U.S. patent application number 15/229970 was filed with the patent office on 2018-01-11 for novel peptides and combination of peptides for use in immunotherapy against prostate cancer and other cancers.
The applicant listed for this patent is immatics biotechnology GmbH. Invention is credited to Jens FRITSCHE, Valentina GOLDFINGER, Julia LEIBOLD, Andrea MAHR, Phillip MUELLER, Oliver SCHOOR, Harpreet SINGH, Toni WEINSCHENK.
Application Number | 20180009858 15/229970 |
Document ID | / |
Family ID | 54200350 |
Filed Date | 2018-01-11 |
United States Patent
Application |
20180009858 |
Kind Code |
A9 |
MAHR; Andrea ; et
al. |
January 11, 2018 |
NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY
AGAINST PROSTATE CANCER AND OTHER CANCERS
Abstract
The present invention relates to peptides, proteins, nucleic
acids and cells for use in immunotherapeutic methods. In
particular, the present invention relates to the immunotherapy of
cancer. The present invention furthermore relates to
tumor-associated T-cell peptide epitopes, alone or in combination
with other tumor-associated peptides that can for example serve as
active pharmaceutical ingredients of vaccine compositions that
stimulate anti-tumor immune responses, or to stimulate T cells ex
vivo and transfer into patients. Peptides bound to molecules of the
major histocompatibility complex (MHC), or peptides as such, can
also be targets of antibodies, soluble T-cell receptors, and other
binding molecules.
Inventors: |
MAHR; Andrea; (Tuebingen,
DE) ; WEINSCHENK; Toni; (Aichwald, DE) ;
SCHOOR; Oliver; (Tuebingen, DE) ; FRITSCHE; Jens;
(Dusslingen, DE) ; SINGH; Harpreet; (Houston,
TX) ; MUELLER; Phillip; (Kassel, DE) ;
LEIBOLD; Julia; (Tuebingen, DE) ; GOLDFINGER;
Valentina; (Tuebingen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
immatics biotechnology GmbH |
Tuebingen |
|
DE |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20170037089 A1 |
February 9, 2017 |
|
|
Family ID: |
54200350 |
Appl. No.: |
15/229970 |
Filed: |
August 5, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62201289 |
Aug 5, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/00 20130101;
C12N 5/0636 20130101; C12Q 2600/16 20130101; A61K 2039/5158
20130101; G01N 33/57407 20130101; C07K 14/7051 20130101; C12N
2310/16 20130101; C07K 14/47 20130101; G01N 2800/52 20130101; C07K
2319/00 20130101; C12Q 2600/158 20130101; A61K 39/0011 20130101;
G01N 2570/00 20130101; C07K 14/4748 20130101; C12Q 2600/156
20130101; G01N 33/5748 20130101; G01N 33/6848 20130101; C12N 15/115
20130101; A61K 2039/572 20130101; C07K 16/30 20130101; C12Q 1/6886
20130101; A61P 35/00 20180101; C12Q 2600/106 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; C12N 15/115 20100101 C12N015/115; A61K 39/00 20060101
A61K039/00; G01N 33/574 20060101 G01N033/574; C07K 16/30 20060101
C07K016/30; C12N 5/0783 20100101 C12N005/0783; C12Q 1/68 20060101
C12Q001/68; C07K 14/725 20060101 C07K014/725 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 6, 2015 |
GB |
1513921.5 |
Claims
1. A peptide comprising an amino acid sequence selected from the
group consisting of SEQ ID No. 1 to SEQ ID No. 48, and variant
sequences thereof which are at least 88% homologous to SEQ ID No. 1
to SEQ ID No. 48, wherein said variant binds to molecule(s) of the
major histocompatibility complex (MHC) and/or induces T cells
cross-reacting with said variant peptide; and a pharmaceutical
acceptable salt thereof, wherein said peptide is not a full-length
polypeptide.
2. The peptide or variant according to claim 1, wherein said
peptide has the ability to bind to a MHC class-I or -II molecule,
and wherein said peptide, when bound to said MHC, is capable of
being recognized by CD4 and/or CD8 T cells.
3. The peptide or variant thereof according to claim 1, wherein the
amino acid sequence thereof comprises a continuous stretch of amino
acids according to the group of SEQ ID No. 1 to SEQ ID No. 48.
4. The peptide or variant thereof according to claim 1, wherein
said peptide or variant thereof has an overall length of from 8 to
100, optionally from 8 to 30, and optionally from 8 to 16 amino
acids, and optionally wherein the peptide consists or consists
essentially of an amino acid sequence according to the group of SEQ
ID No. 1 to SEQ ID No. 48.
5. The peptide comprising an amino acid sequence selected from the
group consisting of SEQ ID No. 1 to SEQ ID No. 48, and variant
sequences thereof which are at least 88% homologous to SEQ ID No. 1
to SEQ ID No. 48, wherein said variant binds to molecule(s) of the
major histocompatibility complex (MHC) and/or induces T cells
cross-reacting with said variant peptide; and a pharmaceutical
acceptable salt thereof, wherein said peptide is not a full-length
polypeptide, wherein said peptide is modified and/or includes
non-peptide bonds.
6. The peptide or variant thereof according to claim 1, wherein
said peptide is part of a fusion protein, optionally comprising
N-terminal amino acids of the HLA-DR antigen-associated invariant
chain (Ii).
7. A nucleic acid, encoding a peptide or variant thereof according
to claim 1, optionally linked to a heterologous promoter
sequence.
8. An expression vector capable of expressing the nucleic acid
according to claim 7.
9. A recombinant host cell comprising the peptide or variant
according to claim 1, a nucleic acid encoding said peptide or
variant or an expression vector capable of expressing said nucleic
acid, wherein said host cell optionally is an antigen presenting
cell optionally a dendritic cell.
10. The peptide or variant thereof according to claim 1, a nucleic
acid encoding said peptide or variant or an expression vector
capable of expressing said nucleic acid, or a host cell comprising
said peptide or variant, capable of being used in medicine.
11. A method for producing the peptide or variant thereof according
to claim 1, the method comprising culturing a host cell that
presents the peptide or variant, or expresses a nucleic acid
encoding said peptide or variant or comprises an expression vector
capable of expressing said nucleic acid, and isolating the peptide
or variant thereof from the host cell or its culture medium.
12. An in vitro method for producing activated T lymphocytes, the
method comprising contacting in vitro T cells with antigen loaded
human class I or II MHC molecules expressed on the surface of a
suitable antigen-presenting cell or an artificial construct
mimicking an antigen-presenting cell for a period of time
sufficient to activate said T cells in an antigen specific manner,
wherein said antigen is a peptide according to claim 1.
13. An activated T cell, optionally an activated T lymphocyte,
produced by the method according to claim 12, that selectively
recognizes a cell which presents a polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID No. 1 to
SEQ ID No. 48, and variant sequences thereof which are at least 88%
homologous to SEQ ID No. 1 to SEQ ID No. 48.
14. A method for killing target cells in a patient which target
cells present a polypeptide comprising an amino acid sequence
selected from the group consisting of SEQ ID No. 1 to SEQ ID No.
48, and variant sequences thereof which are at least 88% homologous
to SEQ ID No. 1 to SEQ ID No. 48, the method comprising
administering to the patient an effective number of activated T
cells as defined in claim 13.
15. An antibody, optionally a soluble or membrane-bound antibody,
that specifically recognizes the peptide or variant thereof
according to claim 1, optionally the peptide or variant are bound
to an MHC molecule, and wherein said antibody optionally carries a
further effector function, optionally an immune stimulating domain
or toxin.
16. A peptide or variant according to claim 1, a nucleic acid
encoding said peptide or variant or an expression vector capable of
expressing said nucleic acid, or a host cell comprising said
peptide or variant, an activated T lymphocyte that selectively
recognizes a cell which presents said peptide or variant or the
antibody optionally a soluble or membrane-bound antibody, that
specifically recognizes the peptide or variant capable of being
used in the treatment of cancer or in the manufacture of a
medicament against cancer, or in a diagnostic method for detection
of cancerous cells.
17. The peptide or variant according to claim 16, wherein said
cancer is selected from the group of lung cancer, kidney cancer,
brain cancer, stomach cancer, colon or rectal cancer, liver cancer,
prostate cancer, leukemia, breast cancer, Merkel cell carcinoma
(MCC), melanoma, ovarian cancer, esophageal cancer, urinary bladder
cancer, endometrial cancer, gall bladder cancer, and bile duct
cancer, and other tumors that show an overexpression of a protein
comprising a peptide sequence of any of SEQ ID No. 1 to SEQ ID No.
48.
18. A kit comprising: (a) a container comprising a pharmaceutical
composition containing the peptide(s) or the variant according to
claim 1, a nucleic acid encoding said peptide or variant or an
expression vector capable of expressing said nucleic acid, or a
host cell comprising said peptide or variant, an activated T
lymphocyte that selectively recognizes a cell which presents said
peptide or variant or the antibody optionally a soluble or
membrane-bound antibody, that specifically recognizes the peptide
or variant, in solution or in lyophilized form; (b) optionally, a
second container containing a diluent or reconstituting solution
for the lyophilized formulation; (c) optionally, at least one more
peptide selected from the group consisting of SEQ ID No. 1 to SEQ
ID No. 59, and (d) optionally, instructions for (i) use of the
solution or (ii) reconstitution and/or use of the lyophilized
formulation.
19. The kit according to claim 18, further comprising one or more
of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or
(v) a syringe.
20. The kit according to claim 18, wherein said peptide is selected
from the group consisting of SEQ ID No. 1 to SEQ ID No. 48.
21. A method for producing a personalized anti-cancer vaccine for
the use as a compound-based and/or cellular therapy for an
individual patient, said method comprising: a) identifying
tumor-associated peptides (TUMAPs) presented by a tumor sample from
the individual patient; b) comparing the peptides as identified in
a) with a warehouse of peptides that have been pre-screened for
immunogenicity and/or over-presentation in tumors as compared to
normal tissues, wherein said warehouse of peptides is one or more
selected from the group consisting of SEQ ID 1-59; c) selecting at
least one peptide from the warehouse that matches a TUMAP
identified in the patient; and d) manufacturing or formulating the
personalized vaccine or compound-based or cellular therapy based on
c).
22. The method according to claim 21, wherein said TUMAPs are
identified by: a1) comparing expression data from the tumor sample
to expression data from a sample of normal tissue corresponding to
a tissue type of the tumor sample to identify proteins that are
over-expressed or aberrantly expressed in the tumor sample; and a2)
correlating the expression data with sequences of MHC ligands bound
to MHC class I and/or class II molecules in the tumor sample to
identify MHC ligands derived from proteins over-expressed or
aberrantly expressed by the tumor.
23. The method according to claim 21, wherein the sequences of MHC
ligands are identified by eluting bound peptides from MHC molecules
isolated from the tumor sample, and sequencing the eluted
ligands.
24. The method according to claim 21, wherein the normal tissue
corresponding to the tissue type of the tumor sample is obtained
from the same patient.
25. The method according to claim 21, wherein the peptides included
in the warehouse are identified based on the following steps: aa.
Performing genome-wide messenger ribonucleic acid (mRNA) expression
analysis by highly parallel methods, such as microarrays or
sequencing-based expression profiling, comprising identify genes
that over-expressed in a malignant tissue, compared with a normal
tissue or tissues; ab. Selecting peptides encoded by selectively
expressed or over-expressed genes as detected in aa, and ac.
Determining an induction of in vivo T-cell responses by the
peptides as selected comprising in vitro immunogenicity assays
using human T cells from healthy donors or said patient; or ba.
Identifying HLA ligands from said tumor sample using mass
spectrometry; bb. Performing genome-wide messenger ribonucleic acid
(mRNA) expression analysis by highly parallel methods, optionally
microarrays or sequencing-based expression profiling, comprising
identify genes that over-expressed in a malignant tissue, compared
with a normal tissue or tissues; bc. Comparing the identified HLA
ligands to said gene expression data; bd. Selecting peptides
encoded by selectively expressed or over-expressed genes as
detected in bc; be. Re-detecting of selected TUMAPs from bd on
tumor tissue and lack of or infrequent detection on healthy tissues
and confirming the relevance of over-expression at the mRNA level;
and bf. Determining an induction of in vivo T-cell responses by the
peptides as selected comprising in vitro immunogenicity assays
using human T cells from healthy donors or said patient.
26. The method according to claim 21, wherein the immunogenicity of
the peptides included in the warehouse is determined by a method
comprising in vitro immunogenicity assays, patient immunomonitoring
for individual HLA binding, MHC multimer staining, ELISPOT assays
and/or intracellular cytokine staining.
27. The method according to claim 21, wherein said warehouse
comprises a plurality of peptides selected from the group
consisting of SEQ ID No. 1 to SEQ ID No. 48.
28. The method according to claim 21, further comprising
identifying at least one mutation that is unique to the tumor
sample relative to normal corresponding tissue from the individual
patient, and selecting a peptide that correlates with the mutation
for inclusion in the vaccine or for the generation of cellular
therapies.
29. The method according to claim 28, wherein said at least one
mutation is identified by whole genome sequencing.
30. A T-cell receptor, optionally soluble or membrane-bound, that
is reactive with an HLA ligand, wherein said ligand has at least
75% identity to an amino acid sequence selected from the group
consisting of SEQ ID No. 1 to SEQ ID No. 48.
31. The T-cell receptor according to claim 30, wherein said amino
acid sequence is at least 88% identical to SEQ ID No. 1 to SEQ ID
No. 48.
32. The T-cell receptor according to claim 30, wherein said amino
acid sequence consists any of SEQ ID No. 1 to SEQ ID No. 59.
33. The T-cell receptor according to claim 30, wherein said T-cell
receptor is provided as a soluble molecule and optionally carries a
further effector function optionally an immune stimulating domain
or toxin.
34. A nucleic acid, encoding for a TCR according to claim 30,
optionally linked to a heterologous promoter sequence.
35. An expression vector expressing the nucleic acid according to
claim 34.
36. A recombinant host cell comprising the nucleic acid according
to claim 34 or a nucleic acid encoding an antibody optionally a
soluble or membrane-bound antibody, that specifically recognizes
the peptide or variant or an expression vector comprising said
nucleic acid, wherein said host cell optionally is a T cell or NK
cell.
37. A method for producing the T cell receptor according to claim
30, said method comprising culturing a host cell, and isolating
said T cell receptor from said host cell and/or its culture
medium.
38. A pharmaceutical composition comprising at least one active
ingredient selected from the group consisting of a) a peptide
selected from the group consisting of SEQ ID No. 1 to SEQ ID No.
48; b) a T-cell receptor reactive with a peptide and/or the
peptide-MHC complex according to a); c) a fusion protein comprising
a peptide according to a), and the N-terminal amino acids 1 to 80
of the HLA-DR antigen-associated invariant chain (Ii); d) a nucleic
acid encoding for any of a) to c) or an expression vector
comprising said nucleic acid, e) a host cell comprising the
expression vector of d, f) an activated T-lymphocyte, obtained by a
method comprising contacting in vitro T cells with a peptide
according to a) expressed on the surface of a suitable antigen
presenting cell for a period of time sufficient to activate said T
cell in an antigen specific manner, as well as a method to transfer
these activated T cells into the autologous or other patients; g)
an antibody, or soluble T-cell receptor, reactive to a peptide
and/or the peptide--MHC complex according to a) and/or a cell
presenting a peptide according to a), and potentially modified by
fusion with optionally immune-activating domains or toxins, h) an
aptamer recognizing a peptide selected from the group consisting of
SEQ ID No. 1 to SEQ ID No. 48, and/or a complex of a peptide
selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 48
with a MHC molecule, i) a conjugated or labelled peptide or
scaffold according to any of a) to h) and a pharmaceutically
acceptable carrier, and optionally, pharmaceutically acceptable
excipients and/or stabilizers.
