RU2807135C2 - Fusion construction containing cell penetrating peptide, polyepitope and tlr peptide agonist, intended for treatment of cancer - Google Patents

Abstract

FIELD: immunology; medicine.

SUBSTANCE: invention is related in particular to a complex intended for use in the prevention and/or treatment of cancer, wherein the complex contains a) a cell-penetrating peptide, b) at least three antigenic epitopes and c) at least one peptide agonist TLR, in which components a)-c) are covalently linked. In particular, compositions are described for use in the prevention and/or treatment of colorectal cancer, such as pharmaceutical compositions and vaccines.

EFFECT: present invention makes it possible to obtain a new complex for use in immunotherapy of colorectal cancer, which is a more effective vaccine, primarily an anti-cancer vaccine, which has improved antitumor activity, intended for the prevention and/or treatment of colorectal cancer.

47 cl, 76 dwg, 46 ex

Description

The present invention relates to the field of vaccination, primarily to vaccines for the prevention and/or treatment of colorectal cancer (CRC).

Colorectal cancer (CRC). Overall, CRC is a common and lethal disease, ranking as the third most common cancer diagnosed (third in men and second in women), with more than 1.36 million new cases reported in 2012 and approximately 694,000 deaths. The risk of developing CRC varies from person to person and is influenced by environmental and genetic factors (Cancer, I.A.f.R.o. GLOBACAN 2012. 2012 [cited 5 July 2015]; available at: http://globocan.iarc.fr/Pages/burden_sel .aspx). Although the incidence of CRC is more common in men than women, it is more dependent on age and lifestyle. Thus, more than 90% of patients diagnosed with CRC were 50 years of age or older (Prevention, C.f.D.C.a. What Are the Risk Factors for Colorectal Cancer? 2015 [cited July 5, 2015]), with 2/3 of cases found in developed countries. There is evidence that overall food consumption, and more specifically red meat, or alcohol consumption, which leads to general obesity, significantly increases the risk of CRC (Stewart W. and S. Wild, World Cancer Report 2014, ed. V. Stewart and S. Wild, 2014, International Agency for Research on Cancer: Geneva). Two other factors that significantly increase the risk of developing colorectal cancer are heredity and inflammatory bowel disease. Familial adenomatous polyposis (FAP), for example, significantly increases the risk of developing CRC in people under 50 years of age (Burt R.W., J.A. DiSario, and L. Cannon-Albright, Genetics of colon cancer: impact of inheritance on colon cancer risk, Annu Rev Med, 46 , 1995, pp. 371-379). FAP, similar to MAP (associated with the MUTYH polyposis gene) (Sieber O.M. et al., Multiple colorectal adenomas, classic adenomatous polyposis, and germ-line mutations in MYH. N Engl J Med, 348(9), 2003, cc. 791 -799) or Lynch syndrome (Lynch H.T. et al., Genetics, natural history, tumor spectrum, and pathology of hereditary nonpolyposis colorectal cancer: an updated review. Gastroenterology, 104(5), 1993, cc. 1535-1549), associated with gene mutations that predispose patients to inherit multiple adenomas of the colon (Dennis J Ahnen D.M., FA. 2015 [cited July 9, 2015]; available at http://www.uptodate.com/contents/colorectal-cancer -epidemiology-risk-factors-and-protective-factors). The influence of diseases such as ulcerative colitis or Crohn's disease on the development of CRC is well known. For example, nonspecific ulcerative colitis is associated with an increased risk of 3-15 times (Ekbom A. et al., Ulcerative colitis and colorectal cancer. A population-based study. N Engl J Med, 323(18), 1990, cc. 1228- 1233). Although less data are available regarding Crohn's disease, the disease is associated with similar relative risks. For patients with a family history of CRC, early screening for adenomatous polyposis or inflammatory bowel disease is strongly recommended (Jemal A. et al., Global cancer statistics. CA Cancer J Clin, 61(2), 2011, pp. 69- 90).

As with many other types of cancer, the life expectancy of patients with CRC depends on the stage of the disease. Patients with earlier stages (stages 0, I and II) have a good prognosis (more than 75%). In stages III, there is greater heterogeneity in the survival rate (from 90% to 50%) depending on tumor invasion into peripheral tissues. Finally, only stage IV has a rapid and dramatic impact on patient survival time: once diagnosed, only about 10% of patients have a survival time greater than 60 months.

Current treatment options are generally based on surgery followed by or without reliable chemotherapy, radiation and/or targeted therapy (Moertel CG, Chemotherapy for colorectal cancer. N Engl J Med, 330(16), 1994, pp. 1136 -1142; Meyerhardt J. A. and R. J. Mayer, Systemic therapy for colorectal cancer. N Engl J Med, 352(5), 2005, pp. 476-487). Depending on the stage of the disease, current treatment options provide survival rates of 30%-95% within two years of diagnosis. For all early stages of CRC, initial treatment strategies include surgery, with or without an adjunctive regimen. The decision on the subsequent therapeutic regimen depends on the stage of disease identified based on preliminary screening results. For advanced CRC, chemotherapy or targeted therapy is generally recommended as first-line treatment. The most commonly used regimens are FOLFOX, CareOX or FOLFIRI. These treatment options may be used in combination with one of the following recommended biologic drugs that target VEGF (bevacizumab/Avastin ^® from Genentech or aflibercept/Zaltrap ^® from Regeneron-Sanofi) or EGFR (cetuximab/Erbitux ^® from Merck-Serono). Other targeted therapies may be offered as stand-alone first-line treatments. Amgen's anti-EGFR monoclonal antibody (panitumumab/Vectibix ^® ) and, in recent years, Bayer's small molecule kinase inhibitor such as regorafenib (Stivarga ^® ) have been demonstrated to improve overall survival time in patients with CRC. If significant spread of the disease to other organs has already occurred, the anti-VEGF monoclonal antibody ramukurimab (Cyramza ^® ) from Eli Lilly can be used. In stage IV, radiation therapy may be used to relieve symptoms such as pain.

Although chemotherapy for CRC is well established (Moertel C.G., Chemotherapy for colorectal cancer. N Engl J Med, 330(16), 1994, cc. 1136-1342), in recent decades important efforts have been made to create more effective and less secondary effects of strategies, namely in the case of stage IV CRC (Gallagher D.J. and N. Kemeny, Metastatic colorectal cancer: from improved survival to potential cure. Oncology, 78(3-4), 2010, cc. 237-248). The first trials focused on complementary adjuvant therapy. However, many years later, the value of postoperative 5-FU-based therapy remains controversial, particularly for patients with stage II CRC (Meyerhardt J.A. and R.J. Mayer, Systemic therapy for colorectal cancer. N Engl J Med, 352(5), 2005 , pp. 476-487).

Immunotherapies have been carefully evaluated in this context. The immune system can recognize and eliminate tumor cells to a certain extent, but the level of this antitumor response is often low and ineffective. Enhancing this weak antitumor response through therapeutic vaccination has long been a goal of anticancer therapy. Thus, modulation of the immune system to enhance immune responses represents a promising therapeutic approach in oncology and can be combined with standard patient care techniques.

Promising results from preclinical studies and advances in clinical trials, including the now FDA-approved Sipuleucel-T vaccine and anti-CTLA-4 antibody, have demonstrated that active immunization is a safe and feasible treatment option for certain types of cancer. Data have been published on the induction of immune responses mediated by tumor-specific cytotoxic T lymphocytes (CTLs) using a variety of approaches, including engineered tumor cell vaccines, peptide vaccines, recombinant viral vectors, DNA-, protein-, or dendritic cell-based vaccines. However, CTL-mediated antitumor immunity only rarely correlates with tumor regression, and only a few projects have reached phase III clinical trials.

Overall, cancer vaccines have demonstrated very limited clinical efficacy to date. Thus, it is known that by the end of 2011, out of 30,000 ongoing clinical trials of cancer vaccines, only 19 reached phase III trials (Global Data, 2012). These include NeuVax, a peptide vaccine for breast cancer, Stimuvax, a liposome-based vaccine for non-small cell lung carcinoma (NSCLC) and breast cancer, TG4010, a vaccinia virus-based vaccine for NSCLC, and GSK1572932A, an adjuvanted liposome for NSCLC. These four cancer vaccines are based on different technologies and what they have in common is that they each target a single antigen.

Therapeutic cancer vaccines can be divided into two main categories: personalized (autologous) and standardized vaccines, and they are further classified depending on the technology platform. Current personalized vaccines include tumor lysate-based vaccines as well as dendritic cell-based vaccines (hereinafter referred to as cell-based vaccines). In the latter case, either pulsed injection of tumor lysates or transfection of RNA extracted from tumors can be used to load the antigen. In this case, the antigens are specific to or associated with the tumor, but are not strictly defined. Dendritic cells can also be loaded with specific antigens, either by pulsed treatment with a peptide or by using a protein such as prostatic acid phosphatase (PAP), which was used to create the Provenge ^® vaccine. However, the production process of these cell-based therapeutic agents is time-consuming and labor-intensive, and quality standards are difficult to achieve and maintain. Immunomonitoring poses additional challenges. In addition, most autologous cancer vaccines do not allow the identification or quantification of the antigens used, which should be monitored, unlike preformulated and standardized vaccines.

Unlike cell-based therapies (APC, T cells, CARs, lysates), subunit vaccines (protein or peptides) allow the development of a standardized vaccine using a simpler production process and with greatly improved batch-to-batch reproducibility that can be administered to a wide range of patients. In addition, the antigens are fully defined, allowing for improved immunomonitoring and reduced risk of adverse effects associated with the vaccine component.

Various approaches that have been evaluated in preclinical and clinical studies include short peptide vaccines (Slingluff C.L., Jr., The present and future of peptide vaccines for cancer: single or multiple, long or short, alone or in combination? Cancer journal 17 (5), 2011, cc. 343-350), vaccines based on long peptides (Melief S.J., van der Burg S.H., Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines, Nature reviews Cancer 8(5 ), 2008, pp. 351-360) and based on proteins. Unlike long peptide and protein vaccines, short peptide vaccines have a very short half-life and may have a negative effect on the immune response.

In the case of protein-based vaccines, the results of targeting MAGE-A3 with a recombinant fusion protein vaccine have been very encouraging following promising phase II data in metastatic melanoma (Kruit W.H., Suciu S., Dreno B., Mortier L. , Robert C., Chiarion-Sileni V. et al., Selection of immunostimulant AS15 for active immunization with MAGE-A3 protein: results of a randomized phase II study of the European Organization for Research and Treatment of Cancer Melanoma Group in Metastatic Melanoma. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 31(19), 2013, cc. 2413-2420) and non-small cell lung cancer (NSLC) (Vansteenkiste J., Zielinski M., binder A., Dahabreh J. , Gonzalez E.E., Malinowski W. et al., Adjuvant MAGE-A3 immunotherapy in resected non-small-cell lung cancer: phase II randomized study results. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 31(19) , 2013, cc. 2396-2403). However, in 2013, the phase III skin trial (DERMA study) in melanoma (NCT00796445) did not confirm the primary endpoint, and then in 2014 the phase III MAGRIT trial in NSCL (NCT00480025) was stopped. Despite these very disappointing clinical trial results, protein-based vaccines certainly have many benefits.

In addition, with respect to colorectal cancer, successful research into tumor immunology has led to a better understanding of the antitumor immune response through cellular and humoral pathways (Smith C. L. et al., Immunotherapy of colorectal cancer. Br Med Bull, 64, 2002, pp. 181-200 ; Koido S. et al., Immunotherapy for colorectal cancer. World J Gastroenterol, 19(46), 2013, pp. 8531-8542) and contributed to better identification of tumor antigens. This progress has opened new perspectives for immunotherapy in CRC. Passive immunotherapies including antibodies have been proposed as the first targeted therapies for the treatment of CRC. As a result of their ability to target tumor growth pathways through the epidermal growth factor receptor (EGFR) or inhibit vascular endothelial growth factor (VEGF), antibodies such as cetuximab, panitumumab and bevacizumab received FDA approval for the treatment of CRC in 2004, 2006 and 2009 respectively.

Unfortunately, clinical trials using adaptive cell transfer (ACT) for the treatment of CRC have thus far produced unsatisfactory results. In fact, the main obstacle appears to be the limited patient population to study treatments using both unengineered and genetically engineered T cells. Likewise, secondary effects found in patients treated with chimeric antigen receptor (CAR)-fused T cells have prevented the demonstration of safety and efficacy of ACT treatment (Koido S. et al., Immunotherapy for colorectal cancer. World J Gastroenterol, 19(46), 2013, pp. 8531-8542; Xiang W. et al., Colorectal cancer immunotherapy. Discov Med, 15(84), 2013, pp. 301-308). Thus, the above circumstances, in addition to the inherent disadvantages of ACT related to feasibility and cost, as well as the memory of the immune response (secondary immune response), appear to block the use of ACT-based treatments at this point in time.

In recent years, positive results from a phase III clinical trial regarding patient survival with aflibercept have led the FDA to approve the use of this anti-VEGF fusion protein in metastatic colorectal cancer (mCRC) (Clarke J.M. and H.I. Hurwitz, Ziv-aflibercept: binding to more than VEGF-A-does more matter? Nat Rev Clin Oncol, 10(1), 2013, pp. 10-11). This targeted therapy paves the way for the development of immunotherapies that do not use antibodies. There are currently no active immunotherapies and no immunomodulators approved for use in CRC. The vast majority of molecules tested for the treatment of CRC are structurally either small molecules, typically kinase inhibitors, or antibodies (52% and 28%, respectively).

Typically, a therapeutic cancer vaccine is given to cancer patients to enhance their immune system's ability to recognize and destroy tumor cells. The main goal of the therapeutic cancer vaccine approach is to generate killer T cells (also referred to as cytotoxic T lymphocytes) specific for tumor cells. To achieve this, and to achieve a strong immune response, the vaccine must contain molecules called antigens, which are also present in the tumor and which must be delivered to antigen presenting cells (APCs), primarily dendritic cells (DCs), to initiate anti-cancer immunity. DCs process these tumor antigens into small peptides that are presented on the surface of cells expressing MHC class I or MHC class II molecules to T cells. Peptides that are then recognized by T cells and thereby induce their stimulation are called epitopes. Presentation of MHC class I and MHC class II molecules mediates the activation of two classes of T cells, cytotoxic CD8 ⁺ T lymphocytes (CTL) and helper CD4 ⁺ T cells (T _h ), respectively. In addition, full activation requires a second signal in addition to antigen-recognizing T cells, the costimulatory signal, which is nonspecific to the antigen and is produced by the interaction between costimulatory molecules expressed on the surface of the APC and the T cell. Thus, the two main requirements for an effective therapeutic cancer vaccine are the specificity of tumor antigens and the ability to efficiently deliver them to DCs.

