US 20160280759A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0280759 A1 MAHR et al. (43) Pub. Date: Sep. 29, 2016
(54) NOVEL PEPTIDES AND COMBINATION OF Publication Classification PEPTDES FOR USE IN IMMUNOTHERAPY AGAINST VARIOUS TUMIORS (51) Int. Cl. C07K I4/74 (2006.01) (71) Applicant: immatics biotechnologies GmbH, C07K 6/28 (2006.01) Tuebingen (DE) C07K 14/725 (2006.01) CI2O I/68 (2006.01) (72) Inventors: Andrea MAHR, Tuebingen (DE); Toni A6139/00 (2006.01) WEINSCHENK, Aichwald (DE); (52) U.S. Cl. Oliver SCHOOR, Tuebingen (DE): CPC ...... C07K 14/70539 (2013.01); C12O 1/6886 Jens FRITSCHE, Dusslingen (DE): (2013.01); A61K 39/00II (2013.01); C07K Harpreet SINGH, Muenchen (DE): 14/7051 (2013.01); C07K 16/2833 (2013.01); Lea STEVERMANN, Tuebingen (DE) CI2O 2600/106 (2013.01); C12O 2600/158 (2013.01); A61K 2039/5158 (2013.01); C07K 2317/34 (2013.01) Assignee: immatics biotechnologies GmbH (73) (57) ABSTRACT The present invention relates to peptides, proteins, nucleic (21) Appl. No.: 15/083,075 acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immuno (22) Filed: Mar. 28, 2016 therapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can Related U.S. Application Data for example serve as active pharmaceutical ingredients of (60) Provisional application No. 62/139,189, filed on Mar. vaccine compositions that stimulate anti-tumor immune 27, 2015. responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histo (30) Foreign Application Priority Data compatibility complex (MHC), or peptides as Such, can also be targets of antibodies, soluble T-cell receptors, and other Mar. 27, 2015 (GB) ...... 1505.305.1 binding molecules. Patent Application Publication Sep. 29, 2016 Sheet 1 of 65 US 2016/0280759 A1
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NOVEL PEPTIDES AND COMBINATION OF and acute myeloid leukemia (AML), non-Small cell and PEPTIDES FOR USE IN IMMUNOTHERAPY small cell lung cancer (NSCLC and SCLC), respectively. AGAINST VARIOUS TUMIORS 0008 GB is the most common central nervous system malignancy with an age-adjusted incidence rate of 3.19 per CROSS REFERENCE TO RELATED 100,000 inhabitants within the United States. GB has a very APPLICATIONS poor prognosis with a 1-year survival rate of 35% and a 0001. This application claims the benefit of U.S. Provi 5-year survival rate lower than 5%. Male gender, older age sional Application Ser. No. 62/139,189, filed Mar. 27, 2015, and ethnicity appear to be risk factors for GB (Thakkar et al., and Great Britain Application No. 1505305.1, filed Mar. 27, 2014). 2015, the content of each of these applications is herein 0009 CLL is the most common leukemia in the Western incorporated by reference in their entirety. world where it comprises about one third of all leukemias. Incidence rates are similar in the US and Europe, and REFERENCE TO SEQUENCE LISTING estimated new cases are about 16,000 per year. CLL is more SUBMITTED ASA COMPLIANT ASCII TEXT common in Caucasians than in Africans, rarer in Hispanics FILE (..txt) and Native Americans and seldom in Asians. In people of Asian origin, CLL incidence rates are 3 fold lower than in 0002 Pursuant to the EFS-Web legal framework and 37 Caucasians (Gunawardana et al., 2008). The five-year over CFR SS 1.821-825 (see MPEP S2442.03(a)), a Sequence all survival for patients with CLL is about 79% (www. Listing in the form of an ASCII-compliant text file (entitled cancer.net/cancer-types/leukemia-chronic-lymphocytic-cll/ “sequence-listing-PCT.TXT, created on Mar. 28, 2016, and statistics). 44,970 bytes in size) is submitted concurrently with the 0010 Lung cancer is the most common type of cancer instant application, and the entire contents of the Sequence worldwide and the leading cause of death from cancer in Listing are incorporated herein by reference. many countries. Lung cancer is Subdivided into Small cell lung cancer and non-Small cell lung cancer. NSCLC FIELD includes the histological types adenocarcinoma, squamous 0003. The present invention relates to peptides, proteins, cell carcinoma and large cell carcinoma and accounts for nucleic acids and cells for use in immunotherapeutic meth 85% of all lung cancers in the United States. The incidence ods. In particular, the present invention relates to the immu of NSCLC is closely correlated with smoking prevalence, notherapy of cancer. The present invention furthermore including current and former Smokers and the five year relates to tumor-associated T-cell peptide epitopes, alone or survival rate was reported to be 15% (World Cancer Report, in combination with other tumor-associated peptides that 2014; Molina et al., 2008). can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune Therapy responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histo Breast Cancer compatibility complex (MHC), or peptides as Such, can also be targets of antibodies, soluble T-cell receptors, and other 0011. The standard treatment for breast cancer patients binding molecules. depends on different parameters: tumor stage, hormone 0004. The present invention relates to several novel pep receptor status and HER2 expression pattern. The standard tide sequences and their variants derived from HLA class I of care includes complete Surgical resection of the tumor molecules of human tumor cells that can be used in vaccine followed by radiation therapy. Chemotherapy with mainly compositions for eliciting anti-tumor immune responses, or anthracyclines and taxanes may be started prior to or after as targets for the development of pharmaceutically/immu resection. Patients with HER2-positive tumors receive the nologically active compounds and cells. anti-HER2 antibody trastuzumab in addition to the chemo therapeutics (S3-Leitlinie Mammakarzinom, 2012). Breast BACKGROUND OF THE INVENTION cancer is an immunogenic cancer entity and different types of infiltrating immune cells in primary tumors exhibit dis 0005 According to the World Health Organization tinct prognostic and predictive significance. A large number (WHO), cancer ranged among the four major non-commu of early phase immunotherapy trials have been conducted in nicable deadly diseases worldwide in 2012. For the same breast cancer patients. Clinical data on the effects of immune year, colorectal cancer, breast cancer and respiratory tract checkpoint modulation with ipilimumab and other T cell cancers were listed within the top 10 causes of death in high activating antibodies in breast cancer patients are emerging income countries (www.who.int/mediacentre/factsheets/ (Emens, 2012). fs310/en/). Chronic Lymphocytic Leukemia Epidemiology 0012 While CLL is not curable at present, many patients 0006. In 2012, 14.1 million new cancer cases, 32.6 mil show only slow progression of the disease or worsening of lion patients suffering from cancer (within 5 years of diag symptoms. For patients with symptomatic or rapidly pro nosis) and 8.2 million cancer deaths were estimated world gressing disease, several treatment options are available. wide (Ferlay et al., 2013: Bray et al., 2013). These include chemotherapy, targeted therapy, immune 0007. Within the groups of brain cancer, leukemia and based therapies like monoclonal antibodies, chimeric anti lung cancer the current invention specifically focuses on gen-receptors (CARS) and active immunotherapy, and stem glioblastoma (GB), chronic lymphocytic leukemia (CLL) cell transplants. US 2016/0280759 A1 Sep. 29, 2016
0013 Several completed and ongoing trials are based on tumors should be treated according to the guidelines for engineered autologous chimeric antigen receptor (CAR)- gastric cancer, as randomized data for targeted therapies in modified T cells with CD19 specificity (Maus et al., 2014). esophageal cancer are very limited (Stahl et al., 2013). So far, only the minority of patients showed detectable or 0021 Data on immunotherapeutic approaches in esopha persistent CARs. One partial response (PR) and two com geal cancer are scarce, as only a very limited number of early plete responses (CR) have been detected in the CART-cell phase clinical trials have been performed. A vaccine con trials by Porter et al. and Kalos et al. (Kalos et al., 2011; sisting of three peptides derived from three different cancer Porter et al., 2011). testis antigens (TTK protein kinase, lymphocyte antigen 6 0014) Active immunotherapy includes the following complex locus K and insulin-like growth factor (IGF)-II strategies: gene therapy, whole modified tumor cell vac mRNA binding protein 3) was administered to patients with cines, DC-based vaccines and tumor associated antigen advanced esophageal cancer in a phase I trial with moderate (TAA)-derived peptide vaccines. results. Intra-tumoral injection of activated T cells after in 0015 Several TAAS are over-expressed in CLL and are vitro challenge with autologous malignant cells elicited suitable for vaccinations. These include fibromodulin (Mayr complete or partial tumor responses in four of eleven et al., 2005), RHAMM/CD168 (Giannopoulos et al., 2006), patients in a phase I/II study (Toomey et al., 2013). MDM2 (Mayr et al., 2006), hTERT (Counter et al., 1995), the oncofetal antigen-immature laminin receptor protein (OFAiLRP) (Siegel et al., 2003), adipophilin (Schmidt et al., Gastric Cancer 2004), survivin (Granziero et al., 2001), KW1 to KW14 (Krackhardt et al., 2002) and the tumor-derived IgVHCDR3 0022 Gastric cancer (GC) begins in the cells lining the region (Harig et al., 2001; Carballido et al., 2012). A phase mucosal layer and spreads through the outer layers as it I clinical trial was conducted using the RHAMM-derived R3 grows. Four types of standard treatment are used. Treatment peptide as a vaccine. 5 of 6 patients had detectable R3-spe for gastric cancer may involve endoscopic or Surgical resec cific CD8+ T-cell responses (Giannopoulos et al., 2010). tion, chemotherapy, radiation therapy or chemoradiation (Leitlinie Magenkarzinom, 2012). Colorectal Cancer 0023 The efficacy of current therapeutic regimens for advanced GC is poor, resulting in low 5-year Survival rates. 0016 Depending on the colorectal cancer (CRC) stage, Immunotherapy might be an alternative approach to ame different standard therapies are available for colon and rectal liorate the survival of GC patients. Adoptive transfer of cancer. Standard procedures include Surgery, radiation tumor-associated lymphocytes and cytokine induced killer therapy, chemotherapy and targeted therapy for CRC (Ber cells, peptide-based vaccines targeting HER2/neu, MAGE-3 man et al., 2015a; Berman et al., 2015b). or vascular endothelial growth factor receptor 1 and 2 and 0017. Latest clinical trials analyze active immunotherapy dendritic cell-based vaccines targeting HER2/neu showed as a treatment option against CRC. Those strategies include promising results in clinical GC trials. Immune checkpoint the vaccination with peptides from tumor-associated anti inhibition and engineered T cells might represent additional gens (TAAS), whole tumor cells, dendritic cell (DC) vac therapeutic options, which is currently evaluated in pre cines and viral vectors (Koido et al., 2013). clinical and clinical studies (Matsueda and Graham, 2014). 0018 Peptide vaccines have so far been directed against carcinoembryonic antigen (CEA), mucin 1, EGFR, squamous cell carcinoma antigen recognized by T cells 3 Glioblastoma (SART3), beta-human chorionic gonadotropin (beta-hCG), Wilms' Tumor antigen 1 (WT1), Survivin-2B, MAGE3, 0024. The therapeutic options for glioblastoma (WHO p53, ring finger protein 43 and translocase of the outer grade IV) are very limited. Different immunotherapeutic mitochondrial membrane 34 (TOMM34), or mutated approaches are investigated for the treatment of GB, includ KRAS. In several phase I and II clinical trials patients ing immune-checkpoint inhibition, vaccination and adoptive showed antigen-specific CTL responses or antibody produc transfer of engineered T cells. tion. In contrast to immunological responses, many patients 0025. Different vaccination strategies for GB patients are did not benefit from peptide vaccines on the clinical level currently investigated, including peptide-based vaccines, (Koido et al., 2013; Miyagi et al., 2001; Moulton et al., heat-shock protein vaccines, autologous tumor cell vaccines, 2002: Okuno et al., 2011). dendritic cell-based vaccines and viral protein-based vac 0019 Dendritic cell vaccines comprise DCs pulsed with cines. In these approaches peptides derived from GB-asso either TAA-derived peptides, tumor cell lysates, apoptotic ciated proteins like epidermal growth factor receptor variant tumor cells, or tumor RNA or DC-tumor cell fusion prod III (EGFRVIII) or heat shock proteins or dendritic cells ucts. While many patients in phase I/II trials showed specific pulsed with autologous tumor cell lysate or cytomegalovirus immunological responses, only the minority had a clinical components are applied to induce an anti-tumor immune benefit (Koido et al., 2013). response in GB patients. Several of these studies reveal good safety and tolerability profiles as well as promising efficacy Esophageal Cancer data. 0020. The primary treatment strategy for esophageal can 0026. Adoptive transfer of genetically modified T cells is cer depends on tumor stage and location, histological type an additional immunotherapeutic approach for the treatment and the medical condition of the patient. Chemotherapeutic of GB. Different clinical trials currently evaluate the safety regimens include oxaliplatin plus fluorouracil, carboplatin and efficacy of chimeric antigen receptor bearing T cells plus paclitaxel, cisplatin plus fluorouracil, FOLFOX and directed against HER2, IL-13 receptor alpha 2 and cisplatin plus irinotecan. Patients with HER2-positive EGFRVIII (Ampie et al., 2015). US 2016/0280759 A1 Sep. 29, 2016
Liver Cancer Non-Small Cell Lung Cancer 0027 Disease management depends on the tumor stage at 0033 Treatment options are determined by the type the time of diagnosis and the overall condition of the liver. (Small cell or non-small cell) and stage of cancer and include Chemotherapy against HCC includes combinations of doxo Surgery, radiation therapy, chemotherapy, and targeted bio rubicin, 5-fluorouracil and cisplatin for systemic therapy and logical therapies such as bevacizumab, erlotinib and gefi doxorubicin, floxuridine and mitomycin C for hepatic artery tinib (S3-Leitlinie Lungenkarzinom, 2011). infusions. However, most HCC show a high resistance to 0034) To expand the therapeutic options for NSCLC, chemotherapeutics (Enguita-German and Fortes, 2014). different immunotherapeutic approaches have been Studied or are still under investigation. While vaccination with Therapeutic options in advanced non-resectable HCC are L-BLP25 or MAGEA3 failed to demonstrate a vaccine limited to Sorafenib, a multi-tyrosine kinase inhibitor mediated Survival advantage in NSCLC patients, an alloge (Chang et al., 2007: Wilhelmet al., 2004). Sorafenib is the neic cell line-derived vaccine showed promising results in only systemic drug confirmed to increase Survival by about clinical studies. Additionally, further vaccination trials tar 3 months and currently represents the only experimental geting gangliosides, the epidermal growth factor receptor treatment option for such patients (Chapiro et al., 2014: and several other antigens are currently ongoing. An alter Llovet et al., 2008). native strategy to enhance the patients anti-tumor T cell 0028. Lately, a limited number of immunotherapy trials response consists of blocking inhibitory T cell receptors or for HCC have been conducted. Cytokines have been used to their ligands with specific antibodies. The therapeutic poten activate Subsets of immune cells and/or increase the tumor tial of several of these antibodies, including ipilimumab, immunogenicity (Reinisch et al., 2002; Sangro et al., 2004). nivolumab, pembrolizumab, MPDL3280A and MEDI-4736, Other trials have focused on the infusion of Tumor-infiltrat in NSCLC is currently evaluated in clinical trials (Reinmuth ing lymphocytes or activated peripheral blood lymphocytes et al., 2015). (Shi et al., 2004: Takayama et al., 1991; Takayama et al., 2000). Ovarian Cancer 0029. So far, a small number of therapeutic vaccination 0035) Surgical resection is the primary therapy in early as trials have been executed. Butterfield et al. conducted two well as advanced stage ovarian carcinoma (S3-Leitlinie trials using peptides derived from alpha-fetoprotein (AFP) maligne Ovarial tumore, 2013). as a vaccine or DCs loaded with AFP peptides ex vivo 0036 Immunotherapy appears to be a promising strategy (Butterfield et al., 2003; Butterfield et al., 2006). In two to ameliorate the treatment of ovarian cancer patients, as the different studies, autologous dendritic cells (DCs) were presence of pro-inflammatory tumor infiltrating lympho pulsed ex vivo with autologous tumor lysate (Lee et al., cytes, especially CD8-positive T cells, correlates with good 2005) or lysate of the hepatoblastoma cell line HepG2 prognosis and T cells specific for tumor-associated antigens (Palmer et al., 2009). So far, vaccination trials have only can be isolated from cancer tissue. shown limited improvements in clinical outcomes. 0037. Therefore, a lot of scientific effort is put into the investigation of different immunotherapies in ovarian can Melanoma cer. A considerable number of pre-clinical and clinical studies has already been performed and further studies are 0030 The standard therapy in melanoma is complete currently ongoing. Clinical data are available for cytokine Surgical resection with Surrounding healthy tissue Therapeu therapy, vaccination, monoclonal antibody treatment, adop tic options include monochemotherapy, polychemotherapy tive cell transfer and immunomodulation. and targeted therapies with specific inhibitors (S3-Leitlinie 0038 Phase I and II vaccination studies, using single or Melanom, 2013). multiple peptides, derived from several tumor-associated proteins (Her2/neu, NY-ESO-1, p53, Wilms tumor-1) or 0031. Several different vaccination approaches have whole tumor antigens, derived from autologous tumor cells already been evaluated in patients with advanced melanoma. revealed good safety and tolerability profiles, but only low So far, phase III trials revealed rather disappointing results to moderate clinical effects. and vaccination strategies clearly need to be improved. 0039 Adoptive transfer of immune cells achieved het Therefore, new clinical trials, like the OncoVEX GM-CSF erogeneous results in clinical trials. Adoptive transfer of trial or the DERMA trial, aim at improving clinical efficacy autologous, in vitro expanded tumor infiltrating T cells was without reducing tolerability (www.cancerresearchuk.org). shown to be a promising approach in a pilot trial. In contrast, 0032. Adoptive T cell transfer shows great promise for transfer of T cells harboring a chimeric antigen receptor the treatment of advanced stage melanoma. In vitro specific for folate receptor alpha did not induce a significant expanded autologous tumor infiltrating lymphocytes as well clinical response in a phase I trial. Dendritic cells pulsed as T cells harboring a high affinity T cell receptor for the with tumor cell lysate or tumor-associated proteins in vitro cancer-testis antigen NY-ESO-1 had significant beneficial were shown to enhance the anti-tumor T cell response upon and low toxic effects upon transfer into melanoma patients. transfer, but the extent of T cell activation did not correlate Unfortunately, T cells with high affinity T cell receptors for with clinical effects. Transfer of natural killer cells caused the melanocyte specific antigens MART1 and gp100 and the significant toxicities in a phase II study. cancer-testis antigen MAGEA3 induced considerable toxic 004.0 Intrinsic anti-tumor immunity as well as immuno effects in clinical trials. Thus, adoptive T cell transfer has therapy are hampered by an immunosuppressive tumor high therapeutic potential, but safety and tolerability of these microenvironment. To overcome this obstacle immuno treatments needs to be further increased (Phan and Rosen modulatory drugs, like cyclophosphamide, anti-CD25 anti berg, 2013: Hinrichs and Restifo, 2013). bodies and pegylated liposomal doxorubicin are tested in US 2016/0280759 A1 Sep. 29, 2016 combination with immunotherapy. Most reliable data are (TKIs) Sunitinib and paZopanib, the monoclonal antibody currently available for ipilimumab, an anti-CTLA4 anti bevacizumab combined with interferon-C. (IFN-O.) and the body, which enhances T cell activity. Ipilimumab was shown mTOR inhibitor temsirolimus. Based on guidelines elabo to exert significant anti-tumor effects in ovarian cancer rated by the US NCCN as well as the European EAU and patients (Mantia-Smaldone et al., 2012). ESMO, the TKIs Sorafenib, paZopanib or recently axitinib are recommended as second-line therapy in RCC patients Pancreatic Cancer who have failed prior therapy with cytokines (IFN-O, IL-2). The NCCN guidelines advise also sunitinib in this setting 0041. Therapeutic options for pancreatic cancer patients (high-level evidence according to NCCN Category I). are very limited. One major problem for effective treatment 0046. The known immunogenity of RCC has represented is the typically advanced tumor stage at diagnosis. the basis Supporting the use of immunotherapy and cancer 0042. Vaccination strategies are investigated as further vaccines in advanced RCC. The interesting correlation innovative and promising alternative for the treatment of between lymphocytes PD-1 expression and RCC advanced pancreatic cancer. Peptide-based vaccines targeting KRAS stage, grade and prognosis, as well as the selective PD-L1 mutations, reactive telomerase, gastrin, Survivin, CEA and expression by RCC tumor cells and its potential association MUC1 have already been evaluated in clinical trials, par with worse clinical outcomes, have led to the development tially with promising results. Furthermore, clinical trials for of new anti PD-1/PD-L1 agents, alone or in combination dendritic cell-based vaccines, allogeneic GM-CSF-secreting with anti-angiogenic drugs or other immunotherapeutic vaccines and algenpantucel-L in pancreatic cancer patients approaches, for the treatment of RCC (Massari et al., 2015). also revealed beneficial effects of immunotherapy. Addi In advanced RCC, a phase III cancer vaccine trial called tional clinical trials further investigating the efficiency