39. An aptamer that specifically recognizes the peptide or variant
thereof according to claim 1, optionally the peptide or variant
thereof being bound to an MHC molecule.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from UK Patent Application
No 1513921.5 filed 6 Aug. 2015 and U.S. Provisional Application No.
62/201,289 filed 5 Aug. 2015 both of which are incorporated herein
by reference in their entireties.
[0002] This application is also related to PCT/EP2016/068727 filed
5 Aug. 2016, the content of which is incorporated herein by
reference in its entirety.
REFERENCE TO SEQUENCE USING 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
"2912919-053001_ST25.txt" created on 4 Aug. 2016, and 9,693 bytes
in size) is submitted concurrently with the instant application,
and the entire contents of the Sequence Listing are incorporated
herein by reference.
FIELD OF THE INVENTION
[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] Prostate cancer is the second most frequently diagnosed
cancer worldwide and the fifth most common cause of cancer death
among men, with an estimated 1.1 million new cases (15% of all
cancers in men) and 0.3 million cancer deaths (7% of all cancer
deaths in men) in 2012. In the same period it was the most frequent
type of cancer among men 84 countries worldwide, largely in
countries that have attained high or very high levels of human
development, but also in several countries in Central and Southern
Africa. An estimated 220,800 new cases of prostate cancer and
27,540 deaths due to prostate cancer are expected in 2015 in the
USA according to the American Cancer Society. Risk factors for
prostate cancer are age, family history, and race (World Cancer
Report, 2014; SEER Stat facts, 2014; American Cancer Society,
2015).
[0007] Almost all prostate cancers are adenocarcinomas which
develop from the gland cells of the prostate. Rare forms of
prostate cancer include sarcomas, small cell carcinomas,
neuroendocrine tumors (other than small cell carcinomas) or
transitional cell carcinomas (American Cancer Society, 2015).
[0008] The therapeutic strategy for prostate cancer mainly depends
on the cancer stage. For locally restricted non-metastasizing
prostate cancer, treatment options include active surveillance
(wait and watch), complete surgical resection of the prostate and
local high dose radiation therapy with or without brachytherapy. In
high risk patients, hormonal ablation therapy and post-operative
local radiation therapy represent further possibilities. Standard
treatments for metastasizing prostate cancer also comprise complete
surgical resection of the prostate, local high dose radiation and
hormonal ablation therapy. Tumors irresponsive to hormonal
deprivation are called castration-resistant prostate cancers
(CRPC). CRPC patients receive docetaxel, abiraterone and the
dendritic cell-based vaccine sipuleucel-T. Bone metastases are
treated with radium-223 alone or a combination of either
radium-223, docetaxel or abiraterone with bisphosphonates or
denosumab (S3-Leitlinie Prostatakarzinom, 2014).
[0009] The dendritic cell-based vaccine sipuleucel-T was the first
anti-cancer vaccine to be approved by the FDA. Due to its positive
effect on survival in patients with CRPC, much effort is put into
the development of further immunotherapies. Regarding vaccination
strategies, the peptide vaccine prostate-specific antigen
(PSA)-TRICOM, the personalized peptide vaccine PPV, the DNA vaccine
pTVG-HP and the whole cell vaccine expressing GM-CSF GVAX showed
promising results in different clinical trials. Furthermore,
dendritic cell-based vaccines other than sipuleucel-T, namely
BPX-101 and DCVAC/Pa were shown to elicited clinical responses in
prostate cancer patients. Immune checkpoint inhibitors like
ipilimumab and nivolumab are currently evaluated in clinical
studies as monotherapy as well as in combination with other
treatments, including androgen deprivation therapy, local radiation
therapy, PSA-TRICOM and GVAX. The immunomodulatory substance
tasquinimod, which significantly slowed progression and increased
progression free survival in a phase II trial, is currently further
investigated in a phase III trial. Lenalidomide, another
immunomodulator, induced promising effects in early phase clinical
studies, but failed to improve survival in a phase III trial.
Despite these disappointing results further lenalidomide trials are
ongoing (Quinn et al., 2015).
[0010] 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 prostate cancer
in particular. There is also a need to identify factors
representing biomarkers for cancer in general and prostate cancer
in particular, leading to better diagnosis of cancer, assessment of
prognosis, and prediction of treatment success.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] Like for all cancer entities evolving from non-vital organs
or tissues, prostate-specific antigens may be a good choice for
cancer immunotherapy because prostate-specific antigens represent
tumor-specific targets in a prostatectomized patient. In a cancer
patient without prostatectomy, such antigens may also be
interesting because prostate is not regarded as a vital organ, and
a similar approach has been taken in melanoma with melanocyte
differentiation antigens. There are several examples showing that
prostate-specific or highly-prostate-associated antigens are safe
targets, e.g. sipuleucel-T (Provenge) from Dendreon comprising
prostate acid phosphatase as used tumor antigen (Westdorp et al.,
2014). This antigen is not exclusively expressed in prostate but
over-expressed at levels of 1-2 orders of magnitude higher in
prostate vs. other tissues (Graddis et al., 2011).
[0015] 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. 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.
[0016] 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.
[0017] 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).
[0018] 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).
[0019] Elongated (longer) peptides of the invention can act as MHC
class II active epitopes. T-helper cells, activated by MHC class II
epitopes, play an important role in 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.
[0020] 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).
[0021] 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).
[0022] 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.
[0023] For an MHC class I peptide to trigger (elicit) a cellular
immune response, it also must bind to an MHC-molecule. This process
is dependent on the allele of the MHC-molecule and specific
polymorphisms of the amino acid sequence of the peptide.
MHC-class-I-binding peptides are usually 8-12 amino acid residues
in length and usually contain two conserved residues ("anchors") in
their sequence that interact with the corresponding binding groove
of the MHC-molecule. In this way each MHC allele has a "binding
motif" determining which peptides can bind specifically to the
binding groove.
[0024] 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).
[0025] For proteins to be recognized by T-lymphocytes as
tumor-specific or -associated antigens, and to be used in a
therapy, particular prerequisites must be fulfilled. The antigen
should be expressed mainly by tumor cells and not, or in comparably
small amounts, by normal healthy tissues. In a preferred
embodiment, the peptide should be over-presented by tumor cells as
compared to normal healthy tissues. It is furthermore desirable
that the respective antigen is not only present in a type of tumor,
but also in high concentrations (i.e. copy numbers of the
respective peptide per cell). Tumor-specific and tumor-associated
antigens are often derived from proteins directly involved in
transformation of a normal cell to a tumor cell due to their
function, e.g. in cell cycle control or suppression of apoptosis.
Additionally, downstream targets of the proteins directly causative
for a transformation may be up-regulated und thus may be indirectly
tumor-associated. Such indirect tumor-associated antigens may also
be targets of a vaccination approach (Singh-Jasuja et al., 2004).
It is essential that epitopes are present in the amino acid
sequence of the antigen, in order to ensure that such a peptide
("immunogenic peptide"), being derived from a tumor associated
antigen, leads to an in vitro or in vivo T-cell-response.
[0026] 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.
[0027] 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").
[0028] In case of targeting peptide-MHC by specific TCRs (e.g.
soluble TCRs) and antibodies or other binding molecules (scaffolds)
according to the invention, the immunogenicity of the underlying
peptides is secondary. In these cases, the presentation is the
determining factor.
SUMMARY OF THE INVENTION
[0029] 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: 48
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: 48, 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.
[0030] 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: 48 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: 48, wherein said peptide or variant thereof has an
overall length of between 8 and 100, preferably between 8 and 30,
and most preferred of between 8 and 14 amino acids.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIGS. 1A-1S depict embodiments as described herein.
[0032] FIGS. 2A-2E depict embodiments as described herein.
[0033] FIGS. 3A, 3B, 4A, 4B, 4C, 5A and 5B depict embodiments as
described herein.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
[0034] 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, Table 3 and Table 5 bind to HLA-A*02. All peptides in Table 2,
Table 4 and Table 6 bind to HLA-A*24. The peptides in Table 3 and
Table 4 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 5 and Table 6 are additional peptides
that may be useful in combination with the other peptides of the
invention. The peptides in Table 7 and Table 8 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 HLA-A*02 peptides according to the present
invention SEQ Official ID Gene No. Sequence GeneID(s) Symbol(s) 1
VTAQIGIVAV 81285 OR51E2 2 SMLGEEIQL 9687 GREB1 3 HLLEDIAHV 4744
NEFH 4 ALLTFVWKL 79054 TRPM8 5 KIFSRLIYI 79054 TRPM8 6 ALLESRVNL
50940 PDE11A 7 TLLQVVGVVSV 10257 ABCC4 8 LLDFSLADA 1674 DES 9
GMLNEAEGKAIKL 4629 MYH11 10 TLWRGPVVV 261729 STEAP2 11 YLEEECPAT
563 AZGP1 12 SLNEEIAFL 1674 DES 13 AMAPNHAVV 4057 LTF 14 KMDEASAQLL
54682 MANSC1 15 KMDEASAQLLA 54682 MANSC1 16 KMDEASAQL 54682 MANSC1
17 RLGIKPESV 1466 CSRP2 18 GLSEFTEYL 4131 MAP1B 19 LLPPPPLLA 23245
ASTN2 20 SLLSHQVLL 57221 KIAA1244 21 YLNDSLRHV 283078 MKX 22
SLYDSIAFI 56978 PRDM8 23 AVAGADVIITV 1428 CRYM
TABLE-US-00002 TABLE 2 HLA-A*24 peptides according to the present
invention. SEQ Official ID Gene No. Sequence GeneID(s) Symbol(s) 24
SYNDALLTF 79054 TRPM8 25 IYEPYLAMF 79054 TRPM8 26 RYADDTFTPAF 5865
RAB3B 27 GYLQGLVSF 9622 KLK4 28 YYAKEIHKF 7043 TGFB3 29 RYGSPINTF
647024 C6orf132 30 SYSPAHARL 2624 GATA2 31 AYTSPPSFF 171024 SYNP02
32 PYQLNASLFTF 171024 SYNP02 33 QYGKDFLTL 79088 ZNF426 34
AFSPDSHYLLF 3679 ITGA7 35 IYTRVTYYL 64499, TPSB2, 7177 TPSAB1 36
RYMWINQEL 374654 KIF7 37 RYLQDLLAW 5339 PLEC 38 VYSDKLWIF 8216
LZTR1 39 SYIDVAVKL 57544 TXNDC16
TABLE-US-00003 TABLE 3 Additional HLA-A*02 peptides according to
the present invention with no known cancer association. J =
phospho-serine prior SEQ ID Official No. Sequence GeneID(s) Gene
Symbol(s) 40 RTFJPTYGL 23336 SYNM
TABLE-US-00004 TABLE 4 Additional HLA-A*24 peptides according to
the present invention with no prior known cancer association SEQ ID
Official No. Sequence GeneID(s) Gene Symbol(s) 41 RYLQKIEEF 3755
KCNG1 42 TYIGQGYII 60681 FKBP10 43 AYIKNGQLF 56978 PRDM8 44
VYNTVSEGTHF 25800 SLC39A6 45 RYFKTPRKF 25792 CIZ1 46 VYEEILHQI
116496 FAM129A 47 SYTPVLNQF 10497 UNC13B 48 AWAPKPYHKF 23043,
50488, TNIK, MINK1, 9448 MAP4K4
TABLE-US-00005 TABLE 5 HLA-A*02 peptides useful for e.g.
personalized cancer therapies SEQ ID Official No. Sequence
GeneID(s) Gene Symbol(s) 49 SLFHPEDTGQV 354 KLK3 50 TLGPASFLV
389816 LRRC26 51 AMFDKKVQL 23336 SYNM 52 ALGDLVQSV 7782 SLC30A4 53
YLLKDKGEYTL 2316 FLNA
TABLE-US-00006 TABLE 6 HLA-A*24 peptides useful for e.g.
personalized cancer therapies SEQ ID Official No. Sequence
GeneID(s) Gene Symbol(s) 54 AYSEKVTEF 3817 KLK2 55 LYFEKGEYF 55
ACPP 56 LFHPEDTGQVF 354 KLK3 57 KYADKIYSI 2346 FOLH1 58 GYIDKVRQL
4744 NEFH 59 IYPDVTYAF 1135 CHRNA2
TABLE-US-00007 TABLE 7 Peptides according to the present invention
and their specific uses in other proliferative diseases, especially
in cancerous diseases. The table shows for selected peptides on
which additional tumor types they were found and either
over-presented on more than 5% of the measured tumor samples, or
presented on more than 5% of the measured tumor samples with a
ratio of geometric means tumor vs normal tissues being larger than
three. Over-presentation is defined as higher presentation on the
tumor sample as compared to the normal sample with highest
presentation. Normal (non-cancerous) tissues against which over-
presentation was tested were selected from: adipose tissue, adrenal
gland, artery, bone marrow, brain, central nerve, colon, duodenum,
esophagus, eye, gallbladder, heart, kidney, liver, lung, lymph
node, mononuclear white blood cells, pancreas, parathyroid gland,
peripheral nerve, peritoneum, pituitary, pleura, rectum, salivary
gland, skeletal muscle, skin, small intestine, spleen,stomach,
thyroid gland, trachea, ureter, urinary bladder, and veins. SEQ ID
No. Sequence Other relevant organs/diseases 1 VTAQIGIVAV SCLC,
Melanoma 2 SMLGEEIQL HCC, BRCA, Melanoma, Uterine Cancer 3
HLLEDIAHV MCC, Uterine Cancer 5 KIFSRLIYI Melanoma 6 ALLESRVNL HCC,
PC 7 TLLQVVGVVSV Uterine Cancer, Gallbladder Cancer, Bile Duct
Cancer 8 LLDFSLADA CRC, BRCA, Melanoma, Urinary bladder cancer,
Uterine Cancer 10 TLWRGPVVV NSCLC, RCC, CRC, HCC, Leukemia, OC,
Esophageal Cancer, Gallbladder Cancer, Bile Duct Cancer, CLL 12
SLNEEIAFL SCLC, PC, Urinary bladder cancer, Gallbladder Cancer,
Bile Duct Cancer 13 AMAPNHAVV Brain Cancer 16 KMDEASAQL Urinary
bladder cancer 17 RLGIKPESV Brain Cancer, HCC, BRCA, Uterine Can-
18 GLSEFTEYL Brain Cancer 19 LLPPPPLLA Brain Cancer, Melanoma,
Urinary bladder cancer 20 SLLSHQVLL SCLC, CRC, HCC, Urinary bladder
can- cer, BRCA, Esophageal Cancer, Uterine Cancer 22 SLYDSIAFI
Brain Cancer, Leukemia, AML 26 RYADDTFTPAF HCC 27 GYLQGLVSF HCC 28
YYAKEIHKF NSCLC, HCC 29 RYGSPINTF NSCLC, GC, HCC 31 AYTSPPSFF GC,
HCC 33 QYGKDFLTL NSCLC, Brain Cancer, HCC 34 AFSPDSHYLLF NSCLC,
RCC, Brain Cancer, HCC 35 IYTRVTYYL NSCLC, GC 36 RYMWINQEL NSCLC,
Brain Cancer, HCC 37 RYLQDLLAW NSCLC, RCC, Brain Cancer 38
VYSDKLWIF NSCLC, Brain Cancer, GC, HCC 40 RTFJPTYGL Urinary bladder
cancer 41 RYLQKIEEF NSCLC, RCC, Brain Cancer 42 TYIGQGYII NSCLC,
Brain Cancer, GC, HCC 43 AYIKNGQLF Brain Cancer 44 VYNTVSEGTHF
NSCLC, Brain Cancer, HCC 45 RYFKTPRKF HCC 47 SYTPVLNQF HCC 48
AWAPKPYHKF NSCLC, RCC, Brain Cancer, HCC kidney NSCLC = non-small
cell lung cancer, SCLC = small cell lung cancer, RCC = kidney
cancer, CRC = colon or rectum cancer, HCC = liver cancer, PC =
pancreatic cancer, BRCA = breast cancer, MCC = Merkel cell
carcinoma, OC = ovarian cancer, AML = acute myeloid leukemia, CLL =
chronic lymphocytic leukemia, GC = stomach cancer; J =
phophoserine
[0035] The present invention furthermore generally relates to the
peptides according to the present invention for use in the
treatment of proliferative diseases, such as, for example, lung
cancer, small cell lung cancer, melanoma, liver cancer, breast
cancer, Uterine cancer, Merkel cell carcinoma, pancreatic cancer,
gallbladder cancer, bile duct cancer, CRC, urinary bladder cancer,
non-small cell lung cancer, kidney cancer, Leukemia (e.g. AML or
CLL), ovarian cancer, esophageal cancer, brain cancer, and gastric
(stomach) cancer.