Thus, taken together, the induction of a tumor-specific immune response requires three main steps: (i) the antigen must be delivered to dendritic cells, which must process it into epitopes, (ii) dendritic cells must receive an appropriate activating signal, and (iii) activated tumor-loaded antigen, dendritic cells must generate T-cell-mediated immune responses in lymphoid organs.

Since tumor cells can evade immune system surveillance by down-regulating the expression of individual antigens (passive immunosurveillance evasion), delivery of an antigen having multiple epitopes (polyepitope antigen) should provide an advantage. Thus, protein-based vaccines provide delivery of a polyepitope antigen to antigen-presenting cells (APCs), such as dendritic cells (DCs), without restriction by a single MHC allele. Another amplification pathway is long-term epitope presentation, currently described for protein-loaded dendritic cells (van Montfoort N., Camps M.G., Khan S., Filippov D.V., Weterings J.J., Griffith J.M. et al., Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity. Proceedings of the National Academy of Sciences of the United States of America 106(16), 2009, pp. 6730-6735). In addition, proteins require uptake and processing by DCs to achieve MHC-restricted presentation of the epitopes present within them. This reduces the risk of induction of peripheral tolerance, which is found after vaccination with short peptides that do not have such strict processing requirements (Toes R.E., Offringa R., Blom R.J., Melief S.J., Kast W.M., Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance induction. Proceedings of the National Academy of Sciences of the United States of America 93(15), 1996, pp. 7855-7860).

However, most soluble proteins tend to be cleaved in endolysosomes and are weakly cross-presented to MHC class I molecules and therefore have poor immunogenicity against CD8 ⁺ T cell responses (Rosalia RA, Quakkelaar ED, Redeker A, Khan S, Camps M., Drijfhout JW, et al., Dendritic cells process synthetic long peptides better than whole protein, improving antigen presentation and T-cell activation. European journal of immunology 43(10), 2013, pp. 2554-2565). In addition, although mature DCs are more effective than immature DCs at priming and inducing T cell responses (Apetoh L., Locher S., Ghiringhelli F., Kroemer G., Zitvogel L., Harnessing dendritic cells in cancer. Semin Immunol. 23, 2011, pp. 42-49), they lose the ability to effectively absorb exogenous antigens, in particular antigens restricted by MHC class II (Banchereau J., Steinman RM, Dendritic cells and the control of immunity. Nature, 392 , 1998, pp. 245-252). As a result, peptide-pulsed DCs are subject to several limitations as vaccines.

For example, peptide degradation, rapid turnover of MHC class I molecules, and peptide dissociation from MHC class I molecules during DC/peptide production and injection may result in short half-lives of MHC class I/peptide complexes on the DC surface, resulting in weak T cell responses. answers.

To increase the efficiency of protein-based vaccine delivery, the use of cell-penetrating peptides for intracellular delivery of cancer peptides to DC has been proposed (Wang R.F., Wang H.Y., Enhancement of antitumor immunity by prolonging antigen presentation on dendritic cells. Nat Biotechnol. 20, 2002, cc. 149 -156). Cell-penetrating peptides (CPPs) are peptides consisting of 8-40 residues that have the ability to cross the cell membrane and penetrate most cell types (Copolovici D.M., Langel K., Eriste E., Langel U., Cell-penetrating peptides : design, synthesis, and applications. ACS nano 8(3), 2014, pp. 1972-1994, Milletti F., Cell-penetrating peptides: classes, origin, and current landscape. Drug Discov Today, 2012). Alternatively, they are also referred to as protein transduction domain (PTD), reflecting their source as naturally occurring proteins. Several effective CPPs have been identified from proteins, including the Tat protein of the human immunodeficiency virus, the VP22 protein of the herpes simplex virus, and fibroblast growth factor (Berry S.S. Intracellular delivery of nanoparticles via the HIV-1 tat peptide. Nanomedicine. 3, 2008, cc. 357 -365; Deshayes S., Morris M.C., Divita G., Heitz F., Cell-penetrating peptides: Tools for intracellular delivery of therapeutics. Cell Mol Life Sci. 62, 2005, pp. 1839-1849; Edenhofer F. Protein transduction revisited: Novel insights into the mechanism underlying intracellular delivery of proteins. Curr Pharm Des. 14, 2008, pp. 3628-3636; Gupta V., Levchenko T.S., Torchilin V.P., Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides. Adv Drug Deliv Rev. 57, 2005, pp. 637-651; Torchilin V.P., Recent approaches to intracellular delivery of drugs and DNA and organelle targeting. Annu Rev Biomed Eng. 8, 2006, pp. 343-375). It was found that T cell activity caused by DC/TAT-TRP2 was 3-10 times higher than the activity induced by DC/TRP2 (Wang H.Y., Fu T., Wang G., Gang Z., Donna M.P.L, Yang J.C., Restifo N.P., Hwu P., Wang R.F., Induction of CD4+ T cell-dependent antitumor immunity by TAT-mediated tumor antigen delivery into dendritic cells. J Clin Invest. 109, 2002a, pp. 1463-1470).

In addition, subunit vaccines (peptides or proteins) have weak immunogenicity. Therefore, from a therapeutic cancer vaccine perspective, a mandatory requirement for them is the addition of an effective adjuvant to the vaccine to increase the level of costimulatory molecules on DCs and therefore enhance the immune system response to target antigens. Adjuvants solve this problem by mimicking stored microbial components that are naturally recognized by the immune system. These include lipopolysaccharide (LPS), bacterial cell wall components, and nucleic acids such as double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), and DNA containing unmethylated CpG dinucleotide. Their presence in combination with a vaccine can significantly enhance the innate immune response to the antigen. In addition, the adjuvant may enhance the adaptive immune response associated with CTL and the polarized Th1 type rather than the humoral immune response leading to antibody production. Various adjuvants have been studied, of which only a limited number have been approved for use in humans. These include alum, MPL (monophosphoryl lipid A) and ASO ₄ (alum and MPL), used in the USA, and MF59 (oil-in-water emulsion), ASO ₄ liposomes, used in Europe (Lim YT, Vaccine adjuvant materials for cancer immunotherapy and control of infectious disease. Clin Exp Vaccine Res, 4(1), 2015, pp. 54-58).

Currently, Toll-like receptor (TLR) ligands are considered as a promising class of adjuvants (Baxevanis CN, IF Voutsas and OE Tsitsilonis, Toll-like receptor agonists: current status and future perspective on their utility as adjuvants in improving anticancer vaccination strategies. Immunotherapy, 5(5), 2013, pp. 497-511). Thus, in important studies aimed at the development of cancer vaccines, various TLR agonists were included in the vaccine composition, such as TLR-3 (poly I:C), TLR-4 (monophosphoryl lipid A; MPL), TLR-5 (flagellin) , TLR-7 (imiquimod) and TLR-9 (CpG) (Duthie MS, Windish HP, Fox S.V., Reed SG, Use of defined TLR ligands as adjuvants within human vaccines. Immunol Rev. 239, 2011, cc. 178-196). The types of signals and cytokines produced by immune cells following TLR stimulation control the differentiation of CD4 ⁺ T cells into Th1, Th2, Th17 and Treg cells. Stimulation of immune cells such as DC and T cells by most TLR-based adjuvants leads to the production of proinflammatory cytokines and enhances Th1 and CD8 ⁺ T cell responses (Manicassamy S., Pulendran B. Modulation of adaptive immunity with Toll-like receptors. Semin Immunol. 21, 2009, pp. 185-193).

Conjugating a vaccine to a TLR ligand is an attractive approach that has several advantages over unconjugated vaccines, including (i) preferential uptake by TLR-expressing immune cells, (ii) enhanced immune response, and (iii) reduced risk of induction of peripheral tolerance. In fact, all antigen-presenting cells loaded with antigen must be activated simultaneously. Various groups of researchers who have studied this approach using different TLR ligands have mainly linked them chemically to a peptide- or protein-based vaccine (Zom G.G., Khan S., Filippov D.V., Ossendorp F., TLR ligand-peptide conjugate vaccines: towards clinical application Adv Immunol 114, 2012, pp. 177-201). Because chemical binding to the peptide is easy to achieve, the most studied TLR ligands for vaccine conjugation have been the TLR2 agonists Pam2Cys and Pam3Cys (Y. Fujita and N. Taguchi, Overview and outlook of Toll-like receptor ligand-antigen conjugate vaccines. Ther Deliv, 3(6), 2012, pp. 749-760).

However, it has been established that most of the cancer vaccines studied to date have limited effectiveness. One explanation for this is the lack of therapies that can simultaneously (I) stimulate cytotoxic polyepitope T cell-mediated immunity, (II) induce T _h cells, and (III) enhance immunological memory. These three parameters are essential for creating effective long-term antitumor immunity. Thus, CTL specific for various epitopes can ensure the destruction of many cancer cells in a heterogeneous tumor mass and avoid the expansion of variants without antigen (tumor evasion from immunological surveillance). T _h cells are involved in maintaining long-term cellular immunity, and tumor infiltration by T _h cells is also an important step in CD8 ⁺ -CTL recruitment and function. Immunological memory is important for protection against tumor recurrence.

In light of the above, the basis of the present invention was to overcome the above-mentioned disadvantages of modern cancer vaccines and to develop a new complex for use in immunotherapy of colorectal cancer, which is a more effective vaccine, primarily an anti-cancer vaccine, which has improved antitumor activity, designed to prevent and /or treatment of colorectal cancer.

This object is achieved by the following subject matter of the invention and the accompanying claims.

Although the present invention is described in detail below, it should be obvious that the present invention is not limited to the specific methodologies, protocols and reagents specified in the present description, and they may vary. It should also be apparent that the terminology used in the context of the description is not intended to limit the scope of the present invention, which is defined only by the appended claims. Unless otherwise indicated, all technical and scientific terms used in the context of this description have the meanings commonly understood and apparent to one of ordinary skill in the art.

Elements of the present invention are described below. These elements are listed by means of specific embodiments of the invention, however, it should be understood that they can be combined in any manner and in any quantity to create additional embodiments of the invention. The various examples and preferred embodiments described are not to be construed as limiting the scope of the present invention, but are intended only to illustrate the described embodiments. It should be obvious that the present description is intended to confirm embodiments of the invention and includes embodiments of the invention that explain the described embodiments of the invention with any number of described and/or preferred elements. In addition, any permutations and combinations of all specified elements in the present description are to be considered as falling within the scope of the present description, unless the context otherwise requires.

In the present specification and in the claims below, unless the context otherwise requires, the term “comprise” and its grammatical forms such as “comprises” and “comprising” are to be considered to include the specified representative, the specified integer, or the specified step. , but not exclusive of any other unspecified representative, any other unspecified integer, or any other unspecified stage. The term "consist of" is a specific variation of the term "comprise", and any other unspecified representative, any other unspecified integer, or any other unspecified step is excluded. In the context of the present invention, the term “comprise” includes the term “consist of”. Thus, the concept of “comprising” also includes “including” as well as “consisting”, for example, a composition “comprising” X may contain only X or may sometimes include an additional element, for example, X+Y.

References in the present specification to the singular include the plural (especially in the context of the claims), unless otherwise indicated in the specification or unless the context clearly contradicts this. The reference to ranges of values in the context of this specification is to serve only as a method of abbreviated reference individually to each individual value falling within the range. Unless specifically stated otherwise, each individual value is included in the specification as if it were individually stated herein. The specification is not limited to any language used to indicate a non-claimed element essential to the practice of the invention.

The term “substantially” does not exclude “completely”; for example, a composition “substantially free” of Y may be a composition completely free of Y. If necessary, the term “substantially” can be omitted from the description of the invention.

The term “about” in relation to the numerical value of x means x±10%.

Complexes proposed in the present invention

The first object of the present invention is a complex containing:

a) cell-penetrating peptide;

b) at least one antigen or antigenic epitope; And

c) at least one TLR peptide agonist,

in which components a)-c), i.e. a cell penetrating peptide, at least one antigen or antigenic epitope and at least one TLR peptide agonist are covalently linked,

intended for use in the prevention and/or treatment of colorectal cancer.

The specified complex proposed for use according to the present invention simultaneously provides (I) stimulation of polyepitope cytotoxic T cell-mediated immunity, (II) induction of T _h cells and (III) enhancement of immunological memory. Thus, the complex proposed in the present invention is an effective vaccine, in particular with improved antitumor activity.

Preferably, the complex proposed for use according to the present invention is a polypeptide or protein, in particular a recombinant polypeptide or recombinant protein, preferably a recombinant fusion protein or recombinant fusion polypeptide. The term “recombinant” as used herein means that a substance (as used herein, a polypeptide or protein) does not occur naturally. Thus, the complex proposed for use according to the present invention, which is a recombinant polypeptide or recombinant protein, typically contains components a) to c), in which components a) to c) are of different origin, i.e. do not occur naturally in this combination.

In the context of the present invention, i.e. As used herein, the terms "peptide", "polypeptide", "protein" and variations of these terms refer to a peptide, oligopeptide, oligomer or protein, including a fusion protein, respectively, containing at least two amino acids linked to each other, preferably with using a conventional peptide bond, or alternatively, using a modified peptide bond, such as the bond in isosteric peptides. The peptide, polypeptide or protein may be composed of L-amino acids and/or D-amino acids. Preferably, the peptide, polypeptide or protein is either (entirely) composed of L-amino acids or (entirely) composed of D-amino acids, thereby forming “retro-inverted peptide sequences”. The term “retro-inverted (peptide) sequences” refers to an isomer of a linear peptide sequence in which the direction of the sequence is reversed and the chirality of each amino acid residue is reversed (inverted) (see, for example, Jameson et al., Nature , 368, 1994, pp. 744-746; Brady et al., Nature, 368, 1994, pp. 692-693). In particular, the terms “peptide”, “polypeptide”, “protein” also include “peptidomimetics”, which denote peptide analogues containing non-peptide structural elements, these peptides have the ability to mimic or antagonize the biological action(s) of a naturally occurring parent peptide. A peptidomimetic lacks the classic characteristics of a peptide, such as enzyme-cleavable peptide bonds. In particular, a peptide, polypeptide or protein may contain amino acids other than the 20 amino acids specified by the genetic code in addition to these amino acids, or it may consist of amino acids other than the 20 amino acids specified by the genetic code. In particular, in the context of the present invention, a peptide, polypeptide or protein may also consist of amino acids modified through natural processes, such as post-translational maturation processes, or through chemical processes that are well known to one skilled in the art. Such modifications are described in detail in the literature. These modifications can affect any region of the polypeptide: the peptide backbone, the amino acid chain, or even the carboxy or amino termini. In particular, the peptide or polypeptide may be branched by ubiquitination or may be cyclic with or without branching. This type of modification may result from natural or artificial post-translational processes that are well known to one skilled in the art. The terms “peptide”, “polypeptide”, “protein” in the context of the present invention also include, in particular, modified peptides, polypeptides and proteins. For example, modifications to a peptide, polypeptide, or protein may include acetylation, acylation, ADP-ribosylation, amidation, covalent binding to a nucleotide or nucleotide derivative, covalent binding to a lipid or lipid derivative, covalent binding to phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, formation disulfide bond, demethylation, glycosylation, including pegylation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, selenoylation, sulfation, addition of amino acids such as arginylation, or ubiquitination. These modifications are described in detail in the literature (Proteins Structure and Molecular Properties, 2nd ed., ed. T.E. Creighton, New York, 1993; Post-translational Covalent Modifications of Proteins, ed. V.S. Johnson, Academic Press, New York, 1983; Seifter et al., Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 182, 1990, pp. 626-646, and Rattan et al., Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci, 663, 1992, pp. 48-62). Thus, the terms "peptide", "polypeptide", "protein" preferably include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, etc. P.

