly Phe Ser
Thr Ile Ala Gly Ser Val1 5 101369PRTHomo sapiens 136Asn Leu Asp Lys
Leu Trp Thr Leu Val1 51379PRTHomo sapiens 137Asn Leu Ile Lys Thr
Val Ile Lys Leu1 513811PRTHomo sapiens 138Asn Leu Leu Asp Ile Asp
Ala Pro Val Thr Val1 5 101399PRTHomo sapiens 139Asn Leu Thr Asp Val
Val Glu Lys Leu1 514010PRTHomo sapiens 140Gln Ile Ala Glu Leu Pro
Ala Thr Ser Val1 5 101419PRTHomo sapiens 141Gln Ile Leu Ser Glu Ile
Val Glu Ala1 51429PRTHomo sapiens 142Gln Leu Asp Glu Pro Ala Pro
Gln Val1 51439PRTHomo sapiens 143Gln Leu Leu Asp Thr Tyr Phe Thr
Leu1 51449PRTHomo sapiens 144Gln Leu Pro Pro Phe Pro Arg Glu Leu1
51459PRTHomo sapiens 145Ser Ala Leu Asp Thr Ile Thr Thr Val1
514610PRTHomo sapiens 146Ser Ile Ile Glu Gly Pro Ile Ile Lys Leu1 5
101479PRTHomo sapiens 147Ser Ile Leu Glu Thr Val Ala Thr Leu1
51489PRTHomo sapiens 148Ser Ile Val Ala Ser Leu Ile Thr Val1
51499PRTHomo sapiens 149Ser Leu Asp Asn Gly Gly Tyr Tyr Ile1
515010PRTHomo sapiens 150Ser Leu Phe Asp Gln Pro Leu Ser Ile Ile1 5
101519PRTHomo sapiens 151Ser Leu Phe Asp Ser Ala Tyr Gly Ala1
51529PRTHomo sapiens 152Ser Leu Ile Arg Ile Leu Gln Thr Ile1
51539PRTHomo sapiens 153Ser Leu Leu Ala Glu Leu His Val Leu1
515411PRTHomo sapiens 154Ser Leu Leu Ala Glu Leu His Val Leu Thr
Val1 5 101559PRTHomo sapiens 155Ser Leu Met Leu Glu Val Pro Ala
Leu1 51569PRTHomo sapiens 156Ser Leu Asn Ile Gly Asp Val Gln Leu1
51579PRTHomo sapiens 157Ser Leu Asn Ile Arg Asp Phe Thr Met1
51589PRTHomo sapiens 158Ser Leu Pro Glu Ala Pro Leu Asp Val1
515910PRTHomo sapiens 159Ser Leu Gln Glu Glu Lys Leu Ile Tyr Val1 5
101609PRTHomo sapiens 160Ser Leu Ser Phe Leu Val Pro Ser Leu1
51619PRTHomo sapiens 161Ser Met Asp Asp Gly Met Ile Asn Val1
51629PRTHomo sapiens 162Ser Met Lys Asp Asp Leu Glu Asn Val1
516311PRTHomo sapiens 163Ser Gln Leu Asp Ile Ser Glu Pro Tyr Lys
Val1 5 101649PRTHomo sapiens 164Ser Val His Lys Gly Phe Ala Phe
Val1 51659PRTHomo sapiens 165Thr Leu Asp Asp Asp Leu Asp Thr Val1
51669PRTHomo sapiens 166Thr Leu Asp Pro Asn Gln Val Ser Leu1
51679PRTHomo sapiens 167Thr Leu Asp Thr Ser Lys Leu Tyr Val1
51689PRTHomo sapiens 168Tyr Leu Leu Asp Gln Ser Phe Val Met1
51699PRTHomo sapiens 169Thr Leu Leu Leu Gly Leu Thr Glu Val1
51709PRTHomo sapiens 170Thr Leu Thr Phe Arg Val Glu Thr Val1
517111PRTHomo sapiens 171Thr Leu Val Pro Pro Ala Ala Leu Ile Ser
Ile1 5 101729PRTHomo sapiens 172Thr Leu Tyr Asp Met Leu Ala Ser
Ile1 51739PRTHomo sapiens 173Thr Val Ile Glu Asn Ile His Thr Ile1
517410PRTHomo sapiens 174Val Leu Ala Glu Leu Pro Ile Ile Val Val1 5
101759PRTHomo sapiens 175Phe Thr Val Pro Arg Val Val Ala Val1
517611PRTHomo sapiens 176Val Leu Ala Glu Gln Asn Ile Ile Pro Ser
Ala1 5 101779PRTHomo sapiens 177Val Leu Asp Asp Arg Glu Leu Leu
Leu1 51789PRTHomo sapiens 178Val Leu Phe Phe Asn Val Gln Glu Val1
51799PRTHomo sapiens 179Val Leu Leu Gly Leu Glu Met Thr Leu1
518010PRTHomo sapiens 180Leu Leu Lys Asp Gly Pro Glu Ile Gly Leu1 5
1018110PRTHomo sapiens 181Val Leu Leu Ser Ile Pro Phe Val Ser Val1
5 1018210PRTHomo sapiens 182Val Leu Leu Ser Val Pro Gly Pro Pro
Val1 5 1018311PRTHomo sapiens 183Val Leu Met Pro Thr Val Tyr Gln
Gln Gly Val1 5 101849PRTHomo sapiens 184Val Leu Ser His Asn Leu Tyr
Thr Val1 51859PRTHomo sapiens 185Val Met Asp Asp Gln Arg Asp Leu