[0036] 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: 48. More preferred
are the peptides--alone or in combination--selected from the group
consisting of SEQ ID No: 1 to SEQ ID No: 6 (see Table 1) and SEQ ID
No: 24 to SEQ ID No: 28 (see Table 2) or SEQ ID No: 1, 4, 5, 6, 49,
and 52 or SEQ ID No: 2, 3, and 54, and their uses in the
immunotherapy of lung cancer, small cell lung cancer, melanoma,
liver cancer, breast cancer, Uterine cancer, Merkel cell carcinoma,
pancreatic cancer, gallbladder cancer, bile duct cancer, CRC,
urinary bladder cancer, non-small cell lung cancer, kidney cancer,
Leukemia (e.g. AML or CLL), ovarian cancer, esophageal cancer,
brain cancer, and gastric (stomach) cancer, and most preferably
prostate cancer. As shown in the above Table 7, many of the
peptides according to the present invention are also found on other
tumor types and can, thus, also be used in the immunotherapy of
other indications. Also refer to FIG. 1 and Example 1.
[0037] 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, 12, and 20 for the--in one
preferred embodiment combined--treatment of non-small cell lung
cancer.
[0038] 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, 5, 8, and 19 for the--in
one preferred embodiment combined--treatment of melanoma.
[0039] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 2, 6, 10, 17, 20, 26, 27, 28,
29, 31, 33, 34, 36, 38, 42, 44, 45, 47, and 48 for the--in one
preferred embodiment combined--treatment of liver cancer.
[0040] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 2, 8, 17, and 20 for the--in one
preferred embodiment combined--treatment of breast cancer.
[0041] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 2, 3, 7, 8, 17, and 20 for
the--in one preferred embodiment combined--treatment of uterine
cancer.
[0042] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to SEQ ID No. 3 for the treatment of MCC.
[0043] 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. 6, and 12 for the--in one
preferred embodiment combined--treatment of PC.
[0044] 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, 10, and 12 for the--in one
preferred embodiment combined--treatment of gallbladder cancer,
and/or bile duct cancer.
[0045] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to any one of SEQ ID No. 8, 10, and 20 for the--in one
preferred embodiment combined--treatment of CRC.
[0046] 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. 10, and 22 for the--in one
preferred embodiment combined--treatment of leukemia.
[0047] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to SEQ ID No. 10 for the treatment of OC.
[0048] 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. 10, and 20 for the--in one
preferred embodiment combined--treatment of esophageal Cancer.
[0049] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to SEQ ID No. 10 for the treatment of CLL.
[0050] 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. 6, and 12 for the--in one
preferred embodiment combined--treatment of PC.
[0051] 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. 10, 28, 29, 33, 34, 35, 36, 37,
38, 41, 42, 44, and 48 for the--in one preferred embodiment
combined--treatment of NSCLC.
[0052] 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. 10, 34, 37, 41, and 48 for
the--in one preferred embodiment combined--treatment of RCC.
[0053] 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, 17, 18, 19, 22, 33, 34, 36,
37, 38, 41, 42, 43, 44, and 48 for the--in one preferred embodiment
combined--treatment of brain cancer.
[0054] Thus, another aspect of the present invention relates to the
use of at least one peptide according to the present invention
according to SEQ ID No. 22 for the treatment of AML.
[0055] 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, 31, 35, 38, and 42 for
the--in one preferred embodiment combined--treatment of GC.
[0056] 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 lung cancer, small cell lung cancer,
melanoma, liver cancer, breast cancer, Uterine cancer, Merkel cell
carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer,
CRC, urinary bladder cancer, non-small cell lung cancer, kidney
cancer, Leukemia (e.g. AML or CLL), ovarian cancer, esophageal
cancer, brain cancer, and gastric (stomach) cancer, and most
preferably prostate cancer.
[0057] 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.
[0058] 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: 48.
[0059] The present invention further relates to the peptides
according to the present invention, wherein said peptide is
modified and/or includes non-peptide bonds.
[0060] 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.
[0061] Another embodiment of the present invention relates to a
non-naturally occurring peptide wherein said peptide consists or
consists essentially of an amino acid sequence according to SEQ ID
No: 1 to SEQ ID No: 48 and has been synthetically produced (e.g.
synthesized) as a pharmaceutically acceptable salt. Methods to
synthetically produce peptides are well known in the art. The salts
of the peptides according to the present invention differ
substantially from the peptides in their state(s) in vivo, as the
peptides as generated in vivo are no salts. The non-natural salt
form of the peptide mediates the solubility of the peptide, in
particular in the context of pharmaceutical compositions comprising
the peptides, e.g. the peptide vaccines as disclosed herein. A
sufficient and at least substantial solubility of the peptide(s) is
required in order to efficiently provide the peptides to the
subject to be treated. Preferably, the salts are pharmaceutically
acceptable salts of the peptides. These salts according to the
invention include alkaline and earth alkaline salts such as salts
of the Hofmeister series comprising as anions PO.sub.4.sup.3-,
SO.sub.4.sup.2-, CH.sub.3COO.sup.-, Cl.sup.-, Br, 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,
KNO.sub.3, KClO.sub.4, KI, KSCN, Na.sub.3PO.sub.4,
Na.sub.2HPO.sub.4, NaH.sub.2PO.sub.4, Na.sub.2SO.sub.4,
NaCH.sub.3COO, NaCl, NaBr, NaNO.sub.3, NaClO.sub.4, NaI, NaSCN,
ZnCl.sub.2 Cs.sub.3PO.sub.4, Cs.sub.2HPO.sub.4, CsH.sub.2PO.sub.4,
Cs.sub.2SO.sub.4, CsCH.sub.3COO, CsCl, CsBr, CsNO.sub.3,
CsClO.sub.4, CsI, CsSCN, Li.sub.3PO.sub.4, Li.sub.2HPO.sub.4,
LiH.sub.2PO.sub.4, Li.sub.2SO.sub.4, LiCH.sub.3COO, LiCl, LiBr,
LiNO.sub.3, LiClO.sub.4, LiI, LiSCN, Cu.sub.2SO.sub.4,
Mg.sub.3(PO.sub.4).sub.2, Mg.sub.2HPO.sub.4,
Mg(H.sub.2PO.sub.4).sub.2, Mg.sub.2SO.sub.4, Mg(CH.sub.3COO).sub.2,
MgCl.sub.2, MgBr.sub.2, Mg(NO.sub.3).sub.2, Mg(ClO.sub.4).sub.2,
MgI.sub.2, Mg(SCN).sub.2, MnCl.sub.2, Ca.sub.3(PO.sub.4),
Ca.sub.2HPO.sub.4, Ca(H.sub.2PO.sub.4).sub.2, CaSO.sub.4,
Ca(CH.sub.3COO).sub.2, CaCl.sub.2, CaBr.sub.2, Ca(NO.sub.3).sub.2,
Ca(ClO.sub.4).sub.2, CaI.sub.2, Ca(SCN).sub.2,
Ba.sub.3(PO.sub.4).sub.2, Ba.sub.2HPO.sub.4,
Ba(H.sub.2PO.sub.4).sub.2, BaSO.sub.4, Ba(CH.sub.3COO).sub.2,
BaCl.sub.2, BaBr.sub.2, Ba(NO.sub.3).sub.2, Ba(ClO.sub.4).sub.2,
BaI.sub.2, and Ba(SCN).sub.2. Particularly preferred are NH
acetate, MgCl.sub.2, KH.sub.2PO.sub.4, Na.sub.2SO.sub.4, KCl, NaCl,
and CaCl.sub.2, such as, for example, the chloride or acetate
(trifluoroacetate) salts.
[0062] 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-hydroxymethylphenoxyacetic 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-dicydohexyl-carbodiimide/1
hydroxybenzotriazole mediated coupling procedure. All coupling and
deprotection reactions are monitored using ninhydrine,
trinitrobenzene sulphonic acid or isotin test procedures. Upon
completion of synthesis, peptides are cleaved from the resin
support with concomitant removal of side-chain protecting groups by
treatment with 95% trifluoroacetic acid containing a 50% scavenger
mix. Scavengers commonly used include ethanedithiol, phenol,
anisole and water, the exact choice depending on the constituent
amino acids of the peptide being synthesized. Also a combination of
solid phase and solution phase methodologies for the synthesis of
peptides is possible (see, for example, (Bruckdorfer et al., 2004),
and the references as cited therein).
[0063] 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).
[0064] Purification may be performed by any one, or a combination
of, techniques such as recrystallization, size exclusion
chromatography, ion-exchange chromatography, hydrophobic
interaction chromatography and (usually) reverse-phase high
performance liquid chromatography using e.g. acetonitril/water
gradient separation.
[0065] 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.
[0066] The present invention further relates to an expression
vector capable of expressing and/or expressing a nucleic acid
according to the present invention.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] The antibodies and TCRs are additional embodiments of the
immunotherapeutic use of the peptides according to the invention at
hand.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.: 48,
preferably containing SEQ ID No: 1 to SEQ ID No: 6 (see Table 1)
and SEQ ID No: 24 to SEQ ID No: 28 (see Table 2), or a variant
amino acid sequence.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] Preferably, said medicament is a cellular therapy, a vaccine
or a protein based on a soluble TCR or antibody.
[0079] The present invention further relates to a use according to
the present invention, wherein said cancer cells are lung cancer,
small cell lung cancer, melanoma, liver cancer, breast cancer,
Uterine cancer, Merkel cell carcinoma, pancreatic cancer,
gallbladder cancer, bile duct cancer, CRC, urinary bladder cancer,
non-small cell lung cancer, kidney cancer, leukemia (e.g. AML or
CLL), ovarian cancer, esophageal cancer, brain cancer, and gastric
(stomach) cancer, and most preferably prostate cancer cells.
[0080] 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
prostate cancer. The marker can be over-presentation of the
peptide(s) themselves, or over-expression of the corresponding
gene(s). The markers may also be used to predict the probability of
success of a treatment, preferably an immunotherapy, and most
preferred an immunotherapy targeting the same target that is
identified by the biomarker. For example, an antibody or soluble
TCR can be used to stain sections of the tumor to detect the
presence of a peptide of interest in complex with MHC.
[0081] Optionally, the antibody carries a further effector function
such as an immune stimulating domain or toxin.
[0082] The present invention also relates to the use of these novel
targets in the context of cancer treatment.
[0083] Both therapeutic and diagnostic uses against additional
cancerous diseases are disclosed in the following more detailed
description of the underlying expression products (polypeptides) of
the peptides according to the invention.
[0084] ATP-binding cassette, sub-family C (CFTR/MRP), member 4
(ABCC4)--ABCC4 is capable of pumping a wide variety of endogenous
and xenobiotic organic anionic compounds out of the cell, including
a wide variety of antiviral, cytostatic, antibiotic and
cardiovascular drugs as well as molecules involved in cellular
signaling, thus giving ABCC4 a key function in cellular protection
and extracellular signaling pathways (Russel et al., 2008). ABCC4
levels are elevated in prostate cancer, and expression has been
shown to be regulated by androgen treatment in vitro. Consequently,
androgen ablation treatment lowers ABCC4 expression in prostate
cancer tissue. Moreover, ABCC4 levels decrease upon prostate cancer
progression (Ho et al., 2008; Montani et al., 2013). ABCC4
overexpression has also been demonstrated for other cancer types,
as for example in lung cancer (Zhao et al., 2014). Suppression of
ABCC4 expression was shown to inhibit proliferation of
ABCC4-overexpressing cancer cells in vitro (Zhao et al., 2014) and
to restore sensitivity to chemotherapeutics in drug-resistant cell
lines (Zhang et al., 2014).
[0085] Astrotactin 2 (ASTN2)--ASTN2 is suggested to function in
neuronal cell migration (Wilson et al., 2010).
[0086] Alpha-2-glycoprotein 1, zinc-binding (AZGP1)--ASTN2
stimulates lipid degradation in adipocytes and causes the extensive
fat losses associated with some advanced cancers (UniProt, 2015).
AZGP1 expression is increased in prostate cancer, which is
detectable also in serum samples and has been suggested as
potential biomarker (Hale et al., 2001). Levels decrease in
high-grade tumors, and AZGP1 expression has been associated with
decreased mortality and decreased tumor recurrence in prostate
cancer patients (Lapointe et al., 2004; Seven et al., 2014). AZGP1
has been suggested as a biomarker for breast cancer, and AZGP1
levels decrease in poorly differentiated tumors (Hassan et al.,
2008).
[0087] Chromosome 6 open reading frame 132 (C6orf132)--C6orf132 is
located on chromosome 6p21.1 (RefSeq, 2002).
[0088] CDKN1A interacting zinc finger protein 1 (CIZ1)--CIZ1
encodes for the CDKN1A interacting zinc finger protein which
functions as a tumor suppressor (Nishibe et al., 2013). In
colorectal cancer CIZ1 was suggested to be involved in cancer
progression by regulating cell proliferation, cell cycle, apoptosis
and colony formation (Yin et al., 2013). A CIZ1 variant that lacks
part of the C-terminal domain is found in lung cancer cells even at
an early stage and has potential as a biomarker (Higgins et al.,
2012).