of TRIST study evaluates whether TroVax (a vaccine using a different vaccination protocols are currently ongoing (Sal tumor-associated antigen, 5T4, with a pox virus vector), man et al., 2013). added to first-line standard of care therapy, prolongs Survival of patients with locally advanced or mRCC. Median survival Prostate Cancer had not been reached in either group with 399 patients (54%) 0043. The therapeutic strategy for prostate cancer mainly remaining on study however analysis of the data confirms depends on the cancer stage. For locally restricted non prior clinical results, demonstrating that TroVax is both metastasizing prostate cancer, treatment options include immunologically active and that there is a correlation active surveillance (wait and watch), complete Surgical between the strength of the 5T4-specific antibody response resection of the prostate and local high dose radiation and improved survival. Further there are several studies therapy with or without brachytherapy (S3-Leitlinie Prosta searching for peptide vaccines using epitopes being over takarzinom, 2014). expressed in RCC. 0044) The dendritic cell-based vaccine sipuleucel-T was 0047 Various approaches of tumor vaccines have been the first anti-cancer vaccine to be approved by the FDA. Due under investigation. Studies using whole-tumor approaches, to its positive effect on survival in patients with CRPC, including tumor cell lysates, fusions of dendritic cells with much effort is put into the development of further immuno tumor cells, or whole-tumor RNA were done in RCC therapies. Regarding vaccination strategies, the peptide vac patients, and remissions of tumor lesions were reported in cine prostate-specific antigen (PSA)-TRICOM, the person some of these trials (Avigan et al., 2004; Holt1 et al., 2002: alized peptide vaccine PPV, the DNA vaccine pTVG-HP and Marten et al., 2002: Su et al., 2003: Wittig et al., 2001). the whole cell vaccine expressing GM-CSF GVAX showed promising results in different clinical trials. Furthermore, Small Cell Lung Cancer dendritic cell-based vaccines other than sipuleucel-T. 0048. The treatment and prognosis of SCLC depend namely BPX-101 and DCVAC/Pa were shown to elicited strongly on the diagnosed cancer stage. The staging of clinical responses in prostate cancer patients. Immune SCLC based on clinical results is more common than the checkpoint inhibitors like ipilimumab and nivolumab are pathologic staging. The clinical staging uses the results of currently evaluated in clinical studies as monotherapy as the physical examination, various imaging tests and biop well as in combination with other treatments, including sies. The standard chemo treatment of SCLC uses the androgen deprivation therapy, local radiation therapy, PSA combination of either etoposide or irinotecan with either TRICOM and GVAX. The immunomodulatory substance cisplatin or carboplatin (American Cancer Society, 2015: tasquinimod, which significantly slowed progression and S3-Leitlinie Lungenkarzinom, 2011). increased progression free Survival in a phase II trial, is 0049. The immune therapy presents an excessively inves currently further investigated in a phase III trial. Lenalido tigated field of cancer therapy. Various approaches are mide, another immunomodulator, induced promising effects studded in the treatment of SCLC. One of the approaches in early phase clinical studies, but failed to improve survival targets the blocking of CTLA-4, a natural human immune in a phase III trial. Despite these disappointing results suppressor. The inhibition of CTLA-4 intends to boost the further lenalidomide trials are ongoing (Quinn et al., 2015). immune system to combat the cancer. Recently, the devel opment of promising immune check point inhibitors for Renal Cell Carcinoma treatment of SCLC has been started. Another approach is based on anti-cancer vaccines which is currently available 0045. Initial treatment is most commonly either partial or for treatment of SCLC in clinical studies (American Cancer complete removal of the affected kidney(s) and remains the Society, 2015; National Cancer Institute, 2015). mainstay of curative treatment (Rini et al., 2008). For first-line treatment of patients with poor prognostic score a Acute Myeloid Leukemia guidance elaborated by several cancer organizations and 0050 AML treatment is divided into two phases: induc Societies recommend the receptor tyrosine kinase inhibitors tion therapy and post-remission/"consolidation therapy'. US 2016/0280759 A1 Sep. 29, 2016
Induction therapy is administered to induce remission and side effects were observed and 3 out of 6 patients showed an consists of combinational chemotherapy. Consolidation immunological response (Coosemans et al., 2013). therapy consists of additional chemotherapy or hematopoi etic cell transplantation (HCT) (Showel and Levis, 2014). Gallbladder Adenocarcinoma and Cholangiocarcinoma 0051 Clinical trials are recommended for patients who belong to the prognostic groups unfavorable and interme 0.058 Cholangiocarcinoma (CCC) is difficult to treat and diate-2. Treatment options include hypomethylating agents is usually lethal. The only curative treatment option is (HMAs) as Azacitidine or decitabine, CPX-351, which is a complete resection (RO). The efficacy of biological therapies liposomal formulation of daunorubicin and cytarabine in a in biliary tract cancers has been mixed. Drugs targeting 1:5 “optimal molar ratio, and volasertib, which is an blood vessel growth such as Sorafenib, bevacizumab, inhibitor of polo kinases. Volasertib is given in combination paZopanib and regorafenib are now studied for the treatment with LDAC (low-dose cytarabine). Several different FLT3 of CCC. Additionally, drugs that target EGFR such as inhibitors can be administered in case of FLT3 mutations. cetuximab and panitumumab are used in clinical studies in These include Sorafenib, which is given in combination with combination with chemotherapy (American Cancer Society, 3+7, quizartinib, a more selective inhibitor of FLT3 ITD that 2015). For most drugs tested so far disease control and also inhibits CKIT, crenolanib, and midostaurin, an unse overall survival were not improved significantly but there lective FLT3 ITD inhibitor. Another treatment option is are further clinical trials ongoing. targeting CD33 with antibody-drug conjugates (anti-CD33+ 0059 Gallbladder cancer (GBC) is the most common and calechiamicin, SGN-CD33a, anti-CD33+ actinium-225), aggressive malignancy of the biliary tract worldwide. Due to bispecific antibodies (recognition of CD33+CD3 (AMG the rarity of carcinomas of the biliary tract in general there 330) or CD33+CD16) and chimeric antigen receptors are only a few GBC or CCC specific studies, while most of (CARs) (Estey, 2014). them include all biliary tract cancers. This is the reason why treatment did not improve during the last decades and RO Non-Hodgkin Lymphoma resection still is the only curative treatment option. 0052 NHL has over 60 subtypes. The three most com Urinary Bladder Cancer mon subtypes are diffuse large B-cell lymphoma (DLBCL, the most common Subtype), follicular lymphoma (FL, the 0060. The standard treatment for bladder cancer includes second most common Subtype) and Small lymphocytic lym Surgery, radiation therapy, chemotherapy and immuno phoma/chronic lymphocytic lymphoma (SLL/CLL, the third therapy (National Cancer Institute, 2015). most common subtype). DLBCL, FL and SLL/CLL account 0061 An effective immunotherapeutic approach is estab for about 85% of NHL (Li et al., 2015). Treatment of NHL lished in the treatment of aggressive non-muscle invasive depends on the histologic type and stage (National Cancer bladder cancer (NMIBC). Thereby, a weakened form of the Institute, 2015). bacterium Mycobacterium bovis (bacillus Calmette 0053 Spontaneous tumor regression can be observed in Guérin-BCG) is applied as an intravesical solution. The lymphoma patients. Therefore, active immunotherapy is a major effect of BCG treatment is a significant long-term (up therapy option (Palomba, 2012). to 10 years) protection from cancer recurrence and reduced 0054 An important vaccination option includes Id vac progression rate. In principle, the treatment with BCG cines. B lymphocytes express Surface immunoglobulins with induces a local inflammatory response which stimulates the a specific amino acid sequence in the variable regions of cellular immune response. The immune response to BCG is their heavy and light chains, unique to each cell clone based on the following key steps: infection of urothelial and (idiotype, Id). The idiotype functions as a tumor associated bladder cancer cells by BCG, followed by increased expres antigen. sion of antigen-presenting molecules, induction of immune 0055 Active immunization includes the injection of response mediated via cytokine release, induction of anti recombinant protein (Id) conjugated to an adjuvant (KLH). tumor activity via involvement of various immune cells given together with GM-CSF as an immune adjuvant. (thereunder cytotoxic T lymphocytes, neutrophils, natural Tumor-specific Id is produced by hybridoma cultures or killer cells, and macrophages) (Fuge et al., 2015; Gandhi et using recombinant DNA technology (plasmids) by bacterial, al., 2013). insect or mammalian cell culture. 0062 Considering the severe side-effects and expense associated with treating cancer, there is a need to identify Uterine Cancer factors that can be used in the treatment of cancer in general and hepatocellular carcinoma (HCC), colorectal carcinoma 0056 More than 80% of endometrial cancers occur as (CRC), glioblastoma (GB), gastric cancer (GC), esophageal endometrioid adenocarcinomas (type I), a form that is asso cancer, non-Small cell lung cancer (NSCLC), pancreatic ciated with estrogen exposure and that is well to moderately cancer (PC), renal cell carcinoma (RCC), benign prostate differentiated. Treatment of endometrial carcinomas and hyperplasia (BPH), prostate cancer (PCA), ovarian cancer cervical cancers is stage-dependent (World Cancer Report, (OC), melanoma, breast cancer, chronic lymphocytic leuke 2014). mia (CLL), Merkel cell carcinoma (MCC), small cell lung 0057 There are also some immunotherapeutic cancer (SCLC), Non-Hodgkin lymphoma (NHL), acute approaches that are currently being tested. In a Phase I/II myeloid leukemia (AML), gallbladder cancer and cholang Clinical Trial patients suffering from uterine cancer were iocarcinoma (GBC, CCC), urinary bladder cancer (UBC), vaccinated with autologous dendritic cells (DCs) electropo uterine cancer (UEC), in particular. There is also a need to rated with Wilms’ tumor gene 1 (WT1) mRNA. Besides one identify factors representing biomarkers for cancer in gen case of local allergic reaction to the adjuvant, no adverse eral and the above-mentioned cancer types in particular, US 2016/0280759 A1 Sep. 29, 2016 leading to better diagnosis of cancer, assessment of prog human papilloma type 16 virus proteins, E6 and E7, which nosis, and prediction of treatment Success. are expressed in cervical carcinoma. 0063. Immunotherapy of cancer represents an option of 006.4 T-cell based immunotherapy targets peptide specific targeting of cancer cells while minimizing side epitopes derived from tumor-associated or tumor-specific effects. Cancer immunotherapy makes use of the existence proteins, which are presented by molecules of the major of tumor associated antigens. The current classification of histocompatibility complex (MHC). The antigens that are tumor associated antigens (TAAS) comprises the following recognized by the tumor specific T lymphocytes, that is, the major groups: epitopes thereof, can be molecules derived from all protein a) Cancer-testis antigens: The first TAAs ever identified that classes, such as enzymes, receptors, transcription factors, can be recognized by T cells belong to this class, which was etc. which are expressed and, as compared to unaltered cells originally called cancer-testis (CT) antigens because of the of the same origin, usually up-regulated in cells of the expression of its members in histologically different human respective tumor. tumors and, among normal tissues, only in spermatocytes/ 0065. There are two classes of MHC-molecules, MHC spermatogonia of testis and, occasionally, in placenta. Since class I and MHC class II. MHC class I molecules are the cells of testis do not express class I and II HLA composed of an alpha heavy chain and beta-2-microglobu molecules, these antigens cannot be recognized by T cells in lin, MHC class II molecules of an alpha and a beta chain. normal tissues and can therefore be considered as immuno Their three-dimensional conformation results in a binding logically tumor-specific. Well-known examples for CT anti groove, which is used for non-covalent interaction with gens are the MAGE family members and NY-ESO-1. peptides. 006.6 MHC class I molecules can be found on most b) Differentiation antigens: These TAAS are shared between nucleated cells. They present peptides that result from tumors and the normal tissue from which the tumor arose. proteolytic cleavage of predominantly endogenous proteins, Most of the known differentiation antigens are found in defective ribosomal products (DRIPs) and larger peptides. melanomas and normal melanocytes. Many of these mel However, peptides derived from endosomal compartments anocyte lineage-related proteins are involved in biosynthesis or exogenous sources are also frequently found on MHC of melanin and are therefore not tumor specific but never class I molecules. This non-classical way of class I presen theless are widely used for cancer immunotherapy. tation is referred to as cross-presentation in literature (Bro Examples include, but are not limited to, tyrosinase and ssart and Bevan, 1997: Rock et al., 1990). MHC class II Melan-A/MART-1 for melanoma or PSA for prostate cancer. molecules can be found predominantly on professional c) Over-expressed TAAs: Genes encoding widely expressed antigen presenting cells (APCs), and primarily present pep TAAs have been detected in histologically different types of tides of exogenous or transmembrane proteins that are taken tumors as well as in many normal tissues, generally with up by APCs e.g. during endocytosis, and are Subsequently lower expression levels. It is possible that many of the processed. epitopes processed and potentially presented by normal 0067 Complexes of peptide and MHC class I are recog tissues are below the threshold level for T-cell recognition, nized by CD8-positive T cells bearing the appropriate T-cell while their over-expression in tumor cells can trigger an receptor (TCR), whereas complexes of peptide and MHC anticancer response by breaking previously established tol class II molecules are recognized by CD4-positive-helper-T erance. Prominent examples for this class of TAAs are cells bearing the appropriate TCR. It is well known that the Her-2/neu, Survivin, telomerase, or WT1. TCR, the peptide and the MHC are thereby present in a d) Tumor-specific antigens: These unique TAAS arise from stoichiometric amount of 1:1:1. mutations of normal genes (such as B-catenin, CDK4, etc.). 0068 CD4-positive helper T cells play an important role Some of these molecular changes are associated with neo in inducing and Sustaining effective responses by CD8 plastic transformation and/or progression. Tumor-specific positive cytotoxic T cells. The identification of CD4-positive antigens are generally able to induce strong immune T-cell epitopes derived from tumor associated antigens responses without bearing the risk for autoimmune reactions (TAA) is of great importance for the development of phar against normal tissues. On the other hand, these TAAS are in maceutical products for triggering anti-tumor immune most cases only relevant to the exact tumor on which they responses (Gnjatic et al., 2003). At the tumor site. T helper were identified and are usually not shared between many cells, support a cytotoxic T cell-(CTL-) friendly cytokine individual tumors. Tumor-specificity (or -association) of a milieu (Mortara et al., 2006) and attract effector cells, e.g. peptide may also arise if the peptide originates from a CTLS. natural killer (NK) cells, macrophages, and granulo tumor-(-associated) exon in case of proteins with tumor cytes (Hwang et al., 2007). specific (-associated) isoforms. 0069. In the absence of inflammation, expression of e) TAAS arising from abnormal post-translational modifica MHC class II molecules is mainly restricted to cells of the tions: Such TAAS may arise from proteins which are neither immune system, especially professional antigen-presenting specific nor overexpressed in tumors but nevertheless cells (APC), e.g., monocytes, monocyte-derived cells, mac become tumor associated by posttranslational processes rophages, dendritic cells. In cancer patients, cells of the primarily active in tumors. Examples for this class arise tumor have been found to express MHC class II molecules from altered glycosylation patterns leading to novel epitopes (Dengel et al., 2006). in tumors as for MUC1 or events like protein splicing during degradation which may or may not be tumor specific. Elongated Peptides of the Invention can Act as MHC Class f) Oncoviral proteins: These TAAs are viral proteins that II Active Epitopes. may play a critical role in the oncogenic process and, (0070 T-helper cells, activated by MHC class II epitopes, because they are foreign (not of human origin), they can play an important role in orchestrating the effector function evoke a T-cell response. Examples of Such proteins are the of CTLs in anti-tumor immunity. T-helper cell epitopes that US 2016/0280759 A1 Sep. 29, 2016 trigger a T-helper cell response of the TH1 type support antigens may also be targets of a vaccination approach effector functions of CD8-positive killer T cells, which (Singh-Jasuja et al., 2004). It is essential that epitopes are include cytotoxic functions directed against tumor cells present in the amino acid sequence of the antigen, in order displaying tumor-associated peptide/MHC complexes on to ensure that such a peptide (“immunogenic peptide'), their cell surfaces. In this way tumor-associated T-helper cell being derived from a tumor associated antigen, leads to an peptide epitopes, alone or in combination with other tumor in vitro or in vivo T-cell-response. associated peptides, can serve as active pharmaceutical (0077 Basically, any peptide able to bind an MHC mol ingredients of vaccine compositions that stimulate anti ecule may function as a T-cell epitope. A prerequisite for the tumor immune responses. induction of an in vitro or in vivo T-cell-response is the 0071. It was shown in mammalian animal models, e.g., presence of a T cell having a corresponding TCR and the mice, that even in the absence of CD8-positive T lympho absence of immunological tolerance for this particular cytes, CD4-positive T cells are sufficient for inhibiting epitope. manifestation of tumors via inhibition of angiogenesis by 0078. Therefore, TAAS are a starting point for the devel secretion of interferon-gamma (IFNY) (Beatty and Paterson, opment of a T cell based therapy including but not limited 2001; Mumberg et al., 1999). There is evidence for CD4 T to tumor vaccines. The methods for identifying and charac cells as direct anti-tumor effectors (Braumuller et al., 2013; terizing the TAAs are usually based on the use of T-cells that Tran et al., 2014). can be isolated from patients or healthy Subjects, or they are 0072 Since the constitutive expression of HLA class II based on the generation of differential transcription profiles molecules is usually limited to immune cells, the possibility or differential peptide expression patterns between tumors of isolating class II peptides directly from primary tumors and normal tissues. However, the identification of genes was previously not considered possible. However, Dengel over-expressed in tumor tissues or human tumor cell lines, et al. were successful in identifying a number of MHC Class or selectively expressed in Such tissues or cell lines, does not II epitopes directly from tumors (WO 2007/028574, EP 1 provide precise information as to the use of the antigens 760 088 B1). being transcribed from these genes in an immune therapy. 0073. Since both types of response, CD8 and CD4 depen This is because only an individual Subpopulation of epitopes dent, contribute jointly and synergistically to the anti-tumor of these antigens are Suitable for Such an application since a effect, the identification and characterization of tumor-asso T cell with a corresponding TCR has to be present and the ciated antigens recognized by either CD8+ T cells (ligand: immunological tolerance for this particular epitope needs to MHC class I molecule+ peptide epitope) or by CD4-positive be absent or minimal. In a very preferred embodiment of the T-helper cells (ligand: MHC class II molecule+ peptide invention it is therefore important to select only those over epitope) is important in the development of tumor vaccines. or selectively presented peptides against which a functional 0074 For an MHC class I peptide to trigger (elicit) a and/or a proliferating T cell can be found. Such a functional cellular immune response, it also must bind to an MHC T cell is defined as a T cell, which upon stimulation with a molecule. This process is dependent on the allele of the specific antigen can be clonally expanded and is able to MHC-molecule and specific polymorphisms of the amino execute effector functions (“effector T cell’). acid sequence of the peptide. MHC-class-I-binding peptides 0079. In case of targeting peptide-MHC by specific TCRs are usually 8-12 amino acid residues in length and usually (e.g. soluble TCRs) and antibodies or other binding mol contain two conserved residues (“anchors’) in their ecules (Scaffolds) according to the invention, the immuno sequence that interact with the corresponding binding genicity of the underlying peptides is secondary. In these groove of the MHC-molecule. In this way each MHC allele cases, the presentation is the determining factor. has a “binding motif determining which peptides can bind specifically to the binding groove. SUMMARY OF THE INVENTION 0075. In the MHC class I dependent immune reaction, 0080. In a first aspect of the present invention, the present peptides not only have to be able to bind to certain MHC invention relates to a peptide comprising an amino acid class I molecules expressed by tumor cells, they Subse sequence selected from the group consisting of SEQID NO: quently also have to be recognized by T cells bearing 1 to SEQ ID NO: 288 or a variant sequence thereof which specific T cell receptors (TCR). is at least 77%, preferably at least 88%, homologous (pref 0076 For proteins to be recognized by T-lymphocytes as erably at least 77% or at least 88% identical) to SEQID NO: tumor-specific or -associated antigens, and to be used in a 1 to SEQ ID NO: 288, wherein said variant binds to MHC therapy, particular prerequisites must be fulfilled. The anti and/or induces T cells cross-reacting with said peptide, or a gen should be expressed mainly by tumor cells and not, or pharmaceutical acceptable salt thereof, wherein said peptide in comparably small amounts, by normal healthy tissues. In is not the underlying full-length polypeptide. a preferred embodiment, the peptide should be over-pre I0081. While the most important criterion for a peptide to sented by tumor cells as compared to normal healthy tissues. function as cancer therapy target is its over-presentation on It is furthermore desirable that the respective antigen is not primary tumor tissues as compared to normal tissues, also only present in a type of tumor, but also in high concentra the RNA expression profile of the corresponding gene can tions (i.e. copy numbers of the respective peptide per cell). help to select appropriate peptides. Particularly, some pep Tumor-specific and tumor-associated antigens are often tides are hard to detect by mass spectrometry, either due to derived from proteins directly involved in transformation of their chemical properties or to their low copy numbers on a normal cell to a tumor cell due to their function, e.g. in cell cells, and a screening approach focusing on detection of cycle control or Suppression of apoptosis. Additionally, peptide presentation may fail to identify these targets. How downstream targets of the proteins directly causative for a ever, these targets may be detected by an alternative transformation may be up-regulated and thus may be indi approach starting with analysis of gene expression in normal rectly tumor-associated. Such indirect tumor-associated tissues and secondarily assessing peptide presentation and US 2016/0280759 A1 Sep. 29, 2016