Ile1 51869PRTHomo sapiens 186Val Met Asp Pro Thr Lys Ile Leu Ile1
51879PRTHomo sapiens 187Val Met Asp Thr His Leu Val Asn Ile1
51889PRTHomo sapiens 188Val Met Gly Asp Ile Pro Ala Ala Val1
51899PRTHomo sapiens 189Val Met Leu Glu Met Thr Pro Glu Leu1
51909PRTHomo sapiens 190Val Val Met Gly Thr Val Pro Arg Leu1
519110PRTHomo sapiens 191Tyr Ile Phe Asp Gly Ser Asp Gly Gly Val1 5
101929PRTHomo sapiens 192Tyr Ile Gln Glu Tyr Leu Thr Leu Leu1
51939PRTHomo sapiens 193Tyr Leu Asp Leu Ser Asn Asn Arg Leu1
51949PRTHomo sapiens 194Tyr Leu Asp Asn Val Leu Ala Glu Leu1
51959PRTHomo sapiens 195Tyr Leu Gly Gly Phe Ala Leu Ser Val1
51969PRTHomo sapiens 196Tyr Leu Leu Leu Gln Thr Tyr Val Leu1
519710PRTHomo sapiens 197Tyr Leu Gln Glu Val Pro Ile Leu Thr Leu1 5
101989PRTHomo sapiens 198Tyr Leu Thr Phe Leu Pro Ala Glu Val1
51999PRTHomo sapiens 199Tyr Leu Val Glu Leu Ser Ser Leu Leu1
520010PRTHomo sapiens 200Tyr Met Phe Glu Glu Val Pro Ile Val Ile1 5
1020110PRTHomo sapiens 201Tyr Gln Leu Glu Leu His Gly Ile Glu Leu1
5 102029PRTHomo sapiens 202Tyr Val Asp Asp Val Phe Leu Arg Val1
52039PRTHomo sapiens 203Ala Leu Leu Ser Ser Gln Leu Ala Leu1
520411PRTHomo sapiens 204Gly Leu Leu Gln Ile Asn Asp Lys Ile Ala
Leu1 5 102059PRTHomo sapiens 205Gly Leu Ser Gln Ala Asn Phe Thr
Leu1 52069PRTHomo sapiens 206His Met Gln Asp Val Arg Val Leu Leu1
520712PRTHomo sapiens 207Ile Ile Ala Asp Leu Asp Thr Thr Ile Met
Phe Ala1 5 1020810PRTHomo sapiens 208Ile Leu Leu Lys Thr Glu Gly
Ile Asn Leu1 5 102099PRTHomo sapiens 209Ile Leu Gln Ala Glu Leu Pro
Ser Leu1 52109PRTHomo sapiens 210Lys Leu Leu Val Gln Asp Phe Phe
Leu1 52119PRTHomo sapiens 211Leu Ile Asp Val Lys Pro Leu Gly Val1
521211PRTHomo sapiens 212Asn Ile Ile Glu Ala Ile Asn Glu Leu Leu
Val1 5 102139PRTHomo sapiens 213Arg Leu Leu Tyr Gln Leu Val Phe
Leu1 52149PRTHomo sapiens 214Arg Leu Gln Glu Leu Thr Glu Lys Leu1
52159PRTHomo sapiens 215Val Met Gln Asp Ile Val Tyr Lys Leu1
52169PRTHomo sapiens 216Trp Leu Ala Gly Asp Val Pro Ala Ala1
521710PRTHomo sapiens 217Ala Leu Asp Glu
Pro Pro Tyr Leu Thr Val1 5 1021811PRTHomo sapiens 218Ala Leu Gly
Glu Glu Trp Lys Gly Tyr Val Val1 5 102199PRTHomo sapiens 219Ala Leu
Leu Asn Leu Leu Glu Ser Ala1 522010PRTHomo sapiens 220Ala Leu Pro
Glu Ile Leu Phe Ala Lys Val1 5 102219PRTHomo sapiens 221Ala Leu Val
Ser Thr Ile Ile Met Val1 52229PRTHomo sapiens 222Ala Leu Trp Glu
Leu Ser Leu Lys Ile1 522310PRTHomo sapiens 223Ala Leu Trp Val Ser
Gln Pro Pro Glu Ile1 5 102249PRTHomo sapiens 224Ala Met Glu Ala Leu
Val Val Glu Val1 522510PRTHomo sapiens 225Ile Leu Gln Glu Arg Glu
Leu Leu Pro Val1 5 102269PRTHomo sapiens 226Ala Met Asn Ile Ser Val
Pro Gln Val1 522711PRTHomo sapiens 227Phe Leu Ala Glu Ala Ser Val
Met Thr Gln Leu1 5 102289PRTHomo sapiens 228Phe Leu Gly Gly Leu Ser
Pro Gly Val1 522910PRTHomo sapiens 229Phe Leu Leu Asn Leu Gln Asn
Cys His Leu1 5 1023010PRTHomo sapiens 230Phe Leu Gln Asp Ser Lys
Val Ile Phe Val1 5 102319PRTHomo sapiens 231Phe Leu Tyr Ile Arg Gln
Leu Ala Ile1 52329PRTHomo sapiens 232Phe Met His Gln Ile Ile Asp
Gln Val1 52339PRTHomo sapiens 233Gly Ile Ile Asp Ile Asn Val Arg
Leu1 52349PRTHomo sapiens 234Gly Leu Asp Asp Ala Glu Tyr Ala Leu1