[0089] Crystallin, mu (CRYM)--CRYM specifically catalyzes the
reduction of imine bonds (Hallen et al., 2011). CRYM is an
androgen-regulated gene whose expression is elevated in prostate
cancer but down-regulated in castration therapy-resistant tumors
(Malinowska et al., 2009). CRYM was overexpressed in non-small cell
lung cancer and in metastasis of uterine leiomyosarcoma as compared
to the primary tumor (Chong et al., 2006; Davidson et al., 2014).
CRYM expression was also found in breast cancer samples, and CRYM
was recognized by breast cancer patients' sera in SEREX analysis
(Forti et al., 2002).
[0090] Cysteine and glycine-rich protein 2 (CSRP2)--CSRP2 belongs
to the family of CSRP genes, encoding LIM domain containing
proteins. Over-expression of CSRP2 is associated with
de-differentiation of hepatocellular carcinoma (Midorikawa et al.,
2002).
[0091] Desmin (DES)--DES are class-Ill intermediate filaments found
in muscle cells. They form a fibrous network connecting myofibrils
to each other and to the plasma membrane from the periphery of the
Z-line structures (Clemen et al., 2015). DES expression is
decreased in prostate cancer tissue and low DES expression has been
associated with a shorter disease-free period (Wu et al., 2014). In
colorectal carcinoma, DES expression was elevated in advanced stage
tumors, possibly due to increased microvessel density and thus
increased levels of pericytes (Arentz et al., 2011). In addition,
elevated DES levels have been associated with decreased survival in
colorectal cancer (Ma et al., 2009).
[0092] Family with sequence similarity 129, member A
(FAM129A)--FAM129A is located on chromosome 1q25 (RefSeq, 2002).
Overexpression of FAM129A has been demonstrated in head and neck
squamous cell carcinoma (Ito et al., 2010a), thyroid tumors
(Matsumoto et al., 2006), and renal cell carcinoma (Adachi et al.,
2004).
[0093] FK506 binding protein 10 (FKBP10)--FKBP10 encodes the FK506
binding protein 10, which belongs to the FKBP-type peptidyl-prolyl
cis/trans isomerase family. The FKBP10 gene product localizes to
the endoplasmic reticulum and acts as a molecular chaperone
(RefSeq, 2002). FKBP10 was identified as a novel gene that
participates in the acquisition and maintenance of the
adriamycin-resistant phenotype in leukemia cells (Sun et al.,
2014). FKBP10 has been associated with colorectal cancer through
its up-regulation (Olesen et al., 2005). In contrast, the
under-expression of FKBP10 was characteristic for epithelial
ovarian carcinomas (Quinn et al., 2013).
[0094] GATA binding protein 2 (GATA2)--GATA2 is critically required
for hematopoiesis, and mutations in GATA2 are associated with
myelodysplastic syndrome and myeloid leukemia (Bresnick et al.,
2012). In solid tumors, overexpression of GATA2 has been described
in breast cancer and was correlated with poor prognosis in
colorectal carcinoma and glioblastoma (Chen et al., 2013; Wang et
al., 2015; Wang et al., 2012). In contrast, other reports rather
suggest decreased expression of GATA2 in neoplastic tissue and
report an association of GATA2 reduction with tumor aggressiveness
and poor outcome in hepatocellular carcinoma, bladder cancer, or
renal cell carcinoma (Kandimalla et al., 2012; Peters et al., 2014;
Li et al., 2014).
[0095] Growth regulation by estrogen in breast cancer
(GREB1)--GREB1 is an estrogen-responsive gene that is an early
response gene in the estrogen receptor-regulated pathway. It is
thought to play an important role in hormone-responsive tissues and
cancer (RefSeq, 2002). GREB1 is overexpressed in prostate cancer
and benign prostate hyperplasia, and is involved in
androgen-induced growth of prostate cancer cells (Rae et al.,
2006). GREB1 also mediates estrogen-stimulated proliferation of
ovarian cancer and breast cancer cells (Laviolette et al.,
2014).
[0096] Integrin alpha 7 (ITGA7)--ITGA7 is the alpha chain of the
laminin-1 receptor dimer integrin alpha-7/beta-1. ITGA7 is a
tumor-suppressor gene that is critical for suppressing the growth
of malignant tumors. Mutational analysis revealed ITGA7 mutations
in prostate cancer, hepatocellular carcinoma, soft tissue
leiomyosarcoma, and glioblastoma multiforme. ITGA7 was
down-regulated in non-metastatic prostate cancer and leiomyosarcoma
(Tan et al., 2013).
[0097] Potassium voltage-gated channel, subfamily G, member 1
(KCNG1)--KCNG1 is abundantly expressed in skeletal muscle (Gutman
et al., 2005).
[0098] KIAA1244--KIAA1244 encodes for ARFGEF family member 3 and is
located on chromosome 6q23.3. (RefSeq, 2002). KIAA1244 was found to
be overexpressed in a majority of breast cancers (Nishidate et al.,
2004).
[0099] Kinesin family member 7 (KIF7)--KIF7 is implicated in a
variety of diseases including Joubert, hydrolethalus and
acrocallosal syndromes. It is also involved in primary cilium
formation (Klejnot and Kozielski, 2012). Aberrant activation of
Hedgehog signaling pathway, in which KIF7 plays an important role,
leads to pathological consequences in a variety of human tumors,
such as basal cell carcinoma of the skin, gastric cancer and
pancreatic cancer (Li et al., 2012; Katoh and Katoh, 2005).
[0100] Kallikrein-related peptidase 4 (KLK4)--KLK4 is one of the
fifteen kallikrein subfamily members whose genes are located in a
cluster on chromosome 19 (Hu et al., 2000). KLK4 is elevated in
breast cancer, ovarian cancer and prostate cancer (Schmitt et al.,
2013). In prostate cancer KLK4 interacts with androgen and mTOR
signaling (Jin et al., 2013).
[0101] Lactotransferrin (LTF)--LTF is a multifunctional protein
that is found at highest levels in milk, and at lower
concentrations in other mucosal fluids. LTF has been shown to exert
antimicrobial and anti-inflammatory activity, is involved in iron
homeostasis, and plays a role in cell growth and differentiation
and in protection against cancer development and metastasis (Ward
et al., 2005). LTF is downregulated in prostate cancer tissue and
sera as compared to benign prostate hyperplasia. In addition,
lowered LTF levels have been correlated with decreased patient
survival (Shaheduzzaman et al., 2007). LTF inhibits cell growth by
blocking cell cycle progression, possibly through several
mechanisms, including inhibition of NF-kappaB and Akt signaling
(Deng et al., 2013; Ye et al., 2014). In addition, LTF also exerts
pro-apoptotic effects, including activation of caspase-3 and JNK
signaling (Sakai et al., 2005; Wang et al., 2011).
[0102] Leucine-zipper-like transcription regulator 1 (LZTR1)--LZTR1
might play a role in regulation of the Ras/MAPK pathway (Yamamoto
et al., 2015). LZTR1 is frequently mutated or deleted in
glioblastoma (Frattini et al., 2013).
[0103] MANSC domain containing 1 (MANSC1)--MANSC1 is located on
chromosome 12p13.2 (RefSeq, 2002). MANSC1 was significantly
downregulated in prostate tumors; it may be critical to initiation
or progression of prostate carcinoma (Kibel et al., 2004). In
patients with different hematologic malignancies MANSC1 shows an
increased expression (Haferlach et al., 2011).
[0104] Microtubule-associated protein 1B (MAP1B)--MAP1B is a
microtubule-stabilizing protein which plays an important role in
central nervous system development and axon function (Halpain and
Dehmelt, 2006). Immunohistochemical analysis of childhood small
round cell tumors suggested that MAP1B is expressed in
neuroblastoma, rhabdomyosarcoma, and Wilms tumor, but not in Ewing
sarcoma (Willoughby et al., 2008)
[0105] Mitogen-activated protein kinase kinase kinase kinase 4
(MAP4K4) MAP4K4 is a member of the serine/threonine protein kinase
family and mediate the TNF-alpha signaling pathway (RefSeq, 2002).
MAP4K4 has been suggested as a biomarker of prostate cancer
aggressiveness (Rizzardi et al., 2014).
[0106] Misshapen-like kinase 1 (MINK1)--MINK1 regulates
cytoskeletal organization and oncogene-induced cell senescence. It
is essential for cytokinesis (Hyodo et al., 2012). MINK1 is
activated by Ras and mediates p38 activation during growth arrest
and senescence (Nicke et al., 2005).
[0107] Mohawk homeobox (MKX)--MKX is a transcription factor with a
reported role in development of bone, skeletal muscle and cartilage
structures (Ito et al., 2010b).
[0108] Myosin, heavy chain 11, smooth muscle (MYH11)--MYH11 is
assigned to chromosome region 16q12 and is expressed in the human
umbilical artery, bladder, esophagus and trachea (Matsuoka et al.,
1993). Inversion at the MYH11 chromosomal locus (inv(16);
CBFB-MYH11) is frequently found in acute myeloid leukemia, leading
to formation of an oncogenic chimeric protein of core binding
factor (CBF-beta) and MYH11 (Liu et al., 1993). Low MYH11
expression levels were linked to poor prognosis in colorectal
cancer (Wang et al., 2014).
[0109] Neurofilament, heavy polypeptide (NEFH)--Neurofilaments are
type IV intermediate filament heteropolymers composed of light,
medium, and heavy chains. Neurofilaments comprise the axoskeleton
and functionally maintain neuronal caliber. They may also play a
role in intracellular transport to axons and dendrites. NEFH
encodes the heavy neurofilament protein (RefSeq, 2002). In patients
with metastatic renal cell carcinoma, NEFH CpG island methylation
demonstrated a tumor-specific increase, association with advanced
disease and distant metastasis and was significantly associated
with a poor progression-free survival and reduced overall survival
(Dubrowinskaja et al., 2014).
[0110] Olfactory receptor, family 51, subfamily E, member2
(OR51E2)--OR51E2 is a member of the G-protein-coupled olfactory
receptor family that is predominantly expressed in the human
prostate and often over-expressed in prostate cancer (Weng et al.,
2005). OR51E2-derived peptides may be used as diagnostic markers as
well as immune targets for development of anticancer vaccines
(Matsueda et al., 2012).
[0111] Phosphodiesterase 11A (PDE11A)--PDE11A catalyzes the
hydrolysis of cAMP and cGMP, thus downregulating the respective
signaling pathways (Fawcett et al., 2000). Mutations in PDE11A have
been associated with adrenocortical hyperplasia as well as with
familial testicular germ cell tumors (Greene et al., 2010; Horvath
and Stratakis, 2008). One study has also identified missense
mutations in PDE11A in 30% of prostate cancer patients. The
variants were characterized as displaying reduced PDE11A activity
in vitro, and their expression levels in vivo were reduced (Faucz
et al., 2011).
[0112] Plectin (PLEC)--PLEC encodes the plakin family member
plectin, a protein involved in the cross-linking and organization
of the cytoskeleton and adhesion complexes (Bouameur et al., 2014).
PLEC is over-expressed in colorectal adenocarcinoma, head and neck
squamous cell carcinoma and pancreatic cancer (Lee et al., 2004;
Katada et al., 2012; Bausch et al., 2011).
[0113] PR domain containing 8 (PRDM8)--PRDM8 is a transcriptional
repressor with histone methyltransferase activity. PRDM8 is
regulated by the Notch-Hes pathway and plays a role in CNS
development (Kinameri et al., 2008)
[0114] RAB3B, member RAS oncogene family (RAB3B)--RAB3B is a low
molecular weight GTP-binding protein that has been implicated in
the regulation of exocytosis (Rotondo et al., 2009). RAB3B is
overexpressed in prostate cancer patients, suggesting that RAB3B
together with AR, FoxA1, and NKX3-1 are important regulators of
prostate cancer progression (Tan et al., 2012).
[0115] Solute carrier family 39 (zinc transporter), member 6
(SLC39A6)--SLC39A6 codes for the Zinc transporter ZIP6, an effector
molecule downstream of soluble growth factors (Lue et al., 2011).
In breast cancer over-expression of SLC39A6 is associated with
shorter recurrence times as well as reduced time to disease-related
mortality (Andres et al., 2013).
[0116] Six transmembrane epithelial antigen of the prostate 2
(STEAP2)--STEAP2 encodes a multi-pass membrane protein that
localizes to the Golgi complex, the plasma membrane, and the
vesicular tubular structures in the cytosol (RefSeq, 2002). STEAP2
expression is elevated in prostate cancer patients with advanced
disease. Overexpression of STEAP2 has been demonstrated to increase
cell proliferation as well as migratory and invasive potential in
vitro, while knock-down inhibited cell growth and might promote
apoptosis (Wang et al., 2010; Whiteland et al., 2014). Together
with other members of the STEAP protein family, STEAP2 is also
overexpressed in other cancer types. In addition, STEAPs have been
investigated as cancer biomarkers and potential targets for cancer
immunotherapy (Grunewald et al., 2012).
[0117] Synemin, intermediate filament protein (SYNM)--SYNM links
desmin with the extracellular matrix and plays an important
structural role in muscle (Bhosle et al., 2006). SYNM expression
was downregulated in breast cancer and hepatocellular carcinoma,
which correlated with decreased survival, lymph node metastases and
advanced grade (Noetzel et al., 2010; Liu et al., 2011).
[0118] Synaptopodin 2 (SYNPO2)--SYNPO2, also known as myopodin, is
an actin-binding protein that plays a role in regulating cell
migration in response to chemotactic stimuli (Kai et al., 2015).
SYNPO2 is considered a tumor-suppressor gene and absence of SYNPO2
in prostate cancer has been correlated with invasiveness and
clinical relapse (Yu et al., 2006).
[0119] Transforming growth factor, beta 3 (TGFB3)--TGFB3 is one of
at least three isoforms of TGF-beta. In normal cells, TGF-beta
signaling stops the cell cycle at the G1 stage to stop
proliferation, induce differentiation, or promote apoptosis
(Hanahan and Weinberg, 2000). In NSCLC TGFB3 is a pro-invasive
factor (Petrella et al., 2012)
[0120] TRAF2 and NCK interacting kinase (TNIK)--TNIK is a germinal
center kinase and is potentially involved in regulation of the
actin cytoskeleton (Fu et al., 1999). TNIK is an essential,
specific activator of Wnt target genes. TNIK was described as a key
player in TRAF6-dependent JNK and NF-kappaB signaling and a
transducer of activating and transforming signals in human B-cells
(Shkoda et al., 2012) In hormone receptor negative breast cancer,
TNIK was identified as a potential cancer gene with impact on
growth and proliferation (Jiao et al., 2013)
[0121] Tryptase alpha/beta 1 (TPSAB1), Tryptase beta 2
(TPSB2)--TPSAB1 and TPSB2 are both serine proteases which are
mainly expressed by mast cells. The presence of tryptase-positive
mast cells in tumor tissue correlates with angiogenesis in several
cancer types, including melanoma, endometrial carcinoma, breast
cancer, gastric cancer and colorectal cancer (Ammendola et al.,
2014).
[0122] Transient receptor potential cation channel, subfamily M,
member 8 (TRPM8)--TRPM8 is a sodium and calcium channel which is
activated in response to cold stimuli. TRPM8 is expressed primarily
in prostate epithelial cells, and in addition also in some sensory
neurons (Prevarskaya et al., 2007). TRPM8 is reported to be
overexpressed in prostate cancer and other cancer types, such as
breast, colon, lung and skin tumors (Tsavaler et al., 2001).
