EA041120B1 - NEW PEPTIDES AND PEPTIDE COMBINATIONS FOR USE IN PROSTATE CANCER AND OTHER CANCER IMMUNOTHERAPY - Google Patents

Description

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to cancer immunotherapy. The present invention further relates to tumor-associated T cell peptide epitopes, alone or in combination with other tumor-associated peptides, which can, for example, serve as active pharmaceutical ingredients in vaccine compositions that stimulate anti-tumor immune responses or stimulate T cells ex vivo with their transfer to the patient's body. Peptides associated with major histocompatibility complex (MHC) molecules, or peptides alone, may also be targets for antibodies, soluble T-cell receptors, and other binding molecules.

The present invention relates to several novel peptide sequences and variants thereof derived from human tumor cell class I HLA molecules that can be used in vaccine compositions to elicit antitumor immune responses or as targets for the development of pharmaceutical/immunologically active compounds and cells.

State of the art

Prostate cancer is the second most commonly diagnosed cancer in the world and the fifth leading cause of cancer death among men, accounting for an estimated 1.1 million new cases in 2012 (15% of all cancer cases). among men) and 0.3 million deaths (7% of deaths among men). During the same period, it was the most common cancer among men in 84 countries around the world, mainly in countries with high or very high levels of development, but also in several countries in Central and Southern Africa. Approximately 220,800 new cases of prostate cancer and 27,540 deaths from prostate cancer are expected in the US in 2015, according to the American Cancer Society. Risk factors include age, family history, and race (World Cancer Report, 2014; SEER Stat facts, 2014; American Cancer Society, 2015).

Almost all prostate cancers are adenocarcinomas that develop from the glandular cells of the prostate. Rare forms of prostate cancer include sarcomas, small cell carcinomas, neuroendocrine tumors (non-small cell carcinomas), or transitional cell carcinomas (American Cancer Society, 2015).

The treatment strategy for prostate cancer mainly depends on the stage of the cancer. For locally confined non-metastatic prostate cancer, treatment options include active surveillance (wait and watch), complete surgical resection of the prostate, and topical high-dose radiotherapy with or without brachytherapy. Hormone suppression therapy and topical postoperative radiotherapy are additional treatment options for patients at high risk. The standard of care for metastatic prostate cancer also includes complete surgical resection of the prostate, topical high-dose radiotherapy, and hormone-suppressive therapy. Tumors that do not respond to hormone deprivation therapy are called castration-resistant prostate cancer (CRPC). Patients with CRPC receive docetaxel, abiraterone, and the dendritic cell vaccine, sipuleucel-T. For the treatment of bone metastases, radium-223 alone or radium-223, docetaxel or abiterone in combination with bisphosphonates or denosumab is used (S3-Leitlinie Prostatakarzinom, 2014).

The dendritic cell-based vaccine, sipuleucel-T, was the first cancer vaccine to be approved by the US Food and Drug Administration (FDA). Due to the positive impact on the survival of patients with castration-resistant prostate cancer (CRPC), many efforts are being made to develop further immunotherapeutic methods. In terms of vaccination strategies, the Prostate Specific Antigen (PSA)-TRICOM Peptide Vaccine, PPV Personalized Peptide Vaccine, pTVG-HP DNA Vaccine, GM-CSF-Expressing Whole Cell Vaccine, have shown promising results in various clinical trials. GVAX. In addition, dendritic cell-based vaccines other than sipuleucel-T, namely BRX-101 and DCVAC/Pa, have been shown to induce clinical responses in patients with prostate cancer. Immune checkpoint inhibitors such as ipilimumab and nivolumab are currently being evaluated in clinical trials as monotherapy as well as in combination with other therapies, including antiandrogen therapy, local radiotherapy, PSA-TRICOM, and GVAX. Studies of the immunomodulatory agent tasquinimod, which significantly reduced progression and improved progression-free survival in a phase II clinical trial, are now ongoing in phase III. Lenalidomide, another immunomodulator, showed promising effects in early phase clinical trials but did not improve survival in phase III clinical trials. Despite these disappointing results, further clinical trials of lenalidomide are ongoing (Quinn et al., 2015).

Given the serious side effects and high costs associated with cancer treatment, there is a need to identify factors that can be used to treat cancer in general and prostate cancer in particular. There is also a need to identify factors that are biomarkers of cancer in general and prostate cancer in particular, which will allow better diagnosis, prognosis and predict the success of treatment.

Cancer immunotherapy is a variant of targeting cancer cells specifically while minimizing side effects. The existence of tumor-associated antigens finds use in cancer immunotherapy.

The current classification of tumor-associated antigens (TAA) includes the following main groups.

a) Cancer-testicular antigens: the first ever identified TAAs that can be recognized by T-cells belong to this class, originally called cancer-testicular antigens (CTs), since its members are expressed in histologically distinct human tumors, and among normal tissues - only in spermatocytes / spermatogonia of the testis and occasionally in the placenta. Since testicular cells do not express HLA class I and II molecules, these antigens cannot be recognized by T cells in normal tissues and therefore can be considered immunologically tumor-specific. Well-known examples of CT antigens are members of the MAGE family and NY-ESO-1.

b) Differentiation antigens: These TAAs are found in tumor and normal tissues from which a tumor develops. Most of the known differentiation antigens are found in melanomas and normal melanocytes. Many of these lineage-specific melanocyte proteins are involved in melanin biosynthesis and therefore are not tumor-specific; however, despite this, they are widely used in anticancer therapy. Examples include, but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma or PSA for prostate cancer.

c) Overexpressed TAAs: Genes encoding widely expressed TAAs have been found in tumors of various histological structures, as well as in many normal tissues, mostly with lower levels of expression. It is possible that many epitopes processed and potentially presented by normal tissues are below the threshold level for recognition by T cells, while their overexpression in tumor cells can initiate an anticancer response, breaking the previously established tolerance. Notable examples of this class of TAAs are Her-2/neu, survivin, telomerase, or WT1.

d) Tumor-specific antigens: These unique TAAs result from mutations in normal genes (such as β-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 carrying the risk of autoimmune reactions against normal tissues. On the other hand, TAA data are in most cases only relevant to the specific tumor on which they were identified and are not usually common to many individual tumors. Tumor specificity (or association) of a peptide can also occur if the peptide is formed from a tumor (tumor associated) exon in the case of proteins with tumor specific (β associated) isoforms.

e) TAAs formed as a result of abnormal post-translational modifications: such TAAs can be formed from proteins that are neither specific nor overexpressed in tumors, however, despite this, become tumor-associated during post-translational processes occurring predominantly in tumors. Examples for this class arise from a change in the pattern of glycosylation leading to new epitopes in tumors, as in the case of MUC1, or from events such as protein splicing during degradation, which may or may not be tumor-specific.

f) Oncoviral proteins: These TAAs are viral proteins and may play a key role in the oncogenic process, and since they are foreign (not of human origin), they may provoke a T-cell response. Examples of such proteins are the human papillomavirus type 16, E6 and E7 viral proteins, which are expressed in cervical carcinoma.

T-cell-based immunotherapy targets are peptide epitopes derived from tumor-associated or tumor-specific proteins, which are presented by molecules of the human major histocompatibility complex (MHC) (MHC). Antigens that are recognized by tumor-specific T-lymphocytes, i.e. their epitopes may be molecules derived from any class of proteins, such as enzymes, receptors, transcription factors, etc., which are expressed and, compared to unaltered cells of the same origin, are usually elevated in the corresponding tumor cells.

As with all other non-vital cancers, prostate-specific antigens may be a good choice for cancer immunotherapy because prostate-specific antigens are tumor-specific targets in prostatectomy patients. In non-prostatectomy cancer patients, such antigens may also be of interest because the prostate is not considered a vital organ, and a similar approach has been used in melanoma using melanocyte differentiating antigens. There are several examples demonstrating that prostate-specific or highly prostate-associated antigens are reliable targets, such as Dendreon's sipuleucel-T (Provenge), which includes prostatic acid phosphatase as the tumor antigen used (Westdorp et al., 2014 ). This antigen is not exclusively expressed in the prostate, but is overexpressed in the prostate at a level that is 1–2 orders of magnitude higher than in other tissues (Graddis et al., 2011).

There are two classes of MHC molecules, MHC class I and MHC class II. Class I MHC molecules consist of an alpha-heavy chain and beta-2-microglobulin, class II MHC molecules consist of alpha and beta chains. Their three-dimensional shape forms a binding groove that is used for non-covalent interaction with peptides.

Class I MHC molecules are found on most cells that have a nucleus. They present peptides resulting from the proteolytic cleavage of predominantly endogenous proteins, defective ribosomal products (DRIP) and larger peptides. However, peptides derived from endosomal compartments or exogenous sources are also frequently found on class I MHC molecules. This non-classical way of presentation by class I in the literature is called cross-presentation. (Brossart and Bevan, 1997; Rock et al., 1990). Class II MHC molecules can occur predominantly on professional antigen-presenting cells (APCs) and primarily present peptides of exogenous or transmembrane proteins that are taken up by APC, for example, during endocytosis and subsequently processed.

Peptide-MHC class I complexes are recognized by CD8-positive T cells carrying the appropriate T cell receptor (TCR), while peptide-MHC class II complexes are recognized by CD4-positive helper T cells carrying the appropriate TCR. It is well known that TCR, peptide and MHC occur in a 1:1:1 stoichiometric ratio.

CD4 positive helper T cells play an important role in inducing and maintaining effective CD8 positive cytotoxic T cell responses. The identification of CD4-positive T-cell epitopes derived from tumor-associated antigens (TAAs) may be extremely important for the development of pharmaceuticals to initiate antitumor immune responses (Gnjatic et al., 2003). At the tumor site, T-helper cells maintain a CTL-friendly cytokine environment (Mortara et al., 2006) and recruit effector cells, such as CTLs, natural killer (NK) cells, macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of class II MHC molecules is predominantly restricted to cells of the immune system, especially professional antigen-presenting cells (APCs), eg monocytes, monocyte-derived cells, macrophages, dendritic cells. Tumor cells of cancer patients have been found to express class II MHC molecules (Dengjel et al., 2006).

Extended (longer) peptides of the invention can act as active MHC class II epitopes. T helper cells activated by class II MHC epitopes play an important role in controlling the CTL effector function in antitumor immunity. T helper cell epitopes that initiate TH1-type T helper cell responses support the effector functions of CD8-positive killer T cells, which include cytotoxic functions directed against tumor cells that exhibit tumor-associated peptide/MHC complexes on their cell surface. Thus, tumor-associated T-helper cell peptide epitopes, alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients in vaccine compositions that stimulate antitumor immune responses.

Even in the absence of CD8-positive T lymphocytes, CD4-positive T cells have been shown in mammalian animal models such as mice to be sufficient to attenuate the clinical manifestations of tumors by inhibiting angiogenesis by interferon-gamma (IFN-gamma) secretion. (Beatty and Paterson, 2001; Mumberg et al., 1999). There is evidence that CD4 T cells are direct antitumor effector cells (Braumuller et al., 2013; Tran et al., 2014).

Since the constitutive expression of HLA class II molecules is usually limited to immune cells, isolation of class II peptides directly from primary tumors was previously considered impossible. However, Dengjel et al have been able to identify a number of class II MHC epitopes directly from tumors (WO 2007/028574, EP 1760088 B1).

Since both types of CD8- and CD4-dependent responses contribute to the antitumor effect together and synergistically, the identification and characterization of tumor-associated antigens recognized by both CD8+ T cells (ligand: MHC class I molecule + peptide epitope) and CD4-positive helper T cells (ligand: MHC class II molecule + peptide epitope) are important in the development of cancer vaccines.

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In order for an MHC class I peptide to initiate (cause) a cellular immune response, it must also bind to an MHC molecule. This process depends on the allele of the MHC molecule and specific polymorphisms of the amino acid sequence of the peptide. Class I MHC binding peptides are typically 8-12 amino acid residues in length and typically contain two conserved residues (anchors) in their sequence that interact with the corresponding binding groove of the MHC molecule. Thus, each MHC allele has a binding motif that determines which peptides can specifically bind to the binding groove.

In an MHC class I dependent immune response, the peptides not only must be able to bind to specific MHC class I molecules expressed by the tumor cells, but they must also then be recognized by T cells bearing specific T cell receptors (TCRs).

In order for proteins to be recognized by T-lymphocytes as tumor-specific or β-associated antigens and to be used in therapy, special prerequisites must be met. The antigen should be predominantly expressed by tumor cells and not expressed or expressed in a relatively small amount by healthy tissues. In a preferred embodiment, the peptide should be over-presented by tumor cells compared to normal healthy tissues. In addition, it is desirable that the corresponding antigen not only be present in some kind of tumor, but also at a high concentration (ie, several copies of the corresponding peptide per cell). Tumor-specific and tumor-associated antigens are often formed from proteins that are directly involved in the transformation of a normal cell into a tumor cell, in connection with their function, for example, in the control of the cell cycle or the suppression of apoptosis. In addition, downstream targets of proteins that directly cause transformation may be present in increased amounts and thus be indirectly tumor-associated. Such indirect tumor-associated antigens may also be targets of the vaccination approach (Singh-Jasuja et al., 2004). It is necessary that epitopes be present in the amino acid sequence of an antigen to ensure that such a peptide (immunogenic peptide) derived from a tumor associated antigen leads to a T cell response in vitro or in vivo.

In essence, any peptide capable of binding to an MHC molecule can function as a T-cell epitope. The prerequisite for inducing a T cell response in vitro or in vivo is the presence of a T cell with the appropriate TCR and lack of immunological tolerance for that particular epitope.

Therefore, TAA antigens are the starting point for the development of T cell-based therapies, including but not limited to cancer vaccines. Methods for identifying and characterizing TAAs are usually based on the use of T cells, which can be isolated from patients or healthy subjects, or they can be based on generating different transcriptional profiles or different peptide expression patterns between tumor and normal tissues. However, identification of genes overexpressed in tumor tissues or human tumor cell lines, or selectively expressed in such tissues or cell lines, does not provide accurate information about the use of antigens transcribed from these genes in immunotherapy. This is because only a subset of the epitopes of these antigens are suitable for such use, since a T cell with the appropriate TCR must be available and there must be no or minimal immunological tolerance for that particular epitope. Therefore, in the most preferred embodiment of the invention, it is important to select only those peptides presented in excess or selectively against which a functional and/or proliferating T cell can be detected. Such a functional T cell is defined as a T cell that, when stimulated with a specific antigen, can be expanded by cloning and is capable of performing effector functions (effector T cell).

In the case of targeting peptide-MHC complexes with specific TCRs (eg, soluble TCRs) and antibodies or other molecules (scaffolds) binding to them, according to the invention, the immunogenicity of the underlying peptides is of secondary importance. In such cases, presentation is the determining factor.

Brief summary of the invention

In a first aspect, the present invention relates to a peptide comprising an amino acid sequence selected from the group consisting of sequences from 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 variant binds to MHC and/or induces T-cell cross-reactivity with said peptide, or a pharmaceutically acceptable salt thereof, wherein said peptide is not a full length basic polypeptide.

The present invention further relates to a peptide of the present invention comprising a sequence selected from the group consisting of sequences from SEQ ID NO: 1 to

SEQ ID NO: 48, or a variant thereof that is at least 77%, preferably at least 88% homologous (preferably at least 77% or at least 88% identical) to the sequence of SEQ ID NO: 1 according to SEQ ID NO: 48, where the specified peptide or variant has a total length of 8-100, preferably 8-30 and most preferably 8-14 amino acids.

The following tables present the peptides of the present invention, their respective SEQ ID NOs, and potential source (underlying) genes for these peptides. All peptides in Table. 1, 3 and 5 bind to HLA-A*02. All peptides in Table. 2, 4 and 6 bind to HLA-A*24. Peptides tab. 3 and 4 have previously been disclosed as extensive lists as results of high throughput screens with a high error rate, or have been computed by algorithms, but have not previously been associated with cancer in any way. Peptides from the table. 5 and 6 are additional peptides that may be useful in combination with other peptides of the invention. Peptides tab. 7 and 8 are also useful for the diagnosis and/or treatment of various other cancers that involve overexpression or overpresentation of the respective base polypeptide.

Table 1

HLA-A*02 binding peptides

in accordance with the present invention SEQID No. Subsequence Ident. gene number(s) Official symbol(s) of the gene 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 TLLQWGWSV 10257 ABCC4 8 LLDFSLADA 1674 DES 9 GMLNEAEGKAIKL 4629 MYH11 10 TLWRGPVW 261729 STEAP2 eleven YLEEECPAT 563 AZGP1 12 SLNEIAFL 1674 DES 13 AMAPNHAW 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 HIC 22 SLYDSIAFI 56978 PRDM8 23 AVAGADVIITV 1428 CRYM table 2 HLA-A*24 binding peptides according to the present invention SEQID No. Subsequence Ident. gene number(s) Official symbol(s) of the gene 24 SYNDALLTF 79054 TRPM8 25 IYEPYLAMF 79054 TRPM8 26 RYADDTTFTPAF 5865 RAB3B 27 GYLQGLVSF 9622 KLK4 28 YYAKEIHKF 7043 TGFB3 29 RYGSPINTF 647024 C6orf132 thirty SYSPAHARL 2624 GATA2 31 AYTSPPSFF 171024 SYNPO2 32 PYQLNASLFTF 171024 SYNPO2 33 QYGKDFLTL 79088 ZNF426 34 AFSPDSHYLLF 3679 ITGA7 35 IYTRVTYYL 64499, 7177 TPSB2, TPSAB1 36 RYMWINQEL 374654 KIF7 37 RYLQDLLAW 5339 PLEC 38 VYSDKLWIF 8216 LZTR1 39 SYIDVAVKL 57544 TXNDC16

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Table 3 Additional HLA-A*O2 binding peptides according to the present invention, whose association with cancer was not previously known. J = phosphoserine

SEQID No. Subsequence Ident. gene number(s) Official symbol(s) of the gene 40 RTFJPTYGL 23336 SYNM

Table 4 Additional HLA-A*24 binding peptides according to the present invention, whose association with cancer _______ was not previously known ________

SEQID No. Subsequence Ident. gene number(s) Official symbol(s) of the gene 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, 9448 TNIK, MINK1, MAP4K4

Table 5 HLA-A*O2 binding peptides useful, for example, for personalized cancer therapy

SEQID No. Subsequence Ident. gene number(s) Official symbol(s) of the gene 49 SLFHPEDTGQV 354 KLK3 50 TLGPASFLV 389816 LRRC26 51 AMFDKKVQL 23336 SYNM 52 ALGDLVQSV 7782 SLC30A4 53 YLLKDKGEYTL 2316 FLNA

Table 6 HLA-A*24 binding peptides useful, for example, for personalized cancer therapy

SEQID No.

Sequence AYSEKUTEF LYFEKGEYF LFHPEDTGQVF KYADKIYSI GYIDKVRQL IYPDVTYAF

Ident. gene number(s)

3817__________

55______

354___________

2346__________

4744

1135

Official symbol(s) of the gene

KLK2___________

ASRR_________

KLK3___________

F0LH1_________

NEFH

CHRNA2

For selected peptides table. 7 shows which additional tumor types they were found on and had either over-representation on more than 5% of tumor samples examined, or over-presentation on more than 5% of tumor samples examined with a geometric mean ratio for tumor and for normal tissues of more than three. Over-presentation is defined as a higher over-representation on a tumor sample compared to a normal tissue sample with the highest presentation. The normal (non-cancerous) tissues against which the overpresentation test was performed were the following: adipose tissue, tissues of the adrenal gland, artery, bone marrow, brain, central nerve, colon, duodenum, esophagus, eye, gallbladder , heart, kidney, liver, lung, lymph nodes, mononuclear leukocytes, pancreatic tissue, parathyroid gland, peripheral nerve, abdominal cavity, pituitary gland, pleura, rectum, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland, trachea, ureter, bladder and veins.

diseases, especially diseases

Table 7

Peptides according to the present invention and their specific use in other proliferative diseases

SEQID No. Subsequence Other relevant organs/diseases 1 VTAQIGIVAV SCLC, melanoma 2 SMLGEEIQL HCC, breast cancer, melanoma, uterine cancer 3 HLLEDIAHV BCM, uterine cancer 5 KIFSRLIYI Melanoma 6 ALLESRVNL GKK, PC 7 TLLQWGWSV Uterine cancer, gallbladder cancer, bile duct cancer

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8 LLDFSLADA CRC, breast cancer, melanoma, bladder cancer, uterine cancer 10 TLWRGPWV NSCLC, RCC, CRC, HCC, leukemia, OC, esophageal cancer, gallbladder cancer, bile duct cancer, CLL 12 SLNEIAFL SCLC, prostate cancer, bladder cancer, gallbladder cancer, bile duct cancer 13 AMAP N HAW Brain cancer 16 KMDEASAQL bladder cancer 17 RLGIKPESV Brain cancer, HCC, breast cancer, uterine cancer 18 GLSEFTEYL Brain cancer 19 LLPPPPLLA Brain cancer, melanoma, bladder cancer 20 SLLSHQVLL SCLC, CRK, HCC, bladder cancer, breast cancer, esophageal cancer, uterine cancer 22 SLYDSIAFI Brain cancer, leukemia, AML 26 RYADDTTFTPAF GKK 27 GYLQGLVSF GKK 28 YYAKEIHKF NSCLC, GCC 29 RYGSPINTF NSCLC, RJ, GCC 31 AYTSPPSFF RJ, GKK 33 QYGKDFLTL NSCLC, brain cancer, HCC 34 AFSPDSHYLLF NSCLC, RCC, brain cancer, HCC 35 IYTRVTYYL NSCLC, RJ 36 RYMWINQEL NSCLC, brain cancer, HCC 37 RYLQDLLAW NSCLC, PCC, brain cancer 38 VYSDKLWIF NSCLC, brain cancer, GC, HCC 40 RTFJPTYGL bladder cancer 41 RYLQKIEEF NSCLC, PCC, brain cancer 42 TYIGQGYII NSCLC, brain cancer, GC, HCC 43 AYIKNGQLF Brain cancer 44 VYNTVSEGTHF NSCLC, brain cancer, HCC 45 RYFKTPRKF GKK 47 SYTPVLNQF GKK 48 AWAPKPYHKF NSCLC, RCC, brain cancer, HCC

NSCLC = non-small cell lung cancer,

SCLC = small cell lung cancer, SCC = kidney cancer, CRC = colon or rectal cancer, HCC = liver cancer, PCa = pancreatic cancer, BC = breast cancer, MCM = Merkel cell carcinoma, OC = ovarian cancer, AML = acute myeloid leukemia, CLL = chronic lymphocytic leukemia, GC = gastric cancer, J = phosphoserine.

The present invention also generally relates to peptides according to the present invention for use in the treatment of proliferative diseases, e.g., lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer. bladder, bile duct cancer, CRC, bladder cancer, non-small cell lung cancer, kidney cancer, leukemia (such as AML or CLL), ovarian cancer, esophageal cancer, brain cancer, and stomach cancer.

Particularly preferred are peptides - alone or in combination - according to the present invention, selected from the group consisting of the sequences of SEQ ID NO: 1 to SEQ ID NO: 48. More preferred are peptides - alone or in combination - selected from a group consisting of sequences from SEQ ID NO: 1 to SEQ ID NO: 6 (see Table 1) and from SEQ ID NO: 24 to SEQ ID NO: 28 (see Table 2) or from SEQ ID NO: 1, 4, 5, 6, 49 and 52 or SEQ ID NOs: 2, 3 and 54 and their use in immunotherapy for lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, cancer pancreatic cancer, gallbladder cancer, bile duct cancer, CRC, bladder cancer, non-small cell lung cancer, kidney cancer, leukemia (eg, AML or CLL), ovarian cancer, esophageal cancer, brain cancer, and gastric cancer, and most preferably, prostate cancer. As shown in Table. 7 above, many of the peptides of the present invention are present in other types of tumors and may thus be used in immunotherapy for other indications. See also FIG. 1 and example 1.

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention in accordance with any of the sequences with

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SEQ ID NOs: 1, 12 and 20 in one preferred embodiment in combination for the treatment of non-small cell lung cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any of the sequences of SEQ ID NOs: 1, 2, 5, 8 and 19 - in one preferred embodiment in combination - for the treatment of melanoma .