gene expression in tumors. This approach was realized in I0082. The present invention further relates to a peptide of this invention using mRNA data from a publicly available the present invention comprising a sequence that is selected database (Lonsdale, 2013) in combination with further gene from the group consisting of SEQID NO: 1 to SEQID NO: expression data (including tumor samples), as well as pep tide presentation data. If the mRNA of a gene is nearly 288 or a variant thereof, which is at least 77%, preferably at absent in normal tissues, especially in vital organ systems, least 88%, homologous (preferably at least 77% or at least targeting the corresponding peptides by even very potent 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 288, strategies (such as bispecific affinity-optimized antibodies or wherein said peptide or variant thereof has an overall length T-cell receptors), is more likely to be safe. Such peptides, of between 8 and 100, preferably between 8 and 30, and even if identified on only a small percentage of tumor most preferred of between 8 and 14 amino acids. tissues, represent interesting targets. Routine mass spec I0083. The following tables show the peptides according trometry analysis is not sensitive enough to assess target to the present invention, their respective SEQ ID NOS, and coverage on the peptide level. Rather, tumor mRNA expres the prospective source (underlying) genes for these peptides. sion can be used to assess coverage. For detection of the All peptides in Table 1 and Table 2 bind to HLA-A*02. The peptide itself, a targeted mass spectrometry approach with peptides in Table 2 have been disclosed before in large higher sensitivity than in the routine screening may be listings as results of high-throughput Screenings with high necessary and may lead to a better estimation of coverage on error rates or calculated using algorithms, but have not been the level of peptide presentation. associated with cancer at all before. TABLE 1. Peptides according to the present invention
SEQ ID No. Sequence Gene ID (S) Official Gene Symbol (s) 1 KLQEKIOEL 1062 CENPE
2. SWLEKEIYSI 1276 O2 DNAH14
3. RWIDDSLWWGW 21.87 FANCB
4 WLFGELPAL 87 O1 DNAH11
5 GLVDIMWHL 87 O1 DNAH11
6 FINAIETAL 87 O1 DNAH11
7 ALLOALMEL 51236, 728 O71 FAM203A, FAM2O3B
8 ALSSSOAEW 38.33 KIFC1
9 SLITGODLLSV 51804 SIX4
O QLIEKNWLL 56992 KIF15
1. LLDPKTIFL 2 6f 62 HAWCR1
2 RLLDPKTIFL 2 6f 62 HAWCR1
3 RLHDENILL 23322 RPGRIP1L
4 YTFSGDVOL 4312 MMP1
GLPSATTTV 94 O25 MUC16
6 SLADISLIL 134391 GPR151
7 GLLPSAESIKL 132989 C4 of 36
8 KTASINONV 81930 KIF18A
9 KWFELDLWTL 1O 63 CENPF
2O ALWEKGEFAL 1O 63 CENPF
21, YLMDDFSSL 1293 COL 6A3
22 LMYPYIYHW 54954 FAM12 OC
23 ALLSPLSIA 4O17, 95.83 LOXL2, ENTPD4
24 KWWSDWTPL 4320, 4322 MMP11, MMP13
25 LLWGHPRWALA. 25878 MXRA5
US 2016/0280759 A1 Sep. 29, 2016 14
TABLE 1 - continued Peptides according to the present invention
SEQ ID No. Sequence Gene ID (S) Official Gene Symbol (s)
(LFSLLCEA 51OSO PI15
ALAKDEISL 12O379
FLFWDPELW 146850
2O4. SEWGSPHAAWP 5539 PPY
2O5 AFGYDDEL 391OO4, 6543.48 PRAMEF17 PRAMEF16
GILDAFRIFL 431704 RGS21
KLFETWEEL 61.21 RPE65
HLNNDRNPL SEMG1
209 WLOTEELVAN SEMG1
GLAGDNIYL 6582 SLC22A2
LLTTWLINA 6582 SLC22A2
MTLSEIHAW 915.3 SLC28A2
ILAWDGWLSW 169026
ALFETLIQL 13942O
OIADIVTSV 13942O
ALSTWTPRI 1663 78 SPATA5
LLWPSSWPA. 24.6777, 794 OO SPESP1, NOX5
SLTGANITW 83932 SPRTN
GVVPTIOKV 6 422O STRA6
22O ALSELERWI 51298 THEG
221 IMLNSWEEI 38.737 THEMIS
222 LLTGVFAQL 3.88564 TMEM238
223 ALHPWOFYL 93.587 TRMT1 OA
224 LLFDWSGTGRADA 794 65 ULBP3
225 FLPOPVPLSV 57695 USP37
226 SLAGNLQEL 11023 WAX1
227 SEMEELPSW 26609, 425 O54, 51481
228 SLLELDGINLRL 2218 O6 WWDE
229 YLYELEHAL 802.17 WDR96
23 O KLLNMIFSI 2829 XCR1
231 LLDDIFIRL 143570 XRRA1
232 LWWGGIATW 84 614
233 SLFESLEYL 1326.25 US 2016/0280759 A1 Sep. 29, 2016
TABLE 2 TABLE 2 - continued Additional peptides according to the present Additional peptides according to the present invention invention
SEQ ID Official SEQ ID Official No. Sequence Gene ID (S) Gene Symbol (s) No. Sequence Gene ID (S) Gene Symbol (s) 234 WLLNEILEOW 641.51 NCAPG 269 GILDCPIFL 2177 FANCD2
235 SLLNOPKAV 63967 CLSPN 27 O TLLTFFHEL 55.215 FANCI
236 KMSELOTYV 1O 63 CENPF 271 WLIEYNFSI 55.215 FANCI
237 ALLEOTGDMSL 1O 63 CENPF 272 FWMEGEPPKL 348.654 GEN1
238 HLQEKLQSL 1O 63 CENPF 273 SLNKOIETV 576.50 KIAA1524
239 WIIKGLEEITW 3832 KIF11 274 TLYNPERTITV 10642, 10643 IGF2BP1, IGF2BP3
24 O SVOENIQOK 3832 KIF11 275 AWPPPPSSW 106.42 IGF2BP1
241 KOFEGTVEI 675 BRCA2 276 RMPTVLOCV 9622 KLK4
242 KLQEEIPWL 1062 CENPE 277 KLOEELNKV 31 61 HMMR
243 GLAEFOENV 57405 SPC25 278 WLEDKWLSW 128239 IQGAP3
244 NWAEIWIHI 8354 O NUF2 279 WLMDEGAWLTL 5.4596 L1TD1
245 ALLEEEEGW 41 O3 MAGEA4 28 O HLWGHALFL 89866 SEC16B
246 ALAGIWTNW 11077 HSF2BP 281 LLLESDPKWYSL 6491 STIL
247 NLLIDDKGTIKL 983 CDK1 282 SLYALHWKA. 79001 WKORC1
248 WLMODSRLYL 983 CDK1 283 ALSELLQQV 9816 URB2
249 YLYOILOGI 983 CDK1 284 KLMDPGSLPPL 2118 ETV4
250 LMODSRLYL 983 CDK1 285 MLLDTWOKV 54892 NCAPG2
251 LWGNLPEI 65382O, 729533 FAM72B, FAM72A 286 FILTEMWHFI 93,517 SDR42E1
252 SLMEKNQSL 241.37, 285643 KIF4A, KIF4B 287 KIOEILTOV 10643 IGF2BP3
253 KLLAWIHEL 25788 RAD54B 288 SLYKGLISW 25788 RAD54B
254 ALGDKFLLRW 4608 MYBPH J = Phospho serine
255 MKNSDLYGA 798 O1 SHCBP1 I0084 Particularly preferred are the peptides—alone or in combination—according to the present invention selected 256 LNDIFERI 337873, 33 7874 from the group consisting of SEQID NO: 1 to SEQID NO: 288. More preferred are the peptides—alone or in combi 27 KLIDHOGLYL 7579 ZSCAN2O nation—selected from the group consisting of SEQID NO: 1 to SEQ ID NO: 126 (see Table 1), and their uses in the 258 QLVORVASW 5683 PSMA2 immunotherapy of hepatocellular carcinoma (HCC), col orectal carcinoma (CRC), glioblastoma (GB), gastric cancer 259 GPGIFPPPPPOP 10879 (GC), esophageal cancer, non-Small cell lung cancer (NSCLC), pancreatic cancer (PC), renal cell carcinoma 26 O ALNESLWEC 551.65 CEP55 (RCC), benign prostate hyperplasia (BPH), prostate cancer 261 GLAALAWHL 21.75 FANCA. (PCA), ovarian cancer (OC), melanoma, breast cancer, chronic lymphocytic leukemia (CLL), Merkel cell carci 262 LLLEAWWHL 21.75 FANCA. noma (MCC), small cell lung cancer (SCLC), Non-Hodgkin 263 SIIEYLPTL 7991.5 ATAD5 lymphoma (NHL), acute myeloid leukemia (AML), gall bladder cancer and cholangiocarcinoma (GBC, CCC), uri 264 2099 ESR1 nary bladder cancer (UBC), uterine cancer (UEC). 265 34.6389 MACC1. I0085 Most preferred are the peptides—alone or in com FLLDKPODLSI bination selected from the group consisting of SEQ ID 266 FLLDKPODL 34.6389 MACC1. NO: 274, 14, 21, 23, 25, 157, 168, 11, 253, 85, 89, 40, 264, 155, 233, and 245 (see Tables 1, 2, and 10), and their uses 267 YLLDMPLWYL 71.53 TOP2A in the immunotherapy of HCC, CRC, GB, GC, esophageal 268 SLDKDIWAL 71.53 TOP2A cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, mela noma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, and CLL. US 2016/0280759 A1 Sep. 29, 2016
I0086. The present invention furthermore relates to pep presenting cell comprises an expression vector capable of tides according to the present invention that have the ability expressing or expressing said peptide containing SEQ ID to bind to a molecule of the human major histocompatibility No. 1 to SEQ ID No.: 288, preferably containing SEQ ID complex (MHC) class-I or in an elongated form, such as a No. 1 to SEQID No.: 126, or a variant amino acid sequence. length-variant MHC class-II. 0100. The present invention further relates to activated T 0087. The present invention further relates to the peptides cells, produced by the method according to the present according to the present invention wherein said peptides invention, wherein said T cell selectively recognizes a cell (each) consist or consist essentially of an amino acid which expresses a polypeptide comprising an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 288. sequence according to the present invention. 0088. The present invention further relates to the peptides 0101 The present invention further relates to a method of according to the present invention, wherein said peptide is killing target cells in a patient which target cells aberrantly modified and/or includes non-peptide bonds. express a polypeptide comprising any amino acid sequence 0089. The present invention further relates to the peptides according to the present invention, the method comprising according to the present invention, wherein said peptide is administering to the patient an effective number of T cells as part of a fusion protein, in particular fused to the N-terminal produced according to the present invention. amino acids of the HLA-DR antigen-associated invariant 0102 The present invention further relates to the use of chain (Ii), or fused to (or into the sequence of) an antibody, any peptide as described, the nucleic acid according to the Such as, for example, an antibody that is specific for den present invention, the expression vector according to the dritic cells. present invention, the cell according to the present inven 0090 The present invention further relates to a nucleic tion, the activated T lymphocyte, the T cell receptor or the acid, encoding the peptides according to the present inven antibody or other peptide- and/or peptide-MHC-binding tion. The present invention further relates to the nucleic acid molecules according to the present invention as a medica according to the present invention that is DNA, cDNA, ment or in the manufacture of a medicament. Preferably, said PNA, RNA or combinations thereof. medicament is active against cancer. 0091. The present invention further relates to an expres 0103 Preferably, said medicament is suitable and used sion vector capable of expressing and/or expressing a for a cellular therapy, a vaccine or a protein based on a nucleic acid according to the present invention. soluble TCR or antibody. 0092. The present invention further relates to a peptide 0104. The present invention further relates to a use according to the present invention, a nucleic acid according according to the present invention, wherein said cancer cells to the present invention or an expression vector according to are HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, the present invention for use in the treatment of diseases and RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, in medicine, in particular in the treatment of cancer. SCLC, NHL, AML, GBC, CCC, UBC, UEC, or CLL cells. 0093. The present invention further relates to antibodies 0105. The present invention further relates to biomarkers that are specific against the peptides according to the present based on the peptides according to the present invention, invention or complexes of said peptides according to the herein called “targets” that can be used in the diagnosis of present invention with MHC, and methods of making these. cancer, preferably HCC, CRC, GB, GC, esophageal cancer, 0094. The present invention further relates to T-cell NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast receptors (TCRs), in particular soluble TCR (sTCRs) and cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, and cloned TCRS engineered into autologous or allogeneic T CLL. The marker can be either over-presentation of the cells, and methods of making these, as well as NK cells or peptide(s) themselves, or over-expression of the correspond other cells bearing said TCR or cross-reacting with said ing gene(s). The markers may also be used to predict the TCRS. probability of Success of a treatment, preferably an immu 0095. The antibodies and TCRs are additional embodi notherapy, and most preferred an immunotherapy targeting ments of the immunotherapeutic use of the peptides accord the same target that is identified by the biomarker. For ing to the invention at hand. example, an antibody or soluble TCR can be used to stain 0096. The present invention further relates to a host cell sections of the tumor to detect the presence of a peptide of comprising a nucleic acid according to the present invention interest in complex with MHC. or an expression vector as described before. The present 0106 Optionally the antibody carries a further effector invention further relates to the host cell according to the function Such as an immune stimulating domain or toxin. present invention that is an antigen presenting cell, and 0107 The present invention also relates to the use of preferably is a dendritic cell. these novel targets in the context of cancer treatment. 0097. The present invention further relates to a method 0.108 CABYR encodes a protein which localizes to the for producing a peptide according to the present invention, principal piece of the sperm flagellum in association with the said method comprising culturing the host cell according to fibrous sheath and exhibits calcium-binding when phospho the present invention, and isolating the peptide from said rylated during the process of capacitation (RefSeq, 2002). host cell or its culture medium. Knock-down of the CABYR isoforms CABYR-a and 0098. The present invention further relates to said method CABYR-b in the non-small cell lung cancer cell lines according to the present invention, wherein the antigen is NCI-H460 and A549 was shown to result in inhibition of loaded onto class I or II MHC molecules expressed on the proliferation and attenuation of constitutively active Akt Surface of a Suitable antigen-presenting cell or artificial phosphorylation (Qian et al., 2014). Silencing of CABYR antigen-presenting cell by contacting a Sufficient amount of expression was shown to impact down-stream components the antigen with an antigen-presenting cell. of the Akt pathways such as phospho-GSK-3beta and the 0099. The present invention further relates to the method p53 and p27 proteins (Qian et al., 2014). Furthermore, according to the present invention, wherein the antigen CABYR knock-down was shown to significantly increase US 2016/0280759 A1 Sep. 29, 2016
chemosensitivity in response to chemotherapeutic drugs and al., 2010). In prostate cancer and ovarian cancer, CYP4Z1 drug-induced apoptosis, both in vitro and in Vivo, and may has been identified as independent predictive marker (Tra thus be a novel method to improve the apoptotic response donsky et al., 2012: Downie et al., 2005). CYP4Z2P is a and chemosensitivity in lung cancer (Qian et al., 2014). pseudogene located on chromosome 1 p33 (RefSeq, 2002). CABYR was described as an initially testis-specific protein 0112 DCAF4L2 encodes the DDB1 and CUL4 associ which was Subsequently shown to be present in brain ated factor 4-like 2. The specific function of this protein tumors, pancreas cancer and lung cancer (Hsu et al., 2005; remains to be elucidated; nevertheless the DCAF4L2 gene Luo et al., 2007: Li et al., 2012). CABYR was shown to be was shown to be associated with optic disc morphology and up-regulated in hepatocellular carcinoma and may play an cleft lip development (Springelkamp et al., 2015; Beaty et oncogenic role in hepatocarcinogenesis as well as its pro al., 2013). gression (Li et al., 2012). 0113 ESR1 encodes an estrogen receptor, a ligand-acti 0109 COL6A3 encodes collagen, type VI, alpha 3, one vated transcription factor important for hormone binding, of the three alpha chains of type VI collagen, a beaded DNA binding and activation of transcription, that is essential filament collagen found in most connective tissues, and for sexual development and reproductive function (RefSeq, important in organizing matrix components (RefSeq, 2002). 2002). Mutations and single nucleotide polymorphisms of COL6A3 encodes the alpha-3 chain of type VI collagen, a ESR1 are associated with risk for different cancer types beaded filament collagen found in most connective tissues, including liver, prostate, gallbladder and breast cancer. The playing an important role in the organization of matrix up-regulation of ESR1 expression is connected with cell components (RefSeq, 2002). COL6A3 is alternatively proliferation and tumor growth but the overall survival of spliced in colon, bladder and prostate cancer. The long patients with ESR1 positive tumors is better due to the isoform of COL6A3 is expressed almost exclusively in Successfully therapy with selective estrogen receptor modu cancer samples and could potentially serve as a new cancer lators (Sun et al., 2015; Hayashi et al., 2003: Bogush et al., marker (Thorsen et al., 2008). COL6A3 is highly expressed 2009: Miyoshi et al., 2010; Xu et al., 2011; Yakimchuket al., in pancreatic ductal adenocarcinoma tissue and undergoes 2013: Fuqua et al., 2014). ESR1 signaling interferes with tumor-specific alternative splicing (Kang et al., 2014). different pathways responsible for cell transformation, COL6A3 has been demonstrated to correlate with high growth and survival like the EGFR/IGFR, PI3K/Akt/mTOR, grade ovarian cancer and contributes to cisplatin resistance. p53, HER2, NFkappaB and TGF-beta pathways (Frasor et COL6A3 was observed to be frequently over-expressed in al., 2015; Band and Laiho, 2011; Berger et al., 2013; gastric cancer tissues (Xie et al., 2014). COL6A3 mutation Skandalis et al., 2014; Mehta and Tripathy, 2014: Ciruelos (s) significantly predicted a better overall Survival in patients Gil, 2014). with colorectal carcinoma independent of tumor differentia 011.4 FMN1 encodes formin1 a protein that has a role in tion and TNM staging (Yu et al., 2015). COL6A3 expression the formation of adherent junctions and the polymerization was reported to be increased in pancreatic cancer, colon of linear actin cables (RefSeq, 2002). A single nucleotide cancer, gastric cancer, mucoepidermoid carcinomas and polymorphism in FMN1 is associated with an increased risk ovarian cancer. Cancer associated transcript variants includ of prostate cancer (Lisitskaia et al., 2010). ing exons 3, 4 and 6 were detected in colon cancer, bladder 0115 HAVCR1, also known as hepatitis A virus cellular cancer, prostate cancer and pancreatic cancer (Arafat et al., receptor 1 or KIM-1, encodes a membrane receptor protein 2011; Smith et al., 2009: Yang et al., 2007: Xie et al., 2014: for both human hepatitis A virus and TIMD4 and may be Leivo et al., 2005; Sherman-Baust et al., 2003; Gardina et involved in the moderation of asthma and allergic diseases al., 2006; Thorsen et al., 2008). In ovarian cancer COL6A3 (RefSeq, 2002). HAVCR1 was described as a novel bio levels correlated with higher tumor grade and in pancreatic marker candidate associated with ovarian clear cell carci cancer COL6A3 was shown to represent a suitable diagnos noma and renal cell carcinoma (Bonventre, 2014; Kobayashi tic serum biomarker (Sherman-Baust et al., 2003; Kang et et al., 2015). HAVCR1 was shown to activate the IL-6/ al., 2014). STAT-3/HIF-1A axis in clear cell renal cell carcinoma 0110 CXorf61, also known as CT83, encodes the cancer/ derived cell lines and determines tumor progression and testis antigen 83 and is located on chromosome Xq23 patient outcome (Cuadros et al., 2014). Constitutive expres (RefSeq, 2002). Expression of CXorf61 has been described sion of HAVCR1 in the kidney was described as a potential in different cancer types, including breast cancer and lung susceptibility trait for clear cell renal cell carcinoma devel cancer (Yao et al., 2014; Hanagiri et al., 2013; Baba et al., opment (Cuadros et al., 2013). Furthermore, enhanced 2013). CXorf61 was shown to be an immunogenic cancer HAVCR1 ecto-domain shedding was shown to promote an testis antigen in lung cancer. Therefore, it might represent a invasive phenotype in vitro and more aggressive tumors in promising candidate for anti-cancer immunotherapy (Fuku vivo (Cuadros et al., 2013). HAVCR1 was described as yama et al., 2006). being up-regulated in renal cell and ovarian clear cell 0111 CYP4Z1 encodes a member of the cytochrome carcinomas and colorectal cancer (Wang et al., 2013b). P450 superfamily of enzymes. The cytochrome P450 pro HAVCR1 up-regulation was described as a potential diag teins are monooxygenases which catalyze many reactions nostic biomarker for colorectal cancer and a prognostic involved in drug metabolism and synthesis