52359PRTHomo sapiens 235Gly Leu Asp Asp Leu Leu Leu Phe Leu1
523610PRTHomo sapiens 236Gly Leu Leu Glu Ser Gly Arg His Tyr Leu1 5
1023710PRTHomo sapiens 237Gly Leu Gln Glu Asn Leu Asp Val Val Val1
5 102389PRTHomo sapiens 238Gly Leu Val Glu Thr Glu Leu Gln Leu1
52399PRTHomo sapiens 239Ile Leu Ala Gly Glu Met Leu Ser Val1
52409PRTHomo sapiens 240Ile Leu Ala Arg Asp Ile Leu Glu Ile1
52419PRTHomo sapiens 241Ile Leu Gly Asp Ile Leu Leu Lys Val1
52429PRTHomo sapiens 242Ile Leu Leu Gly Ile Gln Glu Leu Leu1
524311PRTHomo sapiens 243Ile Leu Pro Thr Leu Glu Lys Glu Leu Phe
Leu1 5 102449PRTHomo sapiens 244Ile Leu Gln Ala Leu Ala Val His
Leu1 524511PRTHomo sapiens 245Lys Ile Met Asp Tyr Ser Leu Leu Leu
Gly Val1 5 102469PRTHomo sapiens 246Lys Leu Asp Glu Thr Gly Val Ala
Leu1 52479PRTHomo sapiens 247Lys Leu Lys Asp Arg Leu Pro Ser Ile1
524810PRTHomo sapiens 248Lys Thr Val Glu Pro Pro Ile Ser Gln Val1 5
102499PRTHomo sapiens 249Leu Leu Pro Thr Gly Val Phe Gln Val1
525011PRTHomo sapiens 250Leu Leu Val Gln Glu Pro Asp Gly Leu Met
Val1 5 102519PRTHomo sapiens 251Leu Leu Tyr Asp Asn Val Pro Gly
Ala1 525211PRTHomo sapiens 252Asn Leu Leu Asp Pro Gly Ser Ser Tyr
Leu Leu1 5 1025311PRTHomo sapiens 253Asn Leu Trp Ser Val Asp Gly
Glu Val Thr Val1 5 102549PRTHomo sapiens 254Gln Leu Ile Pro Lys Leu
Ile Phe Leu1 52559PRTHomo sapiens 255Tyr Leu Phe Glu Glu Ala Ile
Ser Met1 525611PRTHomo sapiens 256Gln Leu Leu Pro Val Ser Asn Val
Val Ser Val1 5 1025710PRTHomo sapiens 257Arg Ile Ile Asn Gly Ile
Ile Ile Ser Val1 5 102589PRTHomo sapiens 258Arg Leu Asp Tyr Ile Thr
Ala Glu Ile1 525910PRTHomo sapiens 259Arg Leu Leu Asp Glu Gln Phe
Ala Val Leu1 5 1026010PRTHomo sapiens 260Ser Leu Asp Asp Val Glu
Gly Met Ser Val1 5 1026110PRTHomo sapiens 261Ser Leu Val Glu Ala
Gln Gly Trp Leu Val1 5 102629PRTHomo sapiens 262Ser Leu Trp Asn Ala
Gly Thr Ser Val1 52639PRTHomo sapiens 263Ser Gln Trp Glu Asp Ile
His Val Val1 52649PRTHomo sapiens 264Thr Ile Leu Asp Tyr Ile Asn
Val Val1 526511PRTHomo sapiens 265Thr Leu Leu Ala Asp Asp Leu Glu
Ile Lys Leu1 5 1026610PRTHomo sapiens 266Thr Leu Leu Asp Gln Leu
Asp Thr Gln Leu1 5 102679PRTHomo sapiens 267Thr Leu Leu Asp Trp Gln
Asp Ser Leu1 52689PRTHomo sapiens 268Thr Leu Leu Gln Val Phe His
Leu Leu1 52699PRTHomo sapiens 269Thr Leu Thr Asp Glu Gln Phe Leu
Val1 527010PRTHomo sapiens 270Thr Val Leu Pro Val Pro Pro Leu Ser
Val1 5 102719PRTHomo sapiens 271Val Ile Arg Asn Ile Val Glu Ala
Ala1 52729PRTHomo sapiens 272Val Leu Asp Glu Leu Pro Pro Leu Ile1
52739PRTHomo sapiens 273Val Leu Gly Glu Tyr Ser Tyr Leu Leu1
527410PRTHomo sapiens 274Val Leu Leu Glu Tyr His Ile Ala Tyr Leu1 5
102759PRTHomo sapiens 275Val Leu Leu Phe Ile Glu His Ser Val1
52769PRTHomo sapiens 276Val Leu Asn Asp Gly Ala Pro Asn Val1
52779PRTHomo sapiens 277Val Met Ile Leu Lys Leu Pro Phe Leu1
52789PRTHomo sapiens 278Tyr Leu Asp Asp Leu Leu Pro Lys Leu1
52799PRTHomo sapiens 279Tyr Met Ala Pro Glu Val Val Glu Ala1
528010PRTHomo sapiens 280Tyr Thr Leu Asp Ser Leu Tyr Trp Ser Val1 5
1028111PRTHomo sapiens 281Asn Leu Leu Asp Asp Arg Gly Met Thr Ala
Leu1 5 102829PRTHomo sapiens 282Leu Leu Arg Asp Gly Ile Glu Leu
Val1 52839PRTHomo sapiens 283Ile Leu Gln Pro Met Asp Ile His Val1