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention in accordance with any of the sequences of SEQ ID NO: 2, 6, 10, 17, 20, 26, 27, 28, 29, 31, 33 , 34, 36, 38, 42, 44, 45, 47 and 48 in one preferred embodiment in combination for the treatment of liver cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any of the sequences of SEQ ID NOs: 2, 8, 17 and 20 - in one preferred embodiment in combination - for the treatment of breast cancer .

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention in accordance with any of the sequences of SEQ ID NOS: 2, 3, 7, 8, 17 and 20 - in one preferred embodiment in combination - for treatment of uterine cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention in accordance with SEQ ID NO: 3 for the treatment of MCC.

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention in accordance with any of the sequences of SEQ ID NO: 6 and 12 - in one preferred embodiment in combination - for the treatment of prostate cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention in accordance with any of the sequences of SEQ ID NO: 7, 10 and 12 - in one preferred embodiment in combination - for the treatment of gallbladder cancer and/ or bile duct cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any of the sequences of SEQ ID NOS: 8, 10 and 20 - in one preferred embodiment in combination - for the treatment of CRC.

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention in accordance with any of the sequences of SEQ ID NO: 10 and 22 - in one preferred embodiment in combination - for the treatment of leukemia.

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention in accordance with SEQ ID NO: 10 for the treatment of OC.

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any of the sequences of SEQ ID NOs: 10 and 20 - in one preferred embodiment in combination - for the treatment of esophageal cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention in accordance with SEQ ID NO: 10 for the treatment of CLL.

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention in accordance with any of the sequences of SEQ ID NO: 6 and 12 - in one preferred embodiment in combination - for the treatment of prostate cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention in accordance with any of the sequences of SEQ ID NO: 10, 28, 29, 33, 34, 35, 36, 37, 38, 41, 42 , 44 and 48 in one preferred embodiment in combination for the treatment of NSCLC.

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention according to any of the sequences of SEQ ID NOs: 10, 34, 37, 41 and 48 - in one preferred embodiment in combination - for the treatment of RCC .

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention in accordance with any of the sequences of SEQ ID NO: 13, 17, 18, 19, 22, 33, 34, 36, 37, 38, 41 , 42, 43, 44 and 48 in one preferred embodiment in combination for the treatment of brain cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention in accordance with SEQ ID NO: 22 for the treatment of AML.

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Thus, another aspect of the present invention relates to the use of at least one peptide of the present invention in accordance with any of the sequences with

SEQ ID NOs: 29, 31, 35, 38 and 42 in one preferred embodiment in combination for the treatment of gastric cancer.

Thus, another aspect of the present invention relates to the use of the peptides according to the present invention - preferably in combination - in the treatment of a proliferative disease selected from the group: lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, carcinoma Merkel cells, pancreatic cancer, gallbladder cancer, bile duct cancer, CRC, bladder cancer, non-small cell lung cancer, kidney cancer, leukemia (eg, AML or CLL), ovarian cancer, esophageal cancer, brain cancer, and stomach cancer, and most preferably prostate cancer.

The present invention further relates to peptides according to the present invention having the ability to bind to a human major histocompatibility complex (MHC) class I molecule or - in an extended form such as a length variant - MHC class II.

The present invention further relates to 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.

The present invention further relates to peptides in accordance with the present invention, where the specified peptide is modified and/or includes non-peptide bonds.

The present invention further relates to 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 an HLA-DR antigen-associated invariant chain (li), or fused to an antibody (or inserted into a sequence) , such as, for example, an antibody specific for dendritic cells.

Another embodiment of the present invention relates to a non-naturally occurring peptide, where the specified peptide consists or consists essentially of the amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 48 and was obtained synthetically (for example, synthesized) in the form pharmaceutically acceptable salt. Methods for the synthetic production of peptides are well known in the art. The salts of the peptides of the present invention differ substantially from the peptides in their in vivo state(s), since the synthesized peptides are not salts in vivo. The non-naturally occurring salt form of the peptide mediates the solubility of the peptide, particularly in the context of pharmaceutical compositions comprising peptides, such as the peptide-based vaccines disclosed herein. Sufficient and at least substantial solubility of the peptide(s) is necessary for effective administration of the peptides to the subject to be treated. Preferably the salts are pharmaceutically acceptable salts of the peptides. Salts according to the invention include alkali and alkaline earth salts, such as salts of the Hofmeister series, including as anions PO ₄ ³ - , SO4 ^2- , CH3COO ^- , Cl ^- , Br, NO3 ^- , ClO ₄ ^- , I ^- , SCN ^- and as cations NH4+, Rb ⁺ , K ⁺ , Na ⁺ , Cs ⁺ , Li ⁺ , Zn ²⁺ , Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Cu ²⁺ and Ba ²⁺ . In particular, the salts are selected from (NH4)3PO4, (NH4)2HPO4, (NH4)H2PO4, (NH4)2SO4, NH4CH3COO, NH4Cl, NH4Br, NH4NO3, NH4ClO4, NH4I, NH4SCN, Rb3PO4, Rb2HPO4, RbH2PO4, Rb2SO4, Rb4CH3COO, Rb4Cl, Rb4Br, Rb4NO3, Rb4ClO4, Rb4I, Rb4SCN, K3PO4, K2HPO4, KH2PO4, K2SO4, KCH3COO, KCl, KBr, KNO3, KClO4, KI, KSCN, Na3PO4, Na2HPO4, NaH2PO4, Na2SO4, NaCH3COO, NaCl, NaBr, NaNO3, NaClO4, Nal, NaSCN, ZnCl2 Cs3PO4, Cs2HPO4, CsH2PO4, Cs2SO4, CsCH3COO, CsCl, CsBr, CsNO3, CsClO4, CsI, CsSCN, Li3PO4, Li2HPO4, LiH2PO4, Li2SO4, LiCH3COO, LiCl, LiBr, LiNO3, LiClO4, LiI, LiSCN , Cu2SO4, Mg3(PO4)2, Mg2HPO4, Mg(H2PO4)2, Mg2SO4, Mg(CH3COO)2, MgCl2, MgBr2, Mg(NO3)2, Mg(ClO4)2, MgI2, Mg(SCN)2, MnCl2 , Ca3(PO4)2, Ca2HPO4, Ca(H2PO4)2, CaSO4, Ca(CH3COO)2, CaCF, CaBr2, Ca(NO3)2, Ca(ClO4)2, Cab, Ca(SCN)2, Ba3(PO4 )2, Ba2HPO4, Ba(H2PO4)2, BaSO4, Ba(CH3COO)2, BaCk, BaBr2, Ba(NO3)2, Ba(ClO4)2, Bab, and Ba(SCN)2. Especially preferred are NH acetate, MgCl2, KH2PO4, Na2SO4, KCl, NaCl and CaCl2, such as, for example, the chloride or acetate (trifluoroacetate) salts.

Typically, peptides and variants (at least those containing peptide bonds between amino acid residues) can be synthesized by the Fmoc-polyamide solid phase peptide synthesis method as disclosed by Lukas et al. (Lukas et al., 1981) and in the accompanying references. The temporary protection of the N-amino group is provided by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Re-cleavage of this highly alkaline protecting group is carried out using 20% piperidine in N,N-dimethylformamide. The functional groups of the side chain can be protected by obtaining compounds such as their butyl esters (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 a 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivative (in the case of arginine). If glutamine or asparagine are C-terminal residues, a 4,4'-dimethoxybenzhydryl group is used to protect the side chain amido group. The solid phase carrier is based on a polydimethylacrylamide polymer consisting of three monomers: dimethylacrylamide (skeleton monomer), bis-acryloylethylenediamine (crosslinker, linker) and acryloylsarcosine methyl ester (functionalizing agent). To form an easily cleavable bond between the peptide and the resin, an acid-labile derivative of 4-hydroxymethylphenoxyacetic acid is used. All amino acid derivatives are added as pre-synthesized symmetrical anhydride derivatives, except for asparagine and glutamine, which are added using a N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole mediated reverse compound reaction. All coupling and deprotection reactions were monitored using ninhydrin, trinitrobenzenesulfonic acid or isotin controls. After completion of the synthesis, the peptides are cleaved from the carrier resin with concomitant side chain deprotection by treatment with 95% trifluoroacetic acid containing 50% scavenger mixture. Commonly used scavengers include ethanedithiol, phenol, anisole and water, the final choice being dependent on the amino acid constituents of the peptide being synthesized. A combination of solid phase and liquid phase methods for peptide synthesis is also possible (see, for example, (Bruckdorfer et al., 2004) and accompanying references).

The trifluoroacetic acid is removed by vacuum evaporation followed by trituration with diethyl ether to give the crude peptide. Any scavengers present are removed by a simple extraction technique which allows the crude peptide without scavengers to be obtained after lyophilization of the aqueous phase. Reagents for synthesis of peptides are generally available from, for example, Calbiochem-Novabiochem (Nottingham, UK).

Purification may be by any technique or combination of techniques such as recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and (typically) reverse phase high performance liquid chromatography using, for example, acetonitrile/water gradient separation.

The present invention further relates to a nucleic acid encoding peptides in accordance with the present invention. The present invention further relates to a nucleic acid in accordance with the present invention, which is DNA, cDNA, PNA, RNA, or combinations thereof.

The present invention further relates to an expression vector capable of expressing and/or expressing a nucleic acid in accordance with the present invention.

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.

The present invention further relates to antibodies that are specific for peptides according to the present invention or complexes of said peptides according to the present invention and MHC, and methods for their preparation.

The present invention further relates to T cell receptors (TCRs), in particular soluble TCRs and cloned TCRs incorporated into autologous or allogeneic T cells, and methods for their production, as well as natural killer (NK) cells or other cells bearing the specified TCR or cross-reacting with the indicated TCR.

Antibodies and TCRs are further embodiments of the immunotherapeutic use of the peptides of the present invention.

The present invention further relates to a host cell comprising a nucleic acid according to the present invention or an expression vector as described previously. The present invention further relates to a host cell according to the present invention, which is an antigen presenting cell, preferably a dendritic cell.

The present invention further relates to a method for producing a peptide in accordance with the present invention, said method comprising culturing a host cell in accordance with the present invention and isolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to the present invention, wherein the antigen is loaded onto MHC class I or II 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 the antigen presenting cell.

The present invention further relates to a method in accordance with the present invention, where the antigen-presenting cell includes an expression vector capable of expressing or expressing the specified peptide containing the sequences of SEQ ID NO: 1 to SEQ ID NO: 48, preferably containing the sequences of SEQ ID NO: 1 according to SEQ ID NO: 6 (see Table 1) and from SEQ ID NO: 24 to SEQ ID NO: 28 (see Table 2) or a variant amino acid sequence.

The present invention further relates to activated T cells obtained by the method of the present invention, wherein said T cell selectively recognizes a cell that expresses a polypeptide comprising the amino acid sequence of the present invention.

The present invention further relates to a method for killing target cells in a patient whose target cells aberrantly express a polypeptide comprising any amino acid sequence in accordance with the present invention, the method comprising administering to the patient an effective number of T cells obtained in accordance with the present invention. the present invention.

The present invention further relates to the use of any described peptide, nucleic acid according to the present invention, an expression vector according to the present invention, a cell according to the present invention, an activated T-lymphocyte, a T-cell receptor or antibody, or other molecules that bind to peptide and/or peptide-MHC complex according to the present invention as a medicament or in the manufacture of a medicament. Preferably, said medicament has an active anti-cancer effect.

Preferably, said medicament is for cell therapy, is a vaccine or soluble TCR protein or antibody.

The present invention further relates to 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, cancer bile duct cancer, CRC, bladder cancer, non-small cell lung cancer, kidney cancer, leukemia (eg AML or CLL), ovarian cancer, esophageal cancer, brain cancer and gastric cancer, and most preferably prostate cancer cells.

The present invention further relates to peptide-based biomarkers according to the present invention, referred to in the context of the invention as targets, which can be used in the diagnosis of cancer, preferably prostate cancer. The marker may be over-presentation of the peptide(s) itself, or over-expression of the corresponding gene(s). These markers can also be used to predict the likelihood of success of a treatment, preferably an immunotherapy, and most preferably an immunotherapy directed to the same target that has been identified by the biomarker. For example, an antibody or soluble TCR can be used to stain tumor sections to detect the presence of a peptide of interest in complex with MHC.

Optionally, the antibody has an additional effector function, such as carrying an immunostimulatory domain or a toxin.

The present invention also relates to the use of these new targets in the context of cancer treatment.

Both therapeutic uses against other types of cancers and diagnostic uses are disclosed in the following more detailed description of the expression products (polypeptides) underlying the peptides of the invention.

ATP-binding cassette transporters subfamily C (CFTR/MRP), member 4 (ABCC4) ABCC4 is able to pump a wide range of endogenous and xenobiotic anionic organic compounds out of the cell, including a wide range of antivirals, cytostatics, antibiotics and drugs for the treatment of cardiovascular diseases , as well as molecules involved in cell signaling, thus endowing ABCC4 with a key function in cell defense processes and extracellular signaling pathways (Russel et al., 2008). ABCC4 levels are elevated in prostate cancer and expression has been shown to be upregulated by androgen exposure in vitro. Therefore, antiandrogen therapy reduces the expression level of ABCC4 in prostate cancer tissue. Moreover, the level of ABCC4 decreases with the progression of prostate cancer (Ho et al., 2008; Montani et al., 2013). Overexpression of ABCC4 has also been demonstrated in other cancers, such as lung cancer (Zhao et al., 2014). Suppression of ABCC4 expression has been shown to inhibit the proliferation of overexpressing ABCC4 cancer cells in vitro (Zhao et al., 2014) and restore chemotherapeutic agent sensitivity in drug-resistant cell lines (Zhang et al., 2014).

Astrotactin 2 (ASTN2) - ASTN2 is thought to have a function in the process of nerve cell migration (Wilson et al., 2010).

Zinc domain-binding alpha-2 glycoprotein 1 (AZGP1) - ASTN2 stimulates lipid breakdown in adipocytes and causes massive fat loss associated with some advanced cancers (UniProt, 2015). The expression level of AZGP1 is elevated in prostate cancer, which is also found in serum samples and has been proposed as a potential biomarker (Hale et al., 2001). Its levels are reduced in high-grade tumors, and AZGP1 expression is associated with reduced mortality and tumor recurrence in patients with prostate cancer (Lapointe et al., 2004; Severi et al., 2014). AZGP1 has been proposed as a biomarker for breast cancer, and AZGP1 levels are reduced in poorly differentiated tumors (Hassan et al., 2008).

Chromosome 6 open reading frame 132 (C6orf132) - C6orf132 is located on chromosome 6p21.1 (RefSeq, 2002).

Zinc finger protein 1 interacting with CDKN1A (CIZ1) - CIZ1 encodes a protein with

- 11 041120 zinc finger domain interacting with CDKN1A, which functions as a tumor suppressor gene (Nishibe et al., 2013). In colorectal cancer, CIZ1 is thought to be involved in cancer progression by regulating cell proliferation, cell cycle, apoptosis, and cell colony formation (Yin et al., 2013). A CIZ1 variant that lacks part of the C-terminal domain has been found in lung cancer cells even at an early stage and has potential as a biomarker (Higgins et al., 2012).

Crystallin, mu (CRYM) - CRYM specifically catalyses the reduction of imine bonds (Hallen et al., 2011). CRYM is an androgen-regulated gene that is upregulated in prostate cancer and downregulated in castration-resistant tumors (Malinowska et al., 2009). CRYM was overexpressed in non-small cell lung cancer and uterine leiomyosarcoma metastases compared to the primary tumor (Chong et al., 2006; Davidson et al., 2014). CRYM expression has also been detected in breast cancer samples, and CRYM has been detected in the sera of breast cancer patients when analyzed by the SEREX method (Forti et al., 2002).

Cysteine and Glycine Rich Protein 2 (CSRP2) - CSRP2 belongs to the CSRP family of genes encoding proteins containing the LIM domain. Overexpression of CSRP2 is associated with dedifferentiation of hepatocellular carcinoma (Midorikawa et al., 2002).

Desmin (DES) - DES are class III intermediate filament proteins present in muscle cells. They form a fibrous network that connects myofibrils to each other and to the plasma membrane from the periphery of the Z-line structures (Clemen et al., 2015). DES expression is downregulated in prostate cancer tissue, and low DES expression is associated with shorter disease-free survival (Wu et al., 2014). In colorectal carcinoma, DES expression levels are elevated in advanced-stage tumors, possibly due to increased microvascular density and thus higher levels of pericytes (Arentz et al., 2011). In addition, elevated DES levels are associated with reduced survival in colorectal cancer (Ma et al., 2009).

Family with sequence similarity 129, member A (FAM129A) - FAM129A is localized 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).

FK506 binding protein 10 (FKBP10) - FKBP10 encodes FK506 binding protein 10, which belongs to the peptidyl prolyl cis/trans isomerase family of the FKBP type. The FKBP10 gene product is localized in the endoplasmic reticulum and acts as a molecular chaperone (RefSeq, 2002). FKBP10 was identified as a new gene that is involved in the acquisition and maintenance of the adriamycin-resistant phenotype by leukemia cells (Sun et al., 2014). FKBP10 is associated with colorectal cancer by increasing its level (Olesen et al., 2005). On the contrary, FKBP10 underexpression was characteristic of epithelial ovarian carcinomas (Quinn et al., 2013).

GATA-binding protein 2 (GATA2) - GATA2 is an essential element required for hematopoiesis, and GATA2 mutations are associated with myelodysplastic syndrome and myeloid leukemia (Bresnick et al., 2012). In solid tumors, GATA2 overexpression has been described in breast cancer and has been 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 suggest rather downregulated GATA2 expression in tumor tissue and report an association of low GATA2 levels 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).

Estrogen-Dependent Regulator of Breast Cancer Growth (GREB1) - GREB1 is an estrogen-responsive gene that is an early response signaling pathway gene regulated by the estrogen receptor. It is believed to play an important role in hormone-sensitive tissues and in cancer (RefSeq, 2002). GREB1 is overexpressed in prostate cancer and benign prostatic hyperplasia and is involved in androgen-induced growth of prostate cancer cells (Rae et al., 2006). GREB1 also mediates estrogen-stimulated proliferation of ovarian and breast cancer cells (Laviolette et al., 2014).

Integrin alpha 7 (ITGA7) - ITGA7 is the alpha subunit of the alpha-7/beta-1 integrin dimer of the laminin 1 receptor. ITGA7 is a tumor suppressor gene that is critical for suppressing the growth of malignant tumors. Mutation analysis found ITGA7 mutations in prostate cancer, hepatocellular carcinoma, soft tissue leiomyosarcoma, and glioblastoma multiforme. ITGA7 levels were decreased in non-metastatic prostate cancer and leiomyosarcoma (Tan et al., 2013).

Voltage independent potassium channel subfamily G member 1 (KCNG1) - KCNG1 is abundantly expressed in skeletal muscle (Gutman et al., 2005).

KIAA1244 - KIAA1244 encodes member 3 of the ARFGEF family and is located on chromosome 6q23.3.

- 12 041120 (RefSeq, 2002). Overexpression of KIAA1244 has been found in most breast cancers (Nishidate et al., 2004).

A member of the kinesin 7 (KIF7) family, KIF7 has been implicated in several diseases, including Joubert's syndrome, hydrolethal syndrome, and acrocallosal syndrome. It is also involved in the formation of primary cilia (Klejnot and Kozielski, 2012). Aberrant activation of the Hedgehog signaling pathway, in which KIF7 plays an important role, leads to pathological consequences in various human neoplastic diseases, such as basal cell carcinoma of the skin, gastric cancer, and pancreatic cancer (Li et al., 2012; Katoh and Katoh, 2005).

Kallikrein-associated peptidase 4 (KLK4) - KLK4 is one of 15 members of the kallikrein subfamily whose genes are located in a cluster on chromosome 19 (Hu et al., 2000). KLK4 levels are 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).

Lactotransferrin (LTF) - LTF is a multifunctional protein found at highest levels in milk and at lower concentrations in other mucosal fluids. LTF has been shown to induce antibacterial and anti-inflammatory activity, is involved in iron homeostasis, and plays a role in cell growth and differentiation, as well as protection against cancer development and metastasis (Ward et al., 2005). LTF levels are reduced in prostate cancer tissue and serum compared to benign prostatic hyperplasia. In addition, reduced LTF levels correlate with reduced patient survival (Shaheduzzaman et al., 2007). LTF inhibits cell growth by blocking the cell cycle, possibly through several mechanisms, including inhibition of NF-kappaB and Akt signaling pathways (Deng et al., 2013; Ye et al., 2014). In addition, LTF also has pro-aptotic effects, including activation of caspase-3 and JNK signaling pathways (Sakai et al., 2005; Wang et al., 2011).

The leucine zipper domain-containing transcriptional regulator 1 (LZTR1) LZTR1 may play a role in the regulation of the Ras/MAPK signaling pathway (Yamamoto et al., 2015). LZTR1 has frequent mutations or is deleted in glioblastoma (Frattini et al., 2013).

MANSC domain containing protein 1 (MANSC1) - MANSC1 is located on chromosome 12p13.2 (RefSeq, 2002). The level of MANSC1 is significantly reduced in prostate tumors; it may be important for the initiation or progression of prostate carcinoma (Kibel et al., 2004). Increased expression of MANSC1 has been demonstrated in patients with various hematological malignancies (Haferlach et al., 2011).

Microtubule Associated Protein 1B (MAP1 B) - MAP1B is a microtubule stabilizing protein that plays an important role in central nervous system development and axonal function (Halpain and Dehmelt, 2006). Immunohistochemical analysis of small round cell tumors in pediatric patients suggested that MAP1B is expressed in neuroblastoma, rhabdomyosarcoma, and Wilms' tumor, but not in Ewing's sarcoma (Willoughby et al., 2008).

Mitogen-activated protein kinase 4 kinase (MAP4K4) - MAP4K4 is a member of the serine/threonine protein kinase family and mediates the TNF-alpha signaling pathway (RefSeq, 2002). MAP4K4 has been proposed as a biomarker for the aggressiveness of prostate cancer (Rizzardi et al., 2014).

Misshapen-like domain kinase 1 (MINK1) - MINK1 regulates the organization of the cytoskeleton and the oncogene-induced cellular aging process. It is essential for cytokinesis (Hyodo et al., 2012). MINK1 is activated by Ras and mediates p38 activation during growth inhibition and aging (Nicke et al., 2005).

Mohawk homeobox gene (MKX) - MKX is a transcription factor that has been reported to play a role in the development of bone, skeletal muscle, and cartilage structures (Ito et al., 2010b).

Myosin heavy chain 11 smooth muscle (MYH11) - MYH11 is located on chromosome 16q12 and is expressed in the human umbilical artery, bladder, esophagus and trachea (Matsuoka et al., 1993). An inversion at the MYH11 chromosomal locus (inv(16); CBFB-MYH11) is frequently found in acute myeloid leukemia, leading to the formation of an oncogenic chimeric protein of the central binding factor (CBF-beta) and MYH11 (Liu et al., 1993). Low levels of MYH11 expression have been associated with poor prognosis in colorectal cancer (Wang et al., 2014).

Neurofilament, Heavy Polypeptide (NEFH) - Neurofilaments are type IV intermediate filament heteropolymers consisting of light, medium and heavy chains. Neurofilaments include the axoskeleton and functionally support the caliber of neurons. They may also play a role in intracellular transport to axons and dendrites. NEFH encodes a heavy neurofilament protein (RefSeq, 2002). In patients with metastatic renal cell carcinoma, CpG island methylation of the NEFH gene showed a tumor-specific increase, an association with advanced disease and distant metastases, and was significantly associated with poor progression-free survival and reduced overall survival (Dubrowinskaja et al., 2014).

- 13 041120

Olfactory receptor family 51 subfamily E member 2 (OR51E2) - OR51E2 is a member of the G-protein coupled olfactory receptor family that is predominantly expressed in the human prostate and is often overexpressed in prostate cancer (Weng et al., 2005). Peptides derived from OR51E2 can be used as diagnostic markers and also as targets of the immune system in the development of cancer vaccines (Matsueda et al., 2012).