However, in the most preferred embodiment of the invention, the complex proposed for use according to the present invention is a "classical" peptide, polypeptide or protein, where the "classical" peptide, polypeptide or protein generally consists of amino acids selected from the 20 amino acids defined genetic code, which are linked to each other using a common peptide bond.

If the complex proposed for use according to the present invention is a polypeptide or protein, then preferably it contains at least 50, at least 60, at least 70, at least 80, at least 90, more preferably at least 100, at least 110, even more preferably at least 120, at least 130, most preferably at least 140, or most preferably at least 150 amino acid residues.

Component a) - Cell-penetrating peptide

CPP allows for efficient delivery, i.e. transport and load, in particular, at least one antigen or antigenic epitope into antigen presenting cells (APC), in particular, dendritic cells (DC), thereby providing a mechanism for antigen processing by dendritic cells.

The term “cell penetrating peptides” (“CPPs”) is generally used to refer to short peptides that have the ability to transport various types of cargo molecules (“payload”) across the plasma membrane and, as a result, facilitate the uptake of various cargo molecules by cells. (from nanoparticles to small chemical molecules and large DNA fragments). "Cellular internalization" of cargo molecules bound to a cell-penetrating peptide generally means transport of the cargo molecule across the plasma membrane and thus entry of the cargo molecule into the cell. Then, depending on the specific circumstances, the cargo molecule can be released into the cytoplasm, directed to an intracellular organelle, or also presented on the cell surface. The cell penetrating ability or internalization of a cell penetrating peptide or complex containing said cell penetrating peptide of the invention can be assessed by standard methods known to one skilled in the art, including flow cytometry or fluorescence microscopy of living and fixed cells, immunocytochemistry of transduced cells the indicated peptide or complex, and Western blotting.

Cell-penetrating peptides typically have an amino acid composition that either contains high relative amounts of positively charged amino acids, such as lysine or arginine, or have a sequence that contains an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are designated as polycationic or amphipathic, respectively. Cell-penetrating peptides have different sizes, amino acid sequences and charges, but all CPPs have a common property, which is the ability to translocate across the plasma membrane and facilitate the delivery of various cargo molecules into the cytoplasm or organelle of the cell. Currently, theories of CPP translocation are based on three main mechanisms of penetration: direct penetration through the membrane, endocytosis-mediated penetration, and translocation through the formation of a transient structure. CPP transduction is an area that requires further research. Cell-penetrating peptides are known to have numerous applications in medicine as drug delivery agents, treatments for various diseases including cancer, and viral inhibitors, as well as contrast agents for cell labeling and imaging.

Typically, cell penetrating peptides (CPPs) are peptides consisting of 8-50 residues that have the ability to cross the cell membrane and penetrate most cell types. Alternatively, they are also referred to as protein transduction domain (PTD), reflecting their source as naturally occurring proteins. Frankel and Pabo, along with Green and Lowenstein, described the ability of trans-activating transcription activator from human immunodeficiency virus 1 (HIV-TAT) to enter cells (Frankel A.D. and C.O. Pabo, Cellular uptake of the tat protein from human immunodeficiency virus. Cell, 55( 6), 1988, pp. 1189-1193). In 1991, transduction of the Antennapedia homeodomain (DNA-binding domain) from Drosophila melanogaster into nerve cells was described (Joliot A. et al., Antennapedia homeobox peptide regulates neural morphogenesis. Proc Natl Acad Sci USA, 88(5), 1991, cc. 1864-1868). In 1994, the first 16-mer CPP peptide, designated penetratin, which has the amino acid sequence RQIKIYFQNRRMKWKK (SEQ ID NO: 1), was characterized from the third helix of the Antennapedia homeodomain translocates through biological membranes. J Biol Chem, 269(14), 1994, pp. 10444-10450), after which in 1998 the minimal TAT domain was identified, having the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 2), necessary for protein transduction ( Vives E., P. Brodin and B. Lebleu, A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem, 272(25), 1997, pp. 16010-16017 ). Over the past two decades, many peptides of various origins have been described, including viral proteins, for example, VP22 (Elliott G. and P. O'Hare, Intercellular trafficking and protein delivery by a herpesvirus structural protein. Cell, 88(2), 1997, cc . p.223-233) and ZEBRA protein (Rothe R. et al., Characterization of the cell-penetrating properties of the Epstein-Barr virus ZEBRA trans-activator. J Biol Chem, 285(26), 2010, cc. 20224 -20233), or from poisons, for example, melittin (Dempsey S.E., The actions of melittin on membranes. Biochim Biophys Acta, 1031(2), 1990, pp. 143-161), mastoporan (Konno K. et al. ., Structure and biological activities of eumenine mastoparan-AF (EMP-AF), a new mast cell degranulating peptide in the venom of the solitary wasp (Anterhynchium flavomarginatum micado). Toxicon, 38(11), 2000, pp. 1505-1515 ), maurocalcine (Esteve E. et al., Transduction of the scorpion toxin maurocalcine into cells. Evidence that the toxin crosses the plasma membrane. J Biol Chem, 280(13), 2005, cc. 12833-12839), crotamine (Nascimento F.D. et al., Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans. J Biol Chem, 282(29), 2007, cc. 21349-21360) or buforin (Kobayashi S. et al., Membrane translocation mechanism of the antimicrobial peptide buforin 2. Biochemistry, 43(49), 2004, cc.15610-15616). Synthetic CPPs have also been created, including polyarginine (R8, R9, R10 and R12) (Futaki S. et al., Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J Biol Chem, 276(8), 2001, cc. 5836-5840) or transportan (Pooga M. et al., Cell penetration by transportan. FASEB J, 12(1), 1998, cc. 67-77). Any of the CPPs described above can be used as a cell-penetrating peptide, i.e. component a), in the complex proposed for use according to the present invention. In particular, component a), i.e. CPP, in the complex proposed for use according to the present invention, may contain a minimal TAT domain, which has the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 2). In particular, component a), i.e. CPP, in the complex proposed for use according to the present invention, may contain penetratin, which has the amino acid sequence RQIKIYFQNRRMKWKK (SEQ ID NO: 1).

Various CPPs that can be used as a cell penetrating peptide, e.g. component a), in the complex proposed for use according to the present invention, are also described in the review by Milletti F., Cell-penetrating peptides: classes, origin, and current landscape. Drug Discov Today 17, (15-16), 2012, cc. 850-860). In other words, the CPPs described in Milletti F., Cell-penetrating: classes, origin, and current landscape. Drug Discov Today 17 (15-16), 2012, cc. 850-860, can be used as a cell-penetrating peptide, i.e. component a), in the complex proposed for use according to the present invention. These include, in particular, cationic CPPs, amphipathic CPPs and hydrophobic CPPs, as well as CPPs derived from heparan-, RNA- and DNA-binding proteins (see table 1 in Millett F., Cell-penetrating peptides penetrating: classes, origin, and current landscape. Drug Discov Today 17 (15-16), 2012, pp. 850-860), CPPs derived from signal peptides (see table 2 in Milletti F., Cell-penetrating peptides: classes, origin, and current landscape. Drug Discov Today 17 (15-16), 2012, pp. 850-860), CPPs derived from antimicrobial peptides (see table 3 in Millett F., Cell-penetrating peptides penetrating: classes, origin, and current landscape. Drug Discov Today 17 (15-16), 2012, pp. 850-860), CPPs derived from viral proteins (see table 4 in Millett F., Cell-penetrating peptides penetrating: classes, origin, and current landscape. Drug Discov Today 17 (15-16), 2012, pp. 850-860), CPPs derived from various natural proteins (see table 5 in Millett F., Cell-penetrating peptides penetrating: classes, origin, and current landscape. Drug Discov Today 17 (15-16), 2012, cc. 850-860), as well as designed CPPs and CPPs obtained from peptide libraries (see table 6 in Millett F., Cell-penetrating peptides penetrating: classes, origin, and current landscape. Drug Discov Today 17 (15-16), 2012, pp. 850-860).

Preferably, the cell penetrating peptide contained in the complex proposed for use according to the present invention is

I) has an amino acid sequence length of said peptide of a total of 5-50 amino acids, preferably a total of 10-45 amino acids, more preferably a total of 15-45 amino acids; and/or

II) has an amino acid sequence comprising a fragment of a ZEBRA protein minimal domain, wherein said minimal domain extends from residue 170 to residue 220 of the ZEBRA amino acid sequence set forth in SEQ ID NO: 3, in which optionally 1, 2, 3, 4 or 5 amino acids are replaced , removed by deletion and/or added without abolishing the ability of the specified peptide to penetrate the cell, or a sequence variant of the specified fragment.

Thus, preferably, the cell penetrating peptide contained in the complex proposed for use according to the present invention is

I) has an amino acid sequence length of said peptide of a total of 5-50 amino acids, preferably a total of 10-45 amino acids, more preferably a total of 15-45 amino acids; And

II) has an amino acid sequence comprising a fragment of a ZEBRA protein minimal domain, wherein said minimal domain extends from residue 170 to residue 220 of the ZEBRA amino acid sequence set forth in SEQ ID NO: 3, in which optionally 1, 2, 3, 4 or 5 amino acids are replaced , removed by deletion and/or added without abolishing the ability of the specified peptide to penetrate the cell, or a sequence variant of the specified fragment.

Said preferred CPPs are described in WO 2014/041505.

The term "ZEBRA" (which is also referred to as Zta, Z, EB1 or BZLF1) generally refers to the leucine zipper (bZIP) transcriptional activator of Epstein-Barr virus (EBV). The minimal ZEBRA domain that has the ability to enter a cell has been identified as a domain extending from residue 170 to residue 220 of ZEBRA. The amino acid sequence of ZEBRA is presented in NCBI, accession code YP_401673, and contains 245 amino acid residues specified in SEQ ID NO: 3:

(SEQ ID NO: 3 - ZEBRA amino acid sequence (naturally occurring sequence from Epstein-Barr virus (EBV)) (YP_401673)).

It is currently described that CPP from the ZEBRA viral protein transduces cargo proteins through biological membranes both by (I) direct translocation and (II) lipid raft-mediated endocytosis (Rothe R., Liguori L., Villegas- Mendez A., Marques V., Grunwald D., Drouet E. et al. Characterization of the cell-penetrating properties of the Epstein-Barr virus ZEBRA trans-activator. The Journal of biological chemistry 285(26), 2010, cc. 20224-20233). The present invention hypothesized that these two entry mechanisms would enhance both class I and class II MHC-restricted presentation of cargo antigens to CD8 ⁺ and CD4 ⁺ T cells, respectively. Thus, the specified CPP can deliver polyepitope peptides to dendritic cells (DC), and then enhance CTL and Th cell activation and antitumor function. Thus, the specified CPP can effectively deliver the complex proposed for use according to the present invention to antigen presenting cells (APC) and lead to MHC class I and II restricted presentation of polyepitope antigens.

In the context of the present invention, the term "MHC class I" refers to one of the two main classes of major histocompatibility complex molecules. MHC class I molecules (also referred to as “MHC I”) are found on every nucleated cell in the body. The function of MHC class I is to present the epitope to cytotoxic cells (CTLs). In humans, MHC class I molecules consist of two polypeptide chains, α- and β2-microglobulin (b2m). Only the α chain is polymorphic and is encoded by the HLA gene, while the b2m subunit is not polymorphic and is encoded by the beta-2 microglobulin gene. In the context of the present invention, the term "MHC class II" refers to the other major class of major histocompatibility complex molecules. MHC class II molecules (also referred to as MHC II) are found only in a small number of specialized cell types, including macrophages, dendritic cells, and B cells, which are all antigen presenting cells (APCs).

Preferably, the sequence variant of the ZEBRA minimal domain fragment described above is characterized, particularly over its entire length, by an amino acid sequence identity of at least 70%, at least 75%, preferably at least 80%, more preferably at least 85% , even more preferably at least 90%, more preferably at least 95%, most preferably at least 99%, with the amino acid sequence of the ZEBRA minimal domain fragment described above, without eliminating the cell penetrating ability of the cell penetrating peptide. In particular, by “fragment” of the minimal ZEBRA domain described above is preferably meant a truncated sequence thereof, i.e. an amino acid sequence truncated at the N-terminus, C-terminus and/or within the sequence compared to the amino acid sequence of the native sequence. In addition, said ZEBRA minimal domain “fragment” preferably consists of 5-50 amino acids in total, preferably consists of 10-45 amino acids in total, more preferably consists of 15-45 amino acids in total.

Thus, the concept of “sequence variant” in the context of the present invention, i.e. in this application, refers to any change in the reference sequence. The term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. Preferably, the reference sequence is any of the sequences listed in the "Table of Sequences and SEQ ID Numbers" (Sequence Listing), i.e. SEQ ID NO: 1-SEQ ID NO: 94. Preferably, the sequence variant is characterized, particularly over the entire length of the sequence, by at least 70% sequence identity, at least 75%, preferably at least 80%, more preferably by at least 85%, even more preferably at least 90%, more preferably at least 95%, most preferably at least 99%, with the reference sequence, where the sequence identity is calculated by the method below. In particular, the sequence variant retains the specific function of the reference sequence. Sequence identity is calculated using the method described below. In particular, the amino acid sequence variant has an altered sequence in which one or more amino acids in the reference sequence are deleted or replaced, or one or more amino acids are inserted into the sequence of the reference amino acid sequence. As a result of the changes, the amino acid sequence variant has an amino acid sequence that is at least 70%, at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90 %, more preferably at least 95%, most preferably at least 99% identical to the reference sequence. For example, sequence variants that are at least 90% identical have no more than 10 changes, i.e. any combination of deletions, insertions or substitutions within 100 amino acids of the reference sequence.