[0123] Thioredoxin domain containing 16 (TXNDC16)--TXNDC16 encodes
a protein of 858 amino acids with a putative thioredoxin 2 domain;
its function is unknown. Autoantibodies against TXNDC16 have been
detected in meningioma patients (Comtesse et al., 2005).
[0124] Unc-13 homolog B (UNC13B)--UNC13B is a constituent of a
protein complex at the presynaptic active zone that controls
neurotransmitter release by synaptic vesicle exocytosis (Sudhof,
2012).
[0125] Zinc finger protein 426 (ZNF426)--ZNF426 is a transcription
regulatory protein (Yang and Wood, 2007).
DETAILED DESCRIPTION OF THE INVENTION
[0126] 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 hosts 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.
[0127] 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).
[0128] As used herein and except as noted otherwise all terms are
defined as given below.
[0129] 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.
[0130] The term "peptide" is used herein to designate a series of
amino acid residues, connected one to the other typically by
peptide bonds between the alpha-amino and carbonyl groups of the
adjacent amino acids. The peptides are preferably 9 amino acids in
length, but can be as short as 8 amino acids in length, and as long
as 10, 11, 12, or 13 or longer, and in case of MHC class II
peptides (elongated variants of the peptides of the invention) they
can be as long as 14, 15, 16, 17, 18, 19 or 20 or more amino acids
in length.
[0131] 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 (trifluoro-acetate) 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.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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-00008 TABLE 8 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 Allele Population from allele
frequency A*02 Caucasian (North America) 49.1% A*02 African
American (North America) 34.1% A*02 Asian American (North America)
43.2% A*02 Latin American (North American) 48.3% DR1 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%
[0137] The peptides of the invention, preferably when included into
a vaccine of the invention as described herein bind to HLA-A*02 or
HLA-A*24. 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.
[0138] 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).
[0139] In a preferred embodiment, the term "nucleotide sequence"
refers to a heteropolymer of deoxyribonudeotides.
[0140] 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 oligonudeotide linkers, or
from a series of oligonudeotides, 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.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 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).
[0146] 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.
[0147] 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.
[0148] 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.
[0149] The term "promoter" means a region of DNA involved in
binding of RNA polymerase to initiate transcription.
[0150] 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 polynudeotide or polypeptide, separated from
some or all of the coexisting materials in the natural system, is
isolated. Such polynudeotides could be part of a vector and/or such
polynudeotides 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.
[0151] The polynudeotides, 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.
[0152] 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.
[0153] 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 polynudeotides with any of the endonucleases.
[0154] 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.
[0155] 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.
[0156] 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: 48 or a variant thereof
which is 88% homologous to SEQ ID No: 1 to SEQ ID No: 48, 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.
[0157] 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.
[0158] 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).
[0159] 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: 48. 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.
[0160] 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: 48, 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.
[0161] 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."
[0162] 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,
lie, Val, Cys); and Group 5-large, aromatic residues (Phe, Tyr,
Trp).
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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-00009 TABLE 9 Variants and motif of the HLA-A*02 peptides
according to SEQ ID No: 2, 4, and 8 Position 1 2 3 4 5 6 7 8 9 SEQ
ID No. 2 S M L G E E I Q L Variants V I A L L V L I L 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 SEQ ID No 4 A L L T F V W K 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 SEQ ID No. 8 L L D F S L A D A Variants I L V M M I M L
M V A A I A L A V V V I V L V V T T I T L T V Q Q I Q L Q V
TABLE-US-00010 TABLE 10 Variants and motif of the HLA-A*24 peptides
according to SEQ ID No: 25, 30, and 34 Position 1 2 3 4 5 6 7 8 9
10 11 SEQ ID 25 I Y E P Y L A M F Variant I L F F I F L Position 1
2 3 4 5 6 7 8 9 10 11 SEQ ID 30 S Y S P A H A R L Variant I F F F I
F F Position 1 2 3 4 5 6 7 8 9 10 11 SEQ ID 34 A F S P D S H Y L L
F Variant Y I Y L Y I L
[0168] 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.
[0169] 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
11.
TABLE-US-00011 TABLE 11 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
[0170] 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.
[0171] 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.
[0172] 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.
[0173] 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.
[0174] 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.
[0175] 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.
[0176] 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: 48.
[0177] "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 48 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.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] 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.
[0185] 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.
[0186] 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.
[0187] 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.
[0188] 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).
[0189] 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.
[0190] A peptide or variant, wherein the peptide is modified or
includes non-peptide bonds is a preferred embodiment of the
invention. Generally, peptides and variants (at least those
containing peptide linkages between amino acid residues) may be
synthesized by the Fmoc-polyamide mode of solid-phase peptide
synthesis as disclosed by Lukas et al. (Lukas et al., 1981) and by
references as cited therein. Temporary N-amino group protection is
afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group.
Repetitive cleavage of this highly base-labile protecting group is
done using 20% piperidine in N, N-dimethylformamide. Side-chain
functionalities may be protected as their butyl ethers (in the case
of serine threonine and tyrosine), butyl esters (in the case of
glutamic acid and aspartic acid), butyloxycarbonyl derivative (in
the case of lysine and histidine), trityl derivative (in the case
of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl
derivative (in the case of arginine). Where glutamine or asparagine
are C-terminal residues, use is made of the
4,4'-dimethoxybenzhydryl group for protection of the side chain
amido functionalities. The solid-phase support is based on a
polydimethyl-acrylamide polymer constituted from the three monomers
dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine
(cross linker) and acryloylsarcosine methyl ester (functionalizing
agent). The peptide-to-resin cleavable linked agent used is the
acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All
amino acid derivatives are added as their preformed symmetrical
anhydride derivatives with the exception of asparagine and
glutamine, which are added using a reversed N,
N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling
procedure. All coupling and deprotection reactions are monitored
using ninhydrin, trinitrobenzene sulphonic acid or isotin test
procedures. Upon completion of synthesis, peptides are cleaved from
the resin support with concomitant removal of side-chain protecting
groups by treatment with 95% trifluoroacetic acid containing a 50%
scavenger mix. Scavengers commonly used include ethanedithiol,
phenol, anisole and water, the exact choice depending on the
constituent amino acids of the peptide being synthesized. Also a
combination of solid phase and solution phase methodologies for the
synthesis of peptides is possible (see, for example, (Bruckdorfer
et al., 2004), and the references as cited therein).
[0191] 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).
[0192] Purification may be performed by any one, or a combination
of, techniques such as recrystallization, size exclusion
chromatography, ion-exchange chromatography, hydrophobic
interaction chromatography and (usually) reverse-phase high
performance liquid chromatography using e.g. acetonitril/water
gradient separation.
[0193] 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.
[0194] 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).
[0195] 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. As peptides derived from prostate-specific antigens can
be identified in any prostate tissue, regardless of its benign or
malignant state, in addition to prostate cancer tissue specimens
also benign prostate hyperplasia samples were analyzed with the
goal to identify peptides encoded by prostate-specific antigens.
The isolated peptides were separated and sequences were identified
by online nano-electrospray-ionization (nanoESI) liquid
chromatography-mass spectrometry (LC-MS) experiments. The resulting
peptide sequences were verified by comparison of the fragmentation
pattern of natural TUMAPs recorded from prostate cancer samples
(N=34 A*02-positive samples and N=37 A*24-positive samples) as well
as additional benign prostatic hyperplasia (N=10 A*02-positive
samples and N=3 A*24-positive samples) with the fragmentation
patterns of corresponding synthetic reference peptides of identical
sequences. Since the peptides were directly identified as ligands
of HLA molecules of tumorous tissue, these results provide direct
evidence for the natural processing and presentation of the
identified peptides on tumorous tissue obtained from 70 patients
with a prostate tumor.
[0196] 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.
[0197] 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.
[0198] HLA-peptide complexes from prostate tumor 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 prostate
tumor samples confirming their presentation on prostate tumors.
[0199] TUMAPs identified on multiple prostate cancer prostatic
hyperplasia 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.
[0200] Furthermore, the discovery pipeline XPRESIDENT.RTM. v2.x
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).
[0201] The present invention provides peptides that are useful in
treating cancers/tumors, preferably prostate cancer that over- or
exclusively present the peptides of the invention. These peptides
were shown by mass spectrometry to be naturally presented by HLA
molecules on human prostate tumor samples.
[0202] 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
tumor compared with normal tissues--"normal tissues" in relation to
this invention shall mean normal, non-prostatic, 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
a prostate tumor, but not on normal tissues (see Example 1).
[0203] 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. prostate tumor
cells presenting the derived peptides.
[0204] 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, non-prostatic, tissues in comparable copy
numbers, preventing the risk of undesired autoimmune reactions
against normal cells in the patient. Prostate-specific antigens may
be a good choice for cancer immunotherapy of prostate cancer
because prostate-specific antigens represent tumor-specific targets
in a prostatectomized patient. In a prostate cancer patient without
prostatectomy, such antigens may also be interesting because
prostate is not regarded as a vital organ.
[0205] The present description further relates to T-cell receptors
(TCRs) comprising an alpha chain and a beta chain ("alpha/beta
TCRs"). Also provided are inventive peptides capable of binding to
TCRs and antibodies when presented by an MHC molecule. The present
description also relates to nucleic acids, vectors and host cells
for expressing TCRs and peptides of the present description; and
methods of using the same.
[0206] 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.
[0207] 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.
[0208] 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.
[0209] 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.
[0210] 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.
[0211] TCRs of the present description preferably bind to an
inventive 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.
[0212] As used herein in connect with TCRs of the present
description, "specific binding" and grammatical variants thereof
are used to mean a TCR having a binding affinity (KD) for an
inventive peptide-HLA molecule complex of 100 .mu.M or less.
[0213] 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.
[0214] 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.
[0215] 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.
[0216] 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 non-mutated TCR.
[0217] In an embodiment, a TCR comprising at least one mutation in
the TCR alpha chain and/or TCR beta chain has a binding affinity
for, and/or a binding half-life for, an inventive peptide-HLA
molecule complex, which is at least double that of a TCR comprising
the non-mutated TCR alpha chain and/or non-mutated TCR beta chain.
Affinity-enhancement of tumor-specific TCRs, and its exploitation,
relies on the existence of a window for optimal TCR affinities. The
existence of such a window is based on observations that TCRs
specific for HLA-A2-restricted pathogens have KD values that are
generally about 10-fold lower when compared to TCRs specific for
HLA-A2-restricted tumor-associated self-antigens. It is now known,
although tumor antigens have the potential to be immunogenic,
because tumors arise from the individual's own cells only mutated
proteins or proteins with altered translational processing will be
seen as foreign by the immune system. Antigens that are upregulated
or overexpressed (so called self-antigens) will not necessarily
induce a functional immune response against the tumor T-cells
expressing TCRs that are highly reactive to these antigens will
have been negatively selected within the thymus in a process known
as central tolerance, meaning that only T-cells with low-affinity
TCRs for self-antigens remain. Therefore, affinity of TCRs or
variants of the present description to peptides of the invention
can be enhanced by methods well known in the art.
[0218] 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/inventive peptide
monomers, incubating the PBMCs with tetramer-phycoerythrin (PE) and
isolating the high avidity T-cells by fluorescence activated cell
sorting (FACS)-Calibur analysis.
[0219] The present description further relates to a method of
identifying and isolating a TCR according to the present
description, said method comprising obtaining a transgenic mouse
with the entire human TCR.alpha..beta. gene loci (1.1 and 0.7 Mb),
whose T-cells express a diverse human TCR repertoire that
compensates for mouse TCR deficiency, immunizing the mouse with
HAVCR1-001, incubating PBMCs obtained from the transgenic mice with
tetramer-phycoerythrin (PE), and isolating the high avidity T-cells
by fluorescence activated cell sorting (FACS)-Calibur analysis.
[0220] 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.
[0221] 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.
[0222] 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), 1-actin, ubiquitin, and a simian virus 40 (SV40)/CD43
composite promoter, elongation factor (EF)-1a and the spleen
focus-forming virus (SFFV) promoter. In a preferred embodiment, the
promoter is heterologous to the nucleic acid being expressed.
[0223] 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).
[0224] 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.
[0225] 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 bicistronic
constructs in a single vector, which has been shown to be capable
of overcoming this obstacle. The use of a viral intra-ribosomal
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).
[0226] 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
"optimal" than others because of the relative availability of
matching tRNAs as well as other factors (Gustafsson et al., 2004).
Modifying the TCR-alpha and TCR-beta gene sequences such that each
amino acid is encoded by the 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).
[0227] 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).
[0228] 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.xi. (CD3.xi. fusion). (Schmitt et al.
2009).
[0229] 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.
[0230] 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.
[0231] The pharmaceutical compositions comprise the peptides either
in the free form or in the form of a pharmaceutically acceptable
salt (see also above). As used herein, "a pharmaceutically
acceptable salt" refers to a derivative of the disclosed peptides
wherein the peptide is modified by making acid or base salts of the
agent. For example, acid salts are prepared from the free base
(typically wherein the neutral form of the drug has a neutral --NH2
group) involving reaction with a suitable acid. Suitable acids for
preparing acid salts include both organic acids, e.g., acetic acid,
propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic
acid, malonic acid, succinic acid, maleic acid, fumaric acid,
tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic
acid, methane sulfonic acid, ethane sulfonic acid,
p-toluenesulfonic acid, salicylic acid, and the like, as well as
inorganic acids, e.g., hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid phosphoric acid and the like.
Conversely, preparation of basic salts of acid moieties which may
be present on a peptide are prepared using a pharmaceutically
acceptable base such as sodium hydroxide, potassium hydroxide,
ammonium hydroxide, calcium hydroxide, trimethylamine or the
like.
[0232] In an especially preferred embodiment, the pharmaceutical
compositions comprise the peptides as salts of acetic acid
(acetates), trifluoro acetates or hydrochloric acid
(chlorides).
[0233] 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.
[0234] 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.
48, 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.
[0235] A further aspect of the invention provides a nucleic acid
(for example a polynucleotide) encoding a peptide or peptide
variant of the invention. The polynudeotide may be, for example,
DNA, cDNA, PNA, RNA or combinations thereof, either single- and/or
double-stranded, or native or stabilized forms of polynudeotides,
such as, for example, polynudeotides 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 polynudeotide. A still further aspect of
the invention provides an expression vector capable of expressing a
polypeptide according to the invention.
[0236] 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.
[0237] 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.
[0238] 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.
[0239] 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.
[0240] 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.
[0241] 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.
[0242] Alternatively, the gene for such selectable trait can be on
another vector, which is used to co-transform the desired host
cell.
[0243] 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.
[0244] 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.
[0245] 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.
[0246] 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 fl 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.
[0247] In another embodiment two or more peptides or peptide
variants of the invention are encoded and thus expressed in a
successive order (similar to "beads on a string" constructs). In
doing so, the peptides or peptide variants may be linked or fused
together by stretches of linker amino acids, such as for example
LLLLLL, or may be linked without any additional peptide(s) between
them. These constructs can also be used for cancer therapy, and may
induce immune responses both involving MHC I and MHC II.
[0248] 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.
[0249] 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.
[0250] 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.
[0251] 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.
[0252] 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).
[0253] 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.
[0254] 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).
[0255] 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.
[0256] 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, betaglucan, 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).