528410PRTHomo sapiens 284Leu Leu Ser Ala Ala Glu Pro Val Pro Ala1 5
1028511PRTHomo sapiens 285Gly Val Ala Thr Ala Gly Cys Val Asn Glu
Val1 5 1028610PRTHomo sapiens 286Phe Leu Leu Glu Asp Leu Ser Gln
Lys Leu1 5 1028710PRTHomo sapiens 287Phe Leu Trp Glu Glu Lys Phe
Asn Ser Leu1 5 1028811PRTHomo sapiens 288Gly Leu Ala Glu Ser Thr
Gly Leu Leu Ala Val1 5 1028911PRTHomo sapiens 289Ile Leu Glu Glu
Gln Pro Met Asp Met Leu Leu1 5 102909PRTHomo sapiens 290Leu Ala Asn
Pro His Glu Leu Ser Leu1 529111PRTHomo sapiens 291Ile Leu Leu Asn
Glu Asp Asp Leu Val Thr Ile1 5 102929PRTHomo sapiens 292Ala Ala Ala
Leu Ile Ile His His Val1 52939PRTHomo sapiens 293Ala Leu Asp Ile
Met Ile Pro Met Val1 529410PRTHomo sapiens 294Ala Leu Leu Asp Gln
Leu His Thr Leu Leu1 5 102959PRTHomo sapiens 295Ala Leu Leu Gln Lys
Leu Gln Gln Leu1 52969PRTHomo sapiens 296Phe Ile Ala Pro Thr Gly
His Ser Leu1 529711PRTHomo sapiens 297Phe Leu Val Glu Pro Gln Glu
Asp Thr Arg Leu1 5 102989PRTHomo sapiens 298Ile Ile Leu Pro Val Glu
Val Glu Val1 52999PRTHomo sapiens 299Ile Leu Glu Glu Asn Ile Pro
Val Leu1 530011PRTHomo sapiens 300Ile Leu Leu Asn Pro Ala Tyr Asp
Val Tyr Leu1 5 103019PRTHomo sapiens 301Ala Ala Ser Pro Ile Ile Thr
Leu Val1 530210PRTHomo sapiens 302Ile Leu Gln Asp Leu Thr Phe Val
His Leu1 5 103039PRTHomo sapiens 303Ile Leu Ser Gln Pro Thr Pro Ser
Leu1 53049PRTHomo sapiens 304Leu Ala Ile Val Pro Val Asn Thr Leu1
530511PRTHomo sapiens 305Leu Leu Phe Pro Gln Ile Glu Gly Ile Lys
Ile1 5 103069PRTHomo sapiens 306Val Val Ala Glu Glu Leu Glu Asn
Val1 53079PRTHomo sapiens 307Leu Leu Leu Thr Lys Pro Thr Glu Ala1
530810PRTHomo sapiens 308Ser Leu Tyr Asp Val Ser Arg Met Tyr Val1 5
103099PRTHomo sapiens 309Ile Leu Tyr Gly Thr Gln Phe Val Leu1
53109PRTHomo sapiens 310Leu Leu Ser Thr Leu His Leu Leu Val1
53119PRTHomo sapiens 311Leu Leu Val Asp Val Glu Pro Lys Val1
531210PRTHomo sapiens 312Leu Leu Tyr Asn Ser Thr Asp Pro Thr Leu1 5
1031310PRTHomo sapiens 313Leu Met Ala Asp Leu Glu Gly Leu His Leu1
5 103149PRTHomo sapiens 314Leu Met Lys Asp Cys Glu Ala Glu Val1
53159PRTHomo sapiens 315Leu Val Tyr Glu Ala Pro Glu Thr Val1
53169PRTHomo sapiens 316Met Leu Leu Glu His Gly Ile Thr Leu1
53179PRTHomo sapiens 317Asn Leu Leu Ala His Ile Trp Ala Leu1
53189PRTHomo sapiens 318Asn Leu Gln Val Thr Gln Pro Thr Val1
53199PRTHomo sapiens 319Gln Val Ile Pro Gln Leu Gln Thr Val1
53209PRTHomo sapiens 320Thr Ile Ala Pro Val Thr Val Ala Val1
532110PRTHomo sapiens 321Arg Leu Leu Glu Phe Glu Leu Ala Gln Leu1 5
103229PRTHomo sapiens 322Ser Leu Ala Ser Ile His Val Pro Leu1
53239PRTHomo sapiens 323Ser Leu Asp Leu Phe Asn Cys Glu Val1
53249PRTHomo sapiens 324Ser Leu Tyr Ser Ala Leu Gln Gln Ala1
53259PRTHomo sapiens 325Thr Leu Glu Asn Gly Val Pro Cys Val1
53269PRTHomo sapiens 326Val Leu Ala Phe Leu Val His Glu Leu1
53279PRTHomo sapiens 327Val Leu Ile Lys Trp Phe Pro Glu Val1
532812PRTHomo sapiens 328Val Leu Leu Pro Gln Glu Thr Ala Glu Ile
His Leu1 5 103299PRTHomo sapiens 329Val Leu Met Asp Gly Ser Val Lys
Leu1 53309PRTHomo sapiens 330Val Leu Met Trp Glu Ile Tyr Ser Leu1
533111PRTHomo sapiens 331Val Leu Trp Glu Leu Ala His Leu Pro Thr
Leu1 5 103329PRTHomo sapiens 332Val Met Ile Gln His Val Glu Asn
Leu1 533311PRTHomo sapiens 333Val Thr Leu Glu Phe Pro Gln Leu Ile