In the context of the present invention, it is meant that an amino acid sequence “has sequence identity” of, for example, at least 95%, with the query amino acid sequence proposed in the present invention, means that the sequence of the subject amino acid sequence is identical to the query sequence except that the amino acid sequence in question may include up to 5 amino acid changes for every 100 amino acids of the requested amino acid sequence. In other words, to obtain an amino acid sequence having a sequence identical to at least 95% of the query amino acid sequence, up to 5% (5 out of 100) of the amino acid residues in the subject sequence can be inserted or replaced with another amino acid or removed by deletion, preferably in in accordance with the above definitions of variants or fragments. The same, of course, also applies to the similarity of nucleotide sequences.

For (amino acid or nucleotide) sequences that do not have an exact match, the "% identity" of the first sequence can be determined relative to the second sequence. In general, the two sequences to be compared are aligned to obtain maximum correlation between the sequences. This may involve inserting "gaps" into either one or both sequences to increase the degree of alignment. The % identity can then be determined over the entire length of each of the sequences being compared (called global alignment), which is most useful for sequences of the same or similar length, or for shorter defined lengths (called local alignment), which is most useful for sequences of unequal length.

Methods for comparing the identity and homology of two or more sequences are well known in the art. The percentage identity of two sequences can be determined, for example, using a mathematical algorithm. A preferred example of a mathematical algorithm that can be used is, but is not limited to, the algorithm described in Karlin et al., PNAS USA, 90, 1993, cc. 5873-5877. This algorithm is integrated into the BLAST family of programs, such as the BLAST or NBLAST program (see also Altschul et al., J. Mol. Biol. 215, 1990, pp. 403-410 or Altschul et al., Nucleic Acids Res, 25, 1997, pp. 3389-3402), available through the NCBI Internet home page at ncbi.nlm.nih.gov), and FASTA (Pearson, Methods Enzymol. 183, 1990, pp. 63-98; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85, 1988, pp. 2444-2448.). Using these programs, it is possible to identify sequences that are identical to a certain extent to other sequences. In addition, programs are available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux et al., Nucleic Acids Res., 1984, pp. 387-395), such as the BESTFIT and GAP programs, which can be used to determine the % identity between two polynucleotides and % identity and % homology or identity between two polypeptide sequences. BESTFIT uses the "local homology" algorithm proposed by Smith and Waterman, J. Mol. Biol. 147, 1981, cc. 195-197.), and this algorithm allows you to find the best single region of similarity between two sequences.

More preferably, the cell penetrating peptide fragments of the invention, or variants thereof described above, also retain the ability of the peptide to present cargo molecules, such as antigens or antigenic epitopes, on the cell surface in the context of MHC class I and/or MHC class II molecules . The ability of a cell-penetrating peptide or a complex containing said cell-penetrating peptide to present a cargo molecule, such as antigens or antigenic epitopes, on the cell surface, in the context of MHC class I and/or MHC class II molecules, can be assessed by standard methods known to one skilled in the art. field, including assessing the ability to stimulate proliferation and/or function of MHC-restricted CD4 ⁺ or CD8 ⁺ T cells specific for these epitopes.

Preferred is a cell penetrating peptide that

I) has an amino acid sequence length of said peptide of a total of 5-50 amino acids, preferably a total of 10-45 amino acids, more preferably a total of 15-45 amino acids; and/or

ii) has an amino acid sequence comprising a ZEBRA minimal domain fragment, wherein said minimal domain extends from residue 170 to residue 220 of the ZEBRA amino acid sequence set forth in SEQ ID NO: 3, in which optionally 1, 2, 3, 4 or 5 amino acids are replaced, removed by deletion and/or added without abolishing the ability of the specified peptide to penetrate into the cell, or a variant of the specified fragment,

preferably contains an amino acid sequence having at least one conservative amino acid substitution compared to the reference sequence, which means that the amino acid residue is replaced by a residue having similar physicochemical characteristics.

In general, substitutions of one or more amino acids present in the reference amino acid sequence should be conservative substitutions. Examples of conservative substitutions include replacing one aliphatic residue with another, such as replacing Ile, VaI, Leu or Ala with each other, or replacing one polar residue with another, such as replacing Lys and Arg with each other; Glu and Asp or Gln and Asn. Other conservative substitutions mentioned are well known, for example substitutions of entire regions having similar hydrophobic properties (Kyte and Doolittle, 1J. Mol. Biol. 157(1), 1982, pp. 105-132). Substitutions of one or more L-amino acids with one or more D-amino acids may be considered conservative substitutions in the context of the present invention. Examples of amino acid substitutions are presented below in Table 1:

Most preferably, a cell penetrating peptide is preferred which

I) has an amino acid sequence length of said peptide of a total of 5-50 amino acids, preferably a total of 10-45 amino acids, more preferably a total of 15-45 amino acids; and/or

ii) has an amino acid sequence comprising a ZEBRA minimal domain fragment, wherein said minimal domain extends from residue 170 to residue 220 of the ZEBRA amino acid sequence set forth in SEQ ID NO: 3, in which optionally 1, 2, 3, 4 or 5 amino acids are replaced, removed by deletion and/or added without abolishing the ability of the specified peptide to penetrate into the cell, or a variant of the specified fragment,

contains a Cys (C) to Ser (S) substitution at a position equivalent to position 189 in the ZEBRA amino acid sequence shown in SEQ ID NO: 3.

Thus, it is preferred that said preferred cell penetrating peptide have an amino acid sequence comprising the sequence of the following general formula (I):

X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ SX ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇

in which 1, 2, 3, 4 or 5 amino acids are replaced, removed by deletion and/or added without abolishing the ability of said peptide to enter the cell, in which

X ₁ is K, R or H, preferably X ₁ is K or R;

X ₂ is R, K or H, preferably X ₂ is R or K;

X ₃ is Y, W or F, preferably X ₃ is Y, W or F;

X ₄ is K, R or H, preferably X ₄ is K or R;

X ₅ represents N or Q;

X ₆ is R, K or H, preferably X ₆ is R or K;

X ₇ is V, I, M, L, F or A, preferably X ₇ is V, I, M or L;

X ₈ is A, V, L, I or G, preferably X ₈ is A or G;

X ₉ represents S or T;

X ₁₀ is R, K or H, preferably X ₁₀ is R or K;

X ₁₁ is K, R or H, preferably X ₁₁ is K or R;

X ₁₃ is R, K or H, preferably X ₁₃ is R or K;

X ₁₄ is A, V, L, I or G, preferably X ₁₄ is A or G;

X ₁₅ is K, R or H, preferably X ₁₅ is K or R;

X ₁₆ is F, L, V, I, Y, W or M, preferably X ₁₆ is F, Y or W; And

X ₁₇ is K, R or H, preferably X ₁₇ is K or R.

Preferably, said peptide, polypeptide or protein consists of either (entirely) L-amino acids or (entirely) D-amino acids, forming “retro-inverted peptide sequences”. The term “retro-inverted (peptide) sequences” refers to an isomer of a linear peptide sequence in which the direction of the sequence is reversed and the chirality of each amino acid residue is reversed (see, for example, Jameson et al., Nature, 368, 1994, pp. 744-746; Brady et al., Nature, 368, 1994, pp. 692-693).

In a specific embodiment of the invention, the cell penetrating peptide of the invention is typically described by the above general formula (I) in which X ₁ is K.

In a specific embodiment, the cell penetrating peptide of the invention is typically described by the above general formula (I) in which X ₂ is R.

In a specific embodiment, the cell penetrating peptide of the invention is typically described by the above general formula (I) in which X ₃ is Y.

In a specific embodiment, the cell penetrating peptide of the invention is typically described by the above general formula (I) in which X ₄ is K.

In a specific embodiment, the cell penetrating peptide of the invention is generally described by the above general formula (I) in which X ₅ is N.

In a specific embodiment, the cell penetrating peptide of the invention is typically described by the above general formula (I) in which X ₆ is R.

In a specific embodiment, the cell penetrating peptide of the invention is typically described by the above general formula (I) in which X ₇ is V.

In a specific embodiment, the cell penetrating peptide of the invention is typically described by the above general formula (I) in which X ₈ is A.

In a specific embodiment, the cell penetrating peptide of the invention is typically described by the above general formula (I) in which X ₉ is S.

In a specific embodiment, the cell penetrating peptide of the invention is generally described by the above general formula (I) in which X ₁₀ is R.

In a specific embodiment, the cell penetrating peptide of the invention is typically described by the above general formula (I) in which X ₁₁ is K.

In a specific embodiment, the cell penetrating peptide of the invention is generally described by the above general formula (I) in which X ₁₃ is R.

In a specific embodiment, the cell penetrating peptide of the invention is typically described by the above general formula (I) in which X ₁₄ is A.

In a specific embodiment, the cell penetrating peptide of the invention is typically described by the above general formula (I) in which X ₁₅ is K.

In a specific embodiment, the cell penetrating peptide of the invention is typically described by the above general formula (I) in which X ₁₆ is F.

In a specific embodiment, the cell penetrating peptide of the invention is typically described by the above general formula (I) in which X ₁₇ is K.

In a specific embodiment, the cell penetrating peptide of the invention is typically described by the above general formula (I) in which the amino acid residue at a position equivalent to position 12 in the general formula (I) is Ser (S).

Preferably also a preferred cell penetrating peptide that

I) has an amino acid sequence length of said peptide of a total of 5-50 amino acids, preferably a total of 10-45 amino acids, more preferably a total of 15-45 amino acids; and/or

ii) has an amino acid sequence comprising a ZEBRA minimal domain fragment, wherein said minimal domain extends from residue 170 to residue 220 of the ZEBRA amino acid sequence set forth in SEQ ID NO: 3, in which optionally 1, 2, 3, 4 or 5 amino acids are replaced, removed by deletion and/or added without abolishing the ability of the specified peptide to penetrate into the cell, or a variant of the specified fragment,

contains or consists of an amino acid sequence selected from the group consisting of amino acid sequences SEQ ID NO: 4-13 or variants of these sequences that do not abolish the ability of the specified peptide to penetrate the cell, preferably from sequence variants in which 1, 2, 3, 4 or 5 amino acids are replaced, removed by deletion and/or added without abolishing the ability of the specified peptide to penetrate the cell.

CPP1 (Z11):

CPP1 (Z11):

CPP2 (Z12):

CPP3 (Z13):

CPP4 (Z14):

CPP5 (Z15):

CPP6 (Z16):

CPP7 (Z17):

CPP8 (Z18):

CPP9 (Z19):

CPP10 (Z20):

Thus, particularly preferred is a cell penetrating peptide that has an amino acid sequence comprising or consisting of the amino acid sequence SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14), SEQ ID NO: 8 ( CPP5/Z15) or SEQ ID NO: 11 (CPP8/Z18), or variants of this sequence that do not abolish the ability of the specified peptide to penetrate the cell, preferably sequence variants in which 1, 2, 3, 4 or 5 amino acids are replaced, deleted by deletions and/or added without abolishing the ability of the specified peptide to penetrate the cell. Moreover, more preferred is a cell penetrating peptide that has an amino acid sequence containing or consisting of the amino acid sequence SEQ ID NO: 6 (CPP3/Z13) or SEQ ID NO: 7 (CPP4/Z14) or variants of the said sequence that do not cancel the cell penetrating ability of said peptide, preferably from sequence variants in which 1, 2, 3, 4 or 5 amino acids are replaced, deleted and/or added without abolishing the cell penetrating ability of said peptide.

Furthermore, most preferred is a cell penetrating peptide that has an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 6 (CPP3/Z13) or variants of said sequence that do not abrogate the cell penetrating ability of said peptide, preferably sequence variants in which 1, 2, 3, 4 or 5 amino acids are replaced, removed by deletion and/or added without abrogating the ability of the specified peptide to penetrate the cell. Thus, it is most preferred that the cell penetrating peptide contains or consists of the amino acid sequence set forth in SEQ ID NO: 6 (CPP3/Z13) or a functional sequence variant that is at least 70% sequence identical, preferably which is sequence identical at least 75%, more preferably at least 80% sequence identical, even more preferably at least 90% sequence identical, especially preferably at least 95% sequence identical, most preferably at least 95% sequence identical, most preferably at least 95% sequence identical, at least 99%. In this context, a “functional sequence variant” means a sequence variant that retains the ability of the peptide to enter a cell.

In one preferred embodiment, the cell penetrating peptide of the invention has an amino acid sequence that contains or consists of SEQ ID NO: 6 (CPP3/Z13).

In another preferred embodiment, the cell penetrating peptide of the invention has an amino acid sequence that contains or consists of SEQ ID NO: 7 (CPP4/Z14).

In another preferred embodiment, the cell penetrating peptide of the invention has an amino acid sequence that contains or consists of SEQ ID NO: 8 (CPP5/Z15).

In another preferred embodiment, the cell penetrating peptide of the invention has an amino acid sequence that contains or consists of SEQ ID NO: 11 (CPP8/Z18).

As will be apparent to one skilled in the art, the primary amino acid sequence of the cell penetrating peptide of the invention may be subject to additional post-translational modification, such as glycosylation or phosphorylation, without departing from the scope of the invention.

In a further embodiment, the cell penetrating peptide of the invention optionally further contains, in addition to its amino acid sequence described above, any of the following components or any combination of the following components:

(I) nuclear localization signal (NLS). Such signals are well known to one skilled in the art and are described in Nair et al., Nucleic Acids Res. 31(1), 2003, cc. 397-399),

(II) a targeting peptide, including tumor homing peptides such as those described in Kapoor et al., PLoS ONE 7(4), 2012, e35187) and listed in http://crdd.osdd.net/raghava /tumorhope/general.php?

Preferably, the cell penetrating peptide of the invention is associated with an antigen or antigenic epitope and facilitates the cellular internalization of said antigen or antigenic epitope.

The complex proposed for use according to the present invention may contain one cell penetrating peptide or several cell penetrating peptides. Preferably, the complex proposed for use according to the present invention contains no more than five cell penetrating peptides, more preferably the complex proposed for use according to the present invention contains no more than four cell penetrating peptides, even more preferably the complex proposed for use according to the present invention , contains no more than three cell penetrating peptides, most preferably the complex proposed for use according to the present invention contains no more than two cell penetrating peptides, and most preferably the complex proposed for use according to the present invention contains one cell penetrating peptide.

Component b) - Antigen/antigenic epitope

The complex proposed for use according to the present invention contains as component b) at least one antigen or antigenic epitope.

As used herein, an “antigen” is a substance of any structure that serves as a target for receptors involved in the adaptive immune response, in particular as a target for antibodies, T-cell receptors and/or B-cell receptors. An "epitope", also called an "antigenic determinant", is a part (or fragment) of an antigen that is recognized by the immune system, in particular by antibodies, T-cell receptors and/or B-cell receptors. Thus, one antigen has at least one epitope, i.e. one antigen has one or more epitopes. In the context of the present invention, the term “epitope” is used primarily to refer to T cell epitopes that are presented on the surface of an antigen-presenting cell, where they are associated with the major histocompatibility complex (MHC). T cell epitopes that are presented by MHC class I molecules are usually, but not always, peptides of 8-11 amino acids, while MHC class II molecules present longer peptides, usually but not always, consisting of 12-25 amino acids.