of cholesterol, marker for a longer disease-free interval after Surgery, which steroids and other lipids (RefSeq, 2002). CYP4Z1 over may also be involved in the metastatic cascade in colorectal expression in breast cancer is associated with high tumor cancer (Wang et al., 2013b). HAVCR1 was shown to be grade and poor prognosis. Functionally, CYP4Z1 promotes associated with T cell large granular lymphocyte leukemia tumor angiogenesis and growth in breast cancer partly via (Wlodarski et al., 2008). PI3/Akt and ERK1/2 signaling (Yu et al., 2012; Murray et 0116. HORMAD1 (also called CT46) encodes a al., 2010). Additionally, CYP4Z1 was described to play a NORMA domain-containing protein that may play a role in role in non-Small-cell lung cancer progression (Bankovic et meiosis. NORMA domains are involved in chromatin bind US 2016/0280759 A1 Sep. 29, 2016 ing and cell cycle regulation (RefSeq, 2002). HORMAD1 is expression and activities in the development of basal cell a cancer/testis antigen over-expressed in different cancer carcinoma (Noubissi et al., 2014). types including breast, gastric and ovarian cancer and thereby a potential biomarker and immunotherapeutic target I0121 IGF2BP3 encodes insulin-like growth factor II (Yao et al., 2014; Shahzad et al., 2013; Chen et al., 2005; mRNA binding protein 3, an oncofetal protein, which Aung et al., 2006: Adelaide et al., 2007). HORMAD1 represses translation of insulin-like growth factor II (Ref. down-regulation leads to reduction of invasion, migration Seq, 2002). Several studies have shown that IGF2BP3 acts and tumor weight and decreased VEGF protein levels in various important aspects of cell function, such as cell (Shahzad et al., 2013). polarization, migration, morphology, metabolism, prolifera 0117 HSF2BP encodes the HSF2 binding protein which tion and differentiation. In vitro studies have shown that associates with HSF2 and may be involved in modulating IGF2BP3 promotes tumor cell proliferation, adhesion, and HSF2 activation (RefSeq, 2002). invasion. Furthermore, IGF2BP3 has been shown to be 0118 HSF4 encodes heat-shock transcription factor 4, associated with aggressive and advanced cancers (Bell et al., which activates heat-shock response genes under conditions 2013; Gong et al., 2014). IGF2BP3 over-expression has of heat or other stresses (RefSeq, 2002). HSF4 was shown been described in numerous tumor types and correlated with to be down-regulated in glioblastoma (Mustafa et al., 2010). poor prognosis, advanced tumor stage and metastasis, as for 0119 HTR3A encodes a 5-hydroxytryptamine (sero example in neuroblastoma, colorectal carcinoma, intrahe tonin) receptor belonging to the ligand-gated ion channel patic cholangiocarcinoma, hepatocellular carcinoma, pros receptor Superfamily that causes fast, depolarizing responses tate cancer, and renal cell carcinoma (Bell et al., 2013; in neurons after activation (RefSeq, 2002). HTR3A (also Findeis-Hosey and Xu, 2012; Hu et al., 2014; Szarvas et al., called 5-HT3) is de-regulated in several cancer types for 2014; Jeng et al., 2009; Chen et al., 2011; Chen et al., 2013: example a down-regulation in mantle cell lymphomas, a Hoffmann et al., 2008: Lin et al., 2013: Yuan et al., 2009). differential expression in diverse B cell tumors and a 0.122 MAGEA3 encodes melanoma-associated antigen decreased expression in breast cancer cell lines (Pai et al., family member A3. MAGEA3 is widely known as cancer 2009; Rinaldi et al., 2010; Ek et al., 2002). testis antigen (RefSeq, 2002; Pineda et al., 2015; De et al., 0120 IGF2BP1, also known as CRD-BP, encodes a 1994). MAGEA3 has been known long time for being used member of the insulin-like growth factor 2 mRNA-binding in therapeutic vaccination trials of metastatic melanoma protein family which functions by binding to the mRNAs of cancer. The currently performed percutaneous peptide certain genes and regulating their translation (RefSeq, immunization with MAGEA3 and 4 other antigens of 2002). Two members of the IGF2 mRNA binding protein patients with advanced malignant melanoma was shown to family, including IGF2BP1 were described as bona fide contribute significantly to longer overall Survival by com oncofetal proteins which are de novo synthesized in various plete responders compared to incomplete responders (Coulie human cancers and which may be powerful posttranscrip et al., 2002: Fujiyama et al., 2014). In NSCLC, MAGEA3 tional oncogenes enhancing tumor growth, drug-resistance was shown to be frequently expressed. The expression of and metastasis (Lederer et al., 2014). Expression of MAGEA3 correlated with higher number of tumor necrosis IGF2BP1 was reported to correlate with an overall poor in NSCLC tissue samples and was shown to inhibit the prognosis and metastasis in various human cancers (Lederer proliferation and invasion and promote the apoptosis in lung et al., 2014). Thus, IGF2BP1 was suggested to be a powerful cancer cell line. By the patients with adenocarcinomas, the biomarker and candidate target for cancer therapy (Lederer expression of MAGEA3 was associated with better survival. et al., 2014). IGF2BP family members were described to be The whole cell antiMAGEA3 vaccine is currently under the highly associated with cancer metastasis and expression of investigation in the promising phase III clinical trial for oncogenic factors such as KRAS, MYC and MDR1 (Bell et treatment of NSCLC (Perez et al., 2011; Reck, 2012; Hallet al., 2013). IGF2BP1 was shown to interact with C-MYC and al., 2013; Grah et al., 2014; Liu et al., 2015). MAGEA3 was found to be expressed in the vast majority of colon and together with 4 other genes was shown to be frequently breast tumors and sarcomas as well as in benign tumors such expressed in HCC. The expression of those genes was as breast fibroadenomas and meningiomas (loannidis et al., correlated with the number of circulating tumor cells, high 2003). IGF2BP1 was shown to be up-regulated in hepato tumor grade and advanced stage in HCC patients. The cellular carcinoma and basal cell carcinoma (Noubissi et al., frequency of liver metastasis was shown to be significantly 2014: Zhang et al., 2015a). Up-regulation of IGF2BP1 and higher in cases with tumor samples that expressed MAGE3 other genes was shown to be significantly associated with than in those that did not express this gene (Bahnassy et al., poor post-Surgery prognosis in hepatocellular carcinoma 2014; Hasegawa et al., 1998). Cancer stem cell-like side (Zhang et al., 2015a). IGF2BP1 was shown to be a target of populations isolated from a bladder cancer cell line as well the tumor suppressor miR-9 and miR-372 in hepatocellular as from lung, colon, or breast cancer cell lines showed carcinoma and in renal cell carcinoma, respectively (Huang expression of MAGEA3 among other cancer-testis antigens. et al., 2015; Zhang et al., 2015a). Loss of stromal IGF2BP1 In general, cancer stem cells are known for being resistant was shown to promote a tumorigenic microenvironment in to current cancer therapy and cause post-therapeutic cancer the colon, indicating that IGF2BP1 plays a tumor-suppres recurrence and progression. Thus, MAGEA3 may serve as a sive role in colon stromal cells (Hamilton et al., 2015). novel target for immunotherapeutic treatment in particular IGF2BP1 was shown to be associated with stage 4 tumors, of bladder cancer (Yamada et al., 2013: Yin et al., 2014). In decreased patient survival and MYCN gene amplification in head and neck squamous cell carcinoma, the expression of neuroblastoma and may therefore be a potential oncogene MAGEA3 was shown to be associated with better disease and an independent negative prognostic factor in neuroblas free survival (Zamuner et al., 2015). Furthermore, toma (Bell et al., 2015). IGF2BP1 was described as a direct MAGEA3 can be used as a prognostic marker for ovarian target of WNT/B-catenin signaling which regulates GLI1 cancer (Szajnik et al., 2013). US 2016/0280759 A1 Sep. 29, 2016
0123 MAGEA4, also known as MAGE4, encodes a colon, or breast cancer cell lines showed expression of member of the MAGEA gene family and is located on MAGEA6 among other cancer-testis antigens (Yamada et chromosome Xq28 (RefSeq, 2002). MAGEA4 was al., 2013). described as a cancer testis antigen which was found to be 0.125 MAGEA9, also known as MAGE9 or MAGE-A9, expressed in a small fraction of classic seminomas but not in encodes a member of the MAGEA gene family and is non-seminomatous testicular germ cell tumors, in breast located on chromosome Xq28 (RefSeq, 2002). High expres carcinoma, Epstein-Barr Virus-negative cases of Hodgkin’s sion of MAGEA9 in tumor and stromal cells of non-small lymphoma, esophageal carcinoma, lung carcinoma, bladder cell lung cancer was shown to be correlated with poor carcinoma, head and neck carcinoma, and colorectal cancer, survival (Zhang et al., 2015b). MAGEA9 expression was oral squamous cell carcinoma, and hepatocellular carcinoma described as an independent prognostic factor for the five (Ries et al., 2005; Bode et al., 2014; Liet al., 2005; Ottaviani year overall Survival rate in non-small cell lung cancer et al., 2006; Hennard et al., 2006; Chen et al., 2003). (Zhang et al., 2015b). MAGEA9 presence in newly diag MAGEA4 was shown to be frequently expressed in primary nosed cases of multiple myeloma was shown to be associ mucosal melanomas of the head and neck and thus may be ated with shorter overall survival (van et al., 2011). a potential target for cancer testis antigen-based immuno MAGEA9 was described as a renal cell carcinoma antigen therapy (Prasad et al., 2004). MAGEA4 was shown to be whose application in dendritic cell vaccination in BALB/c preferentially expressed in cancer stem-like cells derived mice was shown to result in rejection of low-dose RENCA from LHK2 lung adenocarcinoma cells, SW480 colon MAGEA9 renal cell carcinoma grafts (Herbert et al., 2010). adenocarcinoma cells and MCF7 breast adenocarcinoma MAGEA9 peptide-specific cytotoxic T-lymphocyte lines cells (Yamada et al., 2013). Over-expression of MAGEA4 in were shown to display high cytotoxic activity against pep spontaneously transformed normal oral keratinocytes was tide-loaded T2 cells and naturally MAGEA9 expressing shown to promote growth by preventing cell cycle arrest and renal cell carcinoma cell lines, which makes MAGEA9 a by inhibiting apoptosis mediated by the p53 transcriptional potential Suitable target for immunotherapy of renal cell targets BAX and CDKN1A (Bhan et al., 2012). MAGEA4 carcinoma (Oehlrich et al., 2005). MAGEA9 was shown to was shown to be more frequently expressed in hepatitis C be one of the most commonly expressed cancer testis virus-infected patients with cirrhosis and late-stage hepato antigens in uterine cancers (Risinger et al., 2007). MAGEA9 cellular carcinoma compared to patients with early stage was described as a MAGE family member, which is hepatocellular carcinoma, thus making the detection of expressed in testicular cancer (Zhan et al., 2015). High MAGEA4 transcripts potentially helpful to predict progno MAGEA9 expression was shown to be associated with sis (Hussein et al., 2012). MAGEA4 was shown to be one of venous invasion and lymph node metastasis in colorectal several cancer/testis antigens that are expressed in lung cancer (Zhan et al., 2015). MAGEA9 expression was shown cancer and which may function as potential candidates in to be associated with a lower survival rate in colorectal lung cancer patients for polyvalent immunotherapy (Kim et cancer and high MAGEA9 expression was described as a al., 2012). MAGEA4 was described as being up-regulated in poor prognostic factor in colorectal cancer patients (Zhan et esophageal carcinoma and hepatocellular carcinoma (Zhao al., 2015). Thus, MAGEA9 is expected to become a new et al., 2002: Wu et al., 2011). A MAGEA4-derived native target for colorectal cancer treatment (Zhan et al., 2015). peptide analogue called p286-1Y2L9L was described as a MAGEA9 over-expression was shown to be predictive of novel candidate epitope Suitable to develop peptide vaccines poor prognosis in epithelial ovarian cancer, invasive ductal against esophageal cancer (Wu et al., 2011). Several mem breast cancer, laryngeal squamous cell carcinoma and bers of the MAGE gene family, including MAGEA4, were hepatocellular carcinoma (Gu et al., 2014; Han et al., 2014: shown to be frequently mutated in melanoma (Caballero et Xu et al., 2014; Xu et al., 2015). MAGEA9 was shown to be al., 2010). up-regulated in laryngeal squamous cell carcinoma, invasive 0.124 MAGEA6 encodes melanoma-associated antigen ductal breast cancer, epithelial ovarian cancer, colorectal family member A6. MAGEA3 is widely known as cancer cancer and hepatocellular carcinoma (Gu et al., 2014; Han et testis antigen (RefSeq, 2002; Pineda et al., 2015; De et al., al., 2014; Xu et al., 2014; Xu et al., 2015; Zhan et al., 2015). 1994). MAGEA6 was shown to be frequently expressed in (0.126 MAGEA9B encodes a duplication of the melanoma, advanced myeloma, pediatric rhabdomyosar MAGEA9 protein on the X chromosome (RefSeq, 2002). coma, sarcoma, lung, bladder, prostate, breast, and colorec MAGEA9B expression in tumor stage Ib non-small cell lung tal cancers, head and neck squamous cell, esophageal cancer is correlated with patient Survival (Urgard et al., squamous cell, and oral squamous cell carcinomas (Ries et 2011). al., 2005; Hasegawa et al., 1998; Gibbs et al., 2000; Dalerba (O127 MMP1 encodes a member of the peptidase M10 et al., 2001; Otte et al., 2001; van der Bruggen et al., 2002: family of matrix metalloproteinases (MMPs). Proteins in Lin et al., 2004; Tanaka et al., 1997). MAGEA6 expression this family are involved in the breakdown of extracellular has been associated with shorter progression-free Survival in matrix in normal physiological processes, such as embry multiple myeloma patients. In contrast in head and neck onic development, reproduction, and tissue remodeling, as squamous cell carcinoma, the expression of MAGEA6 was well as in disease processes, such as arthritis and metastasis shown to be associated with better disease-free survival (van (RefSeq, 2002). Many authors have demonstrated a positive et al., 2011; Zamuner et al., 2015). MAGEA6 was among a correlation between the pattern of MMP expression and the set of genes overexpressed in a paclitaxel-resistant ovarian tumor invasive and metastatic potential including: rectal and cancer cell line. Moreover, transfection of MAGEA6 also gastric cancer, lung carcinoma, breast, ovarian, prostate, conferred increased drug resistance to paclitaxel-sensitive thyroid cancer and brain tumors (Velinov et al., 2010). cells (Duan et al., 2003). MAGEA6 can be used as a MMP1 was identified as a biomarker with tumor stage prognostic marker for ovarian cancer (Szajnik et al., 2013). dependent expression in laryngeal Squamous cell carcinoma Cancer stem cell-like side populations isolated from lung, (Hui et al., 2015). Breast cancer patients with circulating US 2016/0280759 A1 Sep. 29, 2016 20 tumor cells with epithelial-mesenchymal transition (CTC tency and re-programming (Son et al., 2013: Mongan et al., EMT) in peripheral blood had significantly increased 2006). The expression of ZFP42 is down-regulated in pros expression of MMP1 in tumor cells (p=0.02) and tumor tate cancer cells and renal cell carcinoma, but in contrast associated stroma (p=0.05) than those of patients without up-regulated in squamous cell carcinoma (Raman et al., CTC EMT (Cierna et al., 2014). In a mouse model MMP1 2006; Lee et al., 2010; Reinisch et al., 2011). ZFP42 inhibits expression and secretion was blocked by a specific anti the JAK/STAT signaling pathway via the regulation of FGFR3 monoclonal antibody which substantially blocked SOCS3 expression, which modulates cell differentiation (Xu tumor progression (Du et al., 2014). et al., 2008). 0128 Proteins of the matrix metalloproteinase (MMP) I0132) Stimulation of an immune response is dependent family are involved in the breakdown of extracellular matrix upon the presence of antigens recognized as foreign by the in normal physiological processes, such as embryonic devel host immune system. The discovery of the existence of opment, reproduction, and tissue remodeling, as well as in tumor associated antigens has raised the possibility of using disease processes, such as arthritis and metastasis. However, a hosts immune system to intervene in tumor growth. the enzyme encoded by this gene is activated intracellularly Various mechanisms of harnessing both the humoral and by furin within the constitutive secretory pathway. Also in cellular arms of the immune system are currently being contrast to other MMPs, this enzyme cleaves alpha 1-pro explored for cancer immunotherapy. teinase inhibitor but weakly degrades structural proteins of 0.133 Specific elements of the cellular immune response the extracellular matrix (RefSeq, 2002). MMP-11, also are capable of specifically recognizing and destroying tumor named Stromely sin-3, is a member of the stromelysin Sub cells. The isolation of T-cells from tumor-infiltrating cell group belonging to MMPs superfamily, which has been populations or from peripheral blood Suggests that such cells detected in cancer cells, stromal cells and adjacent microen play an important role in natural immune defense against vironment. Differently, MMP-11 exerts a dual effect on cancer. CD8-positive T-cells in particular, which recognize tumors. On the one hand, MMP-11 promotes cancer devel class I molecules of the major histocompatibility complex opment by inhibiting apoptosis as well as enhancing migra (MHC)-bearing peptides of usually 8 to 10 amino acid tion and invasion of cancer cells; on the other hand, MMP residues derived from proteins or defect ribosomal products 11 plays a negative role against cancer development via (DRIPS) located in the cytosol, play an important role in this Suppressing metastasis in animal models. Overexpression of response. The MHC-molecules of the human are also des MMP-11 was discovered in sera of cancer patients compared ignated as human leukocyte-antigens (HLA). with normal control group as well as in multiple tumor tissue I0134) The term “T-cell response” means the specific specimens, such as gastric cancer, breast cancer, and pan proliferation and activation of effector functions induced by creatic cancer (Zhang et al., 2016). MMP-11 was demon a peptide in vitro or in vivo. For MHC class I restricted strated to be over-expressed at mRNA level and protein level cytotoxic T cells, effector functions may be lysis of peptide in CRC tissue than paired normal mucosa. Further MMP-11 pulsed, peptide-precursor pulsed or naturally peptide-pre expression was correlated with CRC lymph node metastasis: senting target cells, secretion of cytokines, preferably Inter distant metastasis and TNM stage (Tian et al., 2015). MMP feron-gamma, TNF-alpha, or IL-2 induced by peptide, 11 overexpression is associated with aggressive tumor phe secretion of effector molecules, preferably granzymes or notype and unfavorable clinical outcome in upper urinary perforins induced by peptide, or degranulation. tract urothelial carcinomas (UTUC) and urinary bladder 0.135 The term "peptide' is used herein to designate a urothelial carcinomas (UBUC), Suggesting it may serve as a series of amino acid residues, connected one to the other novel prognostic and therapeutic target (Li et al., 2016). typically by peptide bonds between the alpha-amino and 0129 MXRA5 encodes one of the matrix-remodeling carbonyl groups of the adjacent amino acids. The peptides associated proteins, which contains 7 leucine-rich repeats are preferably 9 amino acids in length, but can be as short and 12 immunoglobulin-like C2-type domains related to as 8 amino acids in length, and as long as 10, 11, 12, or 13 perlecan (RefSeq, 2002). A Chinese study identified amino acids or longer, and in case of MHC class II peptides MXRA5 as the second most frequently mutated gene in (elongated variants of the peptides of the invention) they can non-Small cell lung cancer (Xiong et al., 2012). In colon be as long as 14, 15, 16, 17, 18, 19 or 20 or more amino acids cancer, MXRA5 was shown to be over-expressed and might in length. serve as a biomarker for early diagnosis and omental metas 0.136 Furthermore, the term "peptide' shall include salts tasis (Zou et al., 2002; Wang et al., 2013a). of a series of amino acid residues, connected one to the other 0130 RAD54 encodes a protein belonging to the DEAD typically by peptide bonds between the alpha-amino and like helicase superfamily. It shares similarity with Saccha carbonyl groups of the adjacent amino acids. Preferably, the romyces cerevisiae RAD54 and RDH54, both of which are salts are pharmaceutical acceptable salts of the peptides, involved in homologous recombination and repair of DNA. Such as, for example, the chloride or acetate (trifluoroac This protein binds to double-stranded DNA, and displays etate) salts. It has to be noted that the salts of the peptides ATPase activity in the presence of DNA. This gene is highly according to the present invention differ substantially from expressed in testis and spleen, which Suggests active roles in the peptides in their state(s) in vivo, as the peptides are not meiotic and mitotic recombination (RefSeq, 2002). salts in vivo. Homozygous mutations of RAD54B were observed in pri 0.137 The term "peptide' shall also include "oligopep mary lymphoma and colon cancer (Hiramoto et al., 1999). tide'. The term "oligopeptide' is used herein to designate a RAD54B counteracts genome-destabilizing effects of direct series of amino acid residues, connected one to the other binding of RAD51 to dsDNA in human tumor cells (Mason typically by peptide bonds between the alpha-amino and et al., 2015). carbonyl groups of the adjacent amino acids. The length of 0131 ZFP42 (also called REX1) encodes a zinc finger the oligopeptide is not critical to the invention, as long as the protein used as stem cell marker and essential for pluripo correct epitope or epitopes are maintained therein. The US 2016/0280759 A1 Sep. 29, 2016