Arg Val1 5 1033410PRTHomo sapiens 334Tyr Leu Leu Glu Glu Lys Ile
Ala Ser Leu1 5 103359PRTHomo sapiens 335Tyr Leu Tyr Gln Glu Gln Tyr
Phe Ile1 53369PRTHomo sapiens 336Tyr Met Ala Val Thr Thr Gln Glu
Val1 53379PRTHomo sapiens 337Tyr Met Tyr Glu Lys Glu Ser Glu Leu1
53389PRTHomo sapiens 338Phe Leu Asp Met Thr Asn Trp Asn Leu1
53399PRTHomo sapiens 339Gly Leu Trp Gly Thr Val Val Asn Ile1
53409PRTHomo sapiens 340Lys Leu Leu Glu Glu Ile Cys Asn Leu1
534111PRTHomo sapiens 341Leu Leu Ala Glu Leu Pro Ala Ser Val His
Ala1 5 103429PRTHomo sapiens 342Ser Leu Ile Thr Pro Leu Gln Ala
Val1 53439PRTHomo sapiens 343Thr Leu Leu Glu Ala Leu Asp Cys Ile1
53449PRTHomo sapiens 344Val Leu Ala Phe Glu Asn Pro Gln Val1
534512PRTHomo sapiens 345Tyr Leu Ile Glu Pro Asp Val Glu Leu Gln
Arg Ile1 5 103469PRTHomo sapiens 346Val Leu Val Gln Val Ser Pro Ser
Leu1 53479PRTHomo sapiens 347Tyr Leu Gly Pro Val Ser Pro Ser Leu1
534810PRTHomo sapiens 348Ala Leu Ala Lys Pro Pro Val Val Ser Val1 5
103499PRTHomo sapiens 349Ala Leu Ala Thr His Ile Leu Ser Leu1
53509PRTHomo sapiens 350Ala Leu Glu Asp Arg Val Trp Glu Leu1
53519PRTHomo sapiens 351Ala Leu Ser Glu Lys Leu Ala Arg Leu1
53529PRTHomo sapiens 352Ala Leu Val Phe Glu Leu His Tyr Val1
53539PRTHomo sapiens 353Ala Thr Pro Met Pro Thr Pro Ser Val1
535412PRTHomo sapiens 354Phe Ile Met Asp Asp Pro Ala Gly Asn Ser
Tyr Leu1 5 103559PRTHomo sapiens 355Phe Ile Trp Pro Met Leu Ile His
Ile1 53569PRTHomo sapiens 356Phe Leu His Asp His Gln Ala Glu Leu1
53579PRTHomo sapiens 357Phe Leu Ile Gln Glu Ile Lys Thr Leu1
53589PRTHomo sapiens 358Phe Leu Thr Asp Tyr Leu Asn Asp Leu1
535910PRTHomo sapiens 359Phe Met Gln Asp Pro Met Glu Val Phe Val1 5
1036010PRTHomo sapiens 360His Leu Ile Asp Thr Asn Lys Ile Gln Leu1
5 103619PRTHomo sapiens 361Ile Leu Gln Glu Phe Glu Ser Lys Leu1
536210PRTHomo sapiens 362Ile Leu Thr Glu Leu Gly Gly Phe Glu Val1 5
1036311PRTHomo sapiens 363Ile Thr Thr Glu Val Val Asn Glu Leu Tyr
Val1 5 103649PRTHomo sapiens 364Lys Met Asp Trp Ile Phe His Thr
Ile1 53659PRTHomo sapiens 365Leu Ile Ser Pro Leu Leu Leu Pro Val1
53669PRTHomo sapiens 366Leu Leu Ser Glu Thr Cys Val Thr Ile1
53679PRTHomo sapiens 367Asn Leu Trp Ser Leu Val Ala Lys Val1
536811PRTHomo sapiens 368Gln Leu Gln Pro Thr Asp Ala Leu Leu Cys
Val1 5 1036912PRTHomo sapiens 369Arg Leu Leu Asp Leu Glu Asn Ser
Leu Leu Gly Leu1 5 103709PRTHomo sapiens 370Ser Ile Phe Ala Ser Pro
Glu Ser Val1 537111PRTHomo sapiens 371Ser Leu Ala Asp Asp Ser Val
Leu Glu Arg Leu1 5 1037214PRTHomo sapiens 372Ser Leu Phe Gly Pro
Leu Pro Gly Pro Gly Pro Ala Leu Val1 5 1037311PRTHomo sapiens
373Thr Leu Leu Ala Asp Gln Gly Glu Ile Arg Val1 5 103749PRTHomo
sapiens 374Val Leu Ser Val Ile Thr Glu Glu Leu1 53759PRTHomo
sapiens 375Val Leu Trp Phe Lys Pro Val Glu Leu1 537610PRTHomo
sapiens 376Val Leu Tyr Asn Gln Arg Val Glu Glu Ile1 5 103779PRTHomo
sapiens 377Val Val Asp Gly Thr Cys Val Ala Val1 53789PRTHomo
sapiens 378Tyr Ile Leu Gly Lys Phe Phe Ala Leu1 537910PRTHomo
sapiens 379Tyr Leu Ala Glu Leu Val Thr Pro Ile Leu1 5 103809PRTHomo
sapiens 380Tyr Leu Asp Arg Lys Leu Leu Thr Leu1 538110PRTHomo
sapiens 381Tyr Leu Leu Glu Glu Asn Lys Ile Lys Leu1 5 103829PRTHomo