Preferably, in the complex proposed for use according to the present invention, at least one antigen or antigenic epitope is selected from the group consisting of: (I) peptide, polypeptide or protein, (II) polysaccharide, (III) lipid, (IV) lipoprotein or a lipopeptide, (V) a glycolipid, (VI) a nucleic acid, and (VII) a small molecule drug or toxin. Thus, the at least one antigen or antigenic epitope may be a peptide, a protein, a polysaccharide, a lipid, a combination thereof including lipoproteins and glycolipids, a nucleic acid (e.g., DNA, siRNA, shRNA, antisense oligonucleotides, decoy DNA, plasmid) , or a small molecule drug (for example, cyclosporin A, paclitaxel, doxorubicin, methotrexate, 5-aminolevulinic acid), or any combination thereof, in particular if the complex of the invention contains more than one antigen or antigenic epitope.

It will be appreciated that at least one antigen or antigenic epitope may comprise, for example, at least one, i.e. one or more peptides, polypeptides or proteins linked together, and/or at least one, i.e. one or more nucleic acids, for example, each of which encodes a single peptide or polypeptide. In addition, the at least one antigen or antigenic epitope may be a combination of a protein, a lipid and/or a polysaccharide, including lipoproteins and glycolipids. Thus, in particular, if the complex proposed for use according to the present invention contains more than one antigen or antigenic epitope, then it may contain more than one peptide, polypeptide or protein, more than one polysaccharide, more than one lipid, more than one lipoprotein, more than one glycolipid , more than one nucleic acid, more than one small molecule drug or toxin, or a combination thereof.

Preferably, the complex proposed for use according to the present invention contains at least one antigen or antigenic epitope containing one or more epitope(s) selected from a cancer/tumor associated antigen, a cancer/tumor specific antigen and/or antigenic protein from a pathogen, including antigenic protein from viruses, bacteria, fungi, protozoa and metazoan parasites.

More preferably, the at least one antigen or antigenic epitope contains or consists of (I) at least one pathogen epitope and/or (II) at least one cancer/tumor epitope, in particular at least one tumor epitope. Most preferably, the at least one antigen or antigenic epitope contains or consists of at least one cancer/tumor epitope, in particular at least one tumor epitope.

Most preferably, the complex proposed for use according to the present invention contains only those antigen(s) or antigenic epitope(s) that are cancer/tumor associated antigen(s). s), cancer/tumor specific antigen(s) and/or cancer/tumor epitope(s); in particular representing tumor associated antigen(s), tumor specific antigen(s) and/or tumor epitope(s).

As used herein, “cancer epitope” means an epitope from a cancer-associated antigen or from a cancer-specific antigen. Accordingly, “tumor epitope” means an epitope from a tumor-associated antigen or from a tumor-specific antigen. These epitopes are typically specific to (or associated with) a particular type of cancer/tumor. In particular, cancer/tumor associated (also cancer/tumor associated) antigens are antigens that are expressed on both cancer/tumor cells and healthy cells. Accordingly, such antigens are normally present from birth (or even before birth). Accordingly, there is a chance that the immune system will develop self-tolerance to such antigens. In contrast, cancer/tumor-specific antigens are antigens that are expressed specifically by cancer/tumor cells but not by healthy cells. Cancer/tumor-specific antigens include, in particular, neoantigens. In general, neoantigens are antigens that were not previously present and are therefore “new” to the immune system. Neoantigens are usually the result of somatic mutations. Regarding cancer/tumor-specific neoantigens, they are generally not present until the cancer/tumor develops, and cancer/tumor-specific neoantigens are typically encoded by somatic gene mutations in cancer/tumor cells. Because neoantigens are new to the immune system, the risk of self-tolerance to these antigens is considered to be significantly lower than to cancer/tumor-associated antigens. However, each cancer-specific set of tumor-specific mutations is likely unique. Accordingly, in the context of the present invention, it is preferred that said cancer/tumor-specific antigens, in particular neoantigens, be identified in an individual diagnosed with colorectal cancer using methods known to one skilled in the art, for example, by sequencing the cancer genome. Once identified, the appropriate cancer/tumor-specific neoantigens and/or epitopes of the cancer/tumor-specific neoantigens are used in the complex proposed for use according to the present invention.

Preferably, the complex proposed for use according to the present invention contains one or more cancer/tumor associated epitopes and/or one or more cancer/tumor associated antigens (but preferably does not contain cancer/tumor specific epitopes). It is also preferable that the complex proposed for use according to the present invention contains one or more cancer/tumor specific epitopes and/or one or more cancer/tumor specific antigens (but preferably does not contain cancer/tumor associated epitopes). The complex proposed for use according to the present invention may also preferably contain both (i) one or more cancer/tumor-associated epitopes and/or one or more cancer/tumor-associated antigens, and (ii) one or more cancer-specific /tumor epitopes and/or one or more cancer/tumor-specific antigens.

In particular, the cancer/tumor to which antigens or antigenic epitopes are associated, or for which antigens or antigenic epitopes are specific, is colorectal cancer as defined herein. Thus, the antigens are preferably CRC-associated or CRC-specific antigens, and the epitopes are preferably CRC-associated or CRC-specific epitopes.

Information about acceptable cancer/tumor epitopes can be found, for example, in cancer/tumor epitope databases, for example van der Bruggen P., Stroobant V., Vigneron N., Van den Eynde B. Peptide database: T cell-defined tumor antigens Cancer Immun 2013; URL: http://www.cancerimmunity.org/peptide/, in which human tumor antigens recognized by CD4 ⁺ - or CD8 ⁺ -T cells are classified into 4 main groups based on their expression pattern, or from the Tantigen database " (TANTIGEN, version 1.0, December 1, 2009, created by Bioinformatics Core at Cancer Vaccine Center, Dana-Farber Cancer Institute; URL: http://cvc.dfci.harvard.edu/tadb/). Examples of cancer/tumor epitopes include, for example, TRP2-derived epitopes, melanoma-associated glycoprotein 100 (gp100) antigen epitopes, glycoprotein 70 (gp70) antigen epitopes, survivin epitopes, IEa epitopes, IL13rα2, Epha2 (ephrin receptor type 2 A), their immunogenic fragments and fusions of these antigens and/or fragments. Additionally, examples of cancer/tumor epitopes include those of neoantigens, such as the neoantigen from the MC-38 tumor cell line described in Yadav et al., Nature, 515(7528), November 27, 2014, pp. 572-576. As described above, neoantigens are antigens that are completely absent from the genome of a healthy person. Compared to non-mutant autoantigens, neoantigens are relevant for tumor control because the quality of the T cell pool available to these antigens is not affected by central T cell tolerance. In particular, neoantigens can be based on individual tumor genomes. Potential neoantigens can be predicted by methods known to one of ordinary skill in the art, such as cancer genome sequencing or deep sequencing technologies to identify mutations in the protein coding region of the (cancer) genome.

Specific examples of cancer/tumor associated, in particular tumor associated or tissue specific antigens that can be used in the complex proposed for use according to the present invention include, but are not limited to, the following antigens: Her-2/neu, SPAS- 1, TRP-2, tyrosinase, Melan A/Mart-1, gp1OO, BAGE, GAGE, ganglioside GM2, kinesin 2, TATA element modulating factor 1, tumor protein D52, MAGE D, ING2, HIP-55, TGF-1 anti-apoptotic factor, HOM-Mel-40/SSX2, epithelial antigen (LEA 135), DF31MUC1 antigen (Apostolopoulos et al., Immunol. Cell. Biol. 74, 1996, pp. 457-464; Pandey et al., Cancer Res. 55, 1995, pp. 4000-4003); MAGE-1, HOM-Mel-40/SSX2, NY-ESO-1, EGFR, CEA, Epha2, Epha4, PCDGF, HAAH, mesothelin; EPCAM; NY-ESO-1, MUC1 glycoprotein and NIUC10 p5 mucins (especially mutant versions), EGFR, cancer associated serum antigen (CASA) and cancer antigen 125 (CA 125) (Kierkegaard et al., Gynecol. Oncol. 59, 1995, pp. 251-254), epithelial glycoprotein 40 (EGP40) (Kievit et al., Int. J. Cancer 71, 1997, pp. 237-245), squamous cell carcinoma antigen (SCC) (Lozza et al., Anticancer Res. 17, 1997, pp. 525-529), cathepsin E (Mota et al., Am. J Pathol. 150, 1997, pp. 1223-1229), tyrosinase in melanoma (Fishman et al., Cancer 79, 1997, pp. . 1461-1464), nuclear cell antigen (PCNA) of cerebral cavernomas (Notelet et al., Surg. Neurol. 47, 1997, pp. 364-370), tumor-associated autoantigen 35 kD in papillary thyroid carcinoma (Lucas et al. , Anticancer Res. 16, 1996, pp. 2493-2496), CDC27 (including the mutant form of the protein), triosephosphate isomerase antigens, 707-AP, mycobacterial antigen A60 (Macs et al., J. Cancer Res. Clin. Oncol. 122, 1996, pp. 296-300), annexin II, AFP, ART-4, BAGE, β-catenin/m, BCL-2, bcr-abl, bcr-abl p190, bcr-abl p210, BRCA-1, BRCA- 2, CA 19-9 (Tolliver and O'Brien, South Med. J. 90, 1997, cc. 89-90; Tsuruta et al., Urol. Int. 58, 1997, cc. 20-24), CAMEL, CAP-1, CASP-8, CDC27/m, CDK-4/m, CEA (Huang et al., Exper Rev. Vaccines 1, 2002, pp. 49-63), CT9, CT10 , Cyp-B, Dek-cain, DAM-6 (MAGE-B2), DAM-10 (MAGE-B1), EphA2 (Zantek et al., Cell Growth Differ. 10, 1999, pp. 629-638; Carles- Kinch et al., Cancer Res. 62, 2002, pp. 2840-2847), EphA4 (Cheng et al., Cytokine Growth Factor Rev. 13, 2002, pp. 75-85), tumor-associated antigen Thomsen-Friedenreich ( Dahlenborg et al., Int. J Cancer 70, 1997, pp. 63-71), ELF2M, ETV6-AML1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE- 6, GAGE-7B, GAGE-8, GnT-V, gp100 (Zajac et al., Int. J Cancer 71, 1997, cc. 491-496), HAGE, HER2/neu, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT, hTRT, iCE, apoptosis inhibitors (eg, survivin), KH-1 adenocarcinoma antigen (Deshpande and Danishefsky, Nature 387, 1997, pp. 164-166), KIAA0205, K-ras, LAGE, LAGE-1, LDLR/FUT, MAGE-1, MAGE-2, MAGE-3, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-B5, MAGE-B6, MAGE-C2, MAGE-C3, MAGE D, MART-1, MART-1/Melan-A (Kawakami and Rosenberg, Int. Rev. Immunol. 14, 1997, cc. 173-192), MC1R, MDM-2, myosin/m, MUC1, MUC2, MUM-1, MUM-2, MUM-3, neo-polyA polymerase, NA88-A, NY-ESO-1, NY-ESO -1a (CAG-3), PAGE-4, PAP, proteinase 3 (Molldrem et al., Blood 88, 1996, pp. 2450-2457; Molldrem et al., Blood 90, 1997, pp. 2529-2534), P15, p190, Pm1/RARα, PRAME, PSA, PSM, PSMA, RAGE, RAS, RCAS1, RU1, RU2, SAGE, SART-1, SART-2, SART-3, SP17, SPAS-1, TEL/AML1, TPI/m, tyrosinase, TARP, TRP-1 (gp75), TRP-2, TRP-2/INT2, WT-1 and alternatively translated proteins NY-ESO-ORF2 and CAMEL derived from the NY-ESO-1 and LAGE genes -1. In addition, another specific example of a cancer/tumor associated, in particular tumor associated or tissue specific antigen that can be used in the complex proposed for use according to the present invention is the ASCL2 antigen (Achaete-scute 2 homologue, genes , causing the absence of sensory shield hairs.) Numerous other cancer antigens are well known in the art.

Preferably, the cancer/tumor antigen or cancer/tumor epitope is a recombinant cancer/tumor antigen or recombinant cancer/tumor epitope. The recombinant cancer/tumor antigen or recombinant cancer/tumor epitope can be created by introducing mutations (adding, deleting or substituting) that alter specific amino acids throughout the amino acid sequence of the native cancer/tumor antigen or native cancer/tumor epitope. The introduction of mutations does not change the cancer/tumor antigen or cancer/tumor epitope so much that it can no longer be used as a universal for mammalian species and preferably humans or dogs, but changes it sufficiently that the resulting amino acid sequence breaks tolerance or is considered in as a foreign antigen to create an immune response. Another way may be to create a consensus recombinant cancer/tumor antigen or cancer/tumor epitope, the amino acid sequence of which is at least 85% and up to 99% identical to the sequence of the corresponding native cancer/tumor antigen or native cancer/tumor epitope; preferably the sequence identity is at least 90% and up to 98% and; more preferably, the sequence identity is at least 93% and up to 98%; or even more preferably the sequence identity is at least 95% and up to 98%. In some cases, the amino acid sequence of a recombinant cancer/tumor antigen or recombinant cancer/tumor epitope is 95%, 96%, 97%, 98%, or 99% identical to the sequence of the corresponding native cancer/tumor antigen or native cancer/tumor epitope. A native cancer/tumor antigen is an antigen that is normally associated with a specific cancer or a specific cancerous tumor. Depending on the cancer/tumor antigen, the cancer/tumor antigen consensus sequence may be present in mammalian species or subtypes of species or viral strains or serotypes. Some cancer/tumor antigens do not differ significantly from the amino acid sequence of the wild type cancer/tumor antigen. The above approaches can be combined such that the final recombinant cancer/tumor antigen or cancer/tumor epitope has the above percentage similarity to the amino acid sequence of the native cancer antigen. However, preferably, the amino acid sequence of the epitope of the cancer/tumor antigen specified in the present description is not mutated and, therefore, is identical to the reference sequence of the epitope.