oligopeptides are typically less than about 30 amino acid TABLE 1-continued residues in length, and greater than about 15 amino acids in length. Expression frequencies F of HLA-A*02 and HLA-A*24 and the most frequent 0.138. The term “polypeptide' designates a series of HLA-DR serotypes. Frequencies are deduced from amino acid residues, connected one to the other typically by haplotype frequencies Gf within the peptide bonds between the alpha-amino and carbonyl groups American population adapted from Mori et al. (Mori et al., 1997) employing the Hardy of the adjacent amino acids. The length of the polypeptide is Weinberg formula F = 1-(1-Gf°. Combinations of not critical to the invention as long as the correct epitopes A*02 or A*24 with certain HLA-DR are maintained. In contrast to the terms peptide or oligo alleles might be enriched or less frequent than peptide, the term polypeptide is meant to refer to molecules expected from their single frequencies containing more than about 30 amino acid residues. due to linkage disequilibrium. For details 0.139. A peptide, oligopeptide, protein or polynucleotide refer to Chanock et al. (Chanock et al., 2004). coding for Such a molecule is “immunogenic' (and thus is an Calculated phenotype “immunogen' within the present invention), if it is capable Allele Population from allele frequency of inducing an immune response. In the case of the present DR1 African (North) American 13.20% invention, immunogenicity is more specifically defined as DR2 African (North) American 29.80% the ability to induce a T-cell response. Thus, an “immuno DR3 African (North) American 24.80% DR4 African (North) American 11.10% gen” would be a molecule that is capable of inducing an DRS African (North) American 31.10% immune response, and in the case of the present invention, DR6 African (North) American 33.70% a molecule capable of inducing a T-cell response. In another DR7 African (North) American 19.20% aspect, the immunogen can be the peptide, the complex of DR8 African (North) American 12.10% DR9 African (North) American 5.80% the peptide with MHC, oligopeptide, and/or protein that is DR1 Asian (North) American 6.80% used to raise specific antibodies or TCRS against it. DR2 Asian (North) American 33.80% 0140. A class I T cell "epitope' requires a short peptide DR3 Asian (North) American 9.20% DR4 Asian (North) American 28.60% that is bound to a class I MHC receptor, forming a ternary DRS Asian (North) American 30.00% complex (MHC class I alpha chain, beta-2-microglobulin, DR6 Asian (North) American 25.10% and peptide) that can be recognized by a T cell bearing a DR7 Asian (North) American 13.40% matching T-cell receptor binding to the MHC/peptide com DR8 Asian (North) American 12.70% DR9 Asian (North) American 18.60% plex with appropriate affinity. Peptides binding to MHC DR1 Latin (North) American 15.30% class I molecules are typically 8-14 amino acids in length, DR2 Latin (North) American 21.20% and most typically 9 amino acids in length. DR3 Latin (North) American 15.20% DR4 Latin (North) American 36.80% 0141. In humans there are three different genetic loci that DRS Latin (North) American 20.00% encode MHC class I molecules (the MHC-molecules of the DR6 Latin (North) American 31.10% human are also designated human leukocyte antigens DR7 Latin (North) American 20.20% (HLA)): HLA-A. HLA-B, and HLA-C. HLA-A*01, HLA DR8 Latin (North) American 18.60% DR9 Latin (North) American 2.10% A*02, and HLA-B807 are examples of different MHC class A*24 Philippines 65% I alleles that can be expressed from these loci. A*24 Russia Nenets 61% A*24:O2 Japan 59% TABLE 1. A*24 Malaysia S8% A*24:O2 Philippines S4% Expression frequencies F of HLA-A*02 and A*24 India 47% HLA-A*24 and the most frequent A*24 South Korea 40% HLA-DR serotypes. Frequencies are deduced from A*24 Sri Lanka 37% haplotype frequencies Gf within the A*24 China 32% American population adapted from Mori et al. A*24:O2 India 29% (Mori et al., 1997) employing the Hardy A*24 Australia West 22% Weinberg formula F = 1-(1-Gf°. Combinations of A*24 USA 22% A*02 or A*24 with certain HLA-DR A*24 Russia Samara 20% alleles might be enriched or less frequent than A*24 South America 20% expected from their single frequencies A*24 Europe 18% due to linkage disequilibrium. For details refer to Chanock et al. (Chanock et al., 2004). Calculated phenotype 0142. The peptides of the invention, preferably when Allele Population from allele frequency included into a vaccine of the invention as described herein bind to A*02. A vaccine may also include pan-binding MHC A*02 Caucasian (North America) 49.1% class II peptides. Therefore, the vaccine of the invention can A*02 African American (North America) 34.1% A*02 Asian American (North America) 43.2% be used to treat cancer in patients that are A*02 positive, A*02 Latin American (North American) 48.3% whereas no selection for MHC class II allotypes is necessary DR1 Caucasian (North America) 19.4% due to the pan-binding nature of these peptides. DR2 Caucasian (North America) 28.2% DR3 Caucasian (North America) 20.6% 0.143 If A*02 peptides of the invention are combined DR4 Caucasian (North America) 30.7% with peptides binding to another allele, for example A*24, a DRS Caucasian (North America) 23.3% higher percentage of any patient population can be treated DR6 Caucasian (North America) 26.7% DR7 Caucasian (North America) 24.8% compared with addressing either MHC class I allele alone. DR8 Caucasian (North America) 5.7% While in most populations less than 50% of patients could DR9 Caucasian (North America) 2.1% be addressed by either allele alone, a vaccine comprising HLA-A*24 and HLA-A*02 epitopes can treat at least 60% US 2016/0280759 A1 Sep. 29, 2016 22 of patients in any relevant population. Specifically, the frame, where the same do not interfere with manipulation or following percentages of patients will be positive for at least expression of the coding regions. one of these alleles in various regions: USA 61%, Western 0153. The term “primer’ means a short nucleic acid Europe 62%, China 75%, South Korea 77%, Japan 86% sequence that can be paired with one strand of DNA and (calculated from www.allelefrequencies.net). provides a free 3'-OH end at which a DNA polymerase starts 0144. In a preferred embodiment, the term “nucleotide synthesis of a deoxyribonucleotide chain. sequence” refers to a heteropolymer of deoxyribonucle 0154 The term “promoter” means a region of DNA otides. involved in binding of RNA polymerase to initiate transcrip 0145 The nucleotide sequence coding for a particular tion. peptide, oligopeptide, or polypeptide may be naturally 0155 The term “isolated means that the material is occurring or they may be synthetically constructed. Gener removed from its original environment (e.g., the natural ally, DNA segments encoding the peptides, polypeptides, environment, if it is naturally occurring). For example, a and proteins of this invention are assembled from cDNA naturally-occurring polynucleotide or polypeptide present in fragments and short oligonucleotide linkers, or from a series a living animal is not isolated, but the same polynucleotide of oligonucleotides, to provide a synthetic gene that is or polypeptide, separated from Some or all of the coexisting capable of being expressed in a recombinant transcriptional materials in the natural system, is isolated. Such polynucle unit comprising regulatory elements derived from a micro otides could be part of a vector and/or such polynucleotides bial or viral operon. or polypeptides could be part of a composition, and still be 0146. As used herein the term “a nucleotide coding for isolated in that Such vector or composition is not part of its (or encoding) a peptide' refers to a nucleotide sequence natural environment. coding for the peptide including artificial (man-made) start 0156 The polynucleotides, and recombinant or immuno and stop codons compatible for the biological system the genic polypeptides, disclosed in accordance with the present sequence is to be expressed by, for example, a dendritic cell invention may also be in “purified form. The term “puri or another cell system useful for the production of TCRs. fied' does not require absolute purity; rather, it is intended 0147 As used herein, reference to a nucleic acid as a relative definition, and can include preparations that are sequence includes both single stranded and double stranded highly purified or preparations that are only partially puri nucleic acid. Thus, for example for DNA, the specific fied, as those terms are understood by those of skill in the sequence, unless the context indicates otherwise, refers to relevant art. For example, individual clones isolated from a the single strand DNA of such sequence, the duplex of such cDNA library have been conventionally purified to electro sequence with its complement (double stranded DNA) and phoretic homogeneity. Purification of starting material or the complement of Such sequence. natural material to at least one order of magnitude, prefer 0148. The term “coding region” refers to that portion of ably two or three orders, and more preferably four or five a gene which either naturally or normally codes for the orders of magnitude is expressly contemplated. Further expression product of that gene in its natural genomic more, a claimed polypeptide which has a purity of preferably environment, i.e., the region coding in vivo for the native 99.999%, or at least 99.99% or 99.9%; and even desirably expression product of the gene. 99% by weight or greater is expressly encompassed. 014.9 The coding region can be derived from a non 0157. The nucleic acids and polypeptide expression prod mutated (“normal'), mutated or altered gene, or can even be ucts disclosed according to the present invention, as well as derived from a DNA sequence, or gene, wholly synthesized expression vectors containing Such nucleic acids and/or Such in the laboratory using methods well known to those of skill polypeptides, may be in "enriched form'. As used herein, the in the art of DNA synthesis. term "enriched' means that the concentration of the material 0150. The term “expression product” means the polypep is at least about 2, 5, 10, 100, or 1000 times its natural tide or protein that is the natural translation product of the concentration (for example), advantageously 0.01%, by gene and any nucleic acid sequence coding equivalents weight, preferably at least about 0.1% by weight. Enriched resulting from genetic code degeneracy and thus coding for preparations of about 0.5%, 1%. 5%, 10%, and 20% by the same amino acid(s). weight are also contemplated. The sequences, constructs, 0151. The term “fragment', when referring to a coding vectors, clones, and other materials comprising the present sequence, means a portion of DNA comprising less than the invention can advantageously be in enriched or isolated complete coding region, whose expression product retains form. The term “active fragment’ means a fragment, usually essentially the same biological function or activity as the of a peptide, polypeptide or nucleic acid sequence, that expression product of the complete coding region. generates an immune response (i.e., has immunogenic activ 0152 The term “DNA segment” refers to a DNA poly ity) when administered, alone or optionally with a suitable mer, in the form of a separate fragment or as a component adjuvant or in a vector, to an animal. Such as a mammal, for of a larger DNA construct, which has been derived from example, a rabbit or a mouse, and also including a human, DNA isolated at least once in substantially pure form, i.e., Such immune response taking the form of stimulating a free of contaminating endogenous materials and in a quan T-cell response within the recipient animal. Such as a human. tity or concentration enabling identification, manipulation, Alternatively, the “active fragment” may also be used to and recovery of the segment and its component nucleotide induce a T-cell response in vitro. sequences by standard biochemical methods, for example, 0158. As used herein, the terms “portion”, “segment” and by using a cloning vector. Such segments are provided in the “fragment’, when used in relation to polypeptides, refer to form of an open reading frame uninterrupted by internal a continuous sequence of residues, such as amino acid non-translated sequences, or introns, which are typically residues, which sequence forms a Subset of a larger present in eukaryotic genes. Sequences of non-translated sequence. For example, if a polypeptide were subjected to DNA may be present downstream from the open reading treatment with any of the common endopeptidases, such as US 2016/0280759 A1 Sep. 29, 2016
trypsin or chymotrypsin, the oligopeptides resulting from will be able to cross-react with the peptide itself (Appay et Such treatment would represent portions, segments or frag al., 2006; Colombetti et al., 2006: Fong et al., 2001; ments of the starting polypeptide. When used in relation to Zaremba et al., 1997). polynucleotides, these terms refer to the products produced 0164. By a “variant' of the given amino acid sequence by treatment of said polynucleotides with any of the endo the inventors mean that the side chains of for example, one nucleases. or two of the amino acid residues are altered (for example by 0159. In accordance with the present invention, the term replacing them with the side chain of another naturally "percent identity” or “percent identical, when referring to occurring amino acid residue or Some other side chain) Such a sequence, means that a sequence is compared to a claimed that the peptide is still able to bind to an HLA molecule in or described sequence after alignment of the sequence to be Substantially the same way as a peptide consisting of the compared (the “Compared Sequence') with the described or given amino acid sequence in consisting of SEQ ID NO: 1 claimed sequence (the “Reference Sequence'). The percent to SEQ ID NO: 288. For example, a peptide may be identity is then determined according to the following for modified so that it at least maintains, if not improves, the mula: ability to interact with and bind to the binding groove of a percent identity=1001-(C/R) suitable MHC molecule, such as HLA-A*02 or -DR, and in wherein C is the number of differences between the Refer that way it at least maintains, if not improves, the ability to ence Sequence and the Compared Sequence over the length bind to the TCR of activated T cells. of alignment between the Reference Sequence and the 0.165. These T cells can subsequently cross-react with Compared Sequence, wherein cells and kill cells that express a polypeptide that contains (i) each base or amino acid in the Reference Sequence that the natural amino acid sequence of the cognate peptide as does not have a corresponding aligned base or amino acid in defined in the aspects of the invention. As can be derived the Compared Sequence and from the scientific literature and databases (Rammensee et (ii) each gap in the Reference Sequence and al., 1999; Godkin et al., 1997), certain positions of HLA (iii) each aligned base or amino acid in the Reference binding peptides are typically anchor residues forming a Sequence that is different from an aligned base or amino acid core sequence fitting to the binding motif of the HLA in the Compared Sequence, constitutes a difference and receptor, which is defined by polar, electrophysical, hydro (iv) the alignment has to start at position 1 of the aligned phobic and spatial properties of the polypeptide chains sequences; and R is the number of bases or amino acids in constituting the binding groove. Thus, one skilled in the art the Reference Sequence over the length of the alignment would be able to modify the amino acid sequences set forth with the Compared Sequence with any gap created in the in SEQ ID NO: 1 to SEQ ID NO 288, by maintaining the Reference Sequence also being counted as a base or amino known anchor residues, and would be able to determine acid. whether such variants maintain the ability to bind MHC 0160 If an alignment exists between the Compared class I or II molecules. The variants of the present invention Sequence and the Reference Sequence for which the percent retain the ability to bind to the TCR of activated T cells, identity as calculated above is about equal to or greater than which can Subsequently cross-react with and kill cells that a specified minimum Percent Identity then the Compared express a polypeptide containing the natural amino acid Sequence has the specified minimum percent identity to the sequence of the cognate peptide as defined in the aspects of Reference Sequence even though alignments may exist in the invention. which the herein above calculated percent identity is less 0166 The original (unmodified) peptides as disclosed than the specified percent identity. herein can be modified by the substitution of one or more 0161. As mentioned above, the present invention thus residues at different, possibly selective, sites within the provides a peptide comprising a sequence that is selected peptide chain, if not otherwise stated. Preferably those from the group of consisting of SEQ ID NO: 1 to SEQ ID Substitutions are located at the end of the amino acid chain. NO: 288 or a variant thereof which is 88% homologous to Such substitutions may be of a conservative nature, for SEQ ID NO: 1 to SEQID NO: 288, or a variant thereof that example, where one amino acid is replaced by an amino acid will induce T cells cross-reacting with said peptide. The of similar structure and characteristics, such as where a peptides of the invention have the ability to bind to a hydrophobic amino acid is replaced by another hydrophobic molecule of the human major histocompatibility complex amino acid. Even more conservative would be replacement (MHC) class-I or elongated versions of said peptides to class of amino acids of the same or similar size and chemical II. nature. Such as where leucine is replaced by isoleucine. In 0162. In the present invention, the term “homologous' studies of sequence variations in families of naturally occur refers to the degree of identity (see percent identity above) ring homologous proteins, certain amino acid substitutions between sequences of two amino acid sequences, i.e. peptide are more often tolerated than others, and these are often or polypeptide sequences. The aforementioned "homology’ show correlation with similarities in size, charge, polarity, is determined by comparing two sequences aligned under and hydrophobicity between the original amino acid and its optimal conditions over the sequences to be compared. Such replacement, and Such is the basis for defining “conservative a sequence homology can be calculated by creating an substitutions.” alignment using, for example, the ClustalW algorithm. 0.167 Conservative substitutions are herein defined as Commonly available sequence analysis Software, more spe exchanges within one of the following five groups: Group cifically, Vector NTI, GENETYX or other tools are provided 1-small aliphatic, nonpolar or slightly polar residues (Ala, by public databases. Ser. Thr, Pro, Gly); Group 2-polar, negatively charged 0163 A person skilled in the art will be able to assess, residues and their amides (Asp, ASn, Glu, Gln); Group whether T cells induced by a variant of a specific peptide 3-polar, positively charged residues (His, Arg, Lys); Group US 2016/0280759 A1 Sep. 29, 2016 24
4-large, aliphatic, nonpolar residues (Met, Leu, Ile, Val, TABLE 2 - continued Cys); and Group 5-large, aromatic residues (Phe, Tyr, Trp). 0168 Less conservative substitutions might involve the Variants and motif of the peptides according to replacement of one amino acid by another that has similar SEO ID NO. : 4, 13 and 15 characteristics but is somewhat different in size, such as Position replacement of an alanine by an isoleucine residue. Highly non-conservative replacements might involve Substituting 1 2 3 4 5 6 f 8 9 an acidic amino acid for one that is polar, or even for one that is basic in character. Such “radical substitutions cannot, however, be dismissed as potentially ineffective since chemical effects are not totally predictable and radical Substitutions might well give rise to serendipitous effects not otherwise predictable from simple chemical principles. 0169. Of course, such substitutions may involve struc tures other than the common L-amino acids. Thus, D-amino acids might be substituted for the L-amino acids commonly found in the antigenic peptides of the invention and yet still be encompassed by the disclosure herein. In addition, non standard amino acids (i.e., other than the common naturally i.i.i. occurring proteinogenic amino acids) may also be used for Substitution purposes to produce immunogens and immu i. nogenic polypeptides according to the present invention. 0170 If substitutions at more than one position are found to result in a peptide with Substantially equivalent or greater antigenic activity as defined below, then combinations of SEO ID NO. 15 G L P S A T T T W those substitutions will be tested to determine if the com- Variants I L bined substitutions result in additive or synergistic effects on I the antigenicity of the peptide. At most, no more than four I positions within the peptide would be simultaneously sub- I A. stituted. . L 0171 A peptide consisting essentially of the amino acid M sequence as indicated herein can have one or two non- M A. anchor amino acids (see below regarding the anchor motif) A. L exchanged without that the ability to bind to a molecule of A. the human major histocompatibility complex (MHC) class-I A. or-II is substantially changed or is negatively affected, when A. A. compared to the non-modified peptide. In another embodi- W L ment, in a peptide consisting essentially of the amino acid W sequence as indicated herein, one or two amino acids can be W exchanged with their conservative exchange partners (see W A. herein below) without that the ability to bind to a molecule t L of the human major histocompatibility complex (MHC) T class-I or -II is Substantially changed, or is negatively T A. affected, when compared to the non-modified peptide. Q L 0172. The amino acid residues that do not substantially Q contribute to interactions with the T-cell receptor can be Q modified by replacement with other amino acids whose Q A. incorporation do not substantially- affect T-cell reactivity and Position 1 2 3 4. 5 6 7 8 9 does not eliminate binding to the relevant MHC. Thus, apart SEO ID NO. 13 R L H D E N I L L from the proviso given, the peptide of the invention may be Variants W any peptide (by which term the inventors include oligopep tide or polypeptide), which includes the amino acid A. sequences or a portion or variant thereof as given.