sapiens 382Tyr Leu Leu Pro Leu Leu Gln Arg Leu1 53839PRTHomo
sapiens 383Tyr Leu Leu Arg Glu Trp Val Asn Leu1 538411PRTHomo
sapiens 384Tyr Met Ile Gly Ser Glu Val Gly Asn Tyr Leu1 5
103859PRTHomo sapiens 385Tyr Thr Ile Pro Leu Ala Ile Lys Leu1
53869PRTHomo sapiens 386Phe Leu Gly Asp Tyr Val Glu Asn Leu1
53879PRTHomo sapiens 387Tyr Leu Ile Leu Ser Ser His Gln Leu1
53889PRTHomo sapiens 388Gly Leu Leu Ser Ala Leu Glu Asn Val1
53899PRTHomo sapiens 389Gly Leu Ala Ala Leu Ala Val His Leu1
53909PRTHomo sapiens 390Gly Leu Glu Glu Arg Leu Tyr Thr Ala1
53919PRTHomo sapiens 391Phe Leu Leu Glu Arg Glu Gln Leu Leu1
539210PRTHomo sapiens 392Phe Leu Trp Glu Arg Pro Thr Leu Leu Val1 5
1039310PRTHomo sapiens 393Phe Val Met Glu Gly Glu Pro Pro Lys Leu1
5 103949PRTHomo sapiens 394Ile Leu Ser Thr Glu Ile Phe Gly Val1
53959PRTHomo sapiens 395Ala Leu Tyr Gly Lys Leu Leu Lys Leu1
53969PRTHomo sapiens 396Thr Leu Leu Gly Lys Gln Val Thr Leu1
539711PRTHomo sapiens 397Phe Leu Pro Pro Glu His Thr Ile Val Tyr
Ile1 5 1039811PRTHomo sapiens 398Phe Leu Ala Glu Leu Pro Gly Ser
Leu Ser Leu1 5 1039911PRTHomo sapiens 399Ala Leu Ala Ala Pro Asp
Ile Val Pro Ala Leu1 5 1040010PRTHomo sapiens 400Ala Leu Phe Gln
Pro His Leu Ile Asn Val1 5 1040110PRTHomo sapiens 401Ala Met Ala
Asp Lys Met Asp Met Ser Leu1 5 1040210PRTHomo sapiens 402Leu Leu
Val Ser Asn Leu Asp Phe Gly Val1 5 1040313PRTHomo sapiens 403Phe
Ile Met Pro Ala Thr Val Ala Asp Ala Thr Ala Val1 5 104049PRTHomo
sapiens 404Phe Leu Gln Pro Asp Leu Asp Ser Leu1 540510PRTHomo
sapiens 405Phe Leu Val Glu Lys Gln Pro Pro Gln Val1 5 104069PRTHomo
sapiens 406Phe Leu Trp Pro Lys Glu Val Glu Leu1 540711PRTHomo
sapiens 407Phe Met Ile Asp Ala Ser Val His Pro Thr Leu1 5
1040811PRTHomo sapiens 408Phe Met Val Asp Asn Glu Ala Ile Tyr Asp
Ile1 5 104099PRTHomo sapiens 409Gly Leu Asp Ala Ala Thr Ala Thr
Val1 541010PRTHomo sapiens 410Gly Leu Phe Asp Gly Val Pro Thr Thr
Ala1 5 1041113PRTHomo sapiens 411Gly Leu Phe Glu Asp Val Thr Gln
Pro Gly Ile Leu Leu1 5 1041211PRTHomo sapiens 412Ile Leu Gly Thr
Glu Asp Leu Ile Val Glu Val1 5 1041311PRTHomo sapiens 413Ile Leu
Leu Glu His Gly Ala Asp Pro Asn Leu1 5 104149PRTHomo sapiens 414Ile
Leu Ser Val Val Asn Ser Gln Leu1 54159PRTHomo sapiens 415Ile Leu
Val Thr Ser Ile Phe Phe Leu1 54169PRTHomo sapiens 416Ile Met Glu
Asp Ile Ile Leu Thr Leu1 541711PRTHomo sapiens 417Ile Gln Ile Gly
Glu Glu Thr Val Ile Thr Val1 5 104189PRTHomo sapiens 418Ile Val Thr
Glu Ile Ile Ser Glu Ile1 54199PRTHomo sapiens 419Leu Leu Asn Glu
Ile Leu Glu Gln Val1 542012PRTHomo sapiens 420Lys Leu Ile Asp Asp
Gln Asp Ile Ser Ile Ser Leu1 5 1042111PRTHomo sapiens 421Lys Leu
Trp Thr Gly Gly Leu Asp Asn Thr Val1 5 104229PRTHomo sapiens 422Lys
Gln Phe Glu Gly Thr Val Glu Ile1 54239PRTHomo sapiens 423Leu Leu
Ile Gly Thr Asp Val Ser Leu1 542411PRTHomo sapiens 424Leu Leu Pro
Pro Leu Glu Ser Leu Ala Thr Val1
5 104259PRTHomo sapiens 425Leu Leu Ser Asp Val Arg Phe Val Leu1
54269PRTHomo sapiens 426Leu Leu Trp Gly Asn Leu Pro Glu Ile1
54279PRTHomo sapiens 427Leu Leu Tyr Asp Ala Val His Ile Val1
54289PRTHomo sapiens 428Leu Met Tyr Pro Tyr Ile Tyr His Val1
54299PRTHomo sapiens 429Asn Leu Leu Glu Thr Lys Leu Gln Leu1
54309PRTHomo sapiens 430Gln Leu Ile Glu Lys Asn Trp Leu Leu1