[0257] 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.
[0258] 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 derivatives thereof (e.g.
AmpliGen.RTM., Hiltonol.RTM., poly-(ICLC), poly(IC-R),
poly(I:C12U), non-CpG bacterial DNA or RNA as well as immunoactive
small molecules and antibodies such as cyclophosphamide, sunitinib,
Bevacizumab.RTM., celebrex, NCX-4016, sildenafil, tadalafil,
vardenafil, sorafenib, temozolomide, temsirolimus, XL-999,
CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other
antibodies targeting key structures of the immune system (e.g.
anti-CD40, anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which
may act therapeutically and/or as an adjuvant. The amounts and
concentrations of adjuvants and additives useful in the context of
the present invention can readily be determined by the skilled
artisan without undue experimentation.
[0259] 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.
[0260] 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.
[0261] 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.
[0262] 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 adenomateous or cancerous
diseases. Exemplary formulations can be found in, for example,
EP2112253.
[0263] 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).
[0264] 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.
[0265] "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.
[0266] 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.
[0267] Each scaffold can be conjugated with a second active
molecule such as for example IL-21, anti-CD3, anti-CD28.
[0268] For further information on polypeptide scaffolds see for
example the background section of WO 2014/071978A1 and the
references cited therein.
[0269] 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.
[0270] 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.
[0271] 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.
[0272] Further, investigation of cell-binding behavior with flow
cytometry showed that the aptamers revealed very good apparent
affinities that are within the nanomolar range.
[0273] Aptamers are useful for diagnostic and therapeutic purposes.
Further, it could be shown that some of the aptamers are taken up
by tumor cells and thus can function as molecular vehicles for the
targeted delivery of anti-cancer agents such as siRNA into tumor
cells.
[0274] 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 48, according to the invention at hand with the MHC
molecule, using the cell-SELEX (Systematic Evolution of Ligands by
Exponential enrichment) technique.
[0275] 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.
[0276] 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.
[0277] 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.
[0278] 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.
[0279] 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.
[0280] 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: 48, or a variant thereof which is at least 88%
homologous (preferably identical) to SEQ ID No: 1 to SEQ ID No: 48
or a variant thereof that induces T cells cross-reacting with said
peptide, wherein said peptide is not the underlying full-length
polypeptide.
[0281] 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: 48 or a variant thereof which is at
least 88% homologous (preferably identical) to SEQ ID No: 1 to SEQ
ID No: 48, 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.
[0282] 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.
[0283] 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: 48.
[0284] The present invention further relates to the peptides
according to the invention, wherein the peptide is (chemically)
modified and/or includes non-peptide bonds.
[0285] 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.
[0286] 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.
[0287] The present invention further relates to the nucleic acid
according to the invention that is DNA, cDNA, PNA, RNA or
combinations thereof.
[0288] The present invention further relates to an expression
vector capable of expressing a nucleic acid according to the
present invention.
[0289] 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
prostate cancer.
[0290] 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.
[0291] 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.
[0292] 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.
[0293] 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.
[0294] The present invention further relates to the method
according to the invention, wherein the antigen-presenting cell
comprises an expression vector capable of expressing said peptide
containing SEQ ID No: 1 to SEQ ID No: 48 or said variant amino acid
sequence.
[0295] 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.
[0296] 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.
[0297] 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.
[0298] 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.
[0299] The present invention further relates to a use according to
the invention, wherein said cancer cells preferably are prostate
cancer cells or other solid or hematological tumor cells such as
lung cancer, small cell lung cancer, melanoma, liver cancer, breast
cancer, Uterine cancer, Merkel cell carcinoma, pancreatic cancer,
gallbladder cancer, bile duct cancer, CRC, urinary bladder cancer,
non-small cell lung cancer, kidney cancer, leukemia (e.g. AML or
CLL), ovarian cancer, esophageal cancer, brain cancer, and gastric
(stomach) cancer, most preferably prostate cancer cells.
[0300] 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 prostate cancer. The present
invention also relates to the use of these novel targets for cancer
treatment.
[0301] 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
prostate cancer marker (poly)peptide, delivery of a toxin to a
prostate cancer cell expressing a cancer marker gene at an
increased level, and/or inhibiting the activity of a prostate
cancer marker polypeptide) according to the invention.
[0302] 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 prostate cancer marker
polypeptides or fragments thereof may be used to generate the
antibodies of the invention. A polypeptide to be used for
generating an antibody of the invention may be partially or fully
purified from a natural source, or may be produced using
recombinant DNA techniques.
[0303] 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: 48 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 polydonal antibody preparation that specifically bind
the prostate cancer marker polypeptide used to generate the
antibody according to the invention.
[0304] One of skill in the art will realize that the generation of
two or more different sets of monoclonal or polydonal 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.
[0305] 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).
[0306] 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.
[0307] 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).
[0308] 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.
[0309] 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.
[0310] 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.
[0311] 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.
[0312] 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.
[0313] 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.
[0314] 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.
[0315] 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
prostate cancer, the efficacy of the therapeutic antibody can be
assessed in various ways well known to the skilled practitioner.
For instance, the size, number, and/or distribution of cancer in a
subject receiving treatment may be monitored using standard tumor
imaging techniques. A therapeutically-administered antibody that
arrests tumor growth, results in tumor shrinkage, and/or prevents
the development of new tumors, compared to the disease course that
would occurs in the absence of antibody administration, is an
efficacious antibody for treatment of prostate cancer.
[0316] 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 2013057586A1.
[0317] In addition, the peptides and/or the TCRs or antibodies or
other binding molecules of the present invention can be used to
verify a pathologists diagnosis of a cancer based on a biopsied
sample.
[0318] The antibodies or TCRs may also be used for in vivo
diagnostic assays. Generally, the antibody is labeled with a
radionucleotide (such as .sup.111In, .sup.99Tc, .sup.14C,
.sup.131I, .sup.3H, .sup.32P or .sup.35S) so that the tumor can be
localized using immunoscintiography. In one embodiment, antibodies
or fragments thereof bind to the extracellular domains of two or
more targets of a protein selected from the group consisting of the
above-mentioned proteins, and the affinity value (Kd) is less than
1.times.10 .mu.M.
[0319] 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.
[0320] 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.
[0321] 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.
[0322] 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).
[0323] 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.
[0324] In case of a MHC class I epitope being used as an antigen,
the T cells are CD8-positive T cells.
[0325] 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: 48, or a variant amino acid sequence thereof.
[0326] 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.
[0327] 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.
[0328] 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.
[0329] 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: 48.
[0330] 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.
[0331] 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)).
[0332] 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.
[0333] 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. T cells may be obtained
by methods known in the art, e.g. those described above.
[0334] 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).
[0335] 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.
[0336] 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).
[0337] 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.
[0338] 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.
[0339] 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.
[0340] 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).
[0341] 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.
[0342] 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.
[0343] 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.
[0344] 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.
[0345] 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.
[0346] Since the peptides of the invention were isolated from
prostate tumors, the medicament of the invention is preferably used
to treat prostate tumors.
[0347] 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.
[0348] 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.
[0349] 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 prostate cancer
patients with various HLA-A HLA-B and HLA-C alleles. It may contain
MHC class I and MHC class II peptides or elongated MHC class I
peptides. In addition to the tumor associated peptides collected
from several prostate tumor 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 immune-competence
of the patient.
[0350] 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, prostate cancer samples as well as
additional benign prostatic hyperplasia from patients and blood
from healthy donors were analyzed in a stepwise approach:
1. HLA ligands from the tumorous 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 (prostate cancer) 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 prostate cancer patients.
[0351] 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.
[0352] 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.
[0353] In an aspect, the peptides are selected for inclusion in the
vaccine based on their suitability for the individual patient based
on the method according to the present invention as described
herein, or as below.
[0354] The HLA phenotype, transcriptomic and peptidomic data is
gathered from the patients 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.
[0355] 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.
[0356] 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.
[0357] 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.
[0358] 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.
[0359] 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.
[0360] 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.
[0361] 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.
[0362] 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 prostate tumor 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 tumor.
[0363] 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 prostate cancer. Presence of groups
of peptides can enable classification or sub-classification of
diseased tissues.
[0364] 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.
[0365] 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.
[0366] 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
[0367] FIGS. 1A-C show the over-presentation of various peptides in
normal tissues (white bars) and prostate cancer tissues and benign
prostate hyperplasia tissues (black bars).
[0368] FIGS. 1D-E show all cell lines, normal tissues and cancers
tissues where the exemplary peptides (SLLSHQVLL (A*02) (SEQ ID NO.
20) and SLLSHQVLL (A*24) (SEQ ID NO. 20)) has been detected. FIG.
1A) Gene: OR51E2, Peptide: VTAQIGIVAV (A*02; SEQ ID NO.:1)--Tissues
from left to right: 1 adipose tissues, 3 adrenal glands, 6
arteries, 5 bone marrows, 7 brains, 3 breasts, 1 central nerve, 13
colons, 1 duodenum, 8 esophagi, 2 gallbladders, 5 hearts, 16
kidneys, 21 livers, 46 lungs, 4 lymph nodes, 4 leukocyte samples, 4
ovaries, 7 pancreas, 4 peripheral nerves, 1 peritoneum, 3 pituitary
glands, 4 placentas, 3 pleuras, 6 recti, 7 salivary glands, 4
skeletal muscles, 6 skins, 2 small intestines, 4 spleens, 7
stomachs, 4 testis, 3 thymi, 4 thyroid glands, 10 tracheas, 3
ureters, 6 urinary bladders, 2 uteri, 2 veins, 3 prostate, 44
tumorous prostates. The peptide was also found on small cell lung
cancer (not shown). FIG. 1B) Gene: MANSC1, Peptide: KMDEASAQLL
(A*02; SEQ ID NO.:14)--Tissues from left to right: 1 adipose
tissues, 3 adrenal glands, 6 arteries, 5 bone marrows, 7 brains, 3
breasts, 1 central nerve, 13 colons, 1 duodenum, 8 esophagi, 2
gallbladders, 5 hearts, 16 kidneys, 21 livers, 46 lungs, 4 lymph
nodes, 4 leukocyte samples, 4 ovaries, 7 pancreas, 4 peripheral
nerves, 1 peritoneum, 3 pituitary glands, 4 placentas, 3 pleuras, 6
recti, 7 salivary glands, 4 skeletal muscles, 6 skins, 2 small
intestines, 4 spleens, 7 stomachs, 4 testis, 3 thymi, 4 thyroid
glands, 10 tracheas, 3 ureters, 6 urinary bladders, 2 uteri, 2
veins, 3 prostate, 44 tumorous prostates. FIG. 1C) Gene: TRPM8,
Peptide: SYNDALLTF (A*24; SEQ ID NO.:24)--Tissues from left to
right: 2 adrenal glands, 1 artery, 4 brains, 1 breast, 5 colons, 1
heart, 13 kidneys, 9 livers, 9 lungs, 3 pancreas, 1 pituitary
gland, 2 recti, 3 skins, 1 spleen, 12 stomachs, 1 thymus, 2 uteri,
40 tumorous prostates. The peptide was also found on non-small cell
lung cancer (not shown). FIG. 1D) Gene: KIAA1244, Peptide:
SLLSHQVLL (A*02; SEQ ID NO.:20)--Tissues from left to right 1
pancreatic cell line, 20 cancer tissues (1 brain cancer, 1 breast
cancer, 2 colon cancers, 1 esophageal cancer, 1 kidney cancer, 1
liver cancer, 3 lung cancers, 8 prostate cancers, 1 stomach cancer,
1 urinary bladder cancer). The set of normal tissues was the same
as in A-B, but the peptide was not detected on any normal tissue.
FIG. 1E) Gene: KIAA1244, Peptide: QYGKDFLTL (A*24; SEQ ID
NO.:33)--Tissues from left to right: 3 benign prostate hyperplasia
tissues, 3 normal tissues (1 liver, 1 lung, 1 rectum), 31 cancer
tissues (5 brain cancers, 4 liver cancers, 15 lung cancers, 7
prostate cancers). The set of normal tissues was the same as in C,
but tissues without detection are not shown. FIGS. 1F-K show the
over-presentation of various peptides in normal tissues (white
bars) and prostate cancer tissues and benign prostate hyperplasia
tissues (black bars). FIG. 1L-S show all cell lines, normal tissues
and cancers tissues where various peptides have been detected. FIG.
1F) Gene: NEFH, Peptide: HLLEDIAHV (A*02; SEQ ID NO.: 3)--Tissues
from left to right: 1 adipose tissue, 3 adrenal glands, 6 arteries,
5 bone marrows, 7 brains, 3 breasts, 1 central nerve, 13 colons, 1
duodenum, 8 esophagi, 2 gallbladders, 5 hearts, 16 kidneys, 4
leukocyte samples, 21 livers, 46 lungs, 4 lymph nodes, 3 ovaries, 7
pancreases, 4 peripheral nerves, 1 peritoneum, 3 pituitary glands,
2 placentas, 3 pleuras, 6 rectums, 7 salivary glands, 4 skeletal
muscles, 5 skins, 2 small intestines, 4 spleens, 7 stomachs, 4
testes, 3 thymi, 4 thyroid glands, 9 tracheas, 3 ureters, 6 urinary
bladders, 2 uteri, 2 veins, 3 prostates, 33 prostate cancer tissues
and 10 benign prostate hyperplasia tissues. FIG. 1G) Gene: PDE11A,
Peptide: ALLESRVNL (A*02; SEQ ID NO.: 6)--Tissues from left to
right: 1 adipose tissue, 3 adrenal glands, 6 arteries, 5 bone
marrows, 7 brains, 3 breasts, 1 central nerve, 13 colons, 1
duodenum, 8 esophagi, 2 gallbladders, 5 hearts, 16 kidneys, 4
leukocyte samples, 21 livers, 46 lungs, 4 lymph nodes, 3 ovaries, 7
pancreases, 4 peripheral nerves, 1 peritoneum, 3 pituitary glands,
2 placentas, 3 pleuras, 6 rectums, 7 salivary glands, 4 skeletal
muscles, 5 skins, 2 small intestines, 4 spleens, 7 stomachs, 4
testes, 3 thymi, 4 thyroid glands, 9 tracheas, 3 ureters, 6 urinary
bladders, 2 uteri, 2 veins, 3 prostates, 33 prostate cancer tissues
and 10 benign prostate hyperplasia tissues. FIG. 1H) Gene: KLK4,
Peptide: GYLQGLVSF (A*24; SEQ ID NO.: 27)--Tissues from left to
right: 2 adrenal glands, 1 artery, 4 brains, 1 breast, 5 colons, 1
heart, 13 kidneys, 9 livers, 9 lungs, 3 pancreases, 1 pituitary
gland, 2 rectums, 3 skins, 1 spleen, 12 stomachs, 1 thymus, 2
uteri, 37 prostate cancer tissues and 3 benign prostate hyperplasia
tissues. FIG. 1I) Gene: TGFB3, Peptide: YYAKEIHKF (A*24; SEQ ID
NO.: 28)--Tissues from left to right: 2 adrenal glands, 1 artery, 4
brains, 1 breast, 5 colons, 1 heart, 13 kidneys, 9 livers, 9 lungs,
3 pancreases, 1 pituitary gland, 2 rectums, 3 skins, 1 spleen, 12
stomachs, 1 thymus, 2 uteri, 37 prostate cancer tissues and 3
benign prostate hyperplasia tissues. FIG. 1J) Gene: KLK3, Peptide:
SLFHPEDTGQV (A*02; SEQ ID NO.: 49)--Tissues from left to right: 1
adipose tissue, 3 adrenal glands, 6 arteries, 5 bone marrows, 7
brains, 3 breasts, 1 central nerve, 13 colons, 1 duodenum, 8
esophagi, 2 gallbladders, 5 hearts, 16 kidneys, 4 leukocyte
samples, 21 livers, 46 lungs, 4 lymph nodes, 3 ovaries, 7
pancreases, 4 peripheral nerves, 1 peritoneum, 3 pituitary glands,
2 placentas, 3 pleuras, 6 rectums, 7 salivary glands, 4 skeletal
muscles, 5 skins, 2 small intestines, 4 spleens, 7 stomachs, 4
testes, 3 thymi, 4 thyroid glands, 9 tracheas, 3 ureters, 6 urinary
bladders, 2 uteri, 2 veins, 3 prostates, 33 prostate cancer tissues
and 10 benign prostate hyperplasia tissues. FIG. 1K) Gene: KLK2,
Peptide: AYSEKVTEF (A*24; SEQ ID NO.: 54)--Tissues from left to
right: 2 adrenal glands, 1 artery, 4 brains, 1 breast, 5 colons, 1
heart, 13 kidneys, 9 livers, 9 lungs, 3 pancreases, 1 pituitary
gland, 2 rectums, 3 skins, 1 spleen, 12 stomachs, 1 thymus, 2
uteri, 37 prostate cancer tissues and 3 benign prostate hyperplasia
tissues. FIG. 1L) Gene: GREB1, Peptide: SMLGEEIQL (A*02; SEQ ID
NO.: 2)--Tissues from left to right: 1 benign prostate hyperplasia
tissue (BPH), 3 cell-lines (3 skins), 1 normal tissue (1 uterus),
26 cancer tissues (2 breast cancers, 2 liver cancers, 1 lung
cancer, 1 ovarian cancer, 13 prostate cancers, 6 skin cancers, 1
uterus cancer. FIG. 1M) Gene: TRPM8, Peptide: ALLTFVWKL (A*02; SEQ
ID NO.: 4)--Tissues from left to right: 3 benign prostate
hyperplasia tissues (BPH), 13 cancer tissues (1 brain cancer, 12
prostate cancers). FIG. 1N) Gene: TRPM8, Peptide: KIFSRLIYI (A*02;
SEQ ID NO.: 5)--Tissues from left to right: 4 benign prostate
hyperplasia tissues (BPH), 10 cancer tissues (1 brain cancer, 8
prostate cancers, 1 skin cancer). FIG. 1O) Gene: MANSC1, Peptide:
KMDEASAQL (A*02; SEQ ID NO.: 16)--Tissues from left to right: 21
cancer tissues (20 prostate cancers, 1 urinary bladder cancer).