TABLE 2 Variants and motif of the peptides according to SEO ID NO. : 4, 13 and 15 A. Position
SEO ID NO. 4 W I G. E. L P A. Variants US 2016/0280759 A1 Sep. 29, 2016 25
TABLE 2 - continued acids, in case of the elongated class II binding peptides the length can also be 15, 16, 17, 18, 19, 20, 21 or 22 amino Variants and motif of the peptides according to acids. SEO ID NO. : 4, 13 and 15 0179. Of course, the peptide or variant according to the Position present invention will have the ability to bind to a molecule of the human major histocompatibility complex (MHC) 1. 2 3 4. 5 6 7 8 9 class I or II. Binding of a peptide or a variant to a MHC T A. complex may be tested by methods known in the art. Q W 0180 Preferably, when the T cells specific for a peptide Q I according to the present invention are tested against the Q A. substituted peptides, the peptide concentration at which the Substituted peptides achieve half the maximal increase in lysis relative to background is no more than about 1 mM, 0173 Longer (elongated) peptides may also be suitable. preferably no more than about 1 uM, more preferably no It is possible that MHC class I epitopes, although usually more than about 1 nM, and still more preferably no more between 8 and 11 amino acids long, are generated by peptide than about 100 uM, and most preferably no more than about processing from longer peptides or proteins that include the 10 uM. It is also preferred that the substituted peptide be actual epitope. It is preferred that the residues that flank the recognized by T cells from more than one individual, at least actual epitope are residues that do not Substantially affect two, and more preferably three individuals. proteolytic cleavage necessary to expose the actual epitope 0181. In a particularly preferred embodiment of the during processing. invention the peptide consists or consists essentially of an 0.174. The peptides of the invention can be elongated by amino acid sequence according to SEQID NO: 1 to SEQID up to four amino acids, that is 1, 2, 3 or 4 amino acids can NO: 288. be added to either end in any combination between 4:0 and 0182 “Consisting essentially of shall mean that a pep 0:4. Combinations of the elongations according to the inven tide according to the present invention, in addition to the tion can be found in Table 3. sequence according to any of SEQID NO: 1 to SEQID NO 288 or a variant thereof contains additional N- and/or TABLE 3 C-terminally located stretches of amino acids that are not necessarily forming part of the peptide that functions as an Combinations of the elongations of peptides of the invention epitope for MHC molecules epitope. C-terminus N-terminus 0183 Nevertheless, these stretches can be important to 4 O provide an efficient introduction of the peptide according to 3 O or 1 the present invention into the cells. In one embodiment of 2 0 or 1 or 2 the present invention, the peptide is part of a fusion protein 1 0 or 1 or 2 or 3 which comprises, for example, the 80 N-terminal amino O 0 or 1 or 2 or 3 or 4 acids of the HLA-DR antigen-associated invariant chain N-terminus C-terminus (p33, in the following “Ii') as derived from the NCBI, 4 O GenBank Accession number X00497. In other fusions, the 3 O or 1 peptides of the present invention can be fused to an antibody 2 0 or 1 or 2 as described herein, or a functional part thereof, in particular 1 0 or 1 or 2 or 3 into a sequence of an antibody, so as to be specifically O 0 or 1 or 2 or 3 or 4 targeted by said antibody, or, for example, to or into an antibody that is specific for dendritic cells as described 0.175. The amino acids for the elongation/extension can herein. be the peptides of the original sequence of the protein or any 0184. In addition, the peptide or variant may be modified other amino acid(s). The elongation can be used to enhance further to improve stability and/or binding to MHC mol the stability or solubility of the peptides. ecules in order to elicit a stronger immune response. Meth 0176 Thus, the epitopes of the present invention may be ods for Such an optimization of a peptide sequence are well identical to naturally occurring tumor-associated or tumor known in the art and include, for example, the introduction specific epitopes or may include epitopes that differ by no of reverse peptide bonds or non-peptide bonds. more than four residues from the reference peptide, as long 0185. In a reverse peptide bond amino acid residues are as they have Substantially identical antigenic activity. not joined by peptide ( CO. NH ) linkages but the peptide bond is reversed. Such retro-inverso peptidomimet 0177. In an alternative embodiment, the peptide is elon ics may be made using methods known in the art, for gated on either or both sides by more than 4 amino acids, example such as those described in Meziere et al (1997) preferably to a total length of up to 30 amino acids. This may (Meziere et al., 1997), incorporated herein by reference. lead to MHC class II binding peptides. Binding to MHC This approach involves making pseudopeptides containing class II can be tested by methods known in the art. changes involving the backbone, and not the orientation of 0178. Accordingly, the present invention provides pep side chains. Meziere et al. (Meziere et al., 1997) show that tides and variants of MHC class I epitopes, wherein the for MHC binding and T helper cell responses, these pseu peptide or variant has an overall length of between 8 and dopeptides are useful. Retro-inverse peptides, which contain 100, preferably between 8 and 30, and most preferred NH CO bonds instead of CO. NH peptide bonds, are between 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino much more resistant to proteolysis. US 2016/0280759 A1 Sep. 29, 2016 26
0186. A non-peptide bond is, for example, —CH NH, 0191 Selective reduction of disulfide bonds in proteins is —CHS , —CHCH , —CH=CH , —COCH . also common. Disulfide bonds can be formed and oxidized —CH(OH)CH , and —CHSO - U.S. Pat. No. 4,897, during the heat treatment of biopharmaceuticals. Wood 445 provides a method for the solid phase synthesis of ward's Reagent K may be used to modify specific glutamic non-peptide bonds (—CH2—NH) in polypeptide chains acid residues. N-(3-(dimethylamino)propyl)-N'-ethylcarbo which involves polypeptides synthesized by Standard pro diimide can be used to form intra-molecular crosslinks cedures and the non-peptide bond synthesized by reacting an between a lysine residue and a glutamic acid residue. For amino aldehyde and an amino acid in the presence of example, diethylpyrocarbonate is a reagent for the modifi NaCNBH, cation of histidyl residues in proteins. Histidine can also be 0187 Peptides comprising the sequences described modified using 4-hydroxy-2-nonenal. The reaction of lysine above may be synthesized with additional chemical groups residues and other C.-amino groups is, for example, useful in present at their amino and/or carboxy termini, to enhance the binding of peptides to Surfaces or the cross-linking of stability, bioavailability, and/or affinity of the peptides. For proteins/peptides. Lysine is the site of attachment of poly example, hydrophobic groups such as carbobenzoxyl, dan (ethylene)glycol and the major site of modification in the Syl, or t-butyloxycarbonyl groups may be added to the glycosylation of proteins. Methionine residues in proteins peptides amino termini. Likewise, an acetyl group or a can be modified with e.g. iodoacetamide, bromoethylamine, 9-fluorenylmethoxy-carbonyl group may be placed at the and chloramine T. peptides amino termini. Additionally, the hydrophobic 0.192 Tetranitromethane and N-acetylimidazole can be group, t-butyloxycarbonyl, or an amido group may be added used for the modification of tyrosyl residues. Cross-linking to the peptides carboxy termini. via the formation of dityrosine can be accomplished with hydrogen peroxide/copper ions. 0188 Further, the peptides of the invention may be 0193 Recent studies on the modification of tryptophan synthesized to alter their steric configuration. For example, have used N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl the D-isomer of one or more of the amino acid residues of bromide or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)- the peptide may be used, rather than the usual L-isomer. Still further, at least one of the amino acid residues of the 3H-indole (BPNS-skatole). peptides of the invention may be substituted by one of the 0194 Successful modification of therapeutic proteins and well-known non-naturally occurring amino acid residues. peptides with PEG is often associated with an extension of Alterations such as these may serve to increase the stability, circulatory half-life while cross-linking of proteins with bioavailability and/or binding action of the peptides of the glutaraldehyde, polyethylene glycol diacrylate and formal invention. dehyde is used for the preparation of hydrogels. Chemical modification of allergens for immunotherapy is often 0189 Similarly, a peptide or variant of the invention may achieved by carbamylation with potassium cyanate. be modified chemically by reacting specific amino acids 0.195 A peptide or variant, wherein the peptide is modi either before or after synthesis of the peptide. Examples for fied or includes non-peptide bonds is a preferred embodi Such modifications are well known in the art and are ment of the invention. Generally, peptides and variants (at Summarized e.g. in R. Lundblad, Chemical Reagents for least those containing peptide linkages between amino acid Protein Modification, 3rd ed. CRC Press, 2004 (Lundblad, residues) may be synthesized by the Fmoc-polyamide mode 2004), which is incorporated herein by reference. Chemical of Solid-phase peptide synthesis as disclosed by Lukas et al. modification of amino acids includes but is not limited to, (Lukas et al., 1981) and by references as cited therein. modification by acylation, amidination, pyridoxylation of Temporary N-amino group protection is afforded by the lysine, reductive alkylation, trinitrobenzylation of amino 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), cleavage of this highly base-labile protecting group is done amide modification of carboxyl groups and Sulphydryl using 20% piperidine in N,N-dimethylformamide. Side modification by performic acid oxidation of cysteine to chain functionalities may be protected as their butyl ethers cysteic acid, formation of mercurial derivatives, formation (in the case of serine threonine and tyrosine), butyl esters (in of mixed disulphides with other thiol compounds, reaction the case of glutamic acid and aspartic acid), butyloxycar with maleimide, carboxymethylation with iodoacetic acid or bonyl derivative (in the case of lysine and histidine), trityl iodoacetamide and carbamoylation with cyanate at alkaline derivative (in the case of cysteine) and 4-methoxy-2,3,6- pH, although without limitation thereto. In this regard, the trimethylbenzenesulphonyl derivative (in the case of argi skilled person is referred to Chapter 15 of Current Protocols nine). Where glutamine or asparagine are C-terminal resi In Protein Science, Eds. Coligan et al. (John Wiley and Sons dues, use is made of the 4,4'-dimethoxybenzhydryl group for NY 1995-2000) (Coligan et al., 1995) for more extensive protection of the side chain amido functionalities. The methodology relating to chemical modification of proteins. Solid-phase Support is based on a polydimethyl-acrylamide 0.190 Briefly, modification of e.g. arginyl residues in polymer constituted from the three monomers dimethylacry proteins is often based on the reaction of vicinal dicarbonyl lamide (backbone-monomer), bisacryloylethylene diamine compounds such as phenylglyoxal, 2,3-butanedione, and (cross linker) and acryloylsarcosine methyl ester (function 1.2-cyclohexanedione to form an adduct. Another example alizing agent). The peptide-to-resin cleavable linked agent is the reaction of methylglyoxal with arginine residues. used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid Cysteine can be modified without concomitant modification derivative. All amino acid derivatives are added as their of other nucleophilic sites such as lysine and histidine. As a preformed symmetrical anhydride derivatives with the result, a large number of reagents are available for the exception of asparagine and glutamine, which are added modification of cysteine. The websites of companies such as using a reversed N,N-dicyclohexyl-carbodiimide/1 hydroxy Sigma-Aldrich (www.sigma-aldrich.com) provide informa benzotriazole mediated coupling procedure. All coupling tion on specific reagents. and deprotection reactions are monitored using ninhydrin, US 2016/0280759 A1 Sep. 29, 2016 27 trinitrobenzene Sulphonic acid or isotin test procedures. than 170 normal (i.e. non-cancerous) samples analyzed, and Upon completion of synthesis, peptides are cleaved from the if the highest normal tissue presentation was less than 30% resin Support with concomitant removal of side-chain pro of the median tumor signal (over all tumor samples). tecting groups by treatment with 95% trifluoroacetic acid 0201 In order to select over-presented peptides, a pre containing a 50% scavenger mix. Scavengers commonly sentation profile is calculated showing the median sample used include ethanedithiol, phenol, anisole and water, the presentation as well as replicate variation. The profile jux exact choice depending on the constituent amino acids of the taposes samples of the tumor entity of interest to a baseline peptide being synthesized. Also a combination of Solid of normal tissue samples. Each of these profiles can then be phase and solution phase methodologies for the synthesis of consolidated into an over-presentation score by calculating peptides is possible (see, for example, (Bruckdorfer et al., the p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2004), and the references as cited therein). 2015) adjusting for multiple testing by False Discovery Rate 0196. Trifluoroacetic acid is removed by evaporation in (Benjamini and Hochberg, 1995). vacuo, with subsequent trituration with diethyl ether afford 0202 For the identification and relative quantitation of ing the crude peptide. Any scavengers present are removed HLA ligands by mass spectrometry, HLA molecules from by a simple extraction procedure which on lyophilization of shock-frozen tissue samples were purified and HLA-asso the aqueous phase affords the crude peptide free of Scaven ciated peptides were isolated. The isolated peptides were gers. Reagents for peptide synthesis are generally available separated and sequences were identified by online nano from e.g. Calbiochem-Novabiochem (Nottingham, UK). electrospray-ionization (nanoESI) liquid chromatography 0197) Purification may be performed by any one, or a mass spectrometry (LC-MS) experiments. The resulting combination of techniques such as re-crystallization, size peptide sequences were verified by comparison of the frag exclusion chromatography, ion-exchange chromatography, mentation pattern of natural TUMAPs recorded from pri hydrophobic interaction chromatography and (usually) mary tumor samples with the fragmentation patterns of reverse-phase high performance liquid chromatography corresponding synthetic reference peptides of identical using e.g. acetonitrile/water gradient separation. sequences. Since the peptides were directly identified as 0198 Analysis of peptides may be carried out using thin ligands of HLA molecules of primary tumors, these results layer chromatography, electrophoresis, in particular capil provide direct evidence for the natural processing and pre lary electrophoresis, solid phase extraction (CSPE), reverse sentation of the identified peptides on primary cancer tissue. phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment 0203 Sample numbers were (altogether/QC-pass (FAB) mass spectrometric analysis, as well as MALDI and samples): for PC N=39 (36), for RCC N=22 (18), for CRC ESI-Q-TOF mass spectrometric analysis. N=31 (28), for esophageal carcinoma N=14 (11), for BPH and prostate cancer N=53 (43), for HCC N=15 (15), for 0199 For the identification of peptides of the present NSCLC N=96 (87), for GC N=35 (33), for GB N=38 (27), invention, the database of publicly available RNA expres for breast cancer N=2 (2), for melanoma N=5 (2), for sion data (Lonsdale, 2013) from about 3000 normal tissue ovarian cancer N=21 (20), for CLL N=5 (4), for SCLC N=18 samples was screened for genes with near-absent expression (17), NHL N=18 (18), AML N=23 (18), GBC, CCC N=18 in vital organ systems, and low expression in other important (17), for UBC N=17 (15), for UEC N=19 (16). Samples have organ systems. In a second step, cancer-associated peptides passed QC if 5 mass spectrometry replicates are acquired or derived from the protein products of these genes were the sample is consumed completely, and peptides used to identified by mass spectrometry using the XPRESIDENTTM calculate the normalization factor (i.e. occurring in technical platform as described herein. replicates of the same sample with less than 50% variance, 0200. In detail, to select genes of interest using RNASeq and occurring at least in 2 independent samples) are at least data from said database, vital organ systems were considered 30% of all peptides measured in the sample. Samples that to be: brain, heart, blood vessel, lung, and liver. The median were subtyped resulting in a rare subtype (such as A*02:05, of reads per kilobase per million reads (RPKM) for vital A*02:06) were excluded for selection of the peptides of this organs was required to be less than 2, and the 75% percentile invention. was required to be less than 5 RPKM for selection of the gene. If the organ systems were covered by more than one (0204. The discovery pipeline XPRESIDENTR) v2.1 (see, sample class, e. g. different brain regions that had been for example, US 2013–0096016, which is hereby incorpo analyzed separately, the maximal median and maximal 75% rated by reference in its entirety) allows the identification percentile over the multiple sample classes was used for the and selection of relevant over-presented peptide vaccine calculation. Other important organ systems were considered candidates based on direct relative quantitation of HLA to be: skin, nerve, pituitary, colon, kidney, adipose tissue, restricted peptide levels on cancer tissues in comparison to adrenal gland, urinary bladder, whole blood, esophagus, several different non-cancerous tissues and organs. This was muscle, pancreas, salivary gland, Small intestine, stomach, achieved by the development of label-free differential quan breast, spleen, thyroid gland. The maximal median RPKM titation using the acquired LC-MS data processed by a for these organs was required to be less than 10 for selection proprietary data analysis pipeline, combining algorithms for of the gene. Other organs were considered as non-vital and sequence identification, spectral clustering, ion counting, thus no cut-off value for gene expression was applied. These retention time alignment, charge state deconvolution and organs were cervix uteri and uterus, fallopian tube, vagina, normalization. prostate, testis, and ovary. Using this screen, around 14.000 0205 Presentation levels including error estimates for candidate genes were selected. Next, presentation profiles of each peptide and sample were established. Peptides exclu peptides derived from the corresponding proteins were ana sively presented on tumor tissue and peptides over-presented lyzed. Peptides were considered interesting if they were in tumor versus non-cancerous tissues and organs have been presented on less than five normal samples in a set of more identified. US 2016/0280759 A1 Sep. 29, 2016 28