Phosphodiesterase 11A (PDE11 A) - PDEIIA catalyzes the hydrolysis of cAMP and cGMP, thereby suppressing the corresponding signaling pathways (Fawcett et al., 2000). PDE11A mutations are associated with adrenocortical hyperplasia as well as familial testicular germ cell tumor (Greene et al., 2010; Horvath and Stratakis, 2008). In one study, missense mutations were also identified in PDE11A in 30% of patients with prostate cancer. Variants have been characterized as showing reduced PDE11A activity in vitro and their expression levels have been reduced in vivo (Faucz et al., 2011).

Plectin (PLEC) - PLEC encodes for plectin, a member of the plakin family, a protein involved in cross-linking and organizing the cytoskeleton and adhesion complexes (Bouameur et al., 2014). PLEC is overexpressed in colorectal adenocarcinoma, head and neck squamous cell carcinoma, and pancreatic cancer (Lee et al., 2004; Katada et al., 2012; Bausch et al., 2011).

PR domain containing protein 8 (PRDM8) - PRDM8 is a transcriptional repressor with histone methyltransferase activity. PRDM8 is regulated by the Notch-Hes signaling pathway and plays a role in CNS development (Kinameri et al., 2008)

RAB3B, a member of the RAS oncogene family (RAB3B) - RAB3B is a low molecular weight GTP-binding protein that is involved 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).

Solute Transporter Family 39 Member 6 (Zinc Transporter) (SLC39A6) SLC39A6 encodes for the zinc transporter ZIP6, an effector molecule located behind soluble growth factors (Lue et al., 2011). In breast cancer, SLC39A6 overexpression is associated with shorter time to recurrence as well as reduced time to disease-related death (Andres et al., 2013).

Prostate epithelial antigen 2 with six transmembrane domains (STEAP2) - STEAP2 encodes a multi-pass membrane protein that is localized to the Golgi complex, plasma membrane, and tubular-vesicular structures in the cytosol (RefSeq, 2002). The expression level of STEAP2 is increased in patients with advanced prostate cancer. STEAP2 overexpression has been shown to increase cell proliferation as well as the ability to migrate and invade in vitro, while its knockdown inhibited cell growth and possibly promoted apoptosis (Wang et al., 2010; Whiteland et al., 2014) . Along with other members of the STEAP protein family, STEAP2 is also overexpressed in other cancers. In addition, STEAP proteins have been investigated as cancer biomarkers and potential targets for cancer immunotherapy (Grunewald et al., 2012).

Synemine, intermediate filament protein (SYNM) - SYNM binds desmin to the extracellular matrix and plays an important structural role in muscle (Bhosle et al., 2006). SYNM expression is downregulated in breast cancer and hepatocellular carcinoma, which correlates with reduced survival, lymph node metastases, and high grade of malignancy (Noetzel et al., 2010; Liu et al., 2011).

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 the absence of SYNPO2 in prostate cancer correlates with invasiveness and clinical recurrence (Yu et al., 2006).

Transforming growth factor beta 3 (TGFB3) - TGFB3 is one of at least three isoforms of TGF-beta. In normal cells, the TGF-beta signaling arrests the cell cycle in the G1 phase, stopping proliferation, inducing differentiation, or promoting apoptosis (Hanahan and Weinberg, 2000). In NSCLC, TGFB3 is a pro-invasive factor (Petrella et al., 2012).

TRAF2 and NCK interacting kinase (TNIK) - TNIK is a germinal center kinase and is potentially involved in the regulation of the actin cytoskeleton (Fu et al., 1999). TNIK is a necessary, specific activator of Wnt target genes. TNIK has been described as a key element of TRAF6-dependent JNK and NF-kappaB signaling pathways and a transmitter of activation and transformation signals in human cells (Shkoda et al., 2012) In hormone receptor-negative breast cancer, TNIK has been identified as a potential oncogene affecting growth and proliferation (Jiao et al., 2013).

Tryptase alpha/beta 1 (TPSAB1), tryptase beta 2 (TPSB2) - TPSAB1 and TPSB2 are serine proteases that are predominantly expressed by mast cells. The presence of tryptase-positive mast cells in tumor tissue correlates with angiogenesis in several types of tumors, including melanoma, endometrial carcinoma, breast cancer, gastric cancer, and colorectal cancer (Ammendola et al., 2014).

Transient receptor potential cation channel, subfamily M, member 8 (TRPM8) TRPM8 is a sodium and calcium channel activated in response to cold stimuli. TRPM8 is expressed predominantly in prostate epithelial cells and, in addition, also in some sensory neurons (Prevarskaya et al., 2007). TRPM8 has been reported to be overexpressed in prostate cancer and other cancers such as breast, colon, lung and skin tumors (Tsavaler et al., 2001).

Thioredoxin domain containing protein 16 (TXNDC16) - TXNDC16 encodes an 858 amino acid protein with a putative thioredoxin domain 2; its function is unknown. Autoantibodies to TXNDC16 have been identified in meningioma patients (Comtesse et al., 2005).

Unc-13 homologue B (UNC13B) - UNC13B is a component of a protein complex in the presynaptic active zone that controls the release of neurotransmitters via exocytosis of synaptic vesicles (Sudhof, 2012).

Protein 426 with a Zinc finger 426 domain (ZNF426) - ZNF426 is a transcriptional regulatory protein (Yang and Wood, 2007).

Detailed description of the invention

Stimulation of immune responses depends on the presence of antigens recognized by the host's immune system as foreign. The discovery of the existence of tumor-associated antigens has raised the possibility of using the host's immune system to interfere with tumor growth. Various mechanisms for controlling both branches of the immune system, both humoral and cellular, are currently being investigated for cancer immunotherapy.

Specific elements of cellular immune responses are capable of specific recognition and destruction of tumor cells. The isolation of T cells from populations of tumor-infiltrating cells or from peripheral blood suggests that such cells play an important role in the natural immune defense against cancer. In particular, CD8-positive T cells that recognize peptides associated with class I molecules of the major histocompatibility complex (MHC) play an important role in this response. These peptides usually consist of 8-10 amino acid residues derived from proteins or defective ribosomal products (DRIPs) found in the cytosol. Human MHC molecules are also referred to as human leukocyte antigens (HLA).

All terms used here, unless otherwise indicated, have the meanings given below.

The term T-cell response refers to the specific proliferation and activation of effector functions induced by a peptide in vitro or in vivo. For class I MHC restricted cytotoxic T cells, the effector functions may be lysis of target cells loaded with peptide, loaded with peptide precursor, or target cells naturally presenting the peptide; secretion of cytokines, preferably interferon-gamma,

TNF-alpha or IL-2 induced by the peptide; peptide-induced secretion of effector molecules, preferably granzymes or perforins, or degranulation.

The concept of a peptide in the context of the present description means a series of amino acid residues connected to each other usually by peptide bonds between alpha-amine and carbonyl groups of adjacent amino acids. The peptides are preferably 9 amino acids in length, but may be shorter - 8 amino acids in length and longer - 10, 11, 12 or 13 or longer, and in the case of peptides associated with MHC class II molecules (long versions of the peptides according to the invention), they may be 14, 15, 16, 17, 18, 19, or 20 or more amino acids in length.

In addition, the term peptide includes salts of a series of amino acid residues linked to each other, usually by peptide bonds between the alpha-amine and carbonyl groups of adjacent amino acids. Preferably, the salts are pharmaceutically acceptable salts of the peptides, such as, for example, chloride or acetate (trifluoroacetate). It has been observed that the salts of the peptides of the present invention differ substantially from the peptides in their in vivo state(s), since the peptides are not salts in vivo.

The term peptide also includes the term oligopeptide. The term oligopeptide in the context of the present description means a series of amino acid residues linked to each other, usually by peptide bonds between alpha-amine and carbonyl groups of adjacent amino acids. The length of the oligopeptide is not particularly important to the invention as long as it retains the proper epitope or epitopes. Oligopeptides are typically less than about 30 amino acid residues in length and greater than about 15 amino acids in length.

The term polypeptide denotes a series of amino acid residues connected to each other by typically peptide bonds between the alpha-amine and carbonyl groups of adjacent amino acids. The length of the polypeptide is not particularly important to the invention as long as the proper epitopes are maintained. In contrast to the terms peptide or oligopeptide, the term polypeptide is introduced to refer to molecules containing more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide encoding such a molecule is immunogenic (and thus an immunogen within the scope of 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 will be a molecule that is capable of inducing an immune response and, in the case of the present invention, a molecule that is capable of inducing a T cell response. In another aspect, the immunogen may be a peptide, a complex of a peptide and an MHC, an oligopeptide and/or a protein used to generate specific antibodies or TCRs against it.

A class I T-cell epitope requires a short peptide that binds to a class I MHC receptor forming a three-membered complex (MHC class I alpha chain, beta-2 microglobulin and peptide) that can be recognized by a T cell bearing a suitable T -cell receptor that binds to the MHC/peptide complex with suitable affinity. Peptides that bind to MHC class I molecules are typically 8-14 amino acids in length, and most typically 9 amino acids in length.

There are three distinct genetic loci in humans that encode class I MHC molecules (human MHC molecules are also called human leukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*O1, HLA-A*02 and HLA-A*07 are examples of the various class I MHC alleles that can be expressed from these loci.

In table. 8 shows the F expression frequencies of HLA-A*02 and HLA-A*24 and the most common HL-DR serotypes. Expression frequencies are derived from the American Gf haplotype frequencies reported by Mori et al. (Mori et al., 1997), using the Hardy-Weinberg formula F=1-(1-Gf) ² . Combinations of A*02 or A*24 with certain HLA-DR alleles, due to uneven distribution of associations, may be enriched or less frequent than expected from their individual detection frequencies. For more information, see Chanock et al. (Chanock et al., 2004).

Table 8

allele population Calculated phenotype by allele frequency A*02 Caucasian race (North America) 49.1% A*02 African Americans (North America) 34.1% A*02 Mongoloids (North America) 43.2% A*02 Hispanics (North America) 48.3% DR1 Caucasian race (North America) 19.4% DR2 Caucasian race (North America) 28.2% DR3 Caucasian race (North America) 20.6% DR4 Caucasian race (North America) 30.7% DR5 Caucasian race (North America) 23.3% DR6 Caucasian race (North America) 26.7% DR7 Caucasian race (North America) 24.8% DR8 Caucasian race (North America) 5.7% DR9 Caucasian race (North America) 2.1% DR1 African Americans (North America) 13.20% DR2 African Americans (North America) 29.80% DR3 African Americans (North America) 24.80% DR4 African Americans (North America) 11.10% DR5 African Americans (North America) 31.10% DR6 African Americans (North America) 33.70% DR7 African Americans (North America) 19.20% DR8 African Americans (North America) 12.10% DR9 African Americans (North America) 5.80% DR1 Mongoloids (North America) 6.80% DR2 Mongoloids (North America) 33.80% DR3 Mongoloids (North America) 9.20% DR4 Mongoloids (North America) 28.60% DR5 Mongoloids (North America) 30.00% DR6 Mongoloids (North America) 25.10% DR7 Mongoloids (North America) 13.40% DR8 Mongoloids (North America) 12.70% DR9 Mongoloids (North America) 18.60% DR1 Hispanics (North America) 15.30% DR2 Hispanics (North America) 21.20% DR3 Hispanics (North America) 15.20% DR4 Hispanics (North America) 36.80% DR5 Hispanics (North America) 20.00% DR6 Hispanics (North America) 31.10% DR7 Hispanics (North America) 20.20% DR8 Hispanics (North America) 18.60% DR9 Hispanics (North America) 2.10%

- 16 041120

A*24 Philippines 65% A*24 Russian 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 Western Australia 22% A*24 USA 22% A*24 Russia, Samara 20% A*24 South America 20% A*24 Europe 18%

The peptides of the invention, preferably when formulated into a vaccine of the invention as described herein, bind to HLAA*02 or HLA-A*24. The vaccine may also include universal peptides that bind to MHC class II. Therefore, the vaccine according to the invention can be used to treat cancer in patients who are A*02-positive, and due to the universal binding nature of these peptides, selection of MHC class II allotypes is not necessary.

When the A*02 peptides of the invention are combined with peptides that bind to a different allele, such as A*24, a higher percentage of any patient population can be treated compared to vaccination for each MHC class I allele alone. While in most populations less than 50% of patients may be covered by any one allele, at least 60% of patients in any relevant population can be treated with a vaccine comprising HLA-A*24 and HLA-A*02 epitopes. Specifically, the following percentages of patients will be positive for at least one of these alleles in different regions: USA 61%, Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculated from www.allelefrequencies.net).

In a preferred embodiment, the term nucleotide sequence refers to a heteropolymer of deoxyribonucleotides.

The nucleotide sequence encoding a particular peptide, oligopeptide or polypeptide may be naturally occurring or may be synthesized. In general, the DNA segments encoding the peptides, polypeptides and proteins of the present invention are assembled from cDNA fragments and short oligonucleotide linkers or from a series of oligonucleotides to obtain a synthetic gene that is capable of being expressed in a recombinant transcription unit comprising regulatory elements derived from a microbial or viral operon.

In the context of the present description, the term nucleotide encoding a peptide refers to a nucleotide sequence encoding a peptide, including artificial (human-made) start stop codons, compatible with the biological system by which the sequence is to be expressed, for example, a dendritic cell or other cellular system suitable for producing TCR. .

As used herein, reference to a nucleic acid sequence includes both single-stranded and double-stranded nucleic acids. Thus, for example, for DNA, a specific sequence, unless otherwise indicated in the context, refers to the single-stranded DNA of such a sequence, the duplex of such a sequence with its complement (double-stranded DNA), and the complement of such a sequence.

The concept of a coding region refers to that region of a gene that, under natural or normal conditions, encodes the expression product of that gene in its natural genomic environment, i.e. the region encoding in vivo the native expression product of the gene.

The coding region may be derived from a non-mutated (normal), mutated or altered gene, or may even be derived from a DNA sequence or an entire gene synthesized in the laboratory using techniques well known to those skilled in the art of DNA synthesis.

The term expression product means a polypeptide or protein that is the natural translation product of a gene and any nucleic acid sequence that encodes equivalents resulting from the degeneration of the genetic code and thus encodes the same amino acid(s).

The term fragment, when referring to a coding sequence, means a stretch of DNA comprising less than the entire coding region, the expression product of which essentially retains the same biological function or activity as the expression product of the entire coding region.

The term DNA segment refers to a DNA polymer as a single fragment or as a component of a larger DNA construct that has been formed from DNA isolated at least once in substantially pure form, i.e. free of contaminating endogenous materials and in an amount or concentration that allows identification, manipulation and recovery of the segment and

- 17 041120 of its constituent nucleotide sequences by standard biochemical methods, for example using a cloning vector. Such fragments are provided in the form of an open reading frame that is not interrupted by internal untranslated sequences or introns that are commonly found in eukaryotic genes. Untranslated DNA sequences may be present outside the open reading frame where it does not interfere with the manipulation or expression of the coding regions.

The term primer means a short nucleic acid sequence that can be paired with a single strand of DNA to form a free 3'OH end at which DNA polymerase begins to synthesize a deoxyribonucleotide chain.

The concept of a promoter means a DNA region involved in the binding of RNA polymerase to initiate transcription.

The concept of isolated means that the material is removed from its original environment (for example, the natural environment, if it occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in living organisms is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials of the natural system is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition and yet could be isolated such that such a vector or composition is not part of its natural environment.

The polynucleotides and recombinant or immunogenic polypeptides disclosed in accordance with the present invention may also be in purified form. Purified does not require absolute purity; rather, it is intended to provide a relative definition and may include highly purified preparations or only partially purified preparations, as those terms are understood by those skilled in the art. For example, individual clones isolated from a cDNA library were purified to electrophoretic homogeneity as usual. Purification of the source material or natural material from impurities at least one order of magnitude, preferably two or three orders of magnitude, and more preferably four or five orders of magnitude is specifically considered in the invention. Moreover, the claimed polypeptide is specifically included, the purity of which is preferably 99.999%, or at least 99.99%, or 99.9%, and even desirably 99% by weight or more.

Nucleic acids and polypeptides as expression products disclosed in accordance with the present invention, as well as expression vectors containing such nucleic acids and/or such polypeptides, may be in enriched form. As used herein, enriched means that the concentration of a material is at least about 2, 5, 10, 100, or 1000 times its natural concentration (for example), preferably 0.01% by weight, preferably at least about 0.1% by weight. mass. Also contemplated are fortified formulations at about 0.5%, 1%, 5%, 10%, and 20% by weight. The sequences, constructs, vectors, clones, and other materials included in the present invention may preferably be in enriched or isolated form. The term active fragment means a fragment - usually of a peptide, polypeptide or nucleic acid sequence - that elicits 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, such as a rabbit or mice, also including humans; wherein such an immune response takes the form of stimulation of a T-cell response in the recipient animal, such as a human. Alternatively, the active fragment can also be used to initiate a T cell response in vitro.

In the context of the present description, the terms plot, segment and fragment, when used in relation to polypeptides, refer to a continuous sequence of residues, such as amino acid residues, the sequence of which forms a subclass of a larger sequence. For example, if the polypeptide has been treated with any of the known endopeptidases such as trypsin or chymotrypsin, the resulting oligopeptides will represent regions, segments, or fragments of the parent polypeptide. When used in relation to polynucleotides, these terms refer to products obtained by processing said polynucleotides with any of the endonucleases.

In accordance with the present invention, the concept of percentage identity or identical to percentage, if it refers to a sequence, means that the sequence is compared with the claimed or described sequence after aligning the compared sequence (Comparison Sequence) with the described or claimed sequence (Control Sequence). The percent identity is then determined by the following formula: percent identity = 100[1 - (C/R)], where C is the number of differences between the Control sequence and the Comparator sequence in the alignment length between the Control sequence and the Compare sequence, where:

(i) each base or amino acid in the Control Sequence that does not have a corresponding aligned base or amino acid in the Compare Sequence; And

- 18 041120 (ii) each gap in the Control Sequence; and (iii) each aligned base or amino acid in the Control Sequence that differs from the aligned base or amino acid in the Comparable Sequence represents a difference; and (iv) the alignment must start at position 1 of the aligned sequences;

R is the number of bases or amino acids in the Control Sequence along the length of the alignment with the Comparing Sequence, with any gap formed in the Control Sequence also counting as a base or amino acid.

If there is a juxtaposition between a Comparison Sequence and a Control Sequence for which the percent identity calculated above is approximately equal to or greater than the specified minimum Percent Identity, then the Comparison Sequence has a specified minimum percentage identity with the Control Sequence, even if alignments may exist, in which the identity percentage calculated here above is less than the identity percentage set.

As mentioned above, the present invention thus provides a peptide comprising a sequence that is selected from the group consisting of sequences from SEQ ID NO: 1 to SEQ ID NO: 48, or a variant thereof, which is 88% homologous to sequences from SEQ ID NO: 1 according to SEQ ID NO: 48 or their variant, which induces a cross-reaction of T cells with the specified peptide. The peptides of the invention have the ability to bind to a class I human major histocompatibility complex (MHC) molecule or, longer versions of said peptides, to a class II MHC.

In the present invention, the term homologous refers to the degree of identity (see above, Percent Identity) between the sequences of two amino acid sequences, i.e. peptide or polypeptide sequences. The previously mentioned homology is determined by comparing two sequences matched under optimal conditions for the compared sequences. Such sequence homology can be calculated by creating an alignment, such as the ClustalW algorithm. Sequence analysis software is widely available, such as Vector NTI, GENETYX, or other tools provided by open source databanks.

One skilled in the art will be able to assess whether T cells induced by a variant of a particular peptide will be capable of cross-reacting with the peptide itself (Arrau et al., 2006; Colombetti et al., 2006; Fong et al., 2001; Zaremba et al., 1997).

By a variant of a given amino acid sequence, the inventors mean that the side chains of, for example, one or two amino acid residues are changed (for example, by replacing them with a side chain of a residue of another naturally occurring amino acid or some other side chain) so that the peptide is still capable of binding to the HLA molecule in essentially the same way as the peptide consisting of the given amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 48. For example, the peptide can be modified such that it at least retains , if not improved, the ability to interact with and bind to the binding groove of an appropriate MHC molecule, such as HLA-A*02 or -DR, and thus it would at least retain, if not improve, the ability to bind to TCR of activated T cells .

These T cells can then cross-react with and kill cells that express a polypeptide that contains a naturally occurring amino acid sequence of a related peptide, as defined in aspects of this invention. According to scientific literature and databanks (Rammensee et al., 1999; Godkin et al., 1997), specific positions of HLA-binding peptides are typical anchor residues forming a central sequence suitable for the HLA receptor connector, which is determined by polar, electrophysical, hydrophobic and spatial properties of polypeptide chains forming a connecting groove. Thus, a person skilled in the art will be able to modify the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 48 while retaining known anchor residues and will be able to determine whether such variants retain the ability to bind to MHC class I or II molecules. The variants of the present invention retain the ability to bind to TCR activated T cells, which may subsequently cross-react and kill cells expressing a polypeptide that contains the naturally occurring amino acid sequence of a related peptide as defined in aspects of the present invention.

The original (unmodified) peptides disclosed in this description, can be modified by replacing one or more residues in various, possibly selected, areas along the length of the peptide chain, unless otherwise stated. Preferably, such substitutions are located at the end of the amino acid chain. Such substitutions may be conservative, for example, when one amino acid is replaced by an amino acid with similar structure and characteristics, as well as when a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative would be to replace amino acids of the same or similar size and chemical nature, such as

- 19 041120 when replacing leucine with isoleucine. In studies of sequence variation within families of naturally occurring homologous proteins, certain amino acid substitutions are allowed more often than others, and they are often associated with similarities in size, charge, polarity, and hydrophobicity between the parent amino acid and its substitution; and such is the basis for defining conservative substitutions.

Conservative substitutions are defined in the context of this specification as exchanges within one of the following five groups:

group 1 - small, aliphatic, non-polar or weakly 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, non-polar residues (Met, Leu, Ile, Val, Cys);

group 5 - large, aromatic residues (Phe, Tyr, Trp).

Less conservative substitutions may involve replacing one amino acid with another having similar characteristics but differing to some extent in size, as in the case of replacing an alanine with an isoleucine residue. Highly non-conservative substitutions may include the substitution of an acidic amino acid for a polar or even one that is basic. Such radical substitutions cannot, however, be dismissed as potentially ineffective due to the fact that chemical effects are not entirely predictable, and radical substitutions may unexpectedly lead to beneficial effects not predictable from conventional chemical principles.

Of course, structures other than conventional L-amino acids may be involved in such substitutions. Thus, D-amino acids may be replaced by L-amino acids commonly found in the antigenic peptides of the invention and also covered by this disclosure. In addition, non-standard amino acids (ie, other than the ubiquitous proteinogenic amino acids) can also be used as substitutions to produce immunogens and immunogenic polypeptides in accordance with the present invention.

If substitutions have been made at more than one position to produce a peptide with substantially equivalent or greater antigenic activity, as defined below, then combinations of such substitutions will be analyzed to determine if these combinations of substitutions will result in additional or synergistic effects with respect to the antigenicity of the peptide. . For the most part, no more than 4 positions within a peptide should be substituted at the same time.

A peptide consisting essentially of an amino acid sequence as defined herein may have one or two non-anchor amino acid substitutions (see below for anchor motif) such that the ability to bind to a class I or II human major histocompatibility complex (MHC) molecule is not will be significantly altered or adversely affected compared to the unmodified peptide. In another embodiment, in a peptide consisting essentially of an amino acid sequence as defined herein, one or two amino acids may be replaced with conservative substitution partners (see information below) such that the ability to bind to a human major histocompatibility complex molecule (MHC) class I or II will not be significantly altered or adversely affected compared to the unmodified peptide.

Amino acid residues that do not significantly contribute to interaction with the T cell receptor can be modified by substitution for another amino acid, the inclusion of which does not significantly affect the reactivity of the T cell and does not eliminate binding to the corresponding MHC. Thus, in addition to this condition, the peptide of the invention may be any peptide (by which term the inventors include oligopeptides or polypeptides) that includes amino acid sequences or a portion or variant thereof, as given.