54319PRTHomo sapiens 431Arg Met Val Ala Glu Ile Gln Asn Val1
54329PRTHomo sapiens 432Ser Ala Val Asp Phe Ile Arg Thr Leu1
543310PRTHomo sapiens 433Ser Ile Leu Asp Arg Asp Asp Ile Phe Val1 5
1043411PRTHomo sapiens 434Ser Ile Gln Gln Ser Ile Glu Arg Leu Leu
Val1 5 104359PRTHomo sapiens 435Ser Leu Phe Asn Gln Glu Val Gln
Ile1 543613PRTHomo sapiens 436Ser Leu Phe Ser Ser Leu Glu Pro Gln
Ile Gln Pro Val1 5 104379PRTHomo sapiens 437Ser Leu Glu Pro Gln Ile
Gln Pro Val1 543811PRTHomo sapiens 438Ser Leu Phe Ile Gly Glu Lys
Ala Val Leu Leu1 5 104399PRTHomo sapiens 439Ser Leu Leu Asp Leu His
Thr Lys Val1 544012PRTHomo sapiens 440Ser Leu Leu Glu Gln Gly Lys
Glu Pro Trp Met Val1 5 1044111PRTHomo sapiens 441Ser Leu Leu Glu
Val Asn Glu Ala Ser Ser Val1 5 1044212PRTHomo sapiens 442Ser Leu
Asn Asp Leu Glu Lys Asp Val Met Leu Leu1 5 1044310PRTHomo sapiens
443Ser Leu Thr Ile Asp Gly Ile Tyr Tyr Val1 5 104449PRTHomo sapiens
444Ser Met Ser Gly Tyr Asp Gln Val Leu1 544511PRTHomo sapiens
445Val Ile Ile Lys Gly Leu Glu Glu Ile Thr Val1 5 104469PRTHomo
sapiens 446Val Ile Leu Thr Ser Ser Pro Phe Leu1 54479PRTHomo
sapiens 447Val Leu Asp Asp Ser Ile Tyr Leu Val1 54489PRTHomo
sapiens 448Val Met Asn Asp Arg Leu Tyr Ala Ile1 54499PRTHomo
sapiens 449Leu Leu Asp Ala Met Asn Tyr His Leu1 54509PRTHomo
sapiens 450Val Leu Gly Pro Gly Pro Pro Pro Leu1 54519PRTHomo
sapiens 451Val Leu Ile Glu Tyr Asn Phe Ser Ile1 54529PRTHomo
sapiens 452Val Leu Leu Ser Leu Leu Glu Lys Val1 54539PRTHomo
sapiens 453Ser Leu Leu Pro Leu Val Trp Lys Ile1 545411PRTHomo
sapiens 454Val Leu Met Asp Glu Gly Ala Val Leu Thr Leu1 5
1045511PRTHomo sapiens 455Val Leu Pro Asp Pro Glu Val Leu Glu Ala
Val1 5 1045610PRTHomo sapiens 456Tyr Ala Ala Pro Gly Gly Leu Ile
Gly Val1 5 1045711PRTHomo sapiens 457Tyr Ile Met Glu Pro Ser Ile
Phe Asn Thr Leu1 5 104589PRTHomo sapiens 458Tyr Ile Gln Glu His Leu
Leu Gln Ile1 54599PRTHomo sapiens 459Tyr Leu Asp Phe Ser Asn Asn
Arg Leu1 54609PRTHomo sapiens 460Tyr Leu Glu Asp Gly Phe Ala Tyr
Val1 546110PRTHomo sapiens 461Tyr Leu Leu Asn Leu Asn His Leu Gly
Leu1 5 1046210PRTHomo sapiens 462Tyr Val Leu Pro Lys Leu Tyr Val
Lys Leu1 5 104639PRTHomo sapiens 463Ala Leu Met Ala Val Val Ser Gly
Leu1 546412PRTHomo sapiens 464Ala Leu Ser Asp Glu Thr Lys Asn Asn
Trp Glu Val1 5 1046512PRTHomo sapiens 465Phe Leu Tyr Asp Glu Ile
Glu Ala Glu Val Asn Leu1 5 1046610PRTHomo sapiens 466Phe Leu Tyr
Asp Glu Ile Glu Ala Glu Val1 5 1046711PRTHomo sapiens 467Ile Met
Gln Asp Phe Pro Ala Glu Ile Phe Leu1 5 104689PRTHomo sapiens 468Lys
Ile Gln Glu Ile Leu Thr Gln Val1 54699PRTHomo sapiens 469Ser Leu
Asp Gly Thr Glu Leu Gln Leu1 54709PRTHomo sapiens 470Thr Leu Thr
Asn Ile Ile His Asn Leu1 547110PRTHomo sapiens 471Val Leu Leu Ala
Ala Gly Pro Ser Ala Ala1 5 1047210PRTHomo sapiens 472Tyr Leu Phe
Glu Ser Glu Gly Leu Val Leu1 5 1047310PRTHomo sapiens 473Ala Leu
Ala Asp Asp Asp Phe Leu Thr Val1 5 1047412PRTHomo sapiens 474Ala
Leu Ala Asp Leu Ile Glu Lys Glu Leu Ser Val1 5 104759PRTHomo
sapiens 475Ala Leu Asp Asp Met Ile Ser Thr Leu1 54769PRTHomo
sapiens 476Ala Leu Ile Thr Glu Val Val Arg Leu1 547714PRTHomo
sapiens 477Ala Leu Leu Asp Gln Thr Lys Thr Leu Ala Glu Ser Ala Leu1