0206 HLA-peptide complexes from primary HCC, CRC, healthy tissues of the tumor-corresponding type (liver, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, colon/rectum, brain, stomach, esophagus, lung, pancreas, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, kidney, prostate, ovary, skin, breast and leukocytes) or other GBC, CCC, UBC, UEC, and CLL samples were purified and normal tissue cells, demonstrating a high degree of tumor HLA-associated peptides were isolated and analyzed by association of the source genes (see Example 2). Moreover, LC-MS (see examples). All TUMAPs contained in the the peptides themselves are strongly over-presented on present application were identified with this approach on tumor tissue "tumor tissue’ in relation to this invention HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, shall mean a sample from a patient Suffering from HCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, NHL, AML, GBC, CCC, UBC, UEC, and/or CLL samples, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, confirming their presentation on these tumor types. NHL, AML, GBC, CCC, UBC, UEC, or CLL, but not on 0207 TUMAPs identified on multiple tumor and normal normal tissues (see Example 1). tissues were quantified using ion-counting of label-free 0212 HLA-bound peptides can be recognized by the LC-MS data. The method assumes that LC-MS signal areas immune system, specifically T lymphocytes. T cells can of a peptide correlate with its abundance in the sample. All destroy the cells presenting the recognized HLA/peptide quantitative signals of a peptide in various LC-MS experi complex, e.g. HCC, CRC, GB, GC, esophageal cancer, ments were normalized based on central tendency, averaged NSCLC, RCC, BPH/PCA, OC, MCC, melanoma, breast per sample and merged into a bar plot, called presentation cancer, PC, SCLC, NHL, AML, GBC, CCC, UBC, UEC, or profile. The presentation profile consolidates different analy CLL cells presenting the derived peptides. sis methods like protein database search, spectral clustering, 0213. The peptides of the present invention have been charge state deconvolution (decharging) and retention time shown to be capable of stimulating T cell responses and/or alignment and normalization. are over-presented and thus can be used for the production 0208. Furthermore, the discovery pipeline XPRESI of antibodies and/or TCRs, such as soluble TCRs, according DENTR) v2.x allows the direct absolute quantitation of to the present invention (see Example 3. Example 4). MHC-, preferably HLA-restricted, peptide levels on cancer Furthermore, the peptides when complexed with the respec or other infected tissues. Briefly, the total cell count was tive MHC can be used for the production of antibodies calculated from the total DNA content of the analyzed tissue and/or TCRs, in particular sTCRs, according to the present sample. The total peptide amount for a TUMAP in a tissue invention, as well. Respective methods are well known to sample was measured by nanoLC-MS/MS as the ratio of the the person of skill, and can be found in the respective natural TUMAP and a known amount of an isotope-labelled literature as well. Thus, the peptides of the present invention version of the TUMAP, the so-called internal standard. The are useful for generating an immune response in a patient by efficiency of TUMAP isolation was determined by spiking which tumor cells can be destroyed. An immune response in peptide: MHC complexes of all selected TUMAPs into the a patient can be induced by direct administration of the tissue lysate at the earliest possible point of the TUMAP described peptides or Suitable precursor Substances (e.g. isolation procedure and their detection by nanoLC-MS/MS elongated peptides, proteins, or nucleic acids encoding these following completion of the peptide isolation procedure. peptides) to the patient, ideally in combination with an agent The total cell count and the amount of total peptide were enhancing the immunogenicity (i.e. an adjuvant). The calculated from triplicate measurements per tissue sample. immune response originating from Such a therapeutic vac The peptide-specific isolation efficiencies were calculated as cination can be expected to be highly specific against tumor an average from 10 spike experiments each measured as a cells because the target peptides of the present invention are triplicate (see Example 6 and Table 11). not presented on normal tissues in comparable copy num 0209. This combined analysis of RNA expression and bers, preventing the risk of undesired autoimmune reactions mass spectrometry data resulted in the 288 peptides of the against normal cells in the patient. present invention. In many cases the peptide was identified only on a low number of tumors. However, due to the limited 0214. The present description further relates to T-cell sensitivity of routine mass spectrometry analysis, RNA data receptors (TCRS) comprising an alpha chain and a beta chain provide a much better basis for coverage estimation (see ("alpha/beta TCRs). Also provided are peptides capable of Example 2). binding to TCRs and antibodies when presented by an MHC molecule. The present description also relates to nucleic 0210. The present invention provides peptides that are acids, vectors and host cells for expressing TCRS and useful in treating cancers/tumors, preferably HCC, CRC, peptides of the present description; and methods of using the GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, SaC. GBC, CCC, UBC, UEC, and CLL that over- or exclusively 0215. The term “T-cell receptor” (abbreviated TCR) present the peptides of the invention. These peptides were refers to a heterodimeric molecule comprising an alpha shown by mass spectrometry to be naturally presented by polypeptide chain (alpha chain) and a beta polypeptide chain HLA molecules on primary human HCC, CRC, GB, GC, (beta chain), wherein the heterodimeric receptor is capable esophageal cancer, NSCLC, RCC, BPH/PCA, OC, MCC, of binding to a peptide antigen presented by an HLA melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, molecule. The term also includes so-called gamma/delta UBC, UEC, CLL samples, and/or on PC samples. TCRS. 0211 Many of the source gene/proteins (also designated 0216. In one embodiment the description provides a “full-length proteins” or “underlying proteins”) from which method of producing a TCR as described herein, the method the peptides are derived were shown to be highly over comprising culturing a host cell capable of expressing the expressed in cancer compared with normal tissues—"nor TCR under conditions suitable to promote expression of the mal tissues' in relation to this invention shall mean either TCR. US 2016/0280759 A1 Sep. 29, 2016 29
0217. The description in another aspect relates to meth TRBC1 or TRBC2 constant domain sequence of the TCR ods according to the description, wherein the antigen is may be linked by the native disulfide bond between Cys4 of loaded onto class I or II MHC molecules expressed on the exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. Surface of a Suitable antigen-presenting cell or artificial 0224 TCRs of the present description may comprise a antigen-presenting cell by contacting a Sufficient amount of detectable label selected from the group consisting of a the antigen with an antigen-presenting cell or the antigen is radionuclide, a fluorophore and biotin. TCRs of the present loaded onto class I or II MHC tetramers by tetramerizing the description may be conjugated to a therapeutically active antigen/class I or II MHC complex monomers. agent, Such as a radionuclide, a chemotherapeutic agent, or 0218. The alpha and beta chains of alpha/beta TCRs, and a toxin. the gamma and delta chains of gamma/delta TCRS, are 0225. In an embodiment, a TCR of the present descrip generally regarded as each having two “domains, namely tion having at least one mutation in the alpha chain and/or variable and constant domains. The variable domain consists having at least one mutation in the beta chain has modified of a concatenation of variable region (V), and joining region glycosylation compared to the unmutated TCR. (J). The variable domain may also include a leader region 0226. In an embodiment, a TCR comprising at least one (L). Beta and delta chains may also include a diversity mutation in the TCR alpha chain and/or TCR beta chain has region (D). The alpha and beta constant domains may also a binding affinity for, and/or a binding half-life for, an include C-terminal transmembrane (TM) domains that peptide-HLA molecule complex, which is at least double anchor the alpha and beta chains to the cell membrane. that of a TCR comprising the unmutated TCR alpha chain 0219. With respect to gamma/delta TCRs, the term “TCR and/or unmutated TCR beta chain. Affinity-enhancement of gamma variable domain” as used herein refers to the con tumor-specific TCRs, and its exploitation, relies on the catenation of the TCR gamma V (TRGV) region without existence of a window for optimal TCR affinities. The leader region (L), and the TCR gamma J (TRGJ) region, and existence of such a window is based on observations that the term TCR gamma constant domain refers to the extra TCRs specific for HLA-A2-restricted pathogens have KD cellular TRGC region, or to a C-terminal truncated TRGC values that are generally about 10-fold lower when com sequence. Likewise the term “TCR delta variable domain pared to TCRs specific for HLA-A2-restricted tumor-asso refers to the concatenation of the TCR delta V (TRDV) ciated self-antigens. It is now known, although tumor anti region without leader region (L) and the TCR delta D/J gens have the potential to be immunogenic, because tumors (TRDD/TRDJ) region, and the term “TCR delta constant arise from the individual’s own cells only mutated proteins domain” refers to the extracellular TRDC region, or to a or proteins with altered translational processing will be seen C-terminal truncated TRDC sequence. as foreign by the immune system. Antigens that are upregu 0220 TCRs of the present description preferably bind to lated or overexpressed (so called self-antigens) will not a peptide-HLA molecule complex with a binding affinity necessarily induce a functional immune response against the (KD) of about 100 uM or less, about 50 uM or less, about tumor: T-cells expressing TCRs that are highly reactive to 25uM or less, or about 10 uM or less. More preferred are these antigens will have been negatively selected within the high affinity TCRs having binding affinities of about 1 uM thymus in a process known as central tolerance, meaning or less, about 100 nM or less, about 50 nM or less, about 25 that only T-cells with low-affinity TCRs for self-antigens nM or less. Non-limiting examples of preferred binding remain. Therefore, affinity of TCRs or variants of the present affinity ranges for TCRs of the present invention include description to the peptides according to the invention can be about 1 nM to about 10 nM; about 10 nM to about 20 nM; enhanced by methods well known in the art. about 20 nM to about 30 nM; about 30 nM to about 40 nM; 0227. The present description further relates to a method about 40 nM to about 50 nM; about 50 nM to about 60 nM; of identifying and isolating a TCR according to the present about 60 nM to about 70 nM; about 70 nM to about 80 nM; description, said method comprising incubating PBMCs about 80 nM to about 90 nM; and about 90 nM to about 100 from HLA-A*02-negative healthy donors with A2/peptide nM monomers, incubating the PBMCs with tetramer-phyco 0221. As used herein in connect with TCRs of the present erythrin (PE) and isolating the high avidity T-cells by description, “specific binding and grammatical variants fluo-rescence activated cell sorting (FACS) Calibur analy thereof are used to mean a TCR having a binding affinity S1S (KD) for a peptide-HLA molecule complex of 100 uM or 0228. The present description further relates to a method less. of identifying and isolating a TCR according to the present 0222 Alpha/beta heterodimeric TCRs of the present description, said method comprising obtaining a transgenic description may have an introduced disulfide bond between mouse with the entire human TCRO.f3 gene loci (1.1 and 0.7 their constant domains. Preferred TCRs of this type include Mb), whose T-cells express a diverse human TCR repertoire those which have a TRAC constant domain sequence and a that compensates for mouse TCR deficiency, immunizing TRBC1 or TRBC2 constant domain sequence except that the mouse with peptide of interest, incubating PBMCs Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2 are obtained from the transgenic mice with tetramer-phyco replaced by cysteine residues, the said cysteines forming a erythrin (PE), and isolating the high avidity T-cells by disulfide bond between the TRAC constant domain fluorescence activated cell sorting (FACS) Calibur analy sequence and the TRBC1 or TRBC2 constant domain S1S sequence of the TCR. 0229. In one aspect, to obtain T-cells expressing TCRs of 0223. With or without the introduced inter-chain bond the present description, nucleic acids encoding TCR-alpha mentioned above, alpha/beta heterodimeric TCRs of the and/or TCR-beta chains of the present description are cloned present description may have a TRAC constant domain into expression vectors, such as gamma retrovirus or lenti sequence and a TRBC1 or TRBC2 constant domain virus. The recombinant viruses are generated and then tested sequence, and the TRAC constant domain sequence and the for functionality, Such as antigen specificity and functional US 2016/0280759 A1 Sep. 29, 2016 30 avidity. An aliquot of the final product is then used to reduce the number of CD3 molecules available to form transduce the target T-cell population (generally purified properly paired TCR complexes, and therefore can signifi from patient PBMCs), which is expanded before infusion cantly decrease the functional avidity of the cells expressing into the patient. the introduced TCR (Kuball et al., 2007). 0230. In another aspect, to obtain T-cells expressing 0237 To reduce mispairing, the C-terminus domain of TCRs of the present description, TCR RNAs are synthesized the introduced TCR chains of the present description may be by techniques known in the art, e.g., in vitro transcription modified in order to promote interchain affinity, while de sys-tems. The in vitro-synthesized TCR RNAs are then creasing the ability of the introduced chains to pair with the introduced into primary CD8+ T-cells obtained from healthy endogenous TCR. These strategies may include replacing donors by electroporation to re-express tumor specific TCR the human TCR-alpha and TCR-beta C-terminus domains alpha and/or TCR-beta chains. with their murine counterparts (murinized C-terminus 0231. To increase the expression, nucleic acids encoding domain); generating a second interchain disulfide bond in TCRs of the present description may be operably linked to the C-terminus domain by introducing a second cysteine strong promoters, such as retroviral long terminal repeats residue into both the TCR-alpha and TCR-beta chains of the (LTRs), cytomegalovirus (CMV), murine stem cell virus introduced TCR (cysteine modification); Swapping interact (MSCV) U3, phosphoglycerate kinase (PGK), B-actin, ubiq ing residues in the TCR-alpha and TCR-beta chain C-ter uitin, and a simian virus 40 (SV40)/CD43 composite pro minus domains ("knob-in-hole'); and fusing the variable moter, elongation factor (EF)-1 a and the spleen focus domains of the TCR-alpha and TCR-beta chains directly to forming virus (SFFV) promoter. In a preferred embodiment, CD3 (CD3 fusion). (Schmitt et al. 2009). the promoter is heterologous to the nucleic acid being 0238. In an embodiment, a host cell is engineered to expressed. express a TCR of the present description. In preferred 0232. In addition to strong promoters, TCR expression embodiments, the host cell is a human T-cell or T-cell cassettes of the present description may contain additional progenitor. In some embodiments the T-cell or T-cell pro elements that can enhance transgene expression, including a genitor is obtained from a cancer patient. In other embodi central polypurine tract (cPPT), which promotes the nuclear ments the T-cell or T-cell progenitor is obtained from a translocation of lentiviral constructs (Follenzi et al., 2000), healthy donor. Host cells of the present description can be and the woodchuck hepatitis virus posttranscriptional regu allogeneic or autologous with respect to a patient to be latory element (wRE), which increases the level of trans treated. In one embodiment, the host is a gamma/delta T-cell gene expression by increasing RNA stability (Zufferey et al., transformed to express an alpha/beta TCR. 1999). 0239. A "pharmaceutical composition' is a composition 0233. The alpha and beta chains of a TCR of the present Suitable for administration to a human being in a medical invention may be encoded by nucleic acids located in setting. Preferably, a pharmaceutical composition is sterile separate vectors, or may be encoded by polynucleotides and produced according to GMP guidelines. located in the same vector. 0240. The pharmaceutical compositions comprise the 0234 Achieving high-level TCR surface expression peptides either in the free form or in the form of a pharma requires that both the TCR-alpha and TCR-beta chains of the ceutically acceptable salt (see also above). As used herein, introduced TCR be transcribed at high levels. To do so, the “a pharmaceutically acceptable salt” refers to a derivative of TCR-alpha and TCR-beta chains of the present description the disclosed peptides wherein the peptide is modified by may be cloned into bi-cistronic constructs in a single vector, making acid or base salts of the agent. For example, acid which has been shown to be capable of over-coming this salts are prepared from the free base (typically wherein the obstacle. The use of a viral intraribosomal entry site (IRES) neutral form of the drug has a neutral —NH2 group) between the TCR-alpha and TCR-beta chains results in the involving reaction with a suitable acid. Suitable acids for coordinated expression of both chains, because the TCR preparing acid salts include both organic acids, e.g., acetic alpha and TCR-beta chains are generated from a single acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, transcript that is broken into two proteins during translation, malic acid, malonic acid. Succinic acid, maleic acid, fumaric ensuring that an equal molar ratio of TCR-alpha and TCR acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, beta chains are produced. (Schmitt et al. 2009). mandelic acid, methane Sulfonic acid, ethane Sulfonic acid, 0235 Nucleic acids encoding TCRs of the present p-toluenesulfonic acid, salicylic acid, and the like, as well as description may be codon optimized to increase expression inorganic acids, e.g., hydrochloric acid, hydrobromic acid, from a host cell. Redundancy in the genetic code allows Sulfuric acid, nitric acid phosphoric acid and the like. Some amino acids to be encoded by more than one codon, Conversely, preparation of basic salts of acid moieties which but certain codons are less “op-timal’ than others because of may be present on a peptide are prepared using a pharma the relative availability of matching tRNAs as well as other ceutically acceptable base Such as Sodium hydroxide, potas factors (Gustafsson et al., 2004). Modifying the TCR-alpha sium hydroxide, ammonium hydroxide, calcium hydroxide, and TCR-beta gene sequences such that each amino acid is trimethylamine or the like. encoded by the optimal codon for mammalian gene expres 0241. In an especially preferred embodiment, the phar sion, as well as eliminating mRNA instability motifs or maceutical compositions comprise the peptides as salts of cryptic splice sites, has been shown to significantly enhance acetic acid (acetates), trifluoro acetates or hydrochloric acid TCR-alpha and TCR-beta gene expression (Scholten et al., (chlorides). 2006). 0242 Preferably, the medicament of the present inven 0236 Furthermore, mispairing between the introduced tion is an immunotherapeutics Such as a vaccine. It may be and endogenous TCR chains may result in the acquisition of administered directly into the patient, into the affected organ specificities that pose a significant risk for autoimmunity. or systemically i.d., i.m., s.c., i.p. and i.v., or applied ex vivo For example, the formation of mixed TCR dimers may to cells derived from the patient or a human cell line which US 2016/0280759 A1 Sep. 29, 2016 are Subsequently administered to the patient, or used in vitro into a Suitable vector, for example by engineering in Suitable to select a subpopulation of immune cells derived from the restriction sites, or it may be used to modify the DNA in patient, which are then re-administered to the patient. If the other useful ways as is known in the art. If viral vectors are nucleic acid is administered to cells in vitro, it may be useful used, pox- or adenovirus vectors are preferred. for the cells to be transfected so as to co-express immune 0248. The DNA (or in the case of retroviral vectors, stimulating cytokines. Such as interleukin-2. The peptide RNA) may then be expressed in a suitable host to produce may be substantially pure, or combined with an immune a polypeptide comprising the peptide or variant of the stimulating adjuvant (see below) or used in combination invention. Thus, the DNA encoding the peptide or variant of with immune-stimulatory cytokines, or be administered with the invention may be used in accordance with known a Suitable delivery system, for example liposomes. The techniques, appropriately modified in view of the teachings peptide may also be conjugated to a suitable carrier Such as contained herein, to construct an expression vector, which is keyhole limpet haemocyanin (KLH) or mannan (see WO then used to transform an appropriate host cell for the 95/18145 and (Longenecker et al., 1993)). The peptide may expression and production of the polypeptide of the inven also be tagged, may be a fusion protein, or may be a hybrid tion. Such techniques include those disclosed, for example, molecule. The peptides whose sequence is given in the in U.S. Pat. Nos. 4,440,859, 4,530,901, 4,582,800, 4,677, present invention are expected to stimulate CD4 or CD8 T 063, 4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, cells. However, stimulation of CD8 T cells is more efficient and 4,810,648. in the presence of help provided by CD4 T-helper cells. 0249. The DNA (or in the case of retroviral vectors, Thus, for MHC Class I epitopes that stimulate CD8 T cells RNA) encoding the polypeptide constituting the compound the fusion partner or sections of a hybrid molecule suitably of the invention may be joined to a wide variety of other provide epitopes which stimulate CD4-positive T cells. DNA sequences for introduction into an appropriate host. CD4- and CD8-stimulating epitopes are well known in the The companion DNA will depend upon the nature of the art and include those identified in the present invention. host, the manner of the introduction of the DNA into the 0243 In one aspect, the vaccine comprises at least one host, and whether episomal maintenance or integration is peptide having the amino acid sequence set forth SEQ ID desired. No. 1 to SEQID No. 288, and at least one additional peptide, 0250 Generally, the DNA is inserted into an expression preferably two to 50, more preferably two to 25, even more vector, such as a plasmid, in proper orientation and correct preferably two to 20 and most preferably two, three, four, reading frame for expression. If necessary, the DNA may be five, six, seven, eight, nine, ten, eleven, twelve, thirteen, linked to the appropriate transcriptional and translational fourteen, fifteen, sixteen, seventeen or eighteen peptides. regulatory control nucleotide sequences recognized by the The peptide(s) may be derived from one or more specific desired host, although Such controls are generally available TAAs and may bind to MHC class I molecules. in the expression vector. The vector is then introduced into 0244. A further aspect of the invention provides a nucleic the host through standard techniques. Generally, not all of acid (for example a polynucleotide) encoding a peptide or the hosts will be transformed by the vector. Therefore, it will peptide variant of the invention. The polynucleotide may be, be necessary to select for transformed host cells. One for example, DNA, cDNA, PNA, RNA or combinations selection technique involves incorporating into the expres thereof, either single- and/or double-stranded, or native or sion vector a DNA sequence, with any necessary control stabilized forms of polynucleotides, such as, for example, elements, that codes for a selectable trait in the transformed polynucleotides with a phosphorothioate backbone and it cell. Such as antibiotic resistance. may or may not contain introns so long as it codes for the 0251 Alternatively, the gene for such selectable trait can peptide. Of course, only peptides that contain naturally be on another vector, which is used to co-transform the occurring amino acid residues joined by naturally occurring desired host cell. peptide bonds are encodable by a polynucleotide. A still 0252) Host cells that have been transformed by the further aspect of the invention provides an expression vector recombinant DNA of the invention are then cultured for a capable of expressing a polypeptide according to the inven Sufficient time and under appropriate conditions known to tion. those skilled in the art in view of the teachings disclosed 0245) A variety of methods have been developed to link herein to permit the expression of the polypeptide, which polynucleotides, especially DNA, to vectors for example via can then be recovered. complementary cohesive termini. For instance, complemen 0253) Many expression systems are known, including tary homopolymer tracts can be added to the DNA segment bacteria (for example E. coli and Bacillus subtilis), yeasts to be inserted to the vector DNA. The vector and DNA (for example Saccharomyces cerevisiae), filamentous fungi segment are then joined by hydrogen bonding between the (for example Aspergillus spec.), plant cells, animal cells and complementary homopolymeric tails to form recombinant insect cells. Preferably, the system can be mammalian cells DNA molecules. such as CHO cells available from the ATCC Cell Biology 0246 Synthetic linkers containing one or more restriction Collection. sites provide an alternative method of joining the DNA 0254. A typical mammalian cell vector plasmid for con segment to vectors. Synthetic linkers containing a variety of stitutive expression comprises the CMV or SV40 promoter restriction endonuclease sites are commercially available with a suitable poly A tail and a resistance marker, Such as from a number of Sources including International Biotech neomycin. One example is pSVL available from Pharmacia, nologies Inc. New Haven, Conn., USA. Piscataway, N.J., USA. An example of an inducible mam 0247 A desirable method of modifying the DNA encod malian expression vector is pMSG, also available from ing the polypeptide of the invention employs the polymerase Pharmacia. Useful yeast plasmid vectors are pRS403-406 chain reaction as disclosed by Saiki R K, et al. (Saiki et al., and pRS413-416 and are generally available from Strata 1988). This method may be used for introducing the DNA gene Cloning Systems, La Jolla, Calif. 