Table 9 Variants and motif of HLA-A*O2 binding peptides according to SEQ ID NOs: 2, 4 and 8

- 20 041120

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 SEQID NO. 4 A L L T F V W TO L Options 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 SEQID No. 8 L L D F S L A D A Options 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 10

Variants and motif of HLA-A*24 binding peptides _____ according to SEQ ID NOs: 25, 30 and 34_____

Position 1 2 3 4 5 6 7 8 9 10 eleven SEQ ID 25 I Y E R Y L A M F Option I L F F I F L Position 1 2 3 4 5 6 7 8 9 10 eleven SEQ ID 30 S Y S R A H A R L Option I F F F I F F Position 1 2 3 4 5 6 7 8 9 10 eleven SEQID 34 A F S R D S H Y L L F Option Y I Y L Y I L

- 21 041120

Longer (elongated) peptides may also be suitable. It is possible that the epitopes that bind to MHC class I molecules, although they are usually between 8 and 11 amino acids in length, were obtained by processing peptides from longer peptides or proteins containing the true epitope. Preferably, residues that are adjacent to the true epitope do not significantly interfere with the proteolytic cleavage required to present the true epitope during processing.

The peptides of the invention can be extended with up to four amino acids, meaning that 1, 2, 3 or 4 amino acids can be added to one of the ends in any combination between 4:0 and 0:4. Combinations of elongations in accordance with the invention can be taken from the table. eleven.

Table 11

Peptide Elongation Combinations 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

Amino acids for elongation/extension can be peptides of the original protein sequence or any(s) other(s) amino acid(s). Elongation can be used to increase the stability or solubility of peptides.

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 control peptide, provided they have substantially identical antigenic activity.

In an alternative embodiment, the peptide is extended on one side or the other, or on both sides at the same time by adding more than 4 amino acids, preferably to a total length of up to 30 amino acids. This can lead to the formation of class II MHC-binding peptides. Binding to MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptide epitopes and peptide variant epitopes that bind to MHC class I molecules, wherein said peptide or variant has an overall length between 8 and 100, preferably between 8 and 30, and most preferably between 8 and 14, namely 8 , 9, 10, 11, 12, 13, 14 amino acids, in the case of extended peptides binding to class II MHC molecules, the length may also be 15, 16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, a peptide or variant of the present invention will have the ability to bind to a class I or II human major histocompatibility complex (MHC) molecule. Binding of a peptide or variant to the MHC complex can be tested by methods known in the art.

Preferably, T cells specific for a peptide of the present invention are tested against substituted peptides; the peptide concentration at which the substituted peptides achieve half the maximum lysis growth relative to background is not more than about 1 mM, preferably not more than about 1 μM, more preferably not more than about 1 nM, even more preferably not 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 of more than one individual, at least two and more preferably three individuals.

In a particularly preferred embodiment, the peptide consists or essentially consists of the amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 48.

Consists essentially of means that the peptide in accordance with the present invention, in addition to any of the sequences from SEQ ID NO: 1 to SEQ ID NO: 48, or a variant thereof, contains additional amino acid sequence fragments located at the N- and/or C-terminus, which are not necessarily forming part of a peptide that functions as an epitope for MHC molecules.

However, these fragments may be important to ensure the effective introduction of the peptide in accordance with the present invention into cells. In one embodiment of the present invention, the peptide is part of a fusion protein, which includes, for example, 80 N-terminal amino acids of the antigen-associated invariant chain (p33, hereinafter li) HLA-DR, as taken from the NCBI databank, accession number - GenBank Accession -number X00497. In other fusions, the peptides of the present invention may be fused to an antibody as described herein, or a functional portion thereof, such as inserted into an antibody sequence so as to be a specific target of said antibody, or, for example, fused to or embedded in an antibody that is specific for the dendritic cells described herein.

In addition, the peptide or variant may be further modified to improve stability and/or binding to MHC molecules in order to obtain a stronger immune response. Techniques for such optimization of the peptide sequence are well known in the art and include, for example, the introduction of reversed peptide or non-peptide bonds.

In a reversed peptide bond, amino acid residues are not attached by peptide bonds (-CO-NH-), and the peptide bond is reversed. Such retro-reverse peptidomimetics can be prepared by methods known in the art, such as those described by Meziere et al. (1997) (Meziere et al., 1997), incorporated herein by reference. This approach encompasses the production of pseudopeptides that contain changes that span the backbone but not the orientation of the side chains. Meziere et al. (Meziere et al., 1997) show that these pseudopeptides are useful for binding to the MHC and inducing T helper cell responses. Retroreverse peptides that contain NH-CO bonds instead of CO-NH peptide bonds are much more resistant to proteolysis.

A non-peptide bond is, for example, -CH2-NH, -CH2S-, -CH2CH2-, -CH=CH-, -COCH2-, -CH(OH)CH ₂ - and -CH2SO-. US Pat. No. 4,897,445 proposes a method for the solid phase synthesis of non-peptide bonds (-CH ₂ -NH) in polypeptide chains, which includes polypeptides synthesized using standard techniques and a non-peptide bond synthesized by the reaction of an aminoaldehyde and an amino acid in the presence of NaCNBH ₃ .

Peptides comprising the sequences described above can be synthesized with additional chemical groups present at their amino and/or carboxyl termini to increase the stability, bioavailability and/or affinity of the peptides. For example, hydrophobic groups such as carbobenzoxyl, dansyl, or tert-butyloxycarbonyl groups can be added to the amino termini of the peptides. Similarly, an acetyl group or a 9-fluorenylmethoxycarbonyl group can be placed at the amino termini of the peptides. In addition, a hydrophobic group, t-butyloxycarbonyl or amide group can be added to the carboxyl ends of the peptides.

In addition, all peptides of the invention can be synthesized in order to change their spatial configuration. For example, the D-isomer of one or more amino acid residues of the peptide may be used, rather than the usual L-isomer. Moreover, at least one of the amino acid residues of the peptides of the invention may be substituted with one of the well-known non-naturally occurring amino acid residues. Alterations such as data may serve to improve the stability, bioavailability and/or binding properties of the peptides of the invention.

Likewise, a peptide or variant of the invention may be chemically modified by reaction of individual amino acids, either before or after the synthesis of the peptide. Examples of such modifications are well known in the art and are summarized in, for example, 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 amine groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS), amide modification of carboxyl groups, and sulfhydryl modification by oxidation of cysteine with performic acid. to cysteic acid, formation of mercury derivatives, formation of mixed disulfides with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide, and carbamoylation with cyanate at alkaline pH, although not limited to them. In this regard, one of skill in the art may consult Chapter 15 of Current Protocols In Protein Science, Eds. Hassan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995) for more information on techniques related to the chemical modification of proteins.

Briefly, the modification of, for example, 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.

Selective reduction of disulfide bonds in proteins is also common. Disulfide bonds can be formed and oxidized during heat treatment of biopharmaceuticals. Woodward's K reagent can be used to modify certain glutamic acid residues. N-(3-(Dimethylaminopropyl)-N'-ethylcarbodiimide can be used to form intramolecular cross-links between the lysine residue and the glutamic acid residue.

- 23 041120

For example, diethyl pyrocarbonate is a reagent for modifying histidyl residues in proteins. Histidine can also be modified using 4-hydroxy-2-nonenal. The reaction of lysine residues and other α-amine groups is useful, for example, in binding peptides to surfaces or cross-linking proteins/peptides. Lysine is the site of polyethylene glycol attachment and the main site of modification in protein glycosylation. Methionine residues in proteins can be modified, for example, with iodoacetamide, bromoethylamine, and chloramine T.

Tetranitromethane and N-acetylimidazole can be used to modify tyrosyl residues. Cross-linking through the formation of dityrosine can be done with hydrogen peroxide/copper ions.

Recent studies on tryptophan modification have used N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNSskatol).

Successful modification of therapeutic proteins and peptides with PEG (polyethylene glycol) is often associated with an increase in the circulation half-life, while cross-linking of proteins with glutaraldehyde, polyethylene glycol diacrylate and formaldehyde is used to obtain hydrogels. Chemical modification of allergens for immunotherapy is often achieved by carbamoylation with potassium cyanate.

A peptide or variant in which the peptide is modified or includes non-peptide bonds is a preferred embodiment of the invention. Typically, peptides and variants (at least those containing peptide bonds between amino acid residues) can be synthesized by the Fmoc-polyamide solid phase peptide synthesis method as disclosed by Lukas et al. (Lukas et al., 1981) and in the accompanying references. The temporary protection of the N-amino group is provided by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Re-cleavage of this highly alkaline protecting group is carried out using 20% piperidine in N,N-dimethylformamide. The functional groups of the side chain can be protected by obtaining compounds such as their butyl esters (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 a 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivative (in the case of arginine). If glutamine or asparagine are C-terminal residues, a 4,4'-dimethoxybenzhydryl group is used to protect the side chain amido group. The solid phase carrier is based on a polydimethylacrylamide polymer consisting of three monomers: dimethylacrylamide (framework monomer), bis-acryloylethylenediamine (crosslinker, linker) and acryloylsarcosine methyl ester (functionalizing agent). To form an easily cleavable bond between the peptide and the resin, an acid-resistant derivative of 4-hydroxymethylphenoxyacetic acid is used. All amino acid derivatives are added as pre-synthesized symmetric anhydride derivatives, except for asparagine and glutamine, which are added using N,N-dicyclohexylcarbodiimide/1hydroxybenzotriazole mediated reverse compound reaction. All coupling and deprotection reactions were monitored using ninhydrin, trinitrobenzenesulfonic acid or isotin controls. After completion of the synthesis, the peptides are cleaved from the carrier resin with concomitant side chain deprotection by treatment with 95% trifluoroacetic acid containing 50% scavenger mixture. Commonly used scavengers include ethanedithiol, phenol, anisole and water, the final choice being dependent on the amino acid constituents of the peptide being synthesized. A combination of solid phase and liquid phase methods for peptide synthesis is also possible (see, for example, (Bruckdorfer et al., 2004) and the accompanying references).

The trifluoroacetic acid is removed by vacuum evaporation followed by trituration with diethyl ether to give the crude peptide. Any scavengers present are removed by a simple extraction technique which allows the crude peptide without scavengers to be obtained after lyophilization of the aqueous phase. Reagents for synthesis of peptides are generally available from, for example, Calbiochem-Novabiochem (Nottingham, UK).

Purification may be by any technique or combination of techniques such as recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and (typically) reverse phase high performance liquid chromatography using, for example, acetonitrile/water gradient separation.

Analysis of peptides can be performed using thin layer chromatography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (SPE), reverse phase high performance liquid chromatography, amino acid analysis after acid hydrolysis and mass spectrometric analysis by fast atom bombardment (FAB), as well as mass spectrometric analysis of MALDI and ESI-Q-TOF.

In order to select over-presented peptides, a presentation profile was calculated to estimate the median value of sample presentation as well as repeated measures variation. IN

- 24 041120 profile compares samples of the tumor form of interest with the background level of normal tissue samples. Each of these profiles can then be consolidated into an overrepresentation measure by calculating a p-value from a linear mixed effects model (Pinheiro et al., 2015), adjusting it for repeat analyzes for the false positive rate (Benjamini and Hochberg, 1995) (cp. example 1).

To identify and relative quantify HLA ligands by mass spectrometric analysis, HLA molecules from blast-frozen tissue samples were purified and HLA-associated peptides isolated from them. Because peptides derived from prostate-specific antigens can be identified in any prostate tissue, whether benign or malignant, in order to identify peptides encoded by prostate-specific antigens, in addition to prostate cancer tissue preparations samples of benign prostatic hyperplasia were also analyzed. The isolated peptides were separated and the sequences were identified using liquid chromatography and mass spectrometry (LC-MS) with real-time electrospray ionization (nanoESI) methods. The resulting peptide sequences were confirmed by comparing the pattern of fragmentation of natural TUMAP peptides recorded on prostate cancer samples (N=34 A*02-positive samples and N=37 A*24-positive samples), as well as additional samples of benign prostatic hyperplasia (N= 10 A*02 positive samples and N=3 A*24 positive samples) with fragmentation patterns of the corresponding synthetic control peptides with identical sequences. Since the peptides were identified directly as ligands of tumor tissue HLA molecules, these results provide direct evidence for the natural processing and presentation of the identified peptides on tumor tissue obtained from 70 patients with prostate tumors.

The drug-in-development technology platform XPRESIDENT® v2.1 (see, for example, US Patent Application 2013-0096016, incorporated herein in its entirety by reference) allows the identification and selection of appropriate over-presented peptides as candidates for vaccines based on direct relative quantification of HLA-restricted peptide levels in cancer tissue compared to several different non-cancerous tissues and organs. This was accomplished by developing differential quantitation based on label-free LC/MS data processed by a proprietary data analysis technology platform that integrates algorithms for sequence identification, spectral clustering, ion counting, retention time alignment, deconvolution by states of charge and normalization.

Presentation levels were calculated for each peptide and sample, including error estimates. Peptides that are exclusively presented on tumor tissue and peptides that are over-presented on tumor tissues compared to non-cancerous tissues and organs have been identified.

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 this patent document have been identified using this approach in prostate tumor samples, which confirms their presentation in prostate tumors.

TUMAP peptides identified on numerous tissues of prostate cancer, prostatic hyperplasia and normal tissues were quantified by unlabelled LC/MS using ion counting. The method is based on the assumption that the peak areas of the peptide when analyzed by LC/MS correlate with its content in the sample. All quantitative peptide signals in the various LC/MS experiments were normalized based on the underlying trend, their average value per sample was calculated, and histogrammed into the so-called presentation profile. The presentation profile consolidates various analysis methods such as protein databank searches, spectral clustering, state-of-charge (discharge) deconvolution, and retention time equalization and normalization.

In addition, the XPRESIDENT® v2.x technology platform allows direct absolute quantitation of MHC-, preferably HLA-restricted peptide, levels on cancerous or other infected tissues. Briefly, the total number of cells was calculated from the total DNA content of the analyzed tissue sample. The total amount of TUMAP peptide in the tissue sample was measured using nano-LC-MS/MS as the ratio of natural TUMAP peptide and a known amount of the isotopically labeled version of the TUMAP peptide, the so-called internal standard. TUMAP peptide isolation efficiency was determined by introducing a standard addition of the peptide-MHC complex of all selected TUMAP peptides into the tissue lysate at the earliest possible time in the TUMAP peptide isolation process and detecting them using nano-LC-MS/MS, followed by

- 25 041120 followed by the completion of the peptide isolation procedure. The total number of cells and the total amount of peptide were calculated from three replicate measurements per tissue sample. Peptide-specific recovery efficiency was calculated as the average of ten experiments with the introduction of standard additives with three repeated measurements for each (see example 6).

The present invention provides peptides that are useful in the treatment of cancers/tumors, preferably prostate cancer, whose cells present in excess or exclusively the peptides of the invention. As shown by mass spectrometric analysis, these peptides were naturally presented by HLA molecules on samples of human prostate tumors.

Many of the original genes/proteins (also referred to as full-length proteins or basic proteins) from which the peptides were derived were highly overexpressed in the tumor compared to normal tissues - the term normal tissues in connection with the present invention refers to cells of normal non-prostatic tissue showing a high degree of association of the original genes with the tumor (see example 2). Moreover, the peptides themselves are highly over-presented on tumor tissue - the term tumor tissue in connection with the present invention refers to a sample of tissue from a patient suffering from prostate cancer, but not on normal tissues (see Example 1).

HLA-associated peptides can be recognized by the immune system, specifically T-lymphocytes. T cells can destroy cells presenting a recognized HLA/peptide complex; for example, tumor cells of the prostate presenting the obtained peptides.

The peptides of the present invention have been shown to be capable of stimulating T cell responses and/or are over-presented and can therefore be used to generate antibodies and/or TCRs, such as soluble TCRs, in accordance with the present invention (see Examples 3, 4). In addition, peptides, when complexed with an appropriate MHC molecule, can be used to generate antibodies and/or TCRs, in particular soluble TCRs, in accordance with the present invention. Appropriate methods are well known to the person skilled in the art and can also be found in the relevant literature. Thus, the peptides of the present invention are suitable for generating an immune response in a patient to kill tumor cells. An immune response in a patient can be induced by direct administration of the described peptides or suitable precursor substances (e.g., extended peptides, proteins, or nucleic acids encoding these peptides) to the patient, ideally in combination with an immunogenicity-enhancing substance (i.e. adjuvant). The immune response elicited by such therapeutic vaccination can be expected to be highly specific against tumor cells, since the target peptides of the present invention are not presented on normal non-prostatic tissues in comparable copy numbers, thus preventing the risk of unwanted autoimmune reactions against normal cells. at the patient. Prostate-specific antigens may be a good choice for prostate cancer immunotherapy because prostate-specific antigens are tumor-specific targets in the post-prostatectomy patient. In prostate cancer patients who have not undergone a prostatectomy, such antigens may also be of interest because the prostate is not considered a vital organ.

The present description further relates to T-cell receptors (TCR), including the alpha chain and the beta chain (alpha/beta TCR). Also provided are peptides of the invention capable of binding to TCR and antibodies when presented by an MHC molecule. The present description also relates to nucleic acids, vectors and host cells for the expression of TCR and peptides of the present invention and methods of their use.

The term T cell receptor 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 the HLA molecule. This concept also includes the so-called gamma/delta TCR.

In one embodiment, a method for producing TCR according to the present disclosure is provided, the method comprising cultivating a host cell capable of expressing TCR under conditions suitable for promoting TCR expression.

The present disclosure in another aspect further relates to methods according to the present disclosure, wherein the antigen is loaded onto MHC class I or II 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 the antigen-presenting cell, or the antigen is loaded to MHC tetramers of class I or II by tetramerization of antigen-monomer complexes of MHC class I or II.

The alpha and beta chains of alpha/beta TCR and the gamma and delta chains of gamma/delta TCR are generally considered to have two domains each, namely variable and constant domains. The variable domain consists of a variable segment (V) and a junction segment (J) in series. The variable domain may also include a leader segment (L). Beta and delta chains may also include diversity segments (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.

With respect to gamma/delta TCR, the term TCR gamma variable domain used in the context of this invention refers to the junction of the TCR gamma V (TRGV) segment without a leader segment (L) and the TCR gamma J (TRGJ) segment, and the concept of the TCR constant domain gamma refers to the extracellular segment of the TRGC or the C-terminal truncated TRGC sequence. Equally, TCR delta variable domain refers to the junction of a TCR delta V (TRDV) segment without a leader segment (L) and a TCR delta D/J segment (TRDD/TRDJ), while TCR delta constant domain refers to the extracellular segment of TRDC or C-terminal truncated sequence.

The TCRs of the present invention preferably bind to the peptide-HLA molecule complex of the invention with a binding affinity (KD) of about 100 μM or less, about 50 μM or less, about 25 μM or less, or about 10 μM or less. More preferred are high affinity TCRs with binding affinities of about 1 μM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less. Non-limiting examples of preferred TCR binding affinity ranges of the present invention include values from about 1 to about 10 nM; about 10 to about 20 nM; about 20 to about 30 nM; about 30 to about 40 nM; about 40 to about 50 nM; about 50 to about 60 nM; about 60 to about 70 nM; about 70 to about 80 nM; about 80 to about 90 nM; and about 90 to about 100 nM.

The term specific binding, as used in connection with the concept of TCR of the present invention, and its grammatical variants are used to denote a TCR with a binding affinity (KD) for a complex of a peptide of the invention and an HLA molecule of 100 μM or lower.

Alpha/beta heterodimeric TCRs as described herein may have an introduced disulfide bond between their constant domains. Preferred TCRs of this kind include those having a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, except when Thr 48 of the TRAC domain and Ser 57 of the TRBC1 or TRBC2 domains are substituted with cysteine residues, said cysteine residues forming a disulfide bond between the sequence a TRAC constant domain and a TRBC1 or TRBC2 TKR constant domain sequence.

With or without the introduction of the interstrand bond mentioned above, the alpha/beta heterodimeric TCRs of the present invention may have a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence and a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence. The TCR may be linked by an occurring in nature of a disulfide bond between Cys4 of exon 2 of the TRAC domain and Cys2 of exon 2 of the TRBC1 or TRBC2 domain.

The TCRs of the present invention may include a detectable label selected from the group consisting of a radionuclide, a fluorophore, and biotin. The TCRs of the present invention may be conjugated to a therapeutically active ingredient such as a radionuclide, chemotherapeutic agent, or toxin.

In one embodiment, a TCR of the present invention having at least one alpha chain mutation and/or having at least one beta chain mutation has modified glycosylation compared to unmutated TCR.

In one embodiment, a TCR containing at least one mutation in the TCR alpha chain and/or the TCR beta chain has a binding affinity for and/or a binding half-life for a complex of a peptide of the invention and an HLA molecule that is at least twice higher than that of TKR containing the TKR alpha chain without mutations and/or the TKR beta chain without mutations. Increasing the affinity of tumor-specific TCR, as well as its use, relies on the existence of a window with optimal affinity for TCR. The existence of such a window is based on the observation that TCRs specific for HLA-A2 restricted pathogens have KD values that are generally about 10-fold lower than TCRs specific for HLA-A2 restricted tumor-associated autoantigens. It is now known that although tumor antigens have immunogenic potential because tumors arise from an individual's own cells, only mutant proteins or proteins with changes in translational processing will be perceived as foreign by the immune system. Antigens that are elevated or overexpressed (so-called self-antigens) will not necessarily elicit a functional immune response against the tumor: T cells expressing TCRs that are highly active for these antigens will be negatively selected within the thymus glands in a process known as central tolerance, which means that only T cells with low affinity TCRs for self-antigens will be left. Therefore, the affinity of TCR or variants according to

- 27 041120 of the present description in relation to the peptides according to the invention can be enhanced by methods well known in the prior art.

The present disclosure further relates to a method for identifying and isolating TCR according to the present disclosure, said method comprising incubating HLA-A*02 negative healthy PBMCs with A2/peptide monomers of the invention, incubating PBMCs with tetramerphycoerythrin (PE), and isolating T cells with high avidity using fluorescence-activated cell sorting (FACS)-Calibur.

The present description further relates to a method for identifying and isolating TCR according to the present description, said method comprising obtaining a transgenic mouse with intact human TCRae gene loci (1.1 and 0.7 Mb) whose T cells express various human TCRs to compensate for mouse TCR deficiency, immunization of mice with HAVCR1-001 peptide, incubation of transgenic mouse PBMCs with tetramerphycoerythrin (PE), and isolation of high avidity T cells by fluorescence-activated cell sorting (FACS)-Calibur .

In one aspect, to obtain T cells expressing TKR as described herein, nucleic acids encoding TKR-alpha and/or TKR-beta chains as described herein are cloned into expression vectors such as gamma-retrovirus or β-lentivirus. Recombinant viruses are prepared and tested for their functionality, such as antigenic specificity and functional avidity. An aliquot of the final product is then used to transduce the target population of T cells (typically purified from the patient's PBMC) which is cultured prior to infusion into the patient.

In another aspect, to obtain T cells expressing TCR as described herein, TCR RNA is synthesized using techniques known in the art, such as in vitro transcription systems. The in vitro synthesized TCR RNAs are then electroporated into primary CD8+ T cells from healthy donors to re-express tumor-specific TCR alpha and/or beta chains.

To increase expression levels, nucleic acids encoding TCRs as described herein can be operably linked to strong promoters such as long terminal repeats of retrovirus (LTR), cytomegalovirus (CMV), mouse stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin, ubiquitin and the combined simian virus 40 (SV40)/CD43 promoter, elongation factor (EF)-1a and spleen necrosis virus (SFFV) promoter. In a preferred embodiment, the promoter is heterologous to the expressed nucleic acid.

In addition to strong promoters, TCR expression cassettes as described herein may contain additional elements that can enhance transgene expression, including the central polypurine tract (cPPT), which promotes nuclear translocation of lentiviral constructs (Follenzi et al., 2000), and a viral post-transcriptional regulatory element marmot hepatitis (wPRE), which increases the level of transgene expression by increasing RNA stability (Zufferey et al., 1999).