5 1047811PRTHomo sapiens 478Ala Met Phe Glu Ser Ser Gln Asn Val Leu
Leu1 5 104799PRTHomo sapiens 479Tyr Leu Ala His Phe Ile Glu Gly
Leu1 548010PRTHomo sapiens 480Phe Leu Ala Glu Asp Pro Lys Val Thr
Leu1 5 104819PRTHomo sapiens 481Phe Leu Ile Asp Thr Ser Ala Ser
Met1 548210PRTHomo sapiens 482Phe Leu Thr Asp Leu Glu Asp Leu Thr
Leu1 5 104839PRTHomo sapiens 483Phe Leu Thr Glu Met Val His Phe
Ile1 548410PRTHomo sapiens 484Phe Leu Val Asp Gly Pro Arg Val Gln
Leu1 5 104859PRTHomo sapiens 485Gly Leu Leu Asp Cys Pro Ile Phe
Leu1 548611PRTHomo sapiens 486Gly Leu Leu Asp Leu Pro Phe Arg Val
Gly Val1 5 104879PRTHomo sapiens 487Gly Leu Ser Asp Gly Asn Pro Ser
Leu1 548810PRTHomo sapiens 488Gly Val Tyr Asp Gly Glu Glu His Ser
Val1 5 104899PRTHomo sapiens 489His Leu Ala Asn Ile Val Glu Arg
Leu1 54909PRTHomo sapiens 490Ile Leu Asp Glu Lys Pro Val Ile Ile1
549112PRTHomo sapiens 491Ile Leu Phe Ser Glu Asp Ser Thr Lys Leu
Phe Val1 5 1049211PRTHomo sapiens 492Ile Leu Leu Asp Asp Asn Met
Gln Ile Arg Leu1 5 1049310PRTHomo sapiens 493Leu Ile Ile Asp Gln
Ala Asp Ile Tyr Leu1 5 104949PRTHomo sapiens 494Leu Leu Gly Asp Ser
Ser Phe Phe Leu1 549511PRTHomo sapiens 495Leu Leu Leu Asp Glu Glu
Gly Thr Phe Ser Leu1 5 1049611PRTHomo sapiens 496Leu Leu Val Glu
Gln Pro Pro Leu Ala Gly Val1 5 1049710PRTHomo sapiens 497Leu Leu
Trp Asp Val Pro Ala Pro Ser Leu1 5 1049811PRTHomo sapiens 498Leu
Thr Ala Pro Pro Glu Ala Leu Leu Met Val1 5 104999PRTHomo sapiens
499Asn Leu Met Glu Met Val Ala Gln Leu1 55009PRTHomo sapiens 500Gln
Ile Ile Thr Ser Val Val Ser Val1 55019PRTHomo sapiens 501Gln Leu
Ala Leu Lys Val Glu Gly Val1 550211PRTHomo sapiens 502Gln Leu Leu
Asp Glu Thr Ser Ala Ile Thr Leu1 5 1050310PRTHomo sapiens 503Gln
Leu Tyr Glu Glu Pro Asp Thr Lys Leu1 5 1050411PRTHomo sapiens
504Arg Leu Tyr Ser Gly Ile Ser Gly Leu Glu Leu1 5 1050511PRTHomo
sapiens 505Arg Val Leu Glu Val Gly Ala Leu Gln Ala Val1 5
1050611PRTHomo sapiens 506Ser Leu Leu Pro Leu Asp Asp Ile Val Arg
Val1 5 1050711PRTHomo sapiens 507Ser Leu Leu Thr Glu Gln Asp Leu
Trp Thr Val1 5 105089PRTHomo sapiens 508Ser Val Asp Ser Ala Pro Ala
Ala Val1 55099PRTHomo sapiens 509Thr Leu Gln Glu Val Val Thr Gly
Val1 55109PRTHomo sapiens 510Thr Val Asp Val Ala Thr Pro Ser Val1
55119PRTHomo sapiens 511Val Leu Ala Tyr Phe Leu Pro Glu Ala1
55129PRTHomo sapiens 512Val Leu Ile Ser Val Leu Gln Ala Ile1
551311PRTHomo sapiens 513Tyr Leu Ile Pro Phe Thr Gly Ile Val Gly
Leu1 5 1051410PRTHomo sapiens 514Tyr Leu Leu Asp Asp Gly Thr Leu
Val Val1 5 105159PRTHomo sapiens 515Tyr Leu Met Pro Gly Phe Ile His
Leu1 551611PRTHomo sapiens 516Tyr Leu Pro Asp Ile Ile Lys Asp Gln
Lys Ala1 5 105179PRTHomo sapiens 517Tyr Leu Gln Leu Thr Gln Ser Glu
Leu1 551810PRTHomo sapiens 518Tyr Leu Thr Glu Val Phe Leu His Val
Val1 5 105199PRTHomo sapiens 519Tyr Leu Val Glu Gly Arg Phe Ser
Val1 55209PRTHomo sapiens 520Tyr Leu Val Tyr Ile Leu Asn Glu Leu1
552110PRTHomo sapiens 521Tyr Leu Tyr Gly Gln Thr Thr Thr Tyr Leu1 5
105229PRTHomo sapiens 522Tyr Val Leu Thr Gln Pro Pro Ser Val1
552310PRTHomo sapiens 523Glu Leu Ala Gly Ile Gly Ile Leu Thr Val1 5
105249PRTHomo sapiens 524Tyr Leu Leu Pro Ala Ile Val His Ile1
55256PRTArtificial SequenceLinker sequence 525Leu Leu Leu Leu Leu
Leu1 5
* * * * *
References