92037, USA. Plas US 2016/0280759 A1 Sep. 29, 2016 32 mids pRS403, pRS404, pRS405 and pRS406 are Yeast 0258 Transformation of appropriate cell hosts with a Integrating plasmids (Ylps) and incorporate the yeast select DNA construct of the present invention is accomplished by able markers HIS3, TRP1, LEU2 and URA3. Plasmids well-known methods that typically depend on the type of pRS413-416 are Yeast Centromere plasmids (Ycps). CMV vector used. With regard to transformation of prokaryotic promoter-based vectors (for example from Sigma-Aldrich) host cells, see, for example, Cohen et al. (Cohen et al., 1972) provide transient or stable expression, cytoplasmic expres and (Green and Sambrook, 2012). Transformation of yeast sion or secretion, and N-terminal or C-terminal tagging in cells is described in Sherman et al. (Sherman et al., 1986). various combinations of FLAG, 3xFLAG, c-myc or MAT. The method of Beggs (Beggs, 1978) is also useful. With These fusion proteins allow for detection, purification and regard to vertebrate cells, reagents useful in transfecting analysis of recombinant protein. Dual-tagged fusions pro such cells, for example calcium phosphate and DEAE vide flexibility in detection. dextran or liposome formulations, are available from Strata 0255. The strong human cytomegalovirus (CMV) pro gene Cloning Systems, or Life Technologies Inc., Gaithers moter regulatory region drives constitutive protein expres burg, Md. 20877, USA. Electroporation is also useful for sion levels as high as 1 mg/L in COS cells. For less potent transforming and/or transfecting cells and is well known in cell lines, protein levels are typically -0.1 mg/L. The the art for transforming yeast cell, bacterial cells, insect cells presence of the SV40 replication origin will result in high and vertebrate cells. levels of DNA replication in SV40 replication permissive 0259 Successfully transformed cells, i.e. cells that con COS cells. CMV vectors, for example, can contain the tain a DNA construct of the present invention, can be pMB1 (derivative of plBR322) origin for replication in identified by well-known techniques such as PCR. Alterna bacterial cells, the b-lactamase gene for amplicillin resistance tively, the presence of the protein in the Supernatant can be selection in bacteria, hCH polyA, and the fl origin. Vectors detected using antibodies. containing the pre-pro-trypsin leader (PPT) sequence can 0260. It will be appreciated that certain host cells of the direct the secretion of FLAG fusion proteins into the culture invention are useful in the preparation of the peptides of the medium for purification using ANTI-FLAG antibodies, res invention, for example bacterial, yeast and insect cells. ins, and plates. Other vectors and expression systems are However, other host cells may be useful in certain thera well known in the art for use with a variety of host cells. peutic methods. For example, antigen-presenting cells. Such 0256 In another embodiment two or more peptides or as dendritic cells, may usefully be used to express the peptide variants of the invention are encoded and thus peptides of the invention such that they may be loaded into expressed in a successive order (similar to “beads on a appropriate MHC molecules. Thus, the current invention string constructs). In doing so, the peptides or peptide provides a host cell comprising a nucleic acid or an expres variants may be linked or fused together by stretches of sion vector according to the invention. linker amino acids, such as for example LLLLLL, or may be 0261. In a preferred embodiment the host cell is an linked without any additional peptide(s) between them. antigen presenting cell, in particular a dendritic cell or These constructs can also be used for cancer therapy, and antigen presenting cell. APCs loaded with a recombinant may induce immune responses both involving MHC I and fusion protein containing prostatic acid phosphatase (PAP) MHC II. were approved by the U.S. Food and Drug Administration 0257 The present invention also relates to a host cell (FDA) on Apr. 29, 2010, to treat asymptomatic or minimally transformed with a polynucleotide vector construct of the symptomatic metastatic HRPC (Sipuleucel-T) (Rini et al., present invention. The host cell can be either prokaryotic or 2006: Small et al., 2006). eukaryotic. Bacterial cells may be preferred prokaryotic host 0262. A further aspect of the invention provides a method cells in Some circumstances and typically are a strain of E. of producing a peptide or its variant, the method comprising coli such as, for example, the E. coli strains DH5 available culturing a host cell and isolating the peptide from the host from Bethesda Research Laboratories Inc., Bethesda, Md., cell or its culture medium. USA, and RR1 available from the American Type Culture 0263. In another embodiment the peptide, the nucleic Collection (ATCC) of Rockville, Md., USA (No ATCC acid or the expression vector of the invention are used in 31343). Preferred eukaryotic host cells include yeast, insect medicine. For example, the peptide or its variant may be and mammalian cells, preferably vertebrate cells such as prepared for intravenous (i.v.) injection, Sub-cutaneous (s.c.) those from a mouse, rat, monkey or human fibroblastic and injection, intradermal (i.d.) injection, intraperitoneal (i.p.) colon cell lines. Yeast host cells include YPH499, YPH500 injection, intramuscular (i.m.) injection. Preferred methods and YPH501, which are generally available from Stratagene of peptide injection include s.c., i.d., i.p., i.m., and i.V. Cloning Systems, La Jolla, Calif. 92037, USA. Preferred Preferred methods of DNA injection include i.d., i.m., s.c., mammalian host cells include Chinese hamster ovary (CHO) i.p. and i.v. Doses of e.g. between 50 g and 1.5 mg. cells available from the ATCC as CCL61, NIH Swiss mouse preferably 125 ug to 500 ug, of peptide or DNA may be embryo cells NIH/3T3 available from the ATCC as CRL given and will depend on the respective peptide or DNA. 1658, monkey kidney-derived COS-1 cells available from Dosages of this range were successfully used in previous the ATCC as CRL 1650 and 293 cells which are human trials (Walter et al., 2012). embryonic kidney cells. Preferred insect cells are Sf9 cells 0264. The polynucleotide used for active vaccination which can be transfected with baculovirus expression vec may be substantially pure, or contained in a suitable vector tors. An overview regarding the choice of suitable host cells or delivery system. The nucleic acid may be DNA, cDNA, for expression can be found in, for example, the textbook of PNA, RNA or a combination thereof. Methods for designing Paulina Balbás and Argelia Lorence “Methods in Molecular and introducing such a nucleic acid are well known in the Biology Recombinant Gene Expression, Reviews and Pro art. An overview is provided by e.g. Teufel et al. (Teufel et tocols, Part One, Second Edition, ISBN 978-1-58829-262 al., 2005). Polynucleotide vaccines are easy to prepare, but 9, and other literature known to the person of skill. the mode of action of these vectors in inducing an immune US 2016/0280759 A1 Sep. 29, 2016
response is not fully understood. Suitable vectors and deliv Freund's adjuvant (IFA) that normally promote a TH2 bias. ery systems include viral DNA and/or RNA, such as systems CpG oligonucleotides show even greater adjuvant activity based on adenovirus, vaccinia virus, retroviruses, herpes when formulated or co-administered with other adjuvants or virus, adeno-associated virus or hybrids containing elements in formulations such as microparticles, nanoparticles, lipid of more than one virus. Non-viral delivery systems include emulsions or similar formulations, which are especially cationic lipids and cationic polymers and are well known in necessary for inducing a strong response when the antigen is the art of DNA delivery. Physical delivery, such as via a relatively weak. They also accelerate the immune response 'gene-gun' may also be used. The peptide or peptides and enable the antigen doses to be reduced by approximately encoded by the nucleic acid may be a fusion protein, for two orders of magnitude, with comparable antibody example with an epitope that stimulates T cells for the responses to the full-dose vaccine without CpG in some respective opposite CDR as noted above. experiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 0265. The medicament of the invention may also include describes the combined use of CpG oligonucleotides, non one or more adjuvants. Adjuvants are substances that non nucleic acid adjuvants and an antigen to induce an antigen specifically enhance or potentiate the immune response specific immune response. A CpG TLR9 antagonist is (e.g., immune responses mediated by CD8-positive T cells dSLIM (double Stem Loop Immunomodulator) by Mologen and helper-T (TH) cells to an antigen, and would thus be (Berlin, Germany) which is a preferred component of the considered useful in the medicament of the present inven pharmaceutical composition of the present invention. Other tion. Suitable adjuvants include, but are not limited to, 1018 TLR binding molecules such as RNA binding TLR 7, TLR ISS, aluminum salts, AMPLIVAX(R), AS15, BCG, CP-870, 8 and/or TLR 9 may also be used. 893, CpG 7909, CyaA, dSLIM, flagellin or TLR5 ligands 0267. Other examples for useful adjuvants include, but derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, are not limited to chemically modified CpGs (e.g. CpR, Imiquimod (ALDARAR), residuimod, ImuFact IMP321, Idera), dsRNA analogues such as Poly(I:C) and derivatives Interleukins as IL-2, IL-13, IL-21, Interferon-alpha or -beta, thereof (e.g. AmpliCenR, Hiltonol R, poly-(ICLC), poly(IC or pegylated derivatives thereof, IS Patch, ISS, ISCOMA R), poly(I:C12U), non-CpG bacterial DNA or RNA as well TRIX, ISCOMs, JuvImmune(R), LipoVac, MALP2, MF59, as immunoactive Small molecules and antibodies such as monophosphoryl lipid A. Montanide IMS 1312, Montanide cyclophosphamide, Sunitinib, Bevacizumab(R), celebrex, ISA 206, Montanide ISA 50V, Montanide ISA-51, water-in NCX-4016, sildenafil, tadalafil. Vardenafil, Sorafenib, temo oil and oil-in-water emulsions, OK-432, OM-174, OM-197 Zolomide, temsirolimus, XL-999, CP-547632, paZopanib, MP-EC, ONTAK, Osp.A, PepTel(R) vector system, poly(lac VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other anti tid co-glycolid) PLG-based and dextran microparticles, bodies targeting key structures of the immune system (e.g. talactoferrin SRL172, Virosomes and other Virus-like par anti-CD40, anti-TGFbeta, anti-TNFalpha receptor) and ticles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, SC58175, which may act therapeutically and/or as an adju Aquila’s QS21 stimulon, which is derived from saponin, vant. The amounts and concentrations of adjuvants and mycobacterial extracts and synthetic bacterial cell wall additives useful in the context of the present invention can mimics, and other proprietary adjuvants such as Ribis readily be determined by the skilled artisan without undue Detox, Quil, or Superfos. Adjuvants such as Freund's or experimentation. GM-CSF are preferred. Several immunological adjuvants 0268 Preferred adjuvants are anti-CD40, imiquimod, (e.g., MF59) specific for dendritic cells and their preparation residuimod, GM-CSF, cyclophosphamide, Sunitinib, beva have been described previously (Allison and Krummel, cizumab, interferon-alpha, CpG oligonucleotides and deri 1995). Also cytokines may be used. Several cytokines have vates, poly-(I:C) and derivates, RNA, sildenafil, and par been directly linked to influencing dendritic cell migration to ticulate formulations with PLG or virosomes. lymphoid tissues (e.g., TNF-), accelerating the maturation of 0269. In a preferred embodiment, the pharmaceutical dendritic cells into efficient antigen-presenting cells for composition according to the invention the adjuvant is T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No. selected from the group consisting of colony-stimulating 5,849,589, specifically incorporated herein by reference in factors, such as Granulocyte Macrophage Colony Stimulat its entirety) and acting as immunoadjuvants (e.g., IL-12, ing Factor (GM-CSF, SargramoStim), cyclophosphamide, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich et al., imiquimod, residuimod, and interferon-alpha. 1996). 0270. In a preferred embodiment, the pharmaceutical 0266 CpG immunostimulatory oligonucleotides have composition according to the invention the adjuvant is also been reported to enhance the effects of adjuvants in a selected from the group consisting of colony-stimulating vaccine setting. Without being bound by theory, CpG oli factors, such as Granulocyte Macrophage Colony Stimulat gonucleotides act by activating the innate (non-adaptive) ing Factor (GM-CSF, SargramoStim), cyclophosphamide, immune system via Toll-like receptors (TLR), mainly TLR9. imiquimod and residuimod. In a preferred embodiment of CpG triggered TLR9 activation enhances antigen-specific the pharmaceutical composition according to the invention, humoral and cellular responses to a wide variety of antigens, the adjuvant is cyclophosphamide, imiquimod or residui including peptide or protein antigens, live or killed viruses, mod. Even more preferred adjuvants are Montanide IMS dendritic cell vaccines, autologous cellular vaccines and 1312, Montanide ISA 206, Montanide ISA 50V, Montanide polysaccharide conjugates in both prophylactic and thera ISA-51, poly-ICLC (HiltonolR) and anti-CD40 mAB, or peutic vaccines. More importantly it enhances dendritic cell combinations thereof. maturation and differentiation, resulting in enhanced activa 0271 This composition is used for parenteral adminis tion of TH1 cells and strong cytotoxic T-lymphocyte (CTL) tration, such as Subcutaneous, intradermal, intramuscular or generation, even in the absence of CD4 T cell help. The TH1 oral administration. For this, the peptides and optionally bias induced by TLR9 stimulation is maintained even in the other molecules are dissolved or Suspended in a pharma presence of vaccine adjuvants such as alum or incomplete ceutically acceptable, preferably aqueous carrier. In addi US 2016/0280759 A1 Sep. 29, 2016 34 tion, the composition can contain excipients, such as buffers, peptide-MHC presentation, or cells loaded with peptides binding agents, blasting agents, diluents, flavors, lubricants, such that naturally occurring peptide-MHC levels are etc. The peptides can also be administered together with reached. immune stimulating Substances, such as cytokines. An 0275 Each scaffold can comprise a labelling which pro extensive listing of excipients that can be used in Such a vides that the bound scaffold can be detected by determining composition, can be, for example, taken from A. Kibbe, the presence or absence of a signal provided by the label. For Handbook of Pharmaceutical Excipients (Kibbe, 2000). The example, the scaffold can be labelled with a fluorescent dye composition can be used for a prevention, prophylaxis or any other applicable cellular marker molecule. Such and/or therapy of adenomatous or cancerous diseases. marker molecules are well known in the art. For example a Exemplary formulations can be found in, for example, fluorescence-labelling, for example provided by a fluores EP2112253. cence dye, can provide a visualization of the boundaptamer 0272. It is important to realize that the immune response by fluorescence or laser Scanning microscopy or flow cytom triggered by the vaccine according to the invention attacks etry. the cancer in different cell-stages and different stages of 0276 Each scaffold can be conjugated with a second development. Furthermore different cancer associated sig active molecule such as for example IL-21, anti-CD3, and naling pathways are attacked. This is an advantage over anti-CD28. vaccines that address only one or few targets, which may 0277 For further information on polypeptide scaffolds cause the tumor to easily adapt to the attack (tumor escape). see for example the background section of WO 2014/ Furthermore, not all individual tumors express the same 07 1978A1 and the references cited therein. pattern of antigens. Therefore, a combination of several 0278. The present invention further relates to aptamers. tumor-associated peptides ensures that every single tumor Aptamers (see for example WO 2014/191359 and the lit bears at least Some of the targets. The composition is erature as cited therein) are short single-stranded nucleic designed in Such a way that each tumor is expected to acid molecules, which can fold into defined three-dimen express several of the antigens and cover several indepen sional structures and recognize specific target structures. dent pathways necessary for tumor growth and maintenance. They have appeared to be suitable alternatives for develop Thus, the vaccine can easily be used “off-the-shelf for a ing targeted therapies. Aptamers have been shown to selec larger patient population. This means that a pre-selection of tively bind to a variety of complex targets with high affinity patients to be treated with the vaccine can be restricted to and specificity. HLA typing, does not require any additional biomarker 0279 Aptamers recognizing cell surface located mol assessments for antigen expression, but it is still ensured that ecules have been identified within the past decade and several targets are simultaneously attacked by the induced provide means for developing diagnostic and therapeutic immune response, which is important for efficacy approaches. Since aptamers have been shown to possess (Banchereau et al., 2001; Walter et al., 2012). almost no toxicity and immunogenicity they are promising 0273. As used herein, the term “scaffold’ refers to a candidates for biomedical applications. Indeed aptamers, for molecule that specifically binds to an (e.g. antigenic) deter example prostate-specific membrane-antigen recognizing minant. In one embodiment, a scaffold is able to direct the aptamers, have been Successfully employed for targeted entity to which it is attached (e.g. a (second) antigen binding therapies and shown to be functional in Xenograft in vivo moiety) to a target site, for example to a specific type of models. Furthermore, aptamers recognizing specific tumor tumor cell or tumor stroma bearing the antigenic determi cell lines have been identified. nant (e.g. the complex of a peptide with MHC, according to 0280 DNA aptamers can be selected to reveal broad the application at hand). In another embodiment a scaffold spectrum recognition properties for various cancer cells, and is able to activate signaling through its target antigen, for particularly those derived from solid tumors, while non example a T cell receptor complex antigen. Scaffolds tumorigenic and primary healthy cells are not recognized. If include but are not limited to antibodies and fragments the identified aptamers recognize not only a specific tumor thereof, antigen binding domains of an antibody, comprising sub-type but rather interact with a series of tumors, this an antibody heavy chain variable region and an antibody renders the aptamers applicable as so-called broad-spectrum light chain variable region, binding proteins comprising at diagnostics and therapeutics. least one ankyrin repeat motif and single domain antigen 0281 Further, investigation of cell-binding behavior with binding (SDAB) molecules, aptamers, (soluble) TCRs and flow cytometry showed that the aptamers revealed very good (modified) cells such as allogenic or autologous T cells. To apparent affinities that are within the nanomolar range. assess whether a molecule is a scaffold binding to a target, 0282 Aptamers are useful for diagnostic and therapeutic binding assays can be performed. purposes. Further, it could be shown that some of the 0274 “Specific' binding means that the scaffold binds aptamers are taken up by tumor cells and thus can function the peptide-MHC-complex of interest better than other natu as molecular vehicles for the targeted delivery of anti-cancer rally occurring peptide-MHC-complexes, to an extent that a agents such as siRNA into tumor cells. scaffold armed with an active molecule that is able to kill a 0283 Aptamers can be selected against complex targets cell bearing the specific target is not able to kill another cell Such as cells and tissues and complexes of the peptides without the specific target but presenting other peptide comprising, preferably consisting of a sequence according MHC complex(es). Binding to other peptide-MHC com to any of SEQID NO 1 to SEQID NO 288, according to the plexes is irrelevant if the peptide of the cross-reactive invention at hand with the MHC molecule, using the cell peptide-MHC is not naturally occurring, i.e. not derived SELEX (Systematic Evolution of Ligands by Exponential from the human HLA-peptidome. Tests to assess target cell enrichment) technique. killing are well known in the art. They should be performed 0284. The peptides of the present invention can be used using target cells (primary cells or cell lines) with unaltered to generate and develop specific antibodies against MHC/