The TCR alpha and beta chains of the present invention may be encoded by nucleic acids located on separate vectors, or may be encoded by polynucleotides located on the same vector.

Achieving high levels of surface TCR expression requires transcription of high levels of both the TCR-alpha and TCR-beta chains introduced by the TCR. To do this, the TKR-alpha and TKR-beta chains of the present disclosure can be cloned into bicistronic constructs in a single vector that has been shown to be able to overcome this hurdle. The use of the viral internal ribosome entry site (IRES) between the TKR-alpha and TKR-beta chains results in coordinated expression of both chains because the TKR-alpha and TKR-beta chains are generated from a single transcript that splits into two proteins during transcription, providing equal molar ratio of TKR-alpha and TKR-beta chains. (Schmitt et al. 2009). (Schmitt et al. 2009).

Nucleic acids encoding TCRs as described herein can be codon-optimized to increase host cell expression. The redundancy of the genetic code allows some amino acids to be coded for by more than one codon, however some particular codons are less optimal than others due to the relative availability of suitable tRNAs as well as other factors (Gustafsson et al., 2004). Sequence modifications of the TKR-alpha and TKR-beta genes so that each amino acid is encoded by the optimal codon for mammalian gene expression, as well as the removal of unstable mRNA motifs or cryptic splicing sites, have been shown to significantly increase the expression of the TKR-alpha and TKR-beta genes ( Scholten et al., 2006).

In addition, disruption of complementarity between introduced and endogenous TCR chains can lead to the acquisition of specificity that would pose a significant risk to autoimmunity. For example, the formation of mixed TCR dimers can reduce the number of CD3 molecules available to form correctly paired TCR complexes and thus can significantly reduce the functional avidity of cells expressing the introduced TCR (Kuball et al., 2007).

To reduce mismatch, the C-terminal domain of the introduced TCR chains as described herein can be modified to promote interchain affinity while reducing the ability of the introduced chains to pair with endogenous TCR. These strategies may include replacing the C-terminal domains of human TKR-alpha and TKR-beta chains with their mouse equivalents (C-terminal murine domain); obtaining a second interchain disulfide bond in the C-terminal domain by introducing a second cysteine residue in both chains: TKR-alpha and TKR-beta of the introduced TKR (modification with cysteine); exchange of interacting residues in the C-terminal domain of the TKR-alpha and TKR-beta chains (ledge-to-bottom) and the fusion of the variable domains of the TKR-alpha and TKR-beta chains directly into CD3Z (CD3Z fusion). (Schmitt et al. 2009).

In one embodiment, the host cell is genetically modified to express TCR as described herein. In preferred embodiments, the host cell is a human T cell or a progenitor T cell. In some embodiments, the T cell or T cell precursor is obtained from a patient with cancer. In other embodiments, the T cell or T cell precursor is obtained from a healthy donor. Host cells as described herein may be allogeneic or autologous for the patient to be treated. In one embodiment, the host cell is a gamma/delta T cell transformed to express alpha/beta TCR.

The pharmaceutical composition is a composition suitable for administration to a human as part of a treatment. Preferably, the pharmaceutical composition is sterile and manufactured in accordance with the rules of GMP (good manufacturing practice).

Pharmaceutical compositions include peptides both in free form and in the form of a pharmaceutically acceptable salt (see also above). Used in the context of this invention, the term pharmaceutically acceptable salt refers to derivatives of the disclosed peptides, and the peptide is modified by obtaining acidic or basic salts of the substance. For example, acid salts are prepared from the free base (generally where the neutral form of the drug has a neutral -NH ₂ group) using a reaction with an appropriate acid. Suitable acids for making 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, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like, and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Conversely, the preparation of basic salts of acidic components that may be present on the peptide is done using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, and the like.

In one particularly preferred embodiment, the pharmaceutical compositions comprise peptides in the form of acetic acid salts (acetates), trifluoroacetates or hydrochloric acid salts (chlorides).

Preferably, the medicament of the present invention is an immunotherapeutic drug such as a vaccine. It can be administered directly to the patient, into the affected organ, or systemically by iv, IM, s/c, ip and iv, or applied ex vivo to cells obtained from the patient or to a human cell line, which can then administered to a patient or used in vitro to select a subpopulation of immune cells derived from a patient, which are then reintroduced to the patient. If the nucleic acid is introduced into the cells in vitro, it may be useful for the cells to be transfected to co-express immunostimulatory cytokines such as interleukin-2. The peptide may be substantially pure or in combination with an immunostimulatory adjuvant (see below), or used in combination with immunostimulatory cytokines, or administered with a suitable delivery system such as liposomes. The peptide may also be conjugated to a suitable carrier such as keyhole hemocyanin (KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). The peptide may also be labeled or may be a fusion protein or fusion molecule. Peptides, the sequence of which is given in the present invention, is expected to stimulate CD4+ or CD8+ T cells. However, stimulation of CD8 T cells is more effective in the presence of support provided by CD4 helper T cells. Thus, for class I MHC epitopes that stimulate CD8 T cells, fusion partners or fusion sites appropriately provide epitopes that stimulate CD4 positive T cells. CD4 and CD8 stimulatory epitopes are well known in the art and include those identified in the present invention.

In one aspect, the vaccine comprises at least one peptide having the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 48 and at least one additional peptide, preferably 2 to 50, more preferably 2 to 25, even more preferably 2 to 20, and most preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 peptides. The peptide(s) may be derived from one or more specific TAAs and may bind to class I MHC molecules.

In yet another aspect of the invention, a nucleic acid (eg, polynucleotide) encoding a peptide or peptide variant of the invention is provided. The polynucleotide may be, for example, DNA, cDNA, PNA, RNA, or combinations thereof, both single and/or double stranded; natural or stabilized forms of polynucleotides, such as, for example, polynucleotides with a phosphorothioate backbone, and may or may not contain introns, provided that the polynucleotide encodes a peptide. Of course, only peptides that contain naturally occurring amino acid residues linked by naturally occurring peptide bonds can be encoded by a polynucleotide. In another aspect of the invention, there is provided an expression vector capable of expressing a polypeptide according to the invention.

A number of methods have been developed for linking polynucleotides, especially DNA, to vectors, for example, using complementary sticky ends. For example, complementary homopolymer tails can be added to a DNA segment for insertion into vector DNA. This vector and the DNA segment are then joined by a hydrogen bond between complementary homopolymer tails, forming recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide an alternative way to attach a DNA segment to vectors. Synthetic linkers containing a number of restriction endonuclease recognition sites are commercially available from various sources including International Biotechnologies Inc, New Haven, CT, USA.

A desirable method for modifying the DNA encoding the polypeptide of the invention uses the polymerase chain reaction as disclosed by Saiki R.K. et al. (Saiki et al., 1988). This method can be used to introduce the DNA into a suitable vector, for example when constructed at suitable restriction sites, or it can be used to modify the DNA in other suitable ways known in the art. If viral vectors are used, poxvirus or adenovirus vectors are preferred.

The DNA (or, in the case of retroviral vectors, RNA) can then be expressed in a suitable host to produce a polypeptide comprising a peptide or variant of the invention. Thus, DNA encoding a peptide or variant of the invention can be used in accordance with known techniques, modified as appropriate to reflect the ideas disclosed herein, to construct an expression vector, which is then used to transform a suitable host cell for expression and production polypeptide according to the invention. Such techniques include those disclosed, for example, in US Pat.

The DNA (or, in the case of retroviral vectors, RNA) encoding the polypeptide of the compound of the invention can be fused to a wide variety of other DNA sequences for introduction into the appropriate host. The satellite DNA will depend on the nature of the host, the manner in which the DNA is introduced into the host, and whether maintenance in episomal or integrative form is desired.

Typically, the DNA is introduced into an expression vector, such as a plasmid, with the correct orientation and correct reading frame for expression. If desired, the DNA may be fused to appropriate nucleotide sequences that coordinate transcription and translation and are recognized by the desired host, although such control elements are usually present in the expression vector. The vector is then administered to the host by standard means. Typically, not all hosts are transformed by the vector. Therefore, it will be necessary to isolate the transformed host cells. One selection technique involves introducing into the expression vector a DNA sequence, with any necessary control elements, that encodes a selected trait in the transformed cell, such as antibiotic resistance.

Alternatively, the gene for such a selectable trait may be on another vector that is used to co-transform the desired host cell.

Host cells that have been transformed with 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 accordance with the teachings disclosed herein, leading to the expression of the polypeptide, which can then be isolated.

Many expression systems are known, including bacteria (eg E. coli and Bacillus subtilis), yeast (eg Saccharomyces cerevisiae), filamentous fungi (eg Aspergillus spec), plant cells, animal and insect cells. Preferably the system is mammalian cells such as CHO cells available from the American Type Culture Collection ATCC.

- 30 041120

A typical mammalian cellular vector plasmid for constitutive expression comprises a CMV or SV40 promoter with a suitable poly-A terminal and a resistance marker such as neomycin. One example is pSVL, available from Pharmacia, Piscateway, New Jersey, USA. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. Suitable yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integration plasmids (Ylps) and include yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are yeast plasmids with centromeres (Ycp). CMV promoter-based vectors (eg, Sigma-Aldrich) provide transient or sustained expression, cytoplasmic expression or secretion, and N-terminal or C-terminal labeling in various combinations of FLAG, 3xFLAG, c-myc, or MAT. These fusion proteins allow the detection, purification and analysis of the recombinant protein. Double-label merges provide flexibility in detection.

The strong regulatory region of the human cytomegalovirus (CMV) promoter increases constitutive protein expression levels up to 1 mg/L in COS cells. For less active cell lines, protein levels are typically ~0.1 mg/l. The presence of the SV40 origin of replication will result in high levels of DNA replication in permissive COS cells. CMV vectors, for example, may contain a pMB1 (derived pBR322) origin of replication in bacterial cells, a beta-lactamase gene for selection of ampicillin resistance in bacteria, human growth hormone polyA, and an f1 origin of replication. Vectors containing a preprotrypsin (PPT) leader sequence can direct the secretion of FLAG fusion proteins in culture media for purification using anti-FLAG antibodies, resins and plates. Other vectors and expression systems for use with various host cells are well known in the art.

In another preferred embodiment, two or more peptides or peptide variants of the invention are encoded and thus expressed sequentially (as in the case of a bead-on-string structure). To this end, peptides or peptide variants may be joined or fused together by linker amino acid fragments such as, for example, LLLLLL, or may be joined without any additional peptide(s) in between. These structures can also be used in anticancer therapy and possibly induce immune responses involving both MHC I and MHC class II molecules.

The present invention also relates to a host cell transformed with the polynucleotide vector construct of the present invention. The host cell can be either prokaryotic or eukaryotic. The bacterial cells may be preferably prokaryotic host cells under certain conditions and are typically an E. coli strain such as, for example, E. coli strain DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1, available from the American Type Culture Collection (ATCC), Rockville, MD, USA (ATCC No. 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells such as fibroblast cell lines. and colon cells from mouse, rat, monkey, or human species Yeast host cells include YPH499, YPH500, and YPH501, which are generally available from Stratagene Cloning Systems, La Jola, CA 92037, USA Preferred Cells -mammalian hosts include Chinese Hamster Ovary (CHO) cells available from ATCC as CCL61, Swiss mouse embryonic cell line NIH/3T3 available from ATCC as CRL 1658, COS-1 cells from monkey kidneys available from 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 selection of suitable host cells for expression is provided, for example, in 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 skilled in the art.

Transformation of appropriate host cells with the DNA construct of the present invention is carried out using well-known methods, which usually depend on the type of vector used. For transformation of prokaryotic host cells, see, for example, Cohen et al. (Cohen et al., 1972) and (Cohen et al., 1972). Yeast cell transformation is described by Sherman et al. (Sherman et al., 1986). The Beggs method (Beggs, 1978) is also suitable. For vertebrate cells, reagents suitable for transfection of such cells, eg calcium phosphate and DEAE-dextran or liposomal formulations, are available from Stratagene Cloning Systems or Life Technologies Inc., Gaithersburg, MD 20877, USA. Electroporation is also suitable for cell transformation and/or transfection and is well known in the art for transformation of yeast cells, bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain the DNA construct of the present invention can be identified by well known methods such as PCR.

Alternatively, the presence of protein in the supernatant can be detected using antibodies.

It should be understood that certain host cells of the invention are suitable for the production of the peptides of the invention, such as bacterial, yeast and insect cells. However, other host cells may be used in specific therapeutic methods. For example, antigen presenting cells, such as dendritic cells, can be usefully used to express the peptides of the invention such that they can be loaded onto suitable MHC molecules. Thus, the present invention provides a host cell comprising a nucleic acid or an expression vector according to the invention.

In a preferred embodiment, the host cell is an antigen presenting cell, in particular a dendritic cell or an antigen presenting cell. APCs loaded with a recombinant prostatic acid phosphatase (PAP) fusion protein were approved by the US Food and Drug Administration (FDA) on April 29, 2010 for use in the treatment of metastatic HRPC (hormone refractory prostate cancer), asymptomatic or with minimal symptoms (sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

In another aspect of the invention, there is provided a method for producing a peptide or variant thereof, the method comprising culturing a host cell and isolating the peptide from the host cell or its culture medium.

In another embodiment, the peptide, nucleic acid or expression vector of the invention is used in medicine. For example, the peptide or variant thereof may be formulated for intravenous (IV) administration, subcutaneous (SC) administration, intradermal (IV) administration, intraperitoneal (IP) administration, intramuscular (IM) administration. Preferred routes of administering the peptides include s.c., i.v., i.p., i.m., and i.v. Preferred routes of DNA administration include i.c., i.m., s.c., i.b., and i.v. For example, doses of 50 μg to 1.5 mg, preferably 125 to 500 μg of the peptide or DNA may be administered, depending on the respective peptide or DNA. Dosages in this range have been used successfully in previous clinical studies (Walter et al., 2012).

The polynucleotide used in 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 constructing and introducing such a nucleic acid are well known in the art. An overview is provided, for example, in Teufel et al. (Teufel et al., 2005). Polynucleotide vaccines are easy to obtain, but the mechanism by which these vectors act to induce an immune response is not fully understood. Suitable vectors and delivery systems include viral DNA and/or RNA, such as those based on adenovirus, vaccinia virus, retroviruses, herpes virus, adeno-associated virus, or hybrids containing elements from 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, eg, with an epitope that stimulates T cells against the corresponding opposite complementarity-determining region of the CDR as described above.

The medicament of the invention may also include one or more adjuvants. Adjuvants are substances that non-specifically enhance or potentiate an immune response (e.g., immune responses mediated by CD8-positive T cells or helper T cells (TH) to an antigen and may thus be considered useful in the medicament of the present invention. Suitable adjuvants include, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligands derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, imiquimod (ALDARA®), remiquimod, ImuFact IMP321, interleukins such as IL-2, IL-13, IL-21, interferon-alpha or beta or their pegylated derivatives, IS Patch, ISS, ISCOMATRIX, ISCOM immunostimulatory complexes, JuvImmune®, 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® vector system based on poly(lactide coglycolide) [PLG] and dextran microparticles, talactoferrin SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, betaglucan, Pam3Cys, Aquila QS21 stimulon, which is derived from saponin, mycobacterial extracts and synthetic mimics of bacterial cell walls and others proprietary adjuvants such as Ribi's Detox, Quil or Superfos. Preferred adjuvants are Freund's adjuvant or GM-CSF. Several immunological adjuvants (eg, MF59) specific for dendritic cells and their preparation have been described previously (Allison and Krummel, 1995). Cytokines may also be used. Several cytokines have been directly correlated with influencing the migration of dendritic cells to lymphoid tissues (eg, TNF-), accelerating the maturation of dendritic cells into efficient, antigen-presenting T-lymphocyte cells (eg, GM-CSF,

IL-1 and IL-4) (U.S. Patent No. 5,849,589, specifically incorporated herein in its entirety by reference) and acting as immunoadjuvants (e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha, INF-beta) (Gabrilovich et al., 1996).

Immunostimulatory CpG oligonucleotides have also been reported to enhance the effects of adjuvants in vaccine formulations. Without wishing to be bound by any particular theory, the authors believe that CpG oligonucleotides, upon activation of the innate (non-acquired) immune system, act via Toll-like receptors (TLRs), mainly TLR9. CpG-induced activation of TLR9 enhances antigen-specific humoral and cellular responses to a wide range of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly, dendritic cell maturation and differentiation is improved, leading to increased TH1 cell activation and increased production of cytotoxic T lymphocytes (CTLs) even in the absence of CD4 T cell assistance. TH1 activation induced by TLR9 stimulation persists even in the presence of vaccine adjuvants such as alum or Incomplete Freund's Adjuvant (IFA), which normally promote TH2 activation. CpG oligonucleotides exhibit even greater adjuvant activity when they are formulated or administered together with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions or similar formulations, which are particularly needed to initiate a strong response if the antigen relatively weak. They also accelerate the immune response and reduce antigen doses by about two orders of magnitude compared to antibody responses to a full-dose vaccine without CpG, which has been observed in some experiments (Krieg, 2006). US Pat. No. 6,406,705 B1 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants, and an antigen to elicit an antigen-specific immune response. The TLR9 CpG antagonist is dSLIM (double-stranded stem-loop immunomodulator) from Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR-binding molecules such as TLR 7, TLR 8 and/or TLR 9 RNA-binding molecules can also be used.

Other examples of useful adjuvants include, but are not limited to, chemically modified CpGs (e.g., CpR, Idera), dsRNA analogs such as poly-(1:C) and their derivatives (e.g., AmpliGen®, Hiltonol®, poly (ICLC), poly(IC-R), poly(I:C12U), bacterial DNA or RNA other than CpG, and immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, antiCTLA4, other antibodies targeting major immune system structures (eg, CD40 antibodies, TGF-beta , TNF-alpha receptor) and SC58175, which may act therapeutically and/or as adjuvants. The amounts and concentrations of adjuvants and additives suitable for use in the context of the present invention can be readily determined by the skilled artisan without undue experimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpG oligonucleotides and their derivatives, poly(1:C) and its derivatives, RNA, sildenafil, and solid microparticulate formulations with PLG or virosomes.

In a preferred embodiment of 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.

In a preferred embodiment of 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 IMS 1312 montanide, ISA 206 montanide, ISA 50 V montanide, ISA-51 montanide, poly-ICLC (Hiltonol®) and anti-CD40 monoclonal antibodies, or combinations thereof.

This composition is used for parenteral administration such as subcutaneous, intradermal, intramuscular or oral administration. To do this, the peptides and, optionally, other molecules are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous, carrier. In addition, the composition may contain adjuvants such as buffers, binders, ballasts, diluents, fragrances, lubricants, and the like. The peptides may also be administered together with immunostimulatory agents such as cytokines. An extensive list of excipients that can be used in such a formulation can be taken from, for example, A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The composition can be used for the prevention, prophylaxis and/or treatment of adenomatous or cancerous diseases. Examples of pharmaceutical compositions can be taken, for example, from EP 2112253.

It is important to understand that the immune response elicited by the vaccine according to the invention is directed to cancer cells at different stages of the cell cycle and different stages of tumor development. In addition, the attack is directed at various signaling pathways associated with cancer. This is an advantage over vaccines that target only one or few targets, which can result in the tumor easily adapting to such an attack (tumor escape). In addition, not all individual tumors have the same patterns of antigen expression. Therefore, the combination of several tumor-associated peptides ensures that each individual tumor has at least some of these targets. The composition is designed on the basis that each tumor is expected to express multiple antigens and encompass multiple independent signaling pathways required for tumor growth and maintenance. Thus, a ready-to-use vaccine can be easily used in a larger patient population. This means that the pre-selection of patients for vaccine treatment can be limited to HLA typing, without requiring any additional analysis of antigen expression biomarkers, while still guaranteeing simultaneous exposure to multiple targets in the form of an induced immune response, which is important for efficacy (Banchereau et al. ., 2001; Walter et al., 2012).

In the context of the present description, the concept of a frame refers to a molecule that specifically binds to a (eg, antigenic) determinant. In one embodiment, the scaffold is capable of directing the entity to which it is attached (e.g., a (second) antigen binding element) to a target site, e.g., a specific type of tumor cell or tumor stroma bearing an antigenic determinant (e.g., a peptide-MHC complex according to this document). In another embodiment, the scaffold is capable of activating signal transduction pathways through its target antigen, eg, a T-cell receptor complex antigen. Frameworks include, but are not limited to, antibodies and fragments thereof, antibody antigen binding domains including 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 allogeneic or autologous T cells. To assess whether a molecule is a scaffold that binds to a target, a binding assay can be performed.

Specific binding means that the scaffold binds to the peptide-MHC complex of interest better than other naturally occurring peptide-MHC complexes, to the extent that the scaffold provided with an active molecule capable of killing a specific target cell is unable to kill another. a cell without a specific target, but presenting another complex(s) of the peptide with MHC. Binding to other peptide-MHC complexes is not important unless the peptide-MHC cross-reacting complex peptide is naturally occurring, ie. not derived from human HLA peptidome. Tests for evaluating the killing potential of a target cell are well known in the art. They should be carried out using target cells (primary cells or cell lines) with an unchanged presentation of peptide-MHC complexes or cells loaded with peptides such that the level of naturally occurring peptide-MHC complexes will be achieved.

Each framework may include a label that enables the associated framework to be detected by determining the presence or absence of a signal supplied by the label. For example, the scaffold can be labeled with a fluorescent dye or any other applicable cell marker molecule. Such marker molecules are well known in the art. For example, fluorescent labeling, for example with a fluorescent dye, may allow visualization of the bound aptamer by fluorescence or laser scanning microscopy or flow cytometry.

Each scaffold can be conjugated to a second active molecule such as, for example, IL-21, anti-CD3, anti-CD28.

For further information on polypeptide scaffolds see, for example, the prior art section of patent application WO 2014/071978 A1 and the literature cited therein.

The present invention further relates to aptamers. Aptamers (see, for example, WO 2014/191359 and the literature cited therein) are short, single-stranded nucleic acid molecules that can fold into specific three-dimensional structures and recognize specific target structures. It turned out that they represent a suitable alternative for the development of targeted therapies. Aptamers have been shown to selectively bind to various complex targets with high affinity and specificity.

Aptamers that recognize molecules found on the cell surface have been identified in the last decade and provide an opportunity for the development of diagnostic and therapeutic approaches. Since aptamers have been shown to have little to no toxicity and immunogenicity, they are promising candidates for biomedical applications. Indeed, aptamers, such as aptamers recognizing the prostate-specific membrane antigen, have been successfully used in targeted therapy and have shown functionality in in vivo xenograft models. In addition, aptamers that recognize specific tumor lineages have been identified.

DNA aptamers can be selected that exhibit a wide range of properties in recognizing various cancer cells and, in particular, cells derived from solid tumors, while non-tumor and primary healthy cells are not recognized. If the identified aptamers recognize not only a specific tumor subtype, but also interact with various tumors, this makes it possible to use aptamers as so-called broad-spectrum diagnostic and therapeutic agents.

Moreover, flow cytometry studies of cell binding behavior showed that the aptamers exhibited very good apparent affinity, which was expressed at the nanomolar level.

Aptamers are useful for diagnostic and therapeutic purposes. In addition, as could be demonstrated, some aptamers are taken up by tumor cells and thus can act as molecular carriers for the targeted delivery of anticancer agents such as siRNA to tumor cells.

Aptamers to complex targets such as cells and tissues and complexes of peptides can be selected, including, preferably consisting of a sequence in accordance with any of the sequences from SEQ ID NO: 1 to SEQ ID NO: 48 in accordance with the present invention with an MHC molecule, using the cell-SELEX method (Systematic Evolution of Ligands by Exponential enrichment).

The peptides of the present invention can be used to generate and develop specific antibodies to MHC/peptide complexes. They can be used in therapy that targets toxins or radioactive substances to diseased tissue. Another use of these antibodies may be to target radionuclides to diseased tissue for imaging purposes such as PET (Positron Emission Tomography). This can help in detecting small metastases or in determining the size and precise localization of diseased tissues.