As used herein, the term “pathogen epitope” means an epitope of an antigenic protein, antigenic polysaccharide, antigenic lipid, antigenic lipoprotein or antigenic glycolipid from a pathogen, including viruses, bacteria, fungi, protozoa and metazoan parasites. Antigenic proteins, polysaccharides, lipids, lipoproteins or glycolipids from pathogens include, as used herein, proteins, polysaccharides, lipids, lipoproteins and glycolipids, respectively, from pathogens responsible for diseases that can be targeted for vaccination, including, for example, amoebiasis, anthrax, Buruli ulcer (Mycobacterium ulcerans), calicivirus-associated diarrhea, campylobacter diarrhea, cervical cancer (human papillomatosis), Chlamydia trachomatis-associated reproductive system diseases, cholera, Crimean-Congo hemorrhagic fever, Dengue fever, diphtheria, Ebola hemorrhagic fever, diarrhea, enteropathogenic Escherichia coli (ETEC), gastric cancer (Helicobacter pylori), gonorrhea, group A Staphylococcus-associated disease, group B Staphylococcus-associated disease, pneumonia and invasive disease associated with Haemophilus influenzae B, virus-associated diarrhea hepatitis A, hepatitis B, hepatitis C, hepatitis E, genital ulcers associated with herpes simplex virus type 2, HIV/AIDS, hookworm disease, influenza, Japanese encephalitis, Lassa fever, leishmaniasis, leptospirosis, liver cancer (hepatitis B ), liver cancer (hepatitis C), Lyme disease, malaria, Marburg hemorrhagic fever, measles, mumps, nasopharyngeal cancer (Epstein-Barr virus), meningitis associated with Neisseria meningitidis, pneumonia associated with parainfluenza virus, whooping cough, plague, polio, rabies, respiratory syncytial virus (RSV) pneumonia, Rift Valley fever, rotavirus-associated diarrhea, rubella, schistosomiasis, severe acute respiratory syndrome (SARS), shigellosis, smallpox, Staphylococcus aureus-associated diseases, stomach cancer (Helicobacter pylori), streptococcal pneumonia and invasive disease, tetanus, tick-borne encephalitis, trachoma, tuberculosis, tularemia, typhoid fever, West Nile virus-associated disease, yellow fever.

Preferably, at least one antigen or antigenic epitope should be presented on the cell surface in the context of MHC class I and/or MHC class II, and/or in the context of CD1, while being presented on the cell surface in the context of MHC class I and/or MHC class II is preferred. The term “epitope presentation in the context of MHC class I” refers, in particular, to the CD8 ⁺ epitope located in the (peptide-binding) groove of the MHC class I molecule on the cell surface. The term “epitope presentation in the context of MHC class II” refers, in particular, to the CD4 ⁺ epitope located in the groove of the MHC class II molecule on the cell surface. The term “epitope presentation in the context of CD1” refers, in particular, to a lipid epitope located in a groove of cluster of differentiation 1 molecules on the cell surface.

It is advisable that the complex proposed for use according to the invention contains a cell penetrating peptide and at least one antigen or antigenic epitope and ensures the transport and presentation of these epitopes on the cell surface of antigen presenting cells in the context of MHC class I and MHC class II, and what does possible its use for vaccination and immunotherapy.

Preferably, the complex for use according to the present invention contains at least one antigen or antigenic epitope, which is at least one CD4 ⁺ epitope and/or at least one CD8 ⁺ epitope.

The terms “CD4 ⁺ -epitope” or “CD4 ⁺ -restricted epitope” in the context of the present description mean an epitope recognized by a CD4 ⁺ -T cell, said epitope, in particular, consisting of a fragment of an antigen located in the groove of the MHC class II molecule. The single CD4 ⁺ epitope contained in the complex proposed for use according to the present invention preferably consists of about 12-25 amino acids. It may consist, for example, of about 8-25 amino acids or about 6-100 amino acids.

The terms “CD8 ⁺ -epitope” or “CD8 ⁺ -restricted epitope” in the context of the present description mean an epitope recognized by a CD8 ⁺ -T cell, said epitope, in particular, consisting of a fragment of an antigen located in the groove of the MHC class I molecule. Single The CD8 ⁺ epitope contained in the complex proposed for use according to the present invention preferably consists of about 8-11 amino acids. It may consist, for example, of about 8-15 amino acids or about 6-100 amino acids.

Preferably, at least one antigen may comprise, or at least one antigenic epitope may consist of, a CD4 ⁺ epitope and/or a CD8 ⁺ epitope corresponding to the antigenic determinant(s) of the cancer/tumor-associated antigen, cancer-specific antigen/ tumor antigen or antigenic protein from a pathogen. More preferably, at least one antigen may comprise, or at least one antigenic epitope may consist of, a CD4 ⁺ epitope and/or a CD8 ⁺ epitope corresponding to the antigenic determinant(s) of a cancer/tumor associated or cancer-specific antigen /tumor antigen. Most preferably, at least one antigen may contain, or at least one antigenic epitope may consist of, a CD4 ⁺ epitope and/or a CD8 ⁺ epitope corresponding to the antigenic determinant(s) of a tumor-associated antigen or a tumor-specific antigen.

Preferably also the complex proposed for use according to the present invention contains at least two antigens or antigenic epitopes, where at least one antigen or antigenic epitope contains or consists of a CD4 ⁺ epitope and at least one antigen or antigenic epitope contains or consists of from the CD8 ⁺ epitope. It has now been established that T _h cells (CD4 ⁺ ) play a major role in the antitumor immune response, participating both in the “licensing” of DCs and in the recruitment and maintenance of CTL (CD8 ⁺ ) in the tumor area. Thus, a complex proposed for use according to the present invention, which contains at least two antigens or antigenic epitopes, wherein at least one antigen or antigenic epitope contains or consists of a CD4 ⁺ epitope and at least one antigen or antigenic epitope contains or consists of a CD8 ⁺ epitope, provides an integrated immune response that allows simultaneous priming of CTL and T _h cells, and is therefore preferable to immunity against only one CD8 ⁺ epitope or only one CD4 ⁺ epitope. For example, the complex proposed for use according to the present invention may preferably contain the Alpha-CD4 ⁺ epitope and the gp100-CD8 ⁺ epitope.

Preferably, the complex proposed for use according to the present invention contains at least two antigens or antigenic epitopes, where at least two antigens or antigenic epitopes contain or consist of at least two, for example, 2, 3, 4, 5, 6, 7, 8, 9 or more CD4 ⁺ epitopes and/or at least two, for example, 2, 3, 4, 5, 6, 7, 8, 9 or more CD8 ⁺ epitopes. In this case, at least two antigens or antigenic epitopes are preferably different antigens or antigenic epitopes, more preferably at least two antigens or antigenic epitopes are different from each other, but belong to the same tumor type. A multiantigen vaccine must (I) prevent overgrowth of non-antigen variants, (II) target different tumor cells within a heterogeneous tumor mass, and (III) overcome patient-to-patient tumor variability. Thus, the complex proposed for use according to the present invention most preferably contains at least four antigens or antigenic epitopes, in particular at least two CD8 ⁺ epitopes and at least two CD4 ⁺ epitopes. This complex, proposed for use according to the present invention, induces the synergistic functional activity of polyepitope CD8-CTL and CD4-T _h cells against tumor cells and enhances the effectiveness of antitumor immunity. T _h cells are also involved in maintaining long-term cellular immunity, which was detected during monitoring after vaccination. The complex proposed for use according to the present invention induces polyclonal polyepitope immune responses and multifunctional CD8 ⁺ and CD4 ⁺ T cells, and therefore has effective antitumor activity.

Preferably, the complex proposed for use according to the present invention contains at least two antigens or antigenic epitopes, more preferably the complex proposed for use according to the present invention contains at least three antigens or antigenic epitopes, even more preferably the complex proposed for use according to the present invention contains at least four antigens or antigenic epitopes, even more preferably the complex proposed for use according to the present invention contains at least five antigens or antigenic epitopes, most preferably the complex proposed for use according to the present invention contains at least six antigens or antigenic epitopes. The antigens or antigenic epitopes included in the complex proposed for use according to the present invention may be the same or different, preferably the antigens or antigenic epitopes included in the complex proposed for use according to the present invention are different from each other. Preferably, the complex proposed for use according to the present invention contains at least one CD4 ⁺ epitope and at least one CD8 ⁺ epitope.

Preferably, the complex proposed for use according to the present invention contains more than one CD4 ⁺ epitope, for example, two or more CD4 ⁺ epitopes from the same antigen or from different antigens, and preferably does not contain a CD8 ⁺ epitope. Preferably, the complex proposed for use according to the present invention contains more than one CD8 ⁺ epitope, for example, two or more CD8 ⁺ epitopes from the same antigen or from different antigens, and preferably does not contain a CD4 ⁺ epitope. However, most preferably, the complex proposed for use according to the present invention contains (I) at least one CD4 ⁺ epitope, for example, two or more CD4 ⁺ epitopes from the same antigen or from different antigens, and (II) at least one CD8 ⁺ epitope, for example, two or more CD8 ⁺ epitopes from the same antigen or from different antigens.

For example, the complex proposed for use according to the present invention may preferably contain a gp100-CD8 ⁺ epitope, an Alpha-CD4 ⁺ epitope and an additional CD4 ⁺ epitope and an additional CD8 ⁺ epitope. Even more preferably, the complex proposed for use according to the present invention may contain a polypeptide or protein containing the gp100-CD8 ⁺ epitope and the Alpha-CD4 ⁺ epitope. For example, the polypeptide or protein contained in the complex proposed for use according to the present invention contains or consists of the amino acid sequence corresponding to SEQ ID NO: 14, or variants of the said sequence specified above:

SEQ ID NO: 14 (MAD5 cargo molecule containing OVA-CD4 ⁺ , gp100-CD8 ⁺ , Ealpha-CD4 ⁺ and OVA-CD8 ⁺ epitopes).

For example, the complex proposed for use according to the present invention may preferably contain a gp70-CD8 ⁺ epitope and/or a gp70-CD4 ⁺ epitope. Even more preferably, the complex proposed for use according to the present invention may contain a polypeptide or protein containing a gp70-CD8 ⁺ epitope and/or a gp70-CD4 ⁺ epitope. For example, the polypeptide or protein contained in the complex proposed for use according to the present invention contains or consists of the amino acid sequence corresponding to SEQ ID NO: 43, or variants of the said sequence specified above:

SEQ ID NO: 43 (Mad8 cargo molecule containing gp70-CD8 ⁺ and gp70-CD4 ⁺ epitopes).

For example, the complex proposed for use according to the present invention may preferably contain at least one survivin epitope, such as a CD8 ⁺ survivin epitope and/or a CD4 ⁺ survivin epitope. More preferably, the complex proposed for use according to the present invention may contain a polypeptide or protein containing a CD8 ⁺ epitope of survivin and/or a CD4 ⁺ epitope of survivin. More preferably, the complex proposed for use according to the present invention may contain a polypeptide or protein containing more than one CD8 ⁺ epitope of survivin and/or more than one CD4 ⁺ epitope of survivin, for example, two different CD8 ⁺ epitopes of survivin. For example, the polypeptide or protein contained in the complex proposed for use according to the present invention contains or consists of the amino acid sequence corresponding to SEQ ID NO: 44, or variants of the said sequence specified above:

SEQ ID NO: 44 (Mad11 cargo molecule containing CD8 ⁺ epitope 1 of survivin and CD8 ⁺ epitope 2 of survivin).

For example, the complex proposed for use according to the present invention may preferably contain at least one epitope from a neoantigen. Even more preferably, the complex proposed for use according to the present invention may contain a polypeptide or protein containing an epitope from a neoantigen, such as the neoantigen from the MC-38 tumor cell line identified by Yadav et al. Nature. 515(7528), November 27, 2014, ss. 572-576. For example, the polypeptide or protein contained in the complex proposed for use according to the present invention contains or consists of the amino acid sequence corresponding to SEQ ID NO: 42, or variants of the said sequence specified above:

SEQ ID NO: 42 (Mad9 cargo molecule containing an epitope from the neoantigen described in Yadav et al. Nature, 515(7528), November 27, 2014, pp. 572-576).

For example, the complex proposed for use according to the present invention may preferably contain more than one, for example two or three neoantigen epitopes. Even more preferably, the complex proposed for use according to the present invention may contain a polypeptide or protein containing more than one, for example two or three epitopes from neoantigens, such as neoantigens from the MC-38 tumor cell line identified by Yadav et al. Nature, 515 (7528), November 27, 2014, ss. 572-576. For example, the polypeptide or protein contained in the complex proposed for use according to the present invention contains or consists of the amino acid sequence corresponding to SEQ ID NO: 63, or variants of the said sequence specified above:

SEQ ID NO: 63 (Mad12 cargo molecule containing an epitope from the neoantigen described in Yadav et al. Nature, 515(7528), November 27, 2014, pp. 572-576). Preferably, at least one antigen or antigenic epitope contained in the complex proposed for use according to the present invention is a peptide, polypeptide or protein. Examples of an antigen or antigenic epitope of a peptide, polypeptide or protein nature that can be used in the invention include cancer/tumor antigens or antigenic epitopes thereof, allergic antigens or antigenic epitopes thereof, autoimmune autoantigens or antigenic epitopes thereof, pathogen antigens or antigenic epitopes thereof, and antigens or antigenic epitopes thereof from viruses, preferably from viruses such as cytomegalovirus (CMV), variola orthopoxyvirus, white pox orthopoxyvirus, ovine parapoxyvirus, molluscum contagiosum virus, herpes simplex virus 1, herpes simplex virus 2, herpes B virus, varicella zoster virus, pseudorabies virus, human cytomegalovirus, human herpes virus 6, human herpes virus 7, Epstein-Barr virus, human herpes virus 8, hepatitis B virus, Chikungunya fever virus, O'Nyong-Nyong fever virus, rubivirus, hepatitis C virus , GB virus C, West Nile virus, Dengue virus, yellow fever virus, sheep encephalomyelitis virus, St. Louis encephalitis virus, Japanese encephalitis B virus, Powassan virus, FSME virus, SARS, SARS-associated coronavirus, human coronavirus 229E, human corona virus Oc43, torovirus, human T-cell lymphotropic virus type I, human T-cell lymphotropic virus type II, HIV (AIDS), i.e. human immunodeficiency virus type 1 or human immunodeficiency virus type 2, influenza virus, Lassa virus, lymphocytic choriomenengitis virus, Tacaribe virus, Junin virus, Machupo virus, Borna disease virus, Bunyamwera virus, Californian encephalitis virus, Rift Valley fever virus, phlebotomy virus fevers, Tuscany virus, Crimean-Congo hemorrhagic fever virus, Hazara virus, Hassan virus, Hantaan virus, Seoul virus, Avenue Hill virus, Puumala virus, Dobrava-Belgrade virus, Tula virus, Sin Nombre virus, Marburg-Lake Victoria virus, virus Ebola Zaire, Ebola virus Sudan, Ebola virus Ivory Coast, influenza A virus, influenza B virus, influenza C virus, parainfluenza virus, malaria parasite (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi), Marburg virus , measles virus, mumps virus, respiratory scintillation virus, human metapneumovirus, Indian vesicular stomatitis virus, rabies virus, Mokola virus, Duvenhage virus, European bat lysavirus 1+2, Australian bat lysavirus, adenovirus A-F, human papillomavirus, condyloma virus 6, condyloma virus 11, polyoma virus, adeno-associated virus 2, rotavirus, orbivirus, varicella virus, including varicella zoster virus, etc., or antigens or antigenic epitopes from leishmania, trypanosomes, amoebae, bacteria, etc. , or can be selected from epitopes or variants of the above antigens or antigenic epitopes. Preferably, the epitopes, as well as variants of the antigens mentioned above, are characterized by homology or sequence identity of about 10%, in particular at least 10%, about 20%, in particular at least 20%, about 30%, in particular , at least 30%, about 40%, in particular at least 40%, about 50%, in particular at least 50%, about 60%, in particular at least 60%, about 70%, in particular at least 70%, about 80%, in particular at least 80%, about 90%, in particular at least 90%, at least 95% or at least 98%, with one of antigens or antigen sequences presented or described above. In this context, the definitions of epitopes and variants are as above.