FIG. 1P) Gene: C6orf132, Peptide: RYGSPINTF (A*24; SEQ ID NO.:
29)--Tissues from left to right: 4 benign prostate hyperplasia
tissues (BPH), 54 cancer tissues (1 liver cancer, 24 lung cancers,
26 prostate cancers, 3 stomach cancers). FIG. 1Q) Gene: ITGA7,
Peptide: AFSPDSHYLLF (A*24; SEQ ID NO.: 34)--Tissues from left to
right: 5 benign prostate hyperplasia tissues (BPH), 44 cancer
tissues (10 brain cancers, 1 kidney cancer, 4 liver cancers, 18
lung cancers, 11 prostate cancers). FIG. 1R) Gene: TPSB2, TPSAB1,
Peptide: IYTRVTYYL (A*24; SEQ ID NO.: 35)--Tissues from left to
right: 3 benign prostate hyperplasia tissues (BPH), 59 cancer
tissues (36 lung cancers, 14 prostate cancers, 9 stomach cancers).
FIG. 1S) Gene: SLC30A4, Peptide: ALGDLVQSV (A*02; SEQ ID NO.:
52)--Tissues from left to right 1 benign prostate hyperplasia
tissues (BPH), 11 cancer tissues (1 lymph node cancer, 9 prostate
cancers, 1 skin cancer).
[0369] FIGS. 2A to E show exemplary expression profiles (relative
expression compared to normal kidney) of source genes of the
present invention that are highly over-expressed or exclusively
expressed in prostate cancer in a panel of normal tissues and 20
prostate cancer samples. Tissues from left to right: adrenal gland,
artery, bone marrow, brain (whole), breast, colon, esophagus,
heart, kidney (triplicate), leukocytes, liver, lung, lymph node,
ovary, pancreas, placenta, prostate, salivary gland, skeletal
muscle, skin, small intestine, spleen, stomach, testis, thymus,
thyroid gland, urinary bladder, uterine cervix, uterus, vein, 20
prostate cancer samples. FIG. 2A) NEFH; FIG. 2B) ABCC4; FIG. 2C)
RAB3B; FIG. 2D) OR51E2; and FIG. 2E) KLK2.
[0370] FIG. 3 shows exemplary immunogenicity data: flow cytometry
results after peptide-specific multimer staining. A) TYIGQGYII
(FKBP10; SEQ ID No. 42); B) IYTRVTYYL (TPSB2, TPSAB1; SEQ ID No.
35).
[0371] FIG. 4A to C show exemplary results of peptide-specific in
vitro CD8+ T cell responses of a healthy HLA-A*02+ donor. CD8+ T
cells were primed using artificial APCs coated with anti-CD28 mAb
and HLA-A*02 in complex with SeqID No 1 peptide (A, left panel),
SeqID No 3 peptide (B, left panel) or SeqID No 5 peptide (C, 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 (A), A*02/SeqID No 3 (B) or
A*02/SeqID No 5 (C). Right panels (A, B and C) 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.
[0372] FIG. 5A to B show exemplary results of peptide-specific in
vitro CD8+ T cell responses of a healthy HLA-A*24+ donor. CD8+ T
cells were primed using artificial APCs coated with anti-CD28 mAb
and HLA-A*24 in complex with SeqID No 24 peptide (A, left panel) or
SeqID No 27 peptide (B, left panel), respectively. After three
cycles of stimulation, the detection of peptide-reactive cells was
performed by 2D multimer staining with A*24/SeqID No 24 (A), or
A*24/SeqID No 27 (B). Right panels (A, and B) show control staining
of cells stimulated with irrelevant A*24/peptide complexes. Viable
singlet cells were gated for CD8+ lymphocytes. Boolean gates helped
excluding false-positive events detected with multimers specific
for different peptides. Frequencies of specific multimer+ cells
among CD8+ lymphocytes are indicated.
EXAMPLES
Example 1
Identification and Quantitation of Tumor Associated Peptides
Presented on the Cell Surface
[0373] Tissue Samples
[0374] Patients' prostate tumor tissues were obtained from Asterand
(Detroit, USA and Royston, Herts, UK); BioServe (Beltsville, Md.,
USA); Geneticist Inc. (Glendale, Calif., USA); Indivumed GmbH
(Hamburg, Germany); Saint Savas Hospital, Athens, Greece,
University Hospital of Tubingen. Normal tissues were obtained from
Asterand (Detroit, USA and Royston, Herts, UK); Bio-Options Inc,
CA, USA; BioServe, Beltsville, Md., USA; Capital BioScience Inc,
Rockville, Md., USA; Geneticist Inc., Glendale, Calif., USA;
University Hospital of Geneva; University Hospital of Heidelberg;
Kyoto Prefectural University of Medicine (KPUM); Osaka City
University (OCU); University Hospital Munich; ProteoGenex Inc.,
Culver City, Calif., USA; Tissue Solutions Ltd., Glasgow, United
Kingdom; University Hospital of Tubingen. 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.
[0375] Isolation of HLA Peptides from Tissue Samples
[0376] 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.
[0377] Mass Spectrometry Analyses
[0378] The HLA peptide pools as obtained were separated according
to their hydrophobicity by reversed-phase chromatography
(nanoAcquity UPLC system, Waters) and the eluting peptides were
analyzed in LTQ-velos and fusion hybrid mass spectrometers
(ThermoElectron) equipped with an ESI source. Peptide pools were
loaded directly onto the analytical fused-silica micro-capillary
column (75 .mu.m i.d..times.250 mm) packed with 1.7 .mu.m C18
reversed-phase material (Waters) applying a flow rate of 400 nL per
minute. Subsequently, the peptides were separated using a two-step
180 minute-binary gradient from 10% to 33% B at a flow rate of 300
nL per minute. The gradient was composed of Solvent A (0.1% formic
acid in water) and solvent B (0.1% formic acid in acetonitrile). A
gold coated glass capillary (PicoTip, New Objective) was used for
introduction into the nanoESI source. The LTQ-Orbitrap mass
spectrometers were operated in the data-dependent mode using a TOPS
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 patter
with the fragmentation pattern of a synthetic sequence-identical
reference peptide.
[0379] 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 prostate cancer samples and
benign prostate hyperplasia samples to a baseline of normal tissue
samples. Presentation profiles of exemplary over-presented peptides
are shown in FIG. 1. Presentation scores for exemplary peptides are
shown in Table 12 and Table 13.
TABLE-US-00012 TABLE 12 Presentation scores. The table lists
HLA-A*02 peptides that are very highly over-presented on tumors
compared to a panel of normal tissues (+++), highly over-presented
on tumors compared to a panel of normal tissues (++) or over-
presented on tumors compared to a panel of normal tissues (+). SEQ
ID No. Sequence Peptide Presentation 1 VTAQIGIVAV +++ 2 SMLGEEIQL
+++ 3 HLLEDIAHV +++ 4 ALLTFVWKL +++ 5 KIFSRLIYI +++ 6 ALLESRVNL +++
7 TLLQVVGVVSV +++ 8 LLDFSLADA +++ 9 GMLNEAEGKAIKL ++ 10 TLWRGPVVV
+++ 11 YLEEECPAT +++ 12 SLNEEIAFL +++ 14 KMDEASAQLL +++ 15
KMDEASAQLLA +++ 17 RLGIKPESV ++ 18 GLSEFTEYL +++ 19 LLPPPPLLA +++
20 SLLSHQVLL +++ 21 YLNDSLRHV +++ 22 SLYDSIAFI +++ 23 AVAGADVIITV +
40 RTFJPTYGL ++
TABLE-US-00013 TABLE 13 Presentation scores. The table lists
HLA-A*24 peptides that are very highly over-presented on tumors
compared to a panel of normal tissues (+++), highly over-presented
on tumors compared to a panel of normal tissues (++) or over-
presented on tumors compared to a panel of normal tissues (+). SEQ
ID No. Sequence Peptide Presentation 24 SYNDALLTF +++ 25 IYEPYLAMF
+ 26 RYADDTFTPAF +++ 27 GYLQGLVSF +++ 28 YYAKEIHKF +++ 29 RYGSPINTF
+++ 30 SYSPAHARL +++ 31 AYTSPPSFF +++ 32 PYQLNASLFTF +++ 34
AFSPDSHYLLF +++ 35 IYTRVTYYL +++ 36 RYMWINQEL ++ 37 RYLQDLLAW +++
38 VYSDKLWIF ++ 39 SYIDVAVKL + 41 RYLQKIEEF +++ 42 TYIGQGYII +++ 43
AYIKNGQLF +++ 44 VYNTVSEGTHF + 45 RYFKTPRKF ++ 46 VYEEILHQI ++ 47
SYTPVLNQF ++ 48 AWAPKPYHKF ++
Example 2
Expression Profiling of Genes Encoding the Peptides of the
Invention
[0380] 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.
[0381] RNA Sources and Preparation
[0382] 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.
[0383] Total RNA from healthy human tissues was obtained
commercially (Ambion, Huntingdon, UK; Clontech, Heidelberg,
Germany; Stratagene, Amsterdam, Netherlands; BioChain, Hayward,
Calif., USA). The RNA from several individuals (between 2 and 123
individuals) was mixed such that RNA from each individual was
equally weighted.
[0384] 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).
[0385] Microarray Experiments
[0386] Gene expression analysis of all tumor and normal tissue RNA
samples was performed by Affymetrix Human Genome (HG) U133A or
HG-U133 Plus 2.0 oligonucleotide microarrays (Affymetrix, Santa
Clara, Calif., USA). All steps were carried out according to the
Affymetrix manual. Briefly, double-stranded cDNA was synthesized
from 5-8 .mu.g of total RNA, using SuperScript RTII (Invitrogen)
and the oligo-dT-T7 primer (MWG Biotech, Ebersberg, Germany) as
described in the manual. In vitro transcription was performed with
the BioArray High Yield RNA Transcript Labelling Kit (ENZO
Diagnostics, Inc., Farmingdale, N.Y., USA) for the U133A arrays or
with the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus
2.0 arrays, followed by cRNA fragmentation, hybridization, and
staining with streptavidin-phycoerythrin and biotinylated
anti-streptavidin antibody (Molecular Probes, Leiden, Netherlands).
Images were scanned with the Agilent 2500A GeneArray Scanner
(U133A) or the Affymetrix Gene-Chip Scanner 3000 (U133 Plus 2.0),
and data were analyzed with the GCOS software (Affymetrix), using
default settings for all parameters. For normalization, 100
housekeeping genes provided by Affymetrix were used. Relative
expression values were calculated from the signal log ratios given
by the software and the normal kidney sample was arbitrarily set to
1.0. Exemplary expression profiles of source genes of the present
invention that are highly over-expressed or exclusively expressed
in prostate cancer are shown in FIGS. 2A to E. Expression scores
for further exemplary genes are shown in Table 14.
TABLE-US-00014 TABLE 14 Expression scores. The table lists peptides
from genes that are very highly over-expressed in tumors compared
to a panel of normal tissues (+++), highly over-expressed in tumors
compared to a panel of normal tissues (++) or over- expressed in
tumors compared to a panel of normal tissues (+). SEQ ID No
Sequence Gene Expression 1 VTAQIGIVAV ++ 2 SMLGEEIQL ++ 3 HLLEDIAHV
+++ 4 ALLTFVWKL + 5 KIFSRLIYI + 7 TLLQVVGVVSV ++ 11 YLEEECPAT + 19
LLPPPPLLA + 24 SYNDALLTF + 25 IYEPYLAMF + 26 RYADDTFTPAF ++ 28
YYAKEIHKF + 44 VYNTVSEGTHF ++
Example 3
In Vitro Immunogenicity for MHC Class I Presented Peptides
[0387] 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 for some
selected TUMAPs immunogenicity for HLA-A*0201 restricted and
HLA-A*24 restricted TUMAPs of the invention, demonstrating that
these peptides are T-cell epitopes against which CD8+ precursor T
cells exist in humans (Table 15 A+B).
[0388] In Vitro Priming of CD8+ T Cells
[0389] 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.
[0390] 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 IL7 (PromoCell, Heidelberg, Germany) and 10
U/ml IL-2 (Novartis Pharma, Numberg, Germany) were also added to
the TCM at this step.
[0391] 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.
[0392] 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).
[0393] pMHC used for positive and negative control stimulations
were A*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID No. 60) from
modified Melan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5,
SEQ ID No. 61), respectively.