Thus, in another aspect of the invention, there is provided a method for producing a recombinant antibody that specifically binds to a human major histocompatibility complex (MHC) class I or II in complex with an HLA-restricted antigen, the method comprising immunizing a genetically modified, non-human mammal containing cells, expressing molecules of said major human histocompatibility complex (MHC) class I or II with a soluble form of the MHC class I or II molecule in complex with said HLA-restricted antigen; isolating mRNA molecules from antibody-producing cells of said non-human mammal; creating a phage display library containing phages displaying protein molecules encoded by said mRNA molecules; and isolating at least one phage from said phage display library, wherein said at least one phage displays said antibody on the surface that specifically binds to said class I or II human major histocompatibility complex (MHC) in complex with said HLA-restricted antigen.

In another aspect of the invention, an antibody is provided that specifically binds to a human major histocompatibility complex (MHC) class I or II in complex with an HLA-restricted antigen, wherein the antibody is preferably a polyclonal antibody, a monoclonal antibody, a bispecific antibody, and/or a chimeric antibody.

Appropriate methods for the production of such antibodies and class I single chain major histocompatibility complexes, as well as other tools for the production of these antibodies, are disclosed in patent applications WO 03/068201, WO 2004/084798, WO 01/72768, WO 03/070752 and in published works (Cohen et al., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), all of which are hereby expressly incorporated by reference in their entirety for the purposes of the present invention.

Preferably, the antibody binds with a binding affinity below 20 nM, preferably below 10 nM, to a complex, also referred to as specific in the context of the present invention.

The present invention relates to a peptide comprising a sequence selected from the group consisting of sequences from SEQ ID NO: 1 to SEQ ID NO: 48, or a variant thereof, which is at least 88% homologous (preferably if identical) to the sequence with SEQ ID NO: 1 to SEQ ID NO: 48, or a variant thereof which induces T cell cross-reactivity with said peptide, wherein said peptide is not a full length basic polypeptide.

The present invention further relates to a peptide comprising a sequence selected from the group consisting of sequences from SEQ ID NO: 1 to SEQ ID NO: 48, or a variant thereof,

- 35 041120 which is at least 88% homologous (preferably identical) SEQ ID NO: 1 to

SEQ ID NO: 48, wherein said peptide or variant has a total length of 8 to 100, preferably 8 to 30, and most preferably 8 to 14 amino acids.

The present invention further relates to peptides according to the invention capable of binding to a human major histocompatibility complex (MHC) class I or II molecule.

The present invention further relates to peptides according to the invention, wherein the peptide consists or consists essentially of the amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 48.

The present invention further relates to peptides in accordance with the invention, where the peptide is modified (chemically) and/or includes non-peptide bonds.

The present invention further relates to peptides according to the invention, wherein the peptide is part of a fusion protein, in particular comprising the N-terminal amino acids of an HLA-DR antigen-associated invariant chain (li), or where the peptide is fused to an antibody (or fused to an antibody sequence ), such as an antibody that is specific for dendritic cells.

The present invention further relates to a nucleic acid encoding peptides in accordance with the invention, provided that the peptide is not entirely (entirely) human protein.

The present invention further relates to a nucleic acid according to the invention, which is DNA, cDNA, PNA, RNA, or combinations thereof.

The present invention further relates to an expression vector capable of expressing a nucleic acid according to the present invention.

The present invention further relates to a 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 esophageal cancer.

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.

The present invention further relates to a host cell according to the present invention, which is an antigen presenting cell, preferably a dendritic cell.

The present invention further relates to a method for producing a peptide in accordance with the present invention, said method comprising culturing a host cell in accordance with the present invention and isolating the peptide from said host cell or its culture medium.

The present invention further relates to a method according to the present invention, wherein the antigen is loaded onto MHC class I or II molecules expressed on the surface of a suitable antigen presenting cell by contacting a sufficient amount of the antigen with the antigen presenting cell.

The present invention further relates to a method according to the invention, wherein the antigen presenting cell comprises an expression vector capable of expressing said peptide comprising the sequence of SEQ ID NO: 1 to SEQ ID NO: 48 or said variant amino acid sequence.

The present invention further relates to activated T cells obtained by the method of the present invention, wherein said T cells selectively recognize a cell that aberrantly expresses a polypeptide comprising the amino acid sequence of the present invention.

The present invention further relates to a method for killing target cells in a patient whose target cells aberrantly express a polypeptide comprising any amino acid sequence in accordance with the present invention, the method comprising administering to the patient an effective number of T cells in accordance with the present invention.

The present invention further relates to the use of any described peptide, 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 method of use according to the present invention, wherein the medicament exhibits anti-cancer activity.

The present invention further relates to a method of administration according to the invention, wherein the medicament is a vaccine. The present invention further relates to a method of use according to the invention, wherein the medicament exhibits anti-cancer activity.

The present invention further relates to use according to the invention, wherein said cancer cells are preferably prostate cancer cells or other solid or hematological tumors, e.g. lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, cell carcinoma Merkel, pancreatic cancer, gallbladder cancer, bile duct cancer, CRC, bladder cancer, non-small cell lung cancer, kidney cancer, leukemia (eg, AML or CLL), ovarian cancer, esophageal cancer, brain cancer, and gastric cancer, most preferably prostate cancer cells.

The present invention further relates to specific marker proteins and peptide-based biomarkers of the present invention, referred to in the context of the invention as targets, which can be used in the diagnosis and/or prognosis of prostate cancer. The present invention also relates to the use of these new targets for the treatment of cancer.

The term antibody or antibodies is used in the context of this invention in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact or complete immunoglobulin molecules, fragments (e.g., CDRs, Fv, Fab, and Fc fragments) or polymers of such immunoglobulin molecules and humanized versions of immunoglobulin molecules are also included in the term antibody, provided that they exhibit any of the desired properties (e.g., specifically bind to a prostate cancer (poly)peptide marker, deliver a toxin to a prostate cancer cell overexpressing the cancer marker gene, and/or inhibit the activity of the prostate cancer marker polypeptide) according to the invention.

If possible, the antibodies of the invention can be purchased from commercial sources. The antibodies of the invention may also be prepared using well known methods. The skilled artisan will appreciate that both full length polypeptide markers of prostate cancer and fragments thereof can be used to generate the antibodies of the invention. The polypeptide required to produce an antibody of the invention may be partially or completely purified from a natural source, or may be obtained using recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the present invention, such as a peptide of SEQ ID NO: 1 to SEQ ID NO: 48, a polypeptide, or a variant or fragment thereof, can be expressed in prokaryotic cells (e.g., bacteria) or eukaryotic cells. cells (e.g., yeast, insect, or mammalian cells), after which the recombinant protein can be purified and used in preparation of monoclonal or polyclonal antibodies that specifically bind to the ovarian cancer polypeptide marker used to produce the antibody of the invention.

One skilled in the art will appreciate that obtaining two or more different sets of monoclonal or polyclonal antibodies increases the likelihood of obtaining an antibody with the specificity and affinity required for its intended use (e.g., for ELISA, immunohistochemistry, in vivo imaging, immunotoxin-based therapy). Antibodies are tested for their desired activity by known methods according to the purpose of the antibody (e.g. ELISA, immunohistochemistry, immunotherapy, etc.; for further information on generating and testing antibodies see, for example, Greenfield, 2014 (Greenfield , 2014)). For example, antibodies can be examined using ELISA or Western-blot method, immunohistochemical staining of formalin-fixed cancer tissue samples or frozen tissue sections. After initially characterizing them in vitro, antibodies intended for in vivo therapeutic or diagnostic use are tested according to known clinical assay methods.

The term monoclonal antibody in the context of the present invention means an antibody derived from a substantially homogeneous population of antibodies, i.e. individual antibodies within a population are identical, with the exception of possible natural mutations, which may be present in small quantities. Monoclonal antibodies in the context of the present invention specifically include chimeric antibodies in which the heavy and/or light chain portion is identical or homologous to the corresponding sequences of antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder(s) i) the chain is identical (s) or homologous (s) to the corresponding sequences of antibodies derived from a different species or belonging to a different class or subclass of antibodies, equally as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (US patent No. 4816567, which is incorporated herein in its entirety).

Monoclonal antibodies according to the invention can be obtained using the hybridoma method. In the hybridoma method, a mouse or other suitable host animal is typically immunized with an immunizing agent to initiate lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

Monoclonal antibodies can also be generated using recombinant DNA technologies such as those described in US Pat. No. 4,816,567. bind to the genes encoding the heavy and light chains of mouse antibodies).

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In vitro methods are also suitable for the production of monovalent antibodies. Cleavage of antibodies to obtain their fragments, in particular Fab fragments, can be carried out using standard techniques known in the art. For example, splitting can be done using papain. Examples of papain-induced cleavage are described in WO 94/29348 and US Pat. No. 4,342,566. Papain-induced cleavage of antibodies typically results in two identical antigen-binding fragments called Fab fragments, each with a separate antigen-binding site and a residual Fc. -fragment. Pepsin treatment results in an F(ab') ₂ fragment and a pFc' fragment.

Antibody fragments, whether linked to other sequences or not, may also include insertions, deletions, substitutions, or other selected modifications of specific regions or amino acid residues, provided that the activity of the fragment is not significantly altered or damaged compared to an unmodified antibody or antibody fragment. These modifications may introduce some additional properties, such as adding/removing amino acids capable of disulfide binding, increasing their biological stability, changing their secretory characteristics, etc. In any case, the antibody fragment must have the property of biological activity, such as binding activity, regulation of binding on the binding domain, and so on. Functional or active sites of an antibody can be identified by mutagenesis of a particular section of the protein, followed by expression and examination of the expressed polypeptide. Such methods are completely obvious to one of skill in the art and may involve site-directed mutagenesis of the nucleic acid encoding the antibody fragment.

Antibodies of the invention may further include humanized antibodies or human antibodies. Humanized forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', or other antibody antigen-binding subsequences) that contain a minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient are replaced by residues from CDRs of non-human species (donor antibody) such as mice, rats or rabbits having the desired specificity, affinity and binding ability. In some instances, the Fv framework (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are not found in either the recipient antibody or the imported CDR or framework sequences. Typically, a humanized antibody will include substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are as such by consensus. human immunoglobulin sequences. Optimally, the humanized antibody also contains at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin.

Methods for humanizing non-human antibodies are well known in the art. In general, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. Such amino acid residues of non-human origin are often referred to as imported residues, which are usually taken from an imported variable domain. Humanization can essentially be accomplished by replacing rodent CDR regions or CDR sequences with corresponding human antibody sequences. Accordingly, such humanized antibodies are chimeric antibodies (US Pat. No. 4,816,567) wherein substantially less than one intact human variable domain has been replaced by the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies in which some of the CDR residues and possibly FR residues are replaced with similar site residues from rodent antibodies.

Transgenic animals (eg, mice) can be used that are capable of producing the full spectrum of human antibodies when immunized, in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the gene encoding the antibody heavy chain attachment site in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene template into such germline mutant mice will result in the production of human antibodies upon antigen challenge. Human antibodies can also be generated in phage display libraries.

The antibodies of the invention are preferably administered to the subject in a pharmaceutically acceptable carrier. A suitable amount of a pharmaceutically acceptable salt is typically used in the formulation to render the composition isotonic. Examples of pharmaceutically acceptable carriers include saline, Ringer's solution and glucose 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 contemplated are carriers that include sustained release formulations such as semi-permeable antibody containing solid hydrophobic polymer matrices whose matrices are in the form of shaped entities such as films, liposomes, or microparticles. It will be apparent to one of skill in the art that certain carriers may be preferred depending on, for example, the route of administration and the concentration of antibody administered.

Antibodies can be administered to a subject, patient, or cell by injection (eg, intravenously, intraperitoneally, subcutaneously, intramuscularly) or by other means, such as infusion, which ensures delivery to the bloodstream in an efficient manner. Antibodies can also be administered by intratumoral or peritumoral routes to elicit local as well as systemic therapeutic effects. Topical or intravenous administration is preferred.

The effective dosage and mode of administration of antibodies can be determined empirically, and such decisions are within the power of a person skilled in the art. Those skilled in the art will appreciate that the dosage of antibodies to be administered will vary depending on, for example, the subject to whom the antibody will be administered, the route of administration, the particular type of antibody used, and other medicaments administered. A typical daily dosage of antibodies as monotherapy may vary from about 1 μg/kg up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above. Following administration of the antibody, preferably for the treatment of prostate cancer, the effectiveness of the therapeutic antibody can be assessed in a variety of ways known to those skilled in the art. For example, the size, number and/or distribution of cancer in a subject being treated can be monitored using standard tumor imaging techniques. A therapeutically administered antibody that blocks tumor growth, reduces the size and/or prevents the development of new tumors compared to the course of the disease that would occur without the administration of the antibody, and is an effective antibody for the treatment of cancer.

In another aspect of the invention, there is provided a method for producing a soluble T cell receptor (TCR) that recognizes a particular peptide-MHC complex. Such soluble T-cell receptors can be obtained from specific T-cell clones and their affinity can be increased by complementarity-determining mutagenesis. Phage display can be used to select the T cell receptor (US 2010/0113300, (Liddy et al., 2012)). In order to stabilize T-cell receptors during phage display and in the case of practical application as a drug, alpha and beta chains can be linked, for example, through unnatural disulfide bonds, other covalent bonds (single-stranded T-cell receptor) or via dimerization domains (Boulter et al., 2003; Card et al., 2004; Willcox et al., 1999). In order to perform certain functions on target cells, the T cell receptor can be associated with toxins, drugs, cytokines (see, for example, US application 2013/0115191), effector cell recruiting domains, such as an anti-CD3 domain, and etc. Moreover, it can be expressed on T cells used for adoptive transfer. Further information can be found in patent applications WO 2004/033685 A1 and WO 2004/074322 A1. A combination of soluble TCRs is described in patent application WO 2012/056407 A1. Other preparation methods are described in patent application WO 2013/057586 A1.

In addition, the peptides and/or TCRs or antibodies or other binding molecules of the present invention can be used to confirm a diagnosis of cancer made by a pathologist based on biopsy examination.

Antibodies or TCR can also be used for in vivo diagnostics. Typically, the antibody is labeled with a radionucleotide (such as ⁿ¹ In, ⁹⁹ Tc, ¹⁴ C, ¹³¹ I, ³ H, ³² P, or ³⁵ S) so that the tumor can be localized by immunoscintigraphy. 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 proteins with an affinity index (Kd) of less than 1x10 μM.

Antibodies for diagnostic purposes can be labeled with probes suitable for detection by various imaging methods. Probe detection methods 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 active radionuclides. Moreover, probes can be bi- or multi-functional and can be detected by more than one of the above methods. These antibodies can be labeled directly or indirectly with these probes. Attaching probes to antibodies includes covalently attaching a probe, inserting a probe into an antibody, and covalently attaching a chelating compound to attach a probe, among other well-recognized techniques in the art. For immunohistochemical studies, the affected 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 is brought into contact with a labeled primary antibody and a secondary antibody, where the antibody is used to detect in situ protein expression.

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Another aspect of the present invention includes a method for producing activated T cells in vitro, the method comprising contacting T cells in vitro 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 antigen is applied to the antigen presenting cell.

Preferably, the mammalian cell does not have a TAP peptide transporter or has a reduced level or reduced functional activity. Suitable cells deficient in the TAP peptide transporter include T2, RMA-S, and Drosophila cells. TAP is a transporter associated with antigen processing.

A T2-deficient human cell line loaded with peptides is available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA under catalog number CRL 1992; a Drosophila cell line, the Schneider 2 line, is available from the ATCC under catalog number CRL 19863; the RMA-S mouse cell line is described by Ljunggren et al. (Ljunggren and Karre, 1985).

Preferably, said host cell does not substantially express MHC class I molecules prior to transfection. It is also preferred if the stimulator cell expresses a molecule important in providing a costimulation signal to T cells, such as any of B7.1, B7.2, ICAM-1 and LFA 3. Nucleic acid sequences of numerous MHC class I molecules and costimulatory molecules are publicly available in the GenBank and EMBL databanks.

When an MHC class I epitope is used as the antigen, the T cells are CD8 positive T cells.

If the antigen-presenting cell is transfected to express such an epitope, it is preferred that the cell includes an expression vector capable of expressing a peptide comprising SEQ ID NO: 1 to SEQ ID NO: 48, or a variant of such an amino acid sequence.

Many other methods can be used to obtain T cells in vitro. For example, autologous tumor-infiltrating lymphocytes are used to obtain CTLs. Plebanski et al. (Plebanski et al., 1995) autologous peripheral blood lymphocytes (PBLs) were used to obtain T cells. In addition, it is possible to obtain autologous T cells by loading dendritic cells with a peptide or polypeptide or by infection with a recombinant virus. B cells can also be used to generate autologous T cells. In addition, macrophages loaded with a peptide or polypeptide or infected with a recombinant virus can be used to generate autologous T cells. S. Walter et al. (Walter et al., 2003) describe in vitro T cell priming using artificial antigen presenting cells (iAPCs), which is also a suitable method for generating T cells against a selected peptide. In the present invention, iAPKs were obtained by attaching pre-formed MHC-peptide complexes to the surface of polystyrene particles (microspheres) using a biochemical method with biotin-streptavidin. This system allows precise control of MHC density on iAPK, which allows selective induction of high or low avidity antigen-specific T cell responses with high efficiency in blood samples. In addition to the MHC-peptide complexes, the APCs must carry other proteins with co-stimulatory activity, such as anti-CD28 antibodies, attached to their surface. In addition, such an iAPK-based system often requires the addition of appropriate soluble factors, such as cytokines such as interleukin-12.

When obtaining T cells can also be used allogeneic cells, and this method is described in detail in patent application WO 97/26328, incorporated here by reference. For example, in addition to Drosophila cells and T2 cells, other cells such as Chinese hamster ovary (CHO) cells, baculovirus-infected insect cells, bacteria, yeast, vaccinia-infected target cells can be used for antigen presentation. In addition, plant viruses can be used (see, for example, Porta et al. (Porta et al., 1994) which describes the development of Chinese cowpea mosaic virus as a highly productive presentation system for foreign peptides).

Activated T cells that are directed against the peptides of the invention are suitable for therapy. Thus, in another aspect of the invention, activated T cells obtainable by the aforementioned methods of the invention are provided.

Activated T cells obtained by the above method will selectively recognize a cell that aberrantly expresses a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 48.

Preferably, the T cell recognizes the cell when interacting via its TCR with the HLA/peptide complex (eg, upon binding). T cells are useful in a method for killing target cells in a patient whose target cells aberrantly express a polypeptide comprising the amino acid sequence of the invention, wherein an effective number of activated T cells are administered to the patient. T cells that are injected into a patient can be obtained from the patient and activated.

- 40 041120 be formed as described above (i.e. they are autologous T cells). Alternatively, T cells are not obtained from the patient, but from another individual. Of course, it is preferable if the individual is a healthy individual. By a healthy individual, the inventors mean that the individual is in good general health, preferably having a competent immune system, and more preferably not suffering from any disease that can be easily monitored and detected.

In vivo target cells for CD8 positive T cells according to the present invention may be tumor cells (which sometimes express MHC class II molecules) and/or stromal cells surrounding the tumor (tumor cells) (which sometimes also express MHC II molecules). class (Dengjel et al., 2006)).

The T cells of the present invention can be used as active ingredients in a therapeutic composition. Thus, the invention also provides a method for killing target cells in a patient whose target cells aberrantly express a polypeptide comprising the amino acid sequence of the invention, the method comprising administering to the patient an effective number of T cells as defined above.

By aberrantly expressed, the inventors also mean that the polypeptide is overexpressed compared to expression levels in normal tissues, or that the gene is silent in the tissue from which the tumor originated, but it is expressed in the tumor. By overexpressed, the inventors understand that the polypeptide is present at a level that is at least 1.2 times higher than that present in normal tissue; preferably at least 2 times and more preferably at least 5 or 10 times the level present in normal tissue.

T cells can be obtained by methods known in the art, such as those described above.

Protocols for this so-called adoptive T cell transfer are well known in the art. Reviews can be found in Gattioni et al. and Morgan et al. (Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention involves the use of peptides in complex with MHC to obtain a T-cell receptor, the nucleic acid of which is cloned and introduced into a host cell, preferably a T cell. This engineered T cell can then be administered to a patient for cancer treatment.

Any molecule according to the invention, i.e. a peptide, nucleic acid, antibody, expression vector, cell, activated T cell, T cell receptor, or nucleic acid encoding the same, is useful in the treatment of disorders characterized by cells escaping the immune response. Therefore, any molecule of the present invention can be used as a drug or in the manufacture of a drug. The molecule may be used alone or in combination with other molecule(s) of the invention or known molecule(s).

The present invention also provides a kit including:

(a) a container that contains a pharmaceutical composition as described above, in solution or lyophilized form;

(b) optionally, a second container containing a diluent or reconstitution solution for the lyophilized formulation; and (c) optionally, instructions for (i) using the solution or (ii) reconstituting the solution and/or using the lyophilized formulation.

In addition, the kit may also include one or more (iii) buffers, (iv) diluents, (v) filters, (vi) needles, or (v) syringes. The container is preferably a bottle, vial, syringe or tube; and it can be a reusable container. The pharmaceutical composition is preferably lyophilized.

The kit according to the present invention preferably includes the freeze-dried composition of the present invention in a suitable container and instructions for its reconstitution and/or its use. Suitable containers include, for example, bottles, vials (eg dual chamber vials), syringes (such as dual chamber syringes) and test tubes. The container can be made from different materials such as glass or plastic. Preferably, the kit and/or container contains(s) instructions for use of the container or associated instructions that provide instructions for reconstitution and/or use. For example, the label may state that the lyophilized formulation must be reconstituted to the peptide concentrations described above. The label may further state that the formulation is being used or intended for subcutaneous administration.

The formulation container may be a refillable vial that allows repeated administration (eg, 2 to 6 injections) of the reconstituted formulation. The kit may further include a second container including a suitable diluent (eg sodium bicarbonate solution).

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After mixing the diluent and the lyophilized formulation, the final peptide concentration in the reconstituted formulation is preferably at least 0.15 mg/ml/peptide (=75 μg) and preferably not more than 3 mg/ml/peptide (=1500 μg). The kit may additionally include other materials desirable from a commercial and user point of view, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

Kits of the present invention may include a single container that contains a dosage form of pharmaceutical compositions in accordance with the present invention with or without other components (for example, other compounds or pharmaceutical compositions of these other compounds) or may have separate containers for each component.

The kit of the invention preferably includes a formulation of the invention packaged for use in combination with co-administration of a second compound (such as adjuvants (e.g. GM-CSF), chemotherapeutic agent, natural product, hormone or antagonist, anti-angiogenesis agent or angiogenesis inhibitor; apoptosis- inducing agent or chelator) or a pharmaceutical composition thereof. The components of the kit may be pre-mixed prior to administration to the patient, or each component may be in a separate container. The components of the kit may be provided as one or more liquid solutions, preferably an aqueous solution, more preferably a sterile aqueous solution. The components of the kit may also be provided in solid form which may be liquefied by the addition of suitable solvents, which are preferably provided in a different, separate container.

The therapeutic kit container may be a vial, tube, flask, bottle, syringe, or any other means containing a solid or liquid. Usually, if there is more than one component, the kit contains a second vial or other container, which allows for a separate administration. The kit may also contain another container for a pharmaceutically acceptable liquid. The treatment kit will preferably include an apparatus (eg, one or more needles, syringes, eyedroppers, pipettes, etc.) that allows the administration of the substances of the invention that are components of this kit.

The present formulation is suitable for administering the peptides by any suitable route, such as oral (enteral), nasal, ocular, subcutaneous, intradermal, intramuscular, intravenous, or transdermal. Preferably, the administration is s.c., and most preferably, s.c. with an infusion pump.

Since the peptides of the invention have been isolated from prostate tumors, the medicament of the invention is preferably used in the treatment of prostate tumors.