Examples of antigens or antigenic epitopes categorized as a peptide, polypeptide or protein include a combination of several glioma epitopes, such as those described in Novellino et al., Cancer Immunol Immunother, 54(3), 2005, cc. 187-207, Vigneron et al. Cancer Immun. 13, 2013, p. 15). However, the individual complex proposed for use according to the present invention may also contain only a subset, i.e. one or more of all specified glioma epitopes. In such a case, preferably, the various complexes of the present invention contain different subsets of all of these glioma epitopes, such that, for example, a vaccine of the present invention containing these various complexes of the present invention contains all of these glioma epitopes, but distributed to various complexes.

In addition, the complex proposed for use according to the invention may also contain at least one antigen or antigenic epitope, wherein said antigen or antigenic epitope is a polysaccharide, lipid, lipoprotein and/or glycolipid, in particular a polysaccharide, lipid, lipoprotein and /or a glycolipid epitope, which may, for example, be pathogen epitopes specified herein.

In particular, the complex proposed for use according to the invention may contain at least one antigen or antigenic epitope, wherein said antigen or antigenic epitope is a polysaccharide, lipid, lipoprotein and/or glycolipid epitope, including antigens or antigenic epitopes of viruses, bacteria, fungi, protozoa and multicellular parasites.

Preferably, said epitopes should be presented on the cell surface in the context of MHC class I and/or MHC class II.

Preferably, said lipid epitopes should be presented on the cell surface in the context of CD1 (cluster of differentiation 1).

The complex proposed for use according to the present invention may also contain at least one antigen or antigenic epitope, where the specified antigen or antigenic epitope is a small molecule drug or toxin.

Examples of small molecule cargo molecules or toxins that can be used according to the invention include cyclosporin A, paclitaxel, doxorubicin, methotrexate, 5-aminolevulinic acid, diphtheria toxin, sunitinib and molecules summarized in De Wit Amer , Neuro Oncol, 12(3), 2010, pp. 304-316.

The complex proposed for use according to the present invention contains at least one antigen or antigenic epitope, preferably the complex proposed for use according to the present invention contains more than one antigen or antigenic epitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens or antigenic epitopes, more preferably the complex proposed for use according to the present invention contains (at least) two or three antigens or antigenic epitopes, even more preferably the complex proposed for use according to the present invention invention, contains (at least) four or five antigens or antigenic epitopes.

If the complex proposed for use according to the present invention contains more than one antigen or antigenic epitope, then it is obvious that said antigen or antigenic epitope, in particular, is also covalently linked in the complex proposed for use according to the present invention, for example, to another antigen or an antigenic epitope and/or with component a), i.e. cell-penetrating peptide, and/or with component c), i.e. TLR peptide agonist.

The various antigens or antigenic epitopes included in the complex proposed for use according to the present invention may be the same or different. Preferably, the various antigens or antigenic epitopes included in the complex proposed for use according to the present invention are different from each other, resulting in the formation of a polyantigenic and/or polyepitope complex.

It is further preferred that more than one antigen or antigenic epitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens or antigenic epitopes, are arranged sequentially in the complex proposed for use according to the present invention. This means, in particular, that all antigens and/or antigenic epitopes included in the complex are located in the form of a region that is not interrupted by component a), i.e. cell-penetrating peptide, nor component c), i.e. TLR peptide agonist. Preferably, component a) and component b) are located in a complex, for example, before or after such a region consisting of all antigens and/or antigenic epitopes. However, antigens and/or antigenic epitopes arranged in such a sequential manner may be linked to each other, for example, by a spacer or linker described below, which is neither component a), i.e. cell-penetrating peptide, nor component c), i.e. TLR peptide agonist.

Moreover, it is also preferred that the complex proposed for use according to the present invention contains one or more antigen epitopes. Thus, the complex preferably contains one or more epitopes of one or more antigens. For example, the complex may contain one or more epitopes of a first antigen and one or more epitopes of a second antigen; or the complex may contain one or more epitopes of a first antigen, one or more epitopes of a second antigen, and one or more epitopes of a third antigen; or the complex may contain one or more epitopes of a first antigen, one or more epitopes of a second antigen, one or more epitopes of a third antigen, and one or more epitopes of a fourth antigen; etc. In a preferred embodiment of the invention, the complex contains more than one epitope of one or more antigens; more preferably, the antigenic epitopes of one antigen are located sequentially (or overlap) in the complex proposed for use according to the present invention. This means, in particular, that all antigenic epitopes of one antigen included in the complex are located in the form of a region that is not interrupted by component a), i.e. cell-penetrating peptide, nor component c), i.e. a TLR peptide agonist, nor any other antigenic epitopes (ie, epitopes from another antigen).

However, alternatively, various antigens and/or antigenic epitopes can also be located in any other way in the complex proposed for use according to the present invention, for example, when component a) and/or component b) are located between two or more antigens and/or antigenic epitopes, i.e. one or more antigens and/or antigenic epitopes are located between component a) and component b) (or vice versa), and optionally one or more antigens and/or antigenic epitopes are located respectively at the other end of component a) and/or component b).

It is obvious that it is advisable to combine several different antigens or antigenic epitopes associated with colorectal cancer in one complex proposed for use according to the present invention. Alternatively, several different antigens or antigenic epitopes associated with colorectal cancer can be subdivided into subsets of different antigens or antigenic epitopes, in particular subsets that complement each other in the context of colorectal cancer, which are included in the various complexes proposed for use according to the present invention wherein said different complexes containing different subsets are advantageously administered simultaneously, for example as a single vaccine, to an individual in need thereof.

Preferably, the complex proposed for use according to the present invention contains at least one tumor epitope, which is an epitope of an antigen selected from the group consisting of EpCAM, HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG , survivin, CEA, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART and IL13Ralpha2. More preferably, the complex proposed for use according to the present invention contains at least one tumor epitope, which is an epitope of an antigen selected from the group consisting of ASCL2, EpCAM, HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, CEA, TGFβR2, p53, KRas, OGT, CASP5, SOA-1, MAGE, SART and IL13Ralpha2. These antigens are particularly valuable in the context of colorectal cancer. Preferably also the complex proposed for use according to the present invention contains at least one antigen selected from the group consisting of EpCAM, HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, CEA, TGFβR2, p53, KRas, OGT, CASP5, SOA-1, MAGE, SART and IL13Ralpha2, or a fragment thereof, or a tumor antigen sequence variant or a sequence variant thereof. Preferably, the complex proposed for use according to the present invention contains at least one tumor antigen selected from the group consisting of ASCL2, EpCAM, HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin , CEA, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART and IL13Ralpha2, or a fragment thereof, or a tumor antigen sequence variant or a sequence variant thereof. As used herein, an antigen "fragment" comprises at least 10 consecutive amino acids of an antigen, preferably at least 15 consecutive amino acids of an antigen, more preferably at least 20 consecutive amino acids of an antigen, even more preferably at least 25 consecutive amino acids of an antigen and most preferably at least 30 consecutive amino acids of the antigen. "Sequence variant" as defined above means a sequence variant having an (amino acid) sequence that is at least 70%, at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, more preferably at least 95%, most preferably at least 99% identical to the reference sequence. A “functional” sequence variant means, in the context of an antigen/antigen fragment/epitope, that the function of the epitope(s), eg, included in the antigen (fragment), is not impaired or lost. In this case, preferably, the amino acid sequence of the epitope(s), for example, included in the antigen (fragment) specified in the present description, is not changed as a result of mutation and is therefore identical to the reference sequence of the epitope.

As described above, suitable cancer/tumor epitopes of these antigens are known from the literature or can be identified using cancer/tumor epitope databases, for example, those described by van der Bruggen P., Stroobant V., Vigneron N., Van den Eynde B. Peptide database: T cell-defined tumor antigens. Cancer Immun, 2013; URL: http://www.cancerimmunity.org/peptide/, in which human tumor antigens recognized by CD4 ⁺ or CD8 ⁺ T cells are classified into 4 main groups based on their expression pattern, or from the Tantigen database " (TANTIGEN, version 1.0, December 1, 2009, created by Bioinformatics Core at Cancer Vaccine Center, Dana-Farber Cancer Institute; URL: http://cvc.dfci.harvard.edu/tadb/).

Ep-CAM

Ep-Cam is a glycoprotein that mediates cell adhesion. The amino acid sequence of EpCAM is presented below:

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 47, or a fragment or variant thereof, specified in the present description.

Several EpCAM epitopes are known to those skilled in the art. The preferred EpCAM epitope that is preferably included in the complex proposed for use according to the present invention includes the following epitope (the epitope sequence shown below is a fragment of the above EpCAM sequence, and it is underlined in the above EpCAM sequence; the epitope sequence below may correspond to one epitope or several (overlapping) epitopes):

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 48, or a fragment or variant thereof, specified in the present description.

HER-2/neu

Her-2 belongs to the EGFR (epidermal growth factor receptor) family. Many HLA-A epitopes are known to one skilled in the art. The amino acid sequence of HER2 is shown below:

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 70, or a fragment or variant thereof, specified in the present description. As described above, suitable Her-2 cancer/tumor epitopes are known from the literature or can be identified using cancer/tumor epitope databases such as those described in van der Bruggen P., Stroobant V., Vigneron N., Van den Eynde B. Peptide database: T cell-defined tumor antigens. Cancer Immun, 2013; URL: http://www.cancerimmunity.org/peptide/, in which human tumor antigens recognized by CD4 ⁺ or CD8 ⁺ T cells are classified into 4 main groups based on their expression pattern, or from the Tantigen database " (TANTIGEN, version 1.0, December 1, 2009, created by Bioinformatics Core at Cancer Vaccine Center, Dana-Farber Cancer Institute; URL: http://cvc.dfci.harvard.edu/tadb/).

Mucin-1 (MUC-1)

MUC-1 is a human epithelial mucin that affects cell adhesion. The amino acid sequence of MUC-1 is given below:

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 49, or a fragment or variant thereof, specified in the present description.

Several epitopes of MUC-1 are known to one skilled in the art. Preferred MUC-1 epitopes that are preferably included in the complex proposed for use according to the present invention include the following epitopes (the epitope sequences presented below are a fragment of the above MUC-1 sequence, and they are underlined in the above MUC-1 sequence; each of the above below epitope sequences may correspond to one epitope or several (overlapping) epitopes):

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 50 and/or the amino acid sequence corresponding to SEQ ID NO: 51.

TOMM34

TOMM34 is involved in the import of precursor proteins into mitochondria. A person skilled in the art knows many of its causes.

RNF43

RNF43 is an E3 ubiquitin ligase and likely contains a transmembrane domain, a protease-associated domain, an ectodomain, and a cytoplasmic RING domain. It is likely that RNF43 provides negative regulation of Wnt signaling, and the expression of RNF43 leads to increased ubiquitination of Frizzled receptors, changes their subcellular distribution, which leads to a reduced level of these receptors on the surface. A person skilled in the art knows many of its causes.

KOC1

KOC1, also known as insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), is an mRNA-binding protein. However, there are no data on its expression.

Vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor (VEGFR)

Vascular endothelial growth factor (VEGF), originally called vascular permeability factor (VPF), is a signaling protein produced by cells that stimulate vasculogenesis and angiogenesis. It is part of a system that restores oxygen supply to tissues when blood circulation is disrupted. The normal function of VEGF is to create new blood vessels during embryonic development, new blood vessels after muscle damage due to exercise, and new blood vessels (collateral circulation) to bypass blocked vessels. There are three main subtypes of VEGF receptors (VEGFR), namely VEGFR1, VEGFR2 and VEGFR3.

Beta subunit of human chorionic gonadotropin (βhCG)

Human chorionic gonadotropin (hCG) is a hormone produced by the embryo after implantation. Some cancers produce this hormone; therefore, elevated levels found in a patient who is not pregnant may contribute to the diagnosis of cancer. hCG is a heterodimer in which the α (alpha) subunit is identical to the subunit of luteinizing hormone (LH), follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and the β (beta) subunit is unique to hCG. The beta subunit of hCG gonadotropin (beta-hCG) contains 145 amino acids and is encoded by six highly homologous genes.

Survivin

Survivin, also called baculovirus inhibitor of apoptosis repeat motif 5 or BIRC5, is a member of the inhibitor of apoptosis (IAP) family. The functions of the survivin protein are to inhibit caspase activation, leading to negative regulation of apoptosis or programmed cell death. The amino acid sequence of survivin is presented below:

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 52, or a fragment or variant thereof, specified in the present description.

Several epitopes of survivin are known to those skilled in the art. The preferred epitope of survivin that is preferably included in the complex proposed for use according to the present invention includes the following epitope (the epitope sequence presented below is a fragment of the above sequence of survivin, and it is underlined in the above sequence of survivin; the epitope sequence below may correspond to one epitope or several (overlapping) epitopes):

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 53.

Thus, preferably, the complex proposed for use according to the present invention contains an epitope of survivin. More preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 52, or a fragment thereof, consisting of at least 10 amino acids (preferably at least 15 amino acids, more preferably at least 20 amino acids, even more preferably at least at least 25 amino acids and most preferably at least 30 amino acids), or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, particularly preferably at least 95%, most preferably at least 99% sequence identity). Even more preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 53. Most preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 95, or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95 %, most preferably at least 99% sequence identity).

Carcinoembryonic antigen (CEA)

CEA is an intracellular adhesion glycoprotein. CEA is normally produced by gastrointestinal tissue during embryonic development, but its production ceases before birth. Thus, CEA is typically present only at very low levels in the blood of healthy adults. The amino acid sequence of CEA is given below:

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 54, or a fragment or variant thereof, specified in the present description.