[0394] 800.000 beads/200 .mu.l were coated in 96-well plates in the
presence of 4.times.12.5 ng different biotin-pMHC, washed and 600
ng biotin anti-CD28 were added subsequently in a volume of 200
.mu.l. Stimulations were initiated in 96-well plates by
co-incubating 1.times.10.sup.6 CD8+ T cells with 2.times.10.sup.5
washed coated beads in 200 .mu.l TCM supplemented with 5 ng/ml
IL-12 (PromoCell) for 3 days at 37.degree. C. Half of the medium
was then exchanged by fresh TCM supplemented with 80 U/ml IL-2 and
incubating was continued for 4 days at 37.degree. C. This
stimulation cycle was performed for a total of three times. For the
pMHC multimer readout using 8 different pMHC molecules per
condition, a two-dimensional combinatorial coding approach was used
as previously described (Andersen et al., 2012) with minor
modifications encompassing coupling to 5 different fluorochromes.
Finally, multimeric analyses were performed by staining the cells
with Live/dead near IR dye (Invitrogen, Karlsruhe, Germany),
CD8-FITC antibody clone SKI (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).
[0395] In Vitro Immunogenicity for Prostate Cancer Peptides
[0396] For tested HLA class I peptides, in vitro immunogenicity
could be demonstrated by generation of peptide specific T-cell
lines. Exemplary flow cytometry results after TUMAP-specific
multimer staining for 2 peptides of the invention are shown in FIG.
3 together with corresponding negative controls. Results for 5
peptides from the invention are summarized in Table 15A. Further
results for 6 peptides from the invention are summarized in Table
15B.
TABLE-US-00015 TABLE 15A 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 donors 17 RLGIKPESV ++
++++ 29 RYGSPINTF + +++ 34 AFSPDSHYLLF + +++ 35 IYTRVTYYL ++ ++++
42 TYIGQGYII + ++++
TABLE-US-00016 TABLE 15B in vitro immunogenicity of HLA class I
peptides of the invention Exemplary results of in vitro
immunogenicity experiments conducted by the applicant for the
peptides of the invention. <20% = +; 20%-49% = ++; 50%-69% =
+++; > = 70% = ++++ SEQ ID Sequence Wells positive [A] 1
VTAQIGIVAV ++++ 3 HLLEDIAHV ++ 5 KIFSRLIYI +++ 6 ALLESRVNL + 24
SYNDALLTF +++ 27 GYLQGLVSF ++
Example 4
Synthesis of Peptides
[0397] 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
[0398] Candidate peptides for T cell based therapies according to
the present invention were further tested for their MHC binding
capacity (affinity). The individual peptide-MHC complexes were
produced by UV-ligand exchange, where a UV-sensitive peptide is
cleaved upon UV-irradiation, and exchanged with the peptide of
interest as analyzed. Only peptide candidates that can effectively
bind and stabilize the peptide-receptive MHC molecules prevent
dissociation of the MHC complexes. To determine the yield of the
exchange reaction, an ELISA was performed based on the detection of
the light chain (.beta.2m) of stabilized MHC complexes. The assay
was performed as generally described in Rodenko et al. (Rodenko et
al., 2006).
[0399] 96 well MAXISorp plates (NUNC) were coated over night with 2
ug/ml streptavidin in PBS at room temperature, washed 4.times. and
blocked for 1 h at 37.degree. C. in 2% BSA containing blocking
buffer. Refolded HLA-A*02:01/MLA-001 monomers served as standards,
covering the range of 15-500 ng/ml. Peptide-MHC monomers of the
UV-exchange reaction were diluted 100 fold in blocking buffer.
Samples were incubated for 1 h at 37.degree. C., washed four times,
incubated with 2 ug/ml HRP conjugated anti-32m for 1 h at
37.degree. C., washed again and detected with TMB solution that is
stopped with NH.sub.2SO.sub.4. Absorption was measured at 450 nm.
Candidate peptides that show a high exchange yield (preferably
higher than 50%, most preferred higher than 75%) are generally
preferred for a generation and production of antibodies or
fragments thereof, and/or T cell receptors or fragments thereof, as
they show sufficient avidity to the MHC molecules and prevent
dissociation of the MHC complexes.
TABLE-US-00017 TABLE 16A MHC class I binding scores Binding of
HLA-class I restricted peptides to HLA-A*24 was evaluated by
peptide ex- change yield: .gtoreq.10% = +; .gtoreq.20% = ++;
.gtoreq.50 = +++; .gtoreq.75% = ++++ SEQ ID Sequence Peptide
exchange 24 SYNDALLTF +++ 25 IYEPYLAMF +++ 26 RYADDTFTPAF +++ 27
GYLQGLVSF ++++ 28 YYAKEIHKF ++++ 29 RYGSPINTF +++ 30 SYSPAHARL +++
31 AYTSPPSFF +++ 32 PYQLNASLFTF ++++ 33 QYGKDFLTL +++ 34
AFSPDSHYLLF +++ 35 IYTRVTYYL ++ 36 RYMWINQEL +++ 41 RYLQKIEEF +++
42 TYIGQGYII +++ 43 AYIKNGQLF +++ 44 VYNTVSEGTHF +++ 45 RYFKTPRKF
++ 47 SYTPVLNQF ++++ 48 AWAPKPYHKF +++ 54 AYSEKVTEF +++ 55
LYFEKGEYF ++++ 56 LFHPEDTGQVF ++ 58 GYIDKVRQL ++ 59 IYPDVTYAF
+++
TABLE-US-00018 TABLE 16B MHC class I binding scores Binding of
HLA-class I restricted peptides to HLA-A*02 was evaluated by
peptide ex- change yield: .gtoreq.10% = +; .gtoreq.20% = ++;
.gtoreq.50 = +++; .gtoreq.75% = ++++ SEQ ID Sequence Peptide
exchange 1 VTAQIGIVAV ++++ 2 SMLGEEIQL ++++ 3 HLLEDIAHV ++++ 4
ALLTFVWKL ++++ 5 KIFSRLIYI +++ 6 ALLESRVNL ++++ 7 TLLQVVGVVSV ++ 9
GMLNEAEGKAIKL +++ 10 TLWRGPVVV +++ 11 YLEEECPAT ++ 13 AMAPNHAVV +++
14 KMDEASAQLL ++ 15 KMDEASAQLLA +++ 16 KMDEASAQL +++ 20 SLLSHQVLL
+++ 21 YLNDSLRHV ++ 22 SLYDSIAFI ++++ 49 SLFHPEDTGQV +++ 52
ALGDLVQSV +++ 53 YLLKDKGEYTL +++
Example 6
Absolute Quantitation of Tumor Associated Peptides Presented on the
Cell Surface
[0400] 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 herein, the inventors did
analyze absolute peptide copies per cell as described. The
quantitation of TUMAP copies per cell in solid tumor samples
requires the absolute quantitation of the isolated TUMAP, the
efficiency of TUMAP isolation, and the cell count of the tissue
sample analyzed.
[0401] Peptide Quantitation by nanoLC-MS/MS
[0402] 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.
[0403] 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.
[0404] 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.
[0405] Efficiency of Peptide/MHC Isolation
[0406] 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.
[0407] 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.
[0408] Determination of the Cell Count in Solid, Frozen Tissue
[0409] 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.
[0410] 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.
[0411] Peptide Copies Per Cell
[0412] 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 17.
TABLE-US-00019 TABLE 17 Absolute copy numbers. The table lists the
results of absolute peptide quantitation in tumor samples. The
median number of copies per cell are indicated for each peptide:
<100 = +; > = 100 = ++; > = 1,000 +++; > = 10,000 =
++++. The number of samples, in which evaluable, high quality MS
data are available is indicated. SEQ ID Peptide Copies per cell
Number of No. Code (median) samples 1 OR51E2-001 + 13 2 GREB-001 ++
14 3 NEFH-001 ++ 11 4 TRPM8-002 +++ 6 5 TRPM8-003 ++ 6 6 PDE11-001
+ 10 24 TRPM8-004 +++ 15 26 RAB3B-001 ++ 15 27 KLK4-001 ++++ 16 28
TGFB3-001 +++ 16 42 FKBP10-002 ++ 19 49 KLK3-004 + 15 50 LRRC26-001
++ 13 54 KLK2-001 +++ 16 55 ACPP-002 +++ 15 56 KLK3-005 +++ 16 57
FOLH1-005 +++ 16
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Sequence CWU 1
1
61110PRTHomo sapiens 1Val Thr Ala Gln Ile Gly Ile Val Ala Val 1 5
10 29PRTHomo sapiens 2Ser Met Leu Gly Glu Glu Ile Gln Leu 1 5
39PRTHomo sapiens 3His Leu Leu Glu Asp Ile Ala His Val 1 5
49PRTHomo sapiens 4Ala Leu Leu Thr Phe Val Trp Lys Leu 1 5
59PRTHomo sapiens 5Lys Ile Phe Ser Arg Leu Ile Tyr Ile 1 5
69PRTHomo sapiens 6Ala Leu Leu Glu Ser Arg Val Asn Leu 1 5
711PRTHomo sapiens 7Thr Leu Leu Gln Val Val Gly Val Val Ser Val 1 5
10 89PRTHomo sapiens 8Leu Leu Asp Phe Ser Leu Ala Asp Ala 1 5
913PRTHomo sapiens 9Gly Met Leu Asn Glu Ala Glu Gly Lys Ala Ile Lys
Leu 1 5 10 109PRTHomo sapiens 10Thr Leu Trp Arg Gly Pro Val Val Val
1 5 119PRTHomo sapiens 11Tyr Leu Glu Glu Glu Cys Pro Ala Thr 1 5
129PRTHomo sapiens 12Ser Leu Asn Glu Glu Ile Ala Phe Leu 1 5
139PRTHomo sapiens 13Ala Met Ala Pro Asn His Ala Val Val 1 5
1410PRTHomo sapiens 14Lys Met Asp Glu Ala Ser Ala Gln Leu Leu 1 5
10 1511PRTHomo sapiens 15Lys Met Asp Glu Ala Ser Ala Gln Leu Leu
Ala 1 5 10 169PRTHomo sapiens 16Lys Met Asp Glu Ala Ser Ala Gln Leu
1 5 179PRTHomo sapiens 17Arg Leu Gly Ile Lys Pro Glu Ser Val 1 5
189PRTHomo sapiens 18Gly Leu Ser Glu Phe Thr Glu Tyr Leu 1 5
199PRTHomo sapiens 19Leu Leu Pro Pro Pro Pro Leu Leu Ala 1 5
209PRTHomo sapiens 20Ser Leu Leu Ser His Gln Val Leu Leu 1 5
219PRTHomo sapiens 21Tyr Leu Asn Asp Ser Leu Arg His Val 1 5
229PRTHomo sapiens 22Ser Leu Tyr Asp Ser Ile Ala Phe Ile 1 5
2311PRTHomo sapiens 23Ala Val Ala Gly Ala Asp Val Ile Ile Thr Val 1
5 10 249PRTHomo sapiens 24Ser Tyr Asn Asp Ala Leu Leu Thr Phe 1 5
259PRTHomo sapiens 25Ile Tyr Glu Pro Tyr Leu Ala Met Phe 1 5
2611PRTHomo sapiens 26Arg Tyr Ala Asp Asp Thr Phe Thr Pro Ala Phe 1
5 10 279PRTHomo sapiens 27Gly Tyr Leu Gln Gly Leu Val Ser Phe 1 5
289PRTHomo sapiens 28Tyr Tyr Ala Lys Glu Ile His Lys Phe 1 5
299PRTHomo sapiens 29Arg Tyr Gly Ser Pro Ile Asn Thr Phe 1 5
309PRTHomo sapiens 30Ser Tyr Ser Pro Ala His Ala Arg Leu 1 5
319PRTHomo sapiens 31Ala Tyr Thr Ser Pro Pro Ser Phe Phe 1 5
3211PRTHomo sapiens 32Pro Tyr Gln Leu Asn Ala Ser Leu Phe Thr Phe 1
5 10 339PRTHomo sapiens 33Gln Tyr Gly Lys Asp Phe Leu Thr Leu 1 5
3411PRTHomo sapiens 34Ala Phe Ser Pro Asp Ser His Tyr Leu Leu Phe 1
5 10 359PRTHomo sapiens 35Ile Tyr Thr Arg Val Thr Tyr Tyr Leu 1 5
369PRTHomo sapiens 36Arg Tyr Met Trp Ile Asn Gln Glu Leu 1 5
379PRTHomo sapiens 37Arg Tyr Leu Gln Asp Leu Leu Ala Trp 1 5
389PRTHomo sapiens 38Val Tyr Ser Asp Lys Leu Trp Ile Phe 1 5
399PRTHomo sapiens 39Ser Tyr Ile Asp Val Ala Val Lys Leu 1 5
409PRTHomo sapiens 40Arg Thr Phe Ser Pro Thr Tyr Gly Leu 1 5
419PRTHomo sapiens 41Arg Tyr Leu Gln Lys Ile Glu Glu Phe 1 5
429PRTHomo sapiens 42Thr Tyr Ile Gly Gln Gly Tyr Ile Ile 1 5
439PRTHomo sapiens 43Ala Tyr Ile Lys Asn Gly Gln Leu Phe 1 5
4411PRTHomo sapiens 44Val Tyr Asn Thr Val Ser Glu Gly Thr His Phe 1
5 10 459PRTHomo sapiens 45Arg Tyr Phe Lys Thr Pro Arg Lys Phe 1 5
469PRTHomo sapiens 46Val Tyr Glu Glu Ile Leu His Gln Ile 1 5
479PRTHomo sapiens 47Ser Tyr Thr Pro Val Leu Asn Gln Phe 1 5
4810PRTHomo sapiens 48Ala Trp Ala Pro Lys Pro Tyr His Lys Phe 1 5
10 4911PRTHomo sapiens 49Ser Leu Phe His Pro Glu Asp Thr Gly Gln
Val 1 5 10 509PRTHomo sapiens 50Thr Leu Gly Pro Ala Ser Phe Leu Val
1 5 519PRTHomo sapiens 51Ala Met Phe Asp Lys Lys Val Gln Leu 1 5
529PRTHomo sapiens 52Ala Leu Gly Asp Leu Val Gln Ser Val 1 5
5311PRTHomo sapiens 53Tyr Leu Leu Lys Asp Lys Gly Glu Tyr Thr Leu 1
5 10 549PRTHomo sapiens 54Ala Tyr Ser Glu Lys Val Thr Glu Phe 1 5
559PRTHomo sapiens 55Leu Tyr Phe Glu Lys Gly Glu Tyr Phe 1 5
5611PRTHomo sapiens 56Leu Phe His Pro Glu Asp Thr Gly Gln Val Phe 1
5 10 579PRTHomo sapiens 57Lys Tyr Ala Asp Lys Ile Tyr Ser Ile 1 5
589PRTHomo sapiens 58Gly Tyr Ile Asp Lys Val Arg Gln Leu 1 5
599PRTHomo sapiens 59Ile Tyr Pro Asp Val Thr Tyr Ala Phe 1 5
6010PRTHomo sapiens 60Glu Leu Ala Gly Ile Gly Ile Leu Thr Val 1 5
10 619PRTHomo sapiens 61Tyr Leu Leu Pro Ala Ile Val His Ile 1 5
* * * * *
References