In addition, the present invention further relates to a method for producing a personalized pharmaceutical preparation for an individual patient, including the production of a pharmaceutical composition comprising at least one peptide selected from a repository of pre-screened TUMAP peptides, where at least one peptide used in the pharmaceutical composition, selected for its suitability for the individual patient. In one embodiment, the pharmaceutical composition is a vaccine. The method can be adapted to obtain T-cell clones for further use, for example in the isolation of TCR or soluble antibodies or other therapies.

A personalized pharmaceutical product refers to therapeutic agents designed specifically for an individual patient that will be used exclusively for the treatment of that patient, including actively personalized cancer vaccines and adoptive cell therapies using the patient's autologous tissue.

In the context of the present invention, the term storage refers to a group or set of peptides that have previously been screened for immunogenicity and/or over-presentation in a particular type of tumor. The concept of storage does not imply that the specific peptides included in the vaccine were prepared in advance and stored in a real room, although this possibility is also taken into account. Consideration is definitely given to the fact that the peptides may be made de novo for each individualized vaccine produced, or may be pre-produced and stored. The repository (eg, in the form of a data bank) consists of tumor-associated peptides that are highly overexpressed in the tumor tissue of prostate cancer patients with different HLA-A, HLA-B, and HLA-C alleles. It may contain peptides associated with MHC class I and MHC class II molecules, or extended peptides associated with MHC class I molecules. In addition to tumor-associated peptides collected from tissues of several prostate tumors, the repository may contain marker peptides associated with HLA-A*02 and HLA-A*24. These peptides allow a quantitative comparison of the intensity of the T-cell immune response induced by the TUMAP peptides, and therefore allow an important conclusion about the ability of the vaccine to induce antitumor responses. Second, they function as important positive control peptides,

- 42 041120 derived from non-self antigen in case the patient does not experience vaccine-induced T-cell responses to TUMAP peptides derived from self antigens. And thirdly, it may allow conclusions to be drawn regarding a patient's immunocompetent status.

TUMAP peptides for storage were identified using an integrated functional genomics approach combining gene expression analysis, mass spectrometry and T-cell immunology (XPresident®). This approach ensures that only those TUMAP peptides that are actually present in a large percentage of tumors, but are not or only minimally expressed on normal tissue, are selected for further analysis. For the purpose of the initial selection of peptides, tissue samples from prostate cancer, as well as benign prostatic hyperplasia of patients and blood from healthy donors were analyzed in stages:

1. HLA ligands from tumor material were identified by mass spectrometry.

2. Whole genome messenger ribonucleic acid (mRNA) expression analysis was used to identify overexpressed genes in malignant tissue (prostate cancer) compared to a number of normal organs and tissues.

3. Identified HLA ligands were compared with gene expression data. Peptides presented in excess or selectively presented on the tumor tissue, preferably encoded by the selectively expressed or overexpressed genes identified in step 2, were considered suitable TUMAP candidates for a multipeptide vaccine.

4. The literature was reviewed to identify additional evidence supporting the relevance of the peptides identified as TUMAP.

5. The relevance of overexpression at the mRNA level was confirmed by re-detection of the TUMAP peptides selected in step 3 on tumor tissue and absence (or infrequent detection) on healthy tissues.

6. In order to assess whether in vivo induction of T cell responses by the selected peptides could be feasible, in vitro immunogenicity assays were performed using human T cells from healthy donors as well as from patients with prostate cancer.

In one aspect, the peptides are pre-screened for immunogenicity prior to inclusion in the repository. By way of example, and not limitation, the immunogenicity of the peptides included in the repository is determined by a method involving in vitro T cell priming by restimulating healthy donor CD8+ T cells with artificial antigen presenting cells loaded with peptide-MHC complexes and anti-CD28 antibodies. .

This method is preferred for rare cancers and patients with a rare expression profile. In contrast to the multipeptide cocktails with a constant composition already developed at the present time, the repository makes it possible to achieve a significantly higher correspondence between the actual expression of antigens in the tumor and the composition of the vaccine. Selected single peptides or combinations of several ready-to-use peptides will be used for each patient in a multi-targeted approach. Theoretically, an approach based on selecting, for example, 5 different antigenic peptides from a library of 50 copies would already lead to approximately 17 million possible drug formulations (MPs).

In one aspect, peptides for inclusion in a vaccine are selected for their suitability for the individual patient based on the method of the present invention, as described herein or as set forth below.

HLA phenotype, transcriptomics and proteomics data are collected from tumor material and patient blood samples to identify the most appropriate peptides for each patient, which include both stored TUMAP peptides and those unique to the patient (i.e., mutated). The peptides selected will be those that are selectively or overexpressed in patient tumors and, where possible, exhibit strong in vitro immunogenicity when assayed with individual patient PBMCs.

Preferably, the peptides included in the vaccine are identified by a method comprising (a) identifying tumor-associated peptides (TUMAP) presented by an individual patient's tumor sample; (b) comparing the peptides identified in step (a) with a peptide repository (databank) as described above; and (c) selecting at least one peptide from the repository (databank) that correlates with a tumor-associated peptide identified in the patient. For example, TUMAP peptides presented by a tumor sample are identified by (a1) comparing expression data in the tumor sample with normal tissue data corresponding to the tissue type of the tumor sample to identify proteins that are overexpressed or aberrantly in the tumor sample; and (a2) correlating expression data and sequences of MHC ligands associated with MHC class I and/or II molecules in the tumor sample in order to identify MHC ligands that are derived from proteins overexpressed or aberrantly expressed by the tumor. Preferably, the MHC ligand sequences are identified by eluting the associated peptides from MHC molecules isolated from

- 43 041120 tumor sample, and sequencing of the eluted ligands. Preferably, the tumor sample and normal tissue are from the same patient.

In addition, or alternatively, by selecting peptides using a database model, TUMAP peptides can be identified de novo in a patient and then included in a vaccine. As one example, candidate TUMAP peptides can be identified in a patient by (a1) comparing expression data in a tumor sample with normal tissue data corresponding to the tissue type of the tumor sample to identify proteins that are overexpressed or aberrantly in the tumor sample; and (a2) correlating expression data and sequences of MHC ligands associated with MHC class I and/or II molecules in the tumor sample in order to identify MHC ligands that are derived from proteins overexpressed or aberrantly expressed by the tumor. As another example, proteins having mutations that are unique to a tumor sample associated with the corresponding normal tissue of an individual patient can be identified, and TUMAP peptides that specifically target the mutation can be identified. For example, the genome of a tumor and the corresponding normal tissue can be sequenced using whole genome sequencing: to detect non-synonymous mutations in protein-coding regions of genes, genomic DNA and RNA are extracted from tumor tissues, and normal germline genomic DNA that does not have mutations is extracted from peripheral blood mononuclear cells ( IPC). The next generation sequencing (NGS) approach used is to resequence protein-coding regions (exome resequencing). For this purpose, exon DNA from human samples is fixed using manufacturer-supplied target fragment enrichment kits, followed by sequencing, for example, using the HiSeq2000 system (Illumina). In addition, tumor mRNA is sequenced to directly quantify gene expression and confirm that mutated genes are expressed in patient tumors. The resulting millions of sequences are read by software algorithms. The resulting list contains mutations and gene expression. Tumor-specific somatic mutations are determined by comparison with germline variants from PBMC and prioritized. The de novo identified peptides can then be tested for immunogenicity as described above for storage, and TUMAP candidate peptides having suitable immunogenicity are selected for inclusion in the vaccine.

In a separate embodiment, the peptides included in the vaccine are identified by (a) identifying tumor-associated peptides (TUMAP) presented by an individual patient's tumor sample by the methods described above; (b) comparing the peptides identified in step (a) with a repository of peptides as described above that have previously been screened for immunogenicity and over-presentation in tumors compared to corresponding normal tissues; (c) selecting at least one peptide from the repository that correlates with a tumor-associated peptide identified in the patient; and (d) optionally, selecting at least one peptide identified de novo in step (a) to confirm its immunogenicity.

In a separate embodiment, the peptides included in the vaccine are identified by (a) identifying tumor-associated peptides (TUMAP) presented by an individual patient's tumor sample; and (b) selecting at least one peptide identified de novo in step (a) and confirming its immunogenicity.

Once peptides have been selected for a personalized peptide-based vaccine, the vaccine is made. The vaccine is preferably a liquid dosage form consisting of individual peptides dissolved in DMSO at a concentration of 20-40%, preferably about 30-35%, such as about 33% DMSO.

Each peptide included in the product is dissolved in DMSO. The concentration of individual peptide solutions should be selected depending on the number of peptides to be included in the product. Solutions of individual peptides in DMSO are mixed in equal proportions to obtain a solution containing all the peptides intended to be included in the product, with a concentration of ~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 sterile filter with a pore size of 0.22 μm. Get the final bulk solution.

The final bulk solution is filled into vials and stored at -20°C until use. One vial contains 700 µl of a solution containing 0.578 mg of each peptide. Of these, 500 μl (approximately 400 μg per peptide) will be administered by intradermal injection.

In addition, the peptides of the present invention are useful not only for cancer treatment, but also as diagnostic agents. Since the peptides were derived from prostate tumor cells and since these peptides were found not to be present or present in small amounts in normal tissues, these peptides can be used to diagnose the presence of a tumor.

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The presence of the claimed peptides on tissue biopsy specimens and in blood samples may assist the pathologist in making a diagnosis of cancer. Detection of specific peptides by antibodies, mass spectrometry, or other methods known in the art can give the pathologist evidence that the tissue sample is malignant, or inflamed, or diseased in general, or can be used as a biomarker for prostate cancer. The presence of groups of peptides may allow the classification or subclassing of affected tissues.

Detection of peptides in diseased tissue samples may allow judgments to be made about the benefit of immune-targeting therapy, particularly if T-lymphocytes are known or expected to be involved in the mechanism of action. Lack of MHC expression is a well-described mechanism by which infected or malignant cells evade immune control. Thus, the presence of peptides indicates that this mechanism is not used by the analyzed cells.

The peptides of the present invention can be used in the analysis of lymphocyte responses to the action of these peptides, such as T-cell responses or responses in the form of antibodies to the peptide or peptide complexed with MHC molecules. These responses of lymphocytes can be used as prognostic markers for deciding on further stages of therapy. These responses can also be used as surrogate response markers in immunotherapeutic approaches aimed at inducing lymphocyte responses by various means, eg protein vaccination, nucleic acids, autologous materials, adoptive lymphocyte transfer. In settings where gene therapy is being performed, lymphocyte responses to peptides can be analyzed to assess side effects. Monitoring of lymphocyte responses may also be a valuable tool for follow-up studies after transplantation, for example, to detect host-versus-graft and graft-versus-host reactions.

The present invention will be described below by way of examples which describe its preferred embodiments with reference to the accompanying figures, without however limiting the scope of the invention. In accordance with the purposes of the present invention, all cited sources are included in this description in its entirety by reference.

Brief description of the figures

In FIG. 1A-1C show over-presentation of various peptides in normal tissues (white bars) and prostate cancer and benign prostatic hyperplasia tissues (black bars).

In FIG. 1D, 1E show all cell lines, normal tissues and cancer tissues in which single peptides were detected (SLLSHQVLL (A*02) (SEQ ID NO: 20) and SLLSHQVLL (A*24) (SEQ ID NO: 20)).

Fig. 1A: gene: OR51E2, peptide: VTAQIGIVAV (A*02; SEQ ID NO: 1) - tissues from left to right: 1 adipose tissue, 3 adrenal glands, 6 arteries, 5 bone marrows, 7 brains, 3 mammary glands, 1 central nerve, 13 colons, 1 duodenum, 8 esophagus, 2 gallbladders, 5 hearts, 16 kidneys, 21 livers, 46 lungs, 4 lymph nodes, 4 white blood cell samples, 4 ovaries, 7 pancreas, 4 peripheral nerves, 1 peritoneum , 3 pituitary glands, 4 placentas, 3 pleurae, 6 rectums, 7 salivary glands, 4 skeletal muscles, 6 skin samples, 2 small intestines, 4 spleens, 7 stomachs, 4 testes, 3 thymus glands, 4 thyroid glands, 10 tracheae, 3 ureters, 6 bladders, 2 uterus, 2 veins, 3 prostates, 44 prostate tumors. The peptide has also been found on small cell lung cancer cells (not shown).

Fig. 1B: gene: MANSC1, peptide: KMDEASAQLL (A*02; SEQ ID NO: 14) - tissues from left to right: 1 adipose tissue, 3 adrenal glands, 6 arteries, 5 bone marrows, 7 brains, 3 mammary glands, 1 central nerve, 13 colons, 1 duodenum, 8 esophagus, 2 gallbladders, 5 hearts, 16 kidneys, 21 livers, 46 lungs, 4 lymph nodes, 4 white blood cell samples, 4 ovaries, 7 pancreas, 4 peripheral nerves, 1 peritoneum , 3 pituitary glands, 4 placentas, 3 pleurae, 6 rectums, 7 salivary glands, 4 skeletal muscles, 6 skin samples, 2 small intestines, 4 spleens, 7 stomachs, 4 testes, 3 thymus glands, 4 thyroid glands, 10 tracheae, 3 ureters, 6 bladders, 2 uterus, 2 veins, 3 prostates, 44 prostate tumors.

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 mammary gland, 5 colons, 1 heart, kidneys, 9 livers, 9 lungs, 3 pancreas, 1 pituitary gland, 2 rectums, 3 skin samples, 1 spleen, 12 stomachs, 1 thymus, 2 uterus, 40 prostate tumors. The peptide has also been found on non-small cell lung cancer cells (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 bladder cancer). The set of normal tissues was the same as in A-B, but the peptide was not detected on any normal tissues.

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Fig. 1E: gene: KIAA1244, peptide: QYGKDFLTL (A*22; SEQ ID NO::33) - tissues from left to right:

benign prostatic hyperplasia tissues, 3 normal tissues (1 liver, lung, 1 rectum), 31 cancerous tissues (5 brain cancers, 4 liver cancers, 15 lung cancers, prostate cancers). The set of normal tissues was the same as in C, but tissues in which the peptide was not detected are not shown.

In FIG. 1F-1K shows over-presentation of various peptides in normal tissues (white bars) and prostate cancer and benign prostatic hyperplasia tissues (black bars).

In FIG. 1L-1S shows all cell lines, normal tissues and cancerous tissues in which 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 mammary glands, 1 central nerve, 13 colons, 1 duodenum, 8 esophaguses, 2 gallbladders, 5 hearts, 16 kidneys, 4 white blood cell samples, 21 livers, 46 lungs, 4 lymph nodes, 3 ovaries, 7 pancreas, 4 peripheral nerves, 1 peritoneum , 3 pituitary glands, 2 placentas, 3 pleurae, 6 rectums, 7 salivary glands, 4 skeletal muscles, 5 skin samples, 2 small intestines, 4 spleens, 7 stomachs, 4 testes, 3 thymus glands, 4 thyroid glands, 9 tracheae, 3 ureters, bladders, 2 uteruses, 2 veins, 3 prostates, 33 prostate cancer tissues and 10 benign prostatic 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 mammary glands, 1 central nerve, 13 colons, 1 duodenum, 8 esophaguses, 2 gallbladders, 5 hearts, 16 kidneys, 4 white blood cell samples, 21 livers, 46 lungs, 4 lymph nodes, 3 ovaries, 7 pancreas, 4 peripheral nerves, 1 peritoneum , 3 pituitary glands, 2 placentas, 3 pleurae, 6 rectums, 7 salivary glands, 4 skeletal muscles, 5 skin samples, 2 small intestines, 4 spleens, 7 stomachs, 4 testes, 3 thymus glands, 4 thyroid glands, 9 tracheae, 3 ureters, 6 bladders, 2 uteruses, 2 veins, 3 prostates, 33 prostate cancer tissues and 10 benign prostatic 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 mammary gland, 5 large intestines, 1 heart, 13 kidneys, 9 livers, 9 lungs, 3 pancreas, 1 pituitary, 2 rectums, 3 skin samples, 1 spleen, 12 stomachs, 1 thymus, 2 uterus, 37 prostate cancer tissue, and 3 benign prostatic hyperplasia tissue.

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 mammary gland, 5 colons, 1 heart, 13 kidneys, 9 livers, 9 lungs, 3 pancreas, 1 pituitary, 2 rectums, 3 skin samples, 1 spleen, 12 stomachs, 1 thymus, 2 uterus, 37 prostate cancer tissue, and 3 benign prostatic hyperplasia tissue.

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 mammary glands, 1 central nerve, 13 colons, 1 duodenum, 8 esophaguses, 2 gallbladders, 5 hearts, 16 kidneys, 4 white blood cell samples, 21 livers, 46 lungs, 4 lymph nodes, 3 ovaries, 7 pancreas, 4 peripheral nerves, 1 peritoneum , 3 pituitary glands, 2 placentas, 3 pleurae, 6 rectums, 7 salivary glands, 4 skeletal muscles, 5 skin samples, 2 small intestines, 4 spleens, 7 stomachs, 4 testes, 3 thymus glands, 4 thyroid glands, 9 tracheae, 3 ureters, 6 bladders, 2 uteruses, 2 veins, 3 prostates, 33 prostate cancer tissues and 10 benign prostatic 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 mammary gland, 5 colons, 1 heart, 13 kidneys, 9 livers, 9 lungs, 3 pancreas, 1 pituitary, 2 rectums, 3 skin samples, 1 spleen, 12 stomachs, 1 thymus, 2 uterus, 37 prostate cancer tissue, and 3 benign prostatic hyperplasia tissue.

Fig. 1L: gene: GREB1, peptide: SMLGEEIQL (A*02; SEQ ID NO: 2) - tissues from left to right: 1 benign prostatic hyperplasia (BPH) tissue, 3 cell lines (3 skins), 1 normal tissue (1 uterus) , 26 cancerous tissues (2 breast cancers, 2 liver cancers, 1 lung cancer, 1 ovarian cancer, 13 prostate cancers, 6 skin cancers, 1 uterine cancer.

Fig. 1M: gene: TRPM8, peptide: ALLTFVWKL (A*02; SEQ ID NO: 4) - tissues from left to right: 3 benign prostatic hyperplasia (BPH) tissues, 13 cancerous 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 prostatic hyperplasia (BPH) tissues, 10 cancerous tissues (1 brain cancer, 8 prostate cancers, 1 skin cancer).

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Fig. 1O: gene: MANSC1, peptide: KMDEASAQLL (A*02; SEQ ID NO: 16) - tissues from left to right:

cancerous tissue (20 prostate cancers, 1 bladder cancer).

Fig. 1P: gene: C6orf132, peptide: RYGSPINTF (A*24; SEQ ID NO: 29) - tissues from left to right: 4 benign prostatic hyperplasia (BPH) tissues, 54 cancerous tissues (1 liver cancer, 24 lung cancers, 26 prostate cancers glands, 3 stomach cancers).

Fig. 1Q: gene: ITGA7, peptide: AFSPDSHYLLF (A*24; SEQ ID NO: 34) - tissues from left to right: 5 benign prostatic hyperplasia (BPH) tissues, 44 cancerous tissues (10 brain cancers, 1 kidney cancer, 4 cancers liver, 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 prostatic hyperplasia (BPH) tissues, 59 cancerous tissues (36 lung cancers, 14 prostate cancers, 9 cancers of the stomach).

Fig. 1S: gene: SLC30A4, peptide: ALGDLVQSV (A*02; SEQ ID NO: 52) - tissues from left to right: 1 benign prostatic hyperplasia (BPH), 11 cancerous tissues (1 lymph node cancer, 9 prostate cancers, 1 skin cancer).

In FIG. 2A-2E are exemplary expression profiles (relative expression versus normal kidney) of the original genes of the present invention that are highly overexpressed or exclusively expressed in prostate cancer cells in a panel of normal tissue samples and 20 prostate cancer samples. Tissues from left to right: adrenal gland, artery, bone marrow, brain (whole), mammary gland, colon, esophagus, heart, kidney (three repeated measurements), leukocytes, liver, lungs, lymph node, ovary, pancreas, placenta , prostate, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus, thyroid, bladder, cervix, uterus, vein, 20 prostate cancer specimens.

Fig. 2A: NEFH.

Fig. 2B: ABCC4.

Fig. 2C: RAB3B.

Fig. 2D: OR51E2.

Fig. 2E: KLK2.

In FIG. 3 shows typical immunogenicity data: flow cytometric analysis results after peptide-specific staining of the multimers. A) TYIGQGYII (FKBP10; SEQ ID NO: 42); B) IYTRVTYYL (TPSB2, TPSAB1; SEQ ID NO: 35).

In FIG. 4A-C are selected results of in vitro peptide-specific responses of CD8 ⁺ T cells from a healthy HLA-A*02+ donor. CD8 ⁺ T cells were primed with artificial APCs stimulated with monoclonal antibodies to CD28 and HLA-A*02 in complex with a peptide with the sequence of SEQ ID NO: 1 (A, left panel), a peptide with the sequence of SEQ ID NO: 3 (B , left panel) or the peptide of SEQ ID NO: 5 (C, left panel), respectively. After three cycles of stimulation, detection of peptide-reactive cells was performed by double staining of multimers using A*02/SEQ ID NO: 1 (A), A*02/SEQ ID NO: 3 (B) or A*02/SEQ ID NO: 5 (C). The right sections (A, B and C) represent control staining of cells stimulated with irrelevant complexes of the peptide and A*02. CD8+ lymphocytes were isolated from individual viable cells by gating. Boole's gates helped eliminate false positive responses detected with multimers specific for different peptides. The frequency of detection of specific multimer-positive cells among CD8+ lymphocytes is indicated.

In FIG. 5A, B are representative results of in vitro responses of peptide-specific CD8 ⁺ T cells from a healthy HLA-A*24+ donor. CD8+ T cells were primed with artificial APC stimulated with anti-CD28 and HLA-A*24 monoclonal antibodies in combination with a peptide of SEQ ID NO: 24 (A, left panel) or a peptide of SEQ ID NO: 27 (B, left section) respectively. After three cycles of stimulation, the detection of cells reactive with the peptide was performed using double staining of multimers A*24/Seq ID No 24 (A) or A*24/SEQ ID NO: 27 (B). The right sections (A and B) represent control staining of cells stimulated with irrelevant complexes of the peptide and A*24. CD8+ lymphocytes were isolated from individual viable cells by gating. Boole's gates helped eliminate false positive responses detected with multimers specific for different peptides. The frequency of detection of specific multimer-positive cells among CD8+ lymphocytes is indicated.

Examples

Example 1

Identification and quantification of tumor-associated peptides presented on the cell surface.

Fabric samples.

Patient prostate tumor tissues were obtained from Asterand (Detroit,

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USA and Royston, Hertfordshire, UK); BioServe (Beltsville, MD, USA); Geneticist Inc. (Glendale, California, USA); Indivumed GmbH (Hamburg, Germany); University Hospital Heidelberg; Saint Savas clinics, Athens, Greece; University Hospital Tübingen. Normal tissues were obtained from Asterand (Detroit, USA and Royston, Hertfordshire, UK); Bio-Options Inc., California, USA; BioServe, Beltsville, Maryland, USA; Geneticist Inc., Glendale, California, USA; University Hospital of Geneva; University Hospital Heidelberg; Kyoto Prefectural Medical University (KPUM); Osaka University (OCU); University Hospital of Munich; ProteoGenex Inc., Culver City, California, USA; Tissue Solutions Ltd., Glasgow, UK; University Hospital Tübingen. Written informed consent was obtained from all patients prior to surgery or autopsy. Immediately after removal, tissues were blast frozen and stored at -70° C. or below until TUMAP peptides were isolated.

Isolation of HLA peptides from tissue samples.

Pools of HLA peptides from shock-frozen tissue samples were obtained by immunoprecipitation from dense tissues according to a slightly modified protocol (Falk et al., 1991; Seeger et al., 1999) using the HLA-A*02-specific antibody BB7.2 or HLA-A, -B, -C-specific antibody W6/32, CNBr-activated sepharose, acid treatment and ultrafiltration.

Mass spectrometric analysis.

The resulting pools of peptide-HLA complexes were separated according to their hydrophobicity by reverse phase chromatography (nanoAcquity UPLC system, Waters) and the eluted peptides were analyzed on LTQ-velos and -fusion hybrid mass spectrometers (ThermoElectron) equipped with an ESI source. Peptide pools were applied directly to an analytical microcapillary fused silica column (75 µm w/d x 250 mm) with 1.7 µm C18 reverse phase sorbent (Waters) using a flow rate of 400 nl/min. The peptides were then separated using a two step 180 minute binary gradient from 10 to 33% solvent B at a flow rate of 300 nl/min. Solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) were used to create the gradient. A gold-plated glass capillary (PicoTip, New Objective) was used to inject into the nano-ESI source. The LTQ-Orbitrap mass spectrometers operated in the information-dependent mode using the TOP5 strategy. Briefly, the scan cycle started with a full high precision mass scan on the Orbitrap spectrometer (R=30000), followed by an MS/MS scan also on the Orbitrap (R=7500) on 5 particularly abundant precursor ions with dynamic exclusion of previously selected ions. Tandem mass spectra were interpreted using the SEQUEST program with additional manual control. The identified peptide sequence was confirmed by comparing the resulting fragmentation pattern of the natural peptide with the fragmentation pattern of a synthetic control peptide with identical sequence.

Relative quantification by LC/MS without isotopic labels was performed by counting ions, i.e. using extraction and analysis of LC/MS results (Mueller et al., 2007). This method is based on the assumption that the LC/MS signal peak area of a peptide correlates with its concentration in the sample. The extracted features were processed by deconvolutional state-of-charge analysis and retention time equalization (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC/MS spectral results were cross-referenced with sequence identification results to combine quantitative data from different samples and tissues into peptide presentation profiles. Quantitative data were normalized using a two-level system according to the central trend in order to account for variability within technical and biological repeated measurements. Thus, each identified peptide can be associated with quantitative data, allowing for relative quantification of samples and tissues. In addition, all quantitative data obtained for the candidate peptides were manually checked to ensure data consistency and to verify the accuracy of the automatic assay method. For each peptide, a presentation profile was calculated showing the average level of presentation in the sample as well as replicate variability. The profile compares prostate cancer samples and benign prostatic hyperplasia samples with a background level of normal tissue samples. Presentation profiles of exemplary over-presented peptides are shown in FIG. 1. The performance of the presentation of individual peptides are shown in table. 12 and 13.

In table. 12 shows HLA-A*02 binding 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 (+).

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Table 12

Presentation metrics

SEQID No. Subsequence Presentation of the peptide 1 VTAQIGIVAV +++ 2 SMLGEEIQL +++ 3 HLLEDIAHV +++ 4 ALLTFVWKL +++ 5 KIFSRLIYI +++ 6 ALLESRVNL +++ 7 TLLQWGWSV +++ 8 LLDFSLADA +++ 9 GMLNEAEGKAIKL ++ 10 TLWRGPVW +++ eleven YLEEECPAT +++ 12 SLNEIAFL +++ 14 KMDEASAQLL +++ 15 KMDEASAQLLA +++ 17 RLGIKPESV ++ 18 GLSEFTEYL +++ 19 LLPPPPLLA +++ 20 SLLSHQVLL +++ 21 YLNDSLRHV +++ 22 SLYDSIAFI +++ 23 AVAGADVIITV + 40 RTFJPTYGL ++

The table shows 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 (++) or presented on tumors compared to a panel of normal tissues (+).

Table 13 ______Presentation Metrics______

SEQID No. Subsequence Presentation of the peptide 24 SYNDALLTF +++ 25 IYEPYLAMF + 26 RYADDTTFTPAF +++ 27 GYLQGLVSF +++ 28 YYAKEIHKF +++ 29 RYGSPINTF +++ thirty 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

Determination of the expression profile of the genes encoding the peptides of the invention.

The over-presentation or specific presentation of a peptide on tumor cells compared to normal cells is sufficient for its suitability in immunotherapy, and some peptides are tumor-specific despite the presence of their parent proteins also in normal tissues. However, the identification of mRNA expression profiles brings an additional level of safety in the selection of peptide targets for immunotherapy. Especially in the case of therapeutic methods with a high safety risk, such as affinity matured TCR, the ideal target peptide will be derived from a protein that is unique to the tumor and not found on normal tissues.

Sources and preparation of RNA.

Surgically removed tissue preparations were provided by the various organizations listed above (see Example 1) after receiving a written informed consent form from each patient. Tumor tissue preparations were flash-frozen after surgery and subsequently homogenized with a mortar and pestle in liquid nitrogen. Total RNA was prepared from these samples using TRI reagent (Ambion, Darmstadt, Germany) followed by RNeasy purification (QIAGEN, Hilden, Germany); both methods were carried out in accordance with the manufacturer's instructions.

Total RNA from healthy human tissues was purchased (Ambion, Huntington, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam, The Netherlands; BioChain, Hayard, CA, USA). The RNA of several individuals (from 2 to 123 individuals) was mixed in such a way that the RNA of each individual had an equal weight.

The 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).

Experiments with microchips.

Gene expression analysis of all RNA samples from tumor and normal tissue was performed using Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0 oligonucleotide microarrays (Affymetrix, Santa Clara, CA, USA). All steps were completed in accordance with the Affymetrix guidelines. Briefly, double-stranded cDNA was synthesized from 5-8 μg total RNA using Superscript RTII (Invitrogen) and oligo-dT-T7 primer (MWG Biotech, Ebersberg, Germany) as described in the manual. In vitro transcription was performed using the BioArray High Yield RNA Transcript Labeling Kit (ENZO Diagnostics, Inc., Farmingdale, NY, USA) for U133A chips or the GeneChip IVT Labeling Kit (Affymetrix) for U133 Plus chips. 2.0 followed by fragmentation, cRNA hybridization and staining with streptavidin phycoerythrin and biotinylated anti-streptavidin antibody (Molecular Probes, Leiden, The Netherlands). Images were scanned on an Agilent 2500A GeneArray Scanner (U133A) or Affymetrix Gene-Chip Scanner 3000 (U133 Plus 2.0) and data was analyzed in GCOS software (Affymetrix) using default settings for all parameters. For normalization, 100 service genes provided by Affymetrix were used. Relative expression values were calculated from the ratios of the logarithms of the recorded signals obtained by the computer program, and the value for the normal kidney sample was arbitrarily set to 1.0. Representative expression profiles of the parent genes of the present invention that are highly overexpressed or exclusively expressed in prostate cancer cells are shown in FIG. 2A-2E. The expression levels of other individual genes are shown in Table 1. 14.

In table. 14 shows peptides derived from genes that are very highly overexpressed in tumors compared to a panel of normal tissues (+++), highly overexpressed in tumors compared to a panel of normal tissues (++), or overexpressed in tumors compared to a panel of normal tissues (+).

Table 14

Presentation metrics

SEQ ID NO Subsequence gene expression 1 VTAQIGIVAV ++ 2 SMLGEEIQL ++ 3 HLLEDIAHV +++ 4 ALLTFVWKL + 5 KIFSRLIYI + 7 TLLQWGWSV ++ eleven YLEEECPAT + 19 LLPPPPLLA + 24 SYNDALLTF + 25 IYEPYLAMF + 26 RYADDTTFTPAF ++ 28 YYAKEIHKF + 44 VYNTVSEGTHF ++

Example 3

In vitro immunogenicity for peptides presented by MHC class I.

To obtain information on the immunogenicity of the TUMAP peptides of the present invention, Applicants conducted in vitro T cell priming studies based on repeated stimulations of CD8+ T cells with artificial antigen presenting cells (iAPCs) loaded with peptide-MHC complexes and an anti-CD28 antibody. Thus, Applicants could show for some selected immunogenicity of the HLA-A*0201 and HLA-A*24 restricted TUMAP peptides of the invention, demonstrating that these peptides are T-cell epitopes against which humans have CD8+ T- precursor cells (Tables 15A, 15B).

Priming of CD8+ T cells in vitro.

In order to perform in vitro stimulations with artificial antigen-presenting cells loaded with a peptide-MHC complex (rMHC) and an anti-CD28 antibody, we first isolated CD8+ T cells from a fresh HLA-A*02 leukapheresis product by positive selection using CD8 microspheres (Miltenyi Biotec, Bergisch Gladbach, Germany). The blood was obtained from healthy donors (after signing an informed consent form) from the University Hospital Mannheim, Germany.

PBMCs and isolated CD8+ lymphocytes were incubated prior to use in T-cell media

- 50 041120 (TCM) consisting of RPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat-inactivated human AB serum (PAN-Biotech, Eidenbach, Germany), 100 U/ml penicillin/100 µg/ml streptomycin ( Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro, Oberdorla, Germany) and 20 μg/ml gentamicin (Cambrex). 2.5 ng/ml IL-7 (PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma, Nuremberg, Germany) were also added at this stage to the TCM medium.

Preparation of rMHC and anti-CD28 antibody-coated microspheres, T cell stimulations and readings were performed on a well established in vitro system using four different rMHC molecules for each stimulation cycle and eight different rMHC molecules for each reading cycle.

Purified co-stimulatory mouse IgG2a to human CD28 Ab 9.3 (Jung et al., 1987) was chemically biotinylated using sulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer (Perbio, Bonn, Germany). The microspheres used were 5.6 μm polystyrene particles coated with streptavidin (Bangs Laboratories, Illinois, USA).

The pMHC used for positive and negative controls were A*O2O1/MLA-OO1 (ELAGIGILTV peptide (SEQ ID NO: 60) from modified Melan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5 , SEQ ID NO: 61), respectively.

800,000 microspheres/200 μl were added to the wells of a 96-well plate in the presence of 4x12.5 ng of another biotinylated pMHC complex, washed and then 600 ng of biotinylated anti-CD28 antibody was added in a volume of 200 μl. Stimulation was performed in 96-well plates by co-incubation of 1x10 ⁶ CD8+ T cells with 2x105 washed coated microspheres in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell) for 3 days at 37°C. Half of the medium was then replaced with fresh TCM medium supplemented with 80 U/ml IL-2 and incubation was continued for 4 days at 37°C. This stimulation cycle was performed a total of three times. Eight different pMHC molecules per run were used to read the pMHC multimers. A two-dimensional combinatorial encoding approach was used as previously described (Andersen et al., 2012) with minor modifications regarding labeling with 5 different fluorochromes. Finally, multimers were analyzed by staining cells with the NIR cell viability kit with Live/dead dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany), and fluorescent pMHC multimers. labels. A BD LSRII SORP cytometer equipped with suitable lasers and filters was used for analysis. Peptide-specific cells were calculated as a percentage of all CD8+ cells. The results of multimer analysis were evaluated using the FlowJo program (Tree Star, Oregon, USA). In vitro priming of specific multimer-positive CD8+ lymphocytes was assessed by comparison with negative control stimulations. Immunogenicity for a given antigen was determined if at least one well of a single healthy donor to be evaluated in vitro stimulated was found to contain a specific CD8+ T cell line after in vitro stimulation (i.e. when that well contained at least 1 % specific multimer-positive among CD8-positive T cells and the percentage of specific multimer-positive cells was at least 10-fold higher than the median negative control stimulations).

In vitro immunogenicity for prostate cancer peptides.

For the analyzed peptides associated with HLA class I molecules, in vitro immunogenicity could be demonstrated by generating peptide-specific T cell lines. Representative results of flow cytometric analysis after TUMAP-specific multimer staining for two peptides of the invention are shown in FIG. 3 along with their respective negative controls. The results for the five peptides according to the invention are summarized in table. 15A. Other results for 6 peptides according to the invention are summarized in table. 15V.

Table 15A

Immunogenicity in vitro of HLA class I peptides according to the invention

Seq ID Subsequence holes donors 17 RLGIKPESV ++ ++++ 29 RYGSPINTF + +++ 34 AFSPDSHYLLF + +++ 35 IYTRVTYYL ++ ++++ 42 TYIGQGYII + ++++

Selected results of in vitro immunogenicity experiments carried out by the Applicant for the peptides of the invention. + =<20%; ++ = 20-49%; +++ = 50-69% ++++ = >70%.

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Table 15B

Immunogenicity in vitro of HLA class I peptides according to the invention

SEQ ID Subsequence Positive wells [%] 1 VTAQIGIVAV ++++ 3 HLLEDIAHV ++ 5 KIFSRLIYI +++ 6 ALLESRVNL + 24 SYNDALLTF +++ 27 GYLQGLVSF ++

Selected results of in vitro immunogenicity experiments carried out by the Applicant for the peptides of the invention. + = <20%; ++ = 20-49%; +++ = 50-69% ++++ = >70%.

Example 4 Synthesis of peptides.

All peptides were synthesized by standard and conventional solid phase peptide synthesis using the Fmoc method. The identity and purity of each individual peptide was determined by mass spectrometry and analytical RP HPLC. Peptides were obtained as a white or off-white lyophilisate (trifluoroacetate salt) with >50% purity. All TUMAP peptides are preferably administered as trifluoroacetate or acetate salts, other salt forms are also possible.

Example 5

MHC binding assay.

Candidate peptides for T cell therapy according to the present invention were further tested for their ability to bind to MHC (affinity). Separate complexes of the peptide and MHC molecules were obtained using a ligand exchange reaction under the influence of UV radiation, in which a UV-sensitive peptide is cleaved under the influence of UV radiation, and an exchange product with the test peptide is obtained. Only candidate peptides that can effectively bind and stabilize peptide-responsive MHC molecules prevent dissociation of complexes with the MHC. To determine the yield of the product of the exchange reaction, an ELISA analysis was performed based on the detection of a light chain (b2t) of stabilized complexes with MHC. This analysis was performed essentially as described by Rodenko et al. (Rodenko et al., 2006).

MAXISorp (NUNC) 96-well plates were loaded overnight with 2 μg/ml streptavidin in PBS at room temperature, washed 4 times, and blocked for 1 hour at 37°C in 2% BSA containing blocking buffer. The refolded HLA-A*02:01/MLA-001 monomers served as a standard covering a range of 15-500 ng/mL. Monomeric peptide-MHC complexes after the exchange reaction under the influence of UV radiation were diluted 100-fold in blocking buffer. Samples were incubated for 1 h at 37°C, washed four times, incubated for 1 h at 37°C with 2 μg/ml horseradish peroxidase conjugated with anti-v2m antibody, washed again and detected with TMB solution; the reaction was stopped with NH2SO4. The absorption value was measured at a wavelength of 450 nm. Candidate peptides that exhibit a high exchange yield (preferably greater than 50%, most preferably greater than 75%) are generally preferred for the generation and production of antibodies or fragments thereof and/or T-cell receptors or fragments thereof, as they exhibit sufficient avidity for MHC molecules and prevent dissociation of MHC complexes.

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Table 16A

peptide exchange: + = >10%; ++ = >20%;+++ = >50;

++++ = >75%.

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Table 16B

Indicators of binding to MHC class I molecules

SEQ ID Subsequence Peptide exchange 1 VTAQIGIVAV ++++ 2 SMLGEEIQL ++++ 3 HLLEDIAHV ++++ 4 ALLTFVWKL ++++ 5 KIFSRLIYI +++ 6 ALLESRVNL ++++ 7 TLLQWGWSV ++ 9 GMLNEAEGKAIKL +++ 10 TLWRGPWV +++ eleven YLEEECPAT ++ 13 AMAP N HAW +++ 14 KMDEASAQLL ++ 15 KMDEASAQLLA +++ 16 KMDEASAQL +++ 20 SLLSHQVLL +++ 21 YLNDSLRHV ++ 22 SLYDSIAFI ++++ 49 SLFHPEDTGQV +++ 52 ALGDLVQSV +++ 53 YLLKDKGEYTL +++

The binding of class I HLA-restricted peptides to HLA-A*24 was assessed by the yield of peptide exchange: + = >10%; ++ = >20%;+++ = >50; ++++ = >75%.

Example 6

Absolute quantification of tumor-associated peptides presented on the cell surface.

The production of binding components, such as antibodies and/or TCR, is a laboratory process that can only be performed on a select number of targets. For tumor-associated and .beta.-specific peptides, selection criteria included, but were not limited to, exclusivity of presentation and density of the peptide presented on the cell surface. In addition to the isolation and relative quantification of the peptides described herein, Applicants analyzed the absolute copy number of the peptide per cell as described. Quantification of TUMAP copies per cell in solid tumor samples requires absolute quantification of the isolated TUMAP peptide, TUMAP isolation efficiency, and cell number of the analyzed tissue sample.

Quantification of the peptide using nano-LC-MS/MS.

For accurate quantitative determination of peptides by mass spectrometry, a calibration curve was constructed for each peptide using the internal standard method. The internal standard is a double isotopically labeled version of each peptide, i.e. during the synthesis of TUMAP, two isotopically labeled amino acids were introduced. It differs from the tumor-associated peptide only in its mass, but it does not differ in other physicochemical properties (Anderson et al., 2012). An internal standard was added in a known amount to each MS sample and all MS signals were normalized to the internal standard MS signal to smooth out potential technical variations in MS experiments.

Calibration curves were plotted in at least three different matrices, i.e. eluates of HLA peptide from natural samples, similar to conventional MS samples, and each preparation underwent two cycles of measurements on MS. For evaluation purposes, the MS signals were normalized with respect to the internal standard signal and the calibration curve was calculated using the logistic regression method.

To quantify tumor-associated peptides from tissue samples, a known amount of internal standard was also added to the corresponding samples; the MS signals were normalized to an internal standard and quantified using a peptide calibration curve.

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Efficiency of isolation of peptide/MHC complexes.

As with any protein purification process, the isolation of proteins from tissue samples involves a certain loss of protein of interest. To determine the recovery efficiency of the TUMAP peptide for all TUMAP peptides selected for absolute quantification, peptide-MHC complexes were obtained. To distinguish peptide-MHC complexes with standard additives from complexes with natural peptide, single isotope labeled versions of TUMAP peptides were used, i.e. during the synthesis of TUMAP, a single isotopically labeled amino acid was introduced. These complexes were added as a standard addition to freshly prepared tissue lysates; at the earliest possible point in the TUMAP peptide isolation process, and then captured as natural peptide-MHC complexes during subsequent affinity purification. Thus, measuring the recovery of singly labeled TUMAP peptides allows conclusions to be drawn regarding the recovery efficiency of individual natural TUMAP peptides.

Recovery efficiency was analyzed on a small number of samples and was comparable among these tissue samples. On the contrary, the efficiency of isolation of individual peptides is different. Therefore, it can be assumed that the recovery efficiency, although it was determined only for a limited number of tissue samples, can be extrapolated to any other tissue preparation. However, it is necessary to analyze each TUMAP peptide individually, since the extraction efficiency cannot be extrapolated from one peptide to others.

Determination of the number of cells in solid frozen tissue.

In order to determine the cell number of tissue samples subject to absolute quantification of the peptide, the inventors used the analysis of DNA content. This method is applicable to a wide range of samples from different origins and, most importantly, frozen samples (Alcoser et al., 2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During the isolation protocol, a tissue sample is processed until a homogeneous lysate is obtained, from which a small aliquot of the lysate is taken. An aliquot is divided into three portions from which DNA is isolated (QiaAmp DNA Mini Kit, Qiagen, Hilden, Germany). The total DNA content from each DNA extraction procedure is quantified by fluorescence-based DNA quantitation (Qubit dsDNA HS quantitation kit, Life Technologies, Darmstadt, Germany) in at least two repetitions.

In order to calculate the number of cells from aliquots of individual healthy blood cells, a standard curve for DNA is constructed with a range of given cell numbers. A standard curve is used to calculate the total cell content from the total DNA content for each DNA extraction procedure. The average total cell number of the tissue sample used to isolate the peptide is extrapolated from the known volume of the lysate aliquots and the total volume of the lysate.

Number of peptide copies per cell.

Using the data from the experiments listed above, the inventors calculated the copy number of TUMAP peptides per cell by dividing the total amount of peptide by the total number of cells in the sample and then dividing by the recovery efficiency. The cell copy number for the selected peptides is shown in Table 1. 17.

Table 17

Absolute number of copies

SEQ ID no. Peptide code Number of copies per cell (median) Number of samples 1 OR51E2-001 + 13 2 GREB-001 ++ 14 3 NEFH-001 ++ eleven 4 TRPM8-002 +++ 6 5 TRPM8-003 ++ 6 6 PDE11-001 + 10 24 TRPM8-004 +++ 15

- 55 041120

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

The table shows the results of the absolute quantitative determination of peptides in tumor samples. The median number of copies per cell is indicated for each peptide: + = <100; ++ = 100; +++ = >1,000; ++++ >10,000. The number of samples for which assessable, high quality MS data are available is indicated.

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Claims (16)

Whiteland, H. et al., Clin Exp. Metastasis 31 (2014): 909-920 Willcox, B. E. et al., Protein Sci. 8 (1999): 2418-2423 Willoughby, V. et al., Appl.lmmunohistochem.Mol.Morphol. 16 (2008): 344-348 Wilson, P. M. et al., J. Neurosci. 30 (2010): 8529-8540 World Cancer Report, (2014) Wu, J. P. et al., Asian J Androl 16 (2014): 710-714 Yamamoto, G. L. et al., J Med. Genet. 52 (2015): 413-421 Yang, Z. et al., J Virol. 81 (2007): 6294-6306 Ye, Q. et al., PLoS.ONE. 9 (2014): e103298 Yin, J. et al., Mol. Med. Rep. 8 (2013): 1630-1634 Yu, Y. P. et al., Urology 68 (2006): 578-582 Zaremba, S. et al., Cancer Res. 57 (1997): 4570-4577 Zhang, G. et al., Oncol Rep. (2014) Zhao, X. et al., Onco.Targets.Ther 7 (2014): 343-351 CLAIM 1. A peptide that binds to major histocompatibility complex (MHC) molecule(s) consisting of the amino acid sequence according to SEQ ID NO: 4, or a pharmaceutically acceptable salt thereof. 2. A peptide according to claim 1, wherein said peptide includes non-peptide bonds. 3. A peptide according to claim 1 or 2, wherein said peptide further comprises the N-terminal amino acids of the antigen-associated invariant chain (li) of HLA-DR. 4. T cell receptor reacting with an HLA ligand consisting of the amino acid sequence of SEQ ID NO: 4. 5. The T cell receptor of claim 4, wherein the ligand is part of a peptide-MHC complex. 6. An antibody that specifically recognizes a peptide according to any one of claims 1-3. 7. An antibody according to claim 6, characterized in that said peptide according to claim 1 is linked to an MHC molecule. 8. Nucleic acid encoding a peptide according to claim 1 or 2. 9. A recombinant host cell comprising a peptide according to claims 1 to 3 or a nucleic acid according to claim 8. 10. A method for producing a T cell receptor according to claim 4, wherein the method includes culturing a host cell that expresses an expression vector encoding a T cell receptor according to claim 4, and isolating said TCR from the host cell or its culture medium. 11. A method for producing activated T-lymphocytes in vitro, comprising contacting T-cells in vitro with antigen-loaded human MHC class I molecules expressed on the surface of a suitable antigen-presenting cell or an artificial construct that mimics an antigen-presenting cell for a period of time sufficient to activate these T cells in an antigen-specific manner, wherein said antigen is a peptide according to claim 1. 12. An activated T lymphocyte obtained by the method of claim 11, which selectively recognizes a cell that presents a polypeptide comprising the amino acid sequence specified in claim 1. 13. Pharmaceutical composition for the treatment of cancer, containing at least one active ingredient selected from the group consisting of a peptide according to any one of claims 1 to 3, a T-cell receptor according to claim 4, an antibody according to claim 6, a nucleic acid according to 8 or an activated T-lymphocyte according to claim 12, and a pharmaceutically acceptable carrier. 14. Use of a peptide according to any one of claims 1 to 3, a T-cell receptor according to claim 4, an antibody according to claim 6, a nucleic acid according to claim 8 or an activated T-lymphocyte according to claim 12 in the diagnosis and/or treatment of cancer . 15. Use according to claim 14, wherein said cancer is selected from the group of brain cancer, prostate cancer and other tumors that overexpress the protein from which the peptide is derived according to SEQ ID NO: 4. 16. A set for the treatment of cancer, including: (a) a container comprising a pharmaceutical composition containing a peptide according to any one of claims 1 to 3, a T-cell receptor according to claim 4, an antibody according to claim 6, a nucleic acid according to claim 8, or an activated -