Several epitopes of CEA are known to those skilled in the art. Preferred CEA epitopes that are preferably included in the complex proposed for use according to the present invention include the following epitopes (the epitope sequences presented below are a fragment of the above CEA sequence, and they are underlined in the above CEA sequence; each of the following epitope sequences may correspond to one epitope or several (overlapping) epitopes):

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 55 and/or the amino acid sequence corresponding to SEQ ID NO: 56.

Thus, preferably the complex proposed for use according to the present invention contains the CEA epitope. More preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 54, or a fragment thereof, consisting of at least 10 amino acids (preferably at least 15 amino acids, more preferably at least 20 amino acids, even more preferably at least at least 25 amino acids and most preferably at least 30 amino acids), or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, particularly preferably at least 95%, most preferably at least 99% sequence identity). Even more preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 55, and/or a peptide that has an amino acid sequence corresponding to SEQ ID NO: 56. Most preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 96, or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity).

Transforming growth factor beta receptor 2 (TGFβR2)

TGFβ receptors are cell membrane-spanning serine/threonine kinase receptors. They are present in several different isoforms. TGFβR2 is a transmembrane protein that contains a protein kinase domain, forms a heterodimeric complex with another receptor protein, and binds to TGF-beta. This receptor/ligand complex phosphorylates proteins that then enter the nucleus and regulate the transcription of a subset of genes associated with cell proliferation.

P53

P53 is a tumor suppressor protein that plays a role in preventing genomic mutations. P53 has several anticancer mechanisms and plays a role in apoptosis, genome stability, and inhibition of angiogenesis. p53 performs its anticancer function through several mechanisms: it activates DNA repair proteins when DNA is significantly damaged; it can arrest growth by arresting the cell cycle at the G1/S regulatory point when recognizing DNA damage; and it can initiate apoptosis.

Kirsten-subtype Ras (KRas)

The GTPase KRas, known as the viral oncogenic homolog of V-Ki-ras2 Kirsten rat sarcoma and KRAS, has an important function in signaling in healthy tissue, and mutation of the KRAS gene is an important step in the development of many types of cancer. Like other members of the ras subfamily, the KRAS protein is a GTPase and is an early participant in many signal transduction pathways. KRAS is typically associated with the cell membrane due to the presence of an isoprene group at its C-terminus. The amino acid sequence of KRas is given below:

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 57, or a fragment or variant thereof, specified in the present description.

Several Kirsten Ras epitopes are known to those skilled in the art. The preferred Kirsten Ras epitope that is preferably included in the complex proposed for use according to the present invention includes the following epitope (the epitope sequence presented below is a fragment of the above Kirsten Ras sequence, and it is underlined in the above Kirsten Ras sequence; the epitope sequence below may correspond to one epitope or several (overlapping) epitopes):

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 58.

O-linked N-acetylglucosamine (GlcNAc)-transferase (OGT)

OGT (O-linked N-acetylglucosamine (GlcNAc)-transferase, O-GlcNAc-transferase, OGTase, O-linked N-acetylglucosaminyltransferase, uridine diphospho-N-acetylglucosamine: beta-N-acetylglucosaminyltransferase polypeptide, O-linked beta-N- acetylglucosamine transferase) is an enzyme with the systematic (nomenclatural) name "UDP-N-acetyl-D-glucosamine:protein-O-beta-N-acetyl-D-glucosaminyl-transferase). OGT catalyzes the addition of one N-acetylglucosamine at the O-glycosidic bond to serine or threonine residues of intracellular proteins. OGT is a component of host biological functions in the human body. OGT is involved in insulin resistance in muscle cells and adipocytes by inhibiting AKT1 threonine 308 phosphorylation, increasing the rate of IRS1 phosphorylation (at serine 307 and serine 632/635), reducing insulin signal transduction and glycosylation of components of the insulin signal transduction pathway. In addition, OGT catalyzes the intracellular glycosylation of serine and threonine residues by adding N-acetylglucosamine. Studies have demonstrated that OGT alleles are vital for embryogenesis, and OGT is required for intracellular glycosylation and embryonic stem cell viability. OGT also catalyzes a post-translational modification that alters transcription factors and RNA polymerase II, but the specific function of this modification is generally unclear.

Caspase 5 (CASP5)

Caspase 5 is an enzyme that proteolytically cleaves other proteins at the aspartic acid residue and belongs to a family of cysteine proteases called caspases. It is a proinflammatory caspase along with caspase 1, caspase 4 and murine caspase 4, a homologue of caspase 11, and plays a role in immune system function.

Colorectal tumor associated antigen-1 (COA-1)

COA-1 was identified in 2003 by Maccalli et al. (Identification of a colorectal tumor-associated antigen (COA-1) recognized by CD4(+) T lymphocytes. Cancer Res, 63(20), 2003, pp. 6735-6743) as a protein with high levels of expression in colorectal and melanoma cells (data not shown). Its mutation may affect the ability to differentiate between tumor and healthy cells.

Melanoma associated antigen (MAGE)

Members of the mammalian MAGE (melanoma-associated antigen) gene family were initially described as completely silent in tissues of healthy adults, except in male germ cells, and occasionally in the placenta. In contrast, these genes are expressed in different types of tumors. Thus, the complex of the present invention preferably contains a MAGE family antigen ("MAGE" antigen) or an epitope thereof. Of the MAGE family, MAGE-A3 and MAGE-D4 are particularly preferred, and MAGE-A3 is most preferred. The normal function of MAGE-A3 in healthy cells is unknown. MAGE-A3 is a tumor-specific protein and has been identified in many tumors. The amino acid sequence of MAGE-A3 is presented below:

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 59.

Several epitopes of MAGE-A3 are known to those skilled in the art. The preferred epitope of MAGE-A3, which is preferably included in the complex proposed for use according to the present invention, includes the following epitope (the epitope sequence presented below is a fragment of the above MAGE-A3 sequence, and it is underlined in the above MAGE-A3 sequence; the following epitope a sequence may correspond to one epitope or several (overlapping) epitopes):

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 60.

Squamous cell carcinoma antigen recognized by T cells (SART)

Among the SART family, SART-3 is the most preferred. Thus, the complex proposed for use according to the present invention preferably contains an antigen from the SART family ("SART" antigen) or an epitope thereof; the complex proposed for use according to the present invention more preferably contains SART-3 or an epitope thereof. Squamous cell carcinoma antigen 3, recognized by T cells, carries tumor epitopes with the ability to induce HLA-A24-restricted and tumor-specific cytotoxic T lymphocytes in cancer patients. SART-3 is likely involved in the regulation of mRNA splicing.

IL13Ralpha2

IL13Ralpha2 binds to interleukin 13 (IL-13) with very high affinity (and can therefore sequester it) but does not mediate binding to IL-4. It acts as a negative regulator of both IL-13 and IL-4, but its mechanism of action has not yet been established. The amino acid sequence of IL13Ralpha2 is given below:

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 61, or a fragment or variant thereof, specified in the present description.

Several IL13Ralpha2 epitopes are known to those skilled in the art. The preferred epitope of IL13Ralpha2, which is preferably included in the complex proposed for use according to the present invention, includes the following epitope (the epitope sequence presented below is a fragment of the above IL13Ralpha2 sequence, and it is underlined in the above IL13Ralpha2 sequence; the following epitope sequence may correspond to one epitope or several (overlapping) epitopes):

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 62.

ASCL2 (Achaete-shita homologue 2)

ASCL2 is a basic helix-loop-helix domain transcription factor important for maintaining proliferating trophoblasts during placental development. It has been established that ASCL2 is a possible regulator of proliferation, which is characterized by overexpression in intestinal neoplasm. The amino acid sequence of ASCL2 is given below:

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 92, or a fragment or variant thereof, specified in the present description.

Several ASCL2 epitopes are known to those skilled in the art. The preferred epitope of ASCL2, which is preferably included in the complex proposed for use according to the present invention, includes the following epitope (the epitope sequence presented below is a fragment of the above ASCL2 sequence, and it is underlined in the above ASCL2 sequence; the epitope sequence below may correspond to one epitope or several (overlapping) epitopes):

Thus, the preferred complex proposed for use according to the present invention contains the amino acid sequence corresponding to SEQ ID NO: 93 and/or the amino acid sequence corresponding to SEQ ID NO: 94.

Thus, preferably, the complex proposed for use according to the present invention contains an epitope of ASCL2. More preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 92, or a fragment thereof, consisting of at least 10 amino acids (preferably at least 15 amino acids, more preferably at least 20 amino acids, even more preferably at least at least 25 amino acids and most preferably at least 30 amino acids), or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, particularly preferably at least 95%, most preferably at least 99% sequence identity). Even more preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 93 and/or a peptide that has an amino acid sequence corresponding to SEQ ID NO: 94. Most preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 97, or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity).

Preferably, the complex proposed for use according to the present invention contains at least one tumor epitope, which is an epitope of an antigen selected from the group consisting of EpCAM, MUC-1, survivin, CEA, KRas, MAGE-A3 and IL13Ralpha2, such as an epitope having any of SEQ ID NOs: 48, 50, 51, 53, 55, 56, 58, 60 and 62; preferably, the complex proposed for use according to the present invention contains at least one tumor epitope, which is an epitope of an antigen selected from the group consisting of EpCAM, MUC-1, survivin, CEA, KRas, MAGE-A3, IL13Ralpha2 and ASCL2 , such as an epitope having any of SEQ ID NO: 48, 50, 51, 53, 55, 56, 58, 60, 62, 93 and 94; more preferably, the at least one tumor epitope is an epitope of an antigen selected from the group consisting of EpCAM, MUC-1, survivin, CEA, KRas and MAGE-A3, such as an epitope having any of SEQ ID NOs 48, 50, 51 , 53, 55, 56, 58 and 60; more preferably, the at least one tumor epitope is an epitope of an antigen selected from the group consisting of EpCAM, MUC-1, survivin, CEA, KRas, MAGE-A3 and ASCL2, such as the epitope having any of SEQ ID NO: 48, 50, 51, 53, 55, 56, 58, 60, 93 and 94; even more preferably, the at least one tumor epitope is an epitope of an antigen selected from the group consisting of EpCAM, MUC-1, survivin and CEA, such as an epitope having any of SEQ ID NO: 48, 50, 51, 53, 55 and 56; even more preferably, the at least one tumor epitope is an epitope of an antigen selected from the group consisting of EpCAM, MUC-1, survivin, CEA and ASCL2, such as an epitope having any of SEQ ID NO: 48, 50, 51, 53 , 55, 56, 93 and 94; and most preferably, the at least one tumor epitope is an epitope of an antigen selected from the group consisting of EpCAM, survivin, CEA and ASCL2, such as an epitope having any of SEQ ID NOs: 48, 53, 55, 56, 93 and 94 .

Also preferred is the complex proposed for use according to the present invention, which contains

I) one or more EpCAM epitopes (eg, the epitope having SEQ ID NO: 48) or functional sequence variants thereof;

ii) one or more MUC-1 epitopes (eg, the epitope having SEQ ID NO: 50 and/or the epitope having SEQ ID NO: 51) or functional sequence variants thereof;

iii) one or more survivin epitopes (eg, the epitope having SEQ ID NO: 53) or functional sequence variants thereof;

IV) one or more CEA epitopes (eg, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof;

V) one or more KRas epitopes (eg, the epitope having SEQ ID NO: 58) or functional sequence variants thereof; and/or

VI) one or more MAGE-A3 epitopes (eg, the epitope having SEQ ID NO: 60) or functional sequence variants thereof.

Also preferred is the complex proposed for use according to the present invention, which contains

VII) one or more EpCAM epitopes (eg, the epitope having SEQ ID NO: 48) or functional sequence variants thereof;

VIII) one or more MUC-1 epitopes (eg, the epitope having SEQ ID NO: 50 and/or the epitope having SEQ ID NO: 51) or functional sequence variants thereof;

IX) one or more survivin epitopes (eg, the epitope having SEQ ID NO: 53) or functional sequence variants thereof;

X) one or more CEA epitopes (eg, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof;

XI) one or more KRas epitopes (eg, the epitope having SEQ ID NO: 58) or functional sequence variants thereof;

XII) one or more MAGE-A3 epitopes (eg, the epitope having SEQ ID NO: 60) or functional sequence variants thereof; and/or

XIII) one or more ASCL2 epitopes (eg, the epitope having SEQ ID NO: 93 and/or the epitope having SEQ ID NO: 94) or functional sequence variants thereof.

As described above, additional epitopes of these antigens (in addition to the exemplary epitopes) can be easily obtained from cancer/tumor epitope databases, for example, those described in van der Bruggen P., Stroobant V., Vigneron N., Van den Eynde B. Peptide database: T cell-defined tumor antigens. Cancer Immun, 2013; URL: http://www.cancerimmunity.org/peptide/, or from the Tantigen database (TANTIGEN, version 1.0, December 1, 2009, created by Bioinformatics Core at Cancer Vaccine Center, Dana-Farber Cancer Institute; URL: http://cvc.dfci.harvard.edu/tadb/).

"Sequence variant" is the variant described above, namely, the sequence variant has an (amino acid) sequence that is at least 70%, at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, most preferably at least 99% identical to the reference sequence. A "functional" sequence variant means, in the context of an epitope, that the function as an epitope is not impaired or eliminated. However, preferably, the amino acid sequence of the epitope of a cancer/tumor antigen specified herein is not changed by mutation, and is therefore identical to the reference sequence of the epitope.

Also preferred is the complex proposed for use according to the present invention, which contains

- an EpCAM fragment containing one or more epitopes or a functional variant of its sequence;

- a fragment of MUC-1 containing one or more epitopes or a functional variant of its sequence;

- a fragment of survivin containing one or more epitopes or a functional variant of its sequence;

- a CEA fragment containing one or more epitopes or a functional variant of its sequence;

- a KRas fragment containing one or more epitopes or a functional variant of its sequence; and/or

- a MAGE-A3 fragment containing one or more epitopes or a functional variant of its sequence.

Also preferred is the complex proposed for use according to the present invention, which contains

- an EpCAM fragment containing one or more epitopes or a functional variant of its sequence;

- a fragment of MUC-1 containing one or more epitopes or a functional variant of its sequence;

- a fragment of survivin containing one or more epitopes or a functional variant of its sequence;

- a CEA fragment containing one or more epitopes or a functional variant of its sequence;

- a KRas fragment containing one or more epitopes or a functional variant of its sequence;

- a MAGE-A3 fragment containing one or more epitopes or a functional variant of its sequence; and/or

- an ASCL2 fragment containing one or more epitopes or a functional variant of its sequence.

As used herein, an antigen "fragment" comprises at least 10 consecutive amino acids of an antigen, preferably at least 15 consecutive amino acids of an antigen, more preferably at least 20 consecutive amino acids of an antigen, even more preferably at least 25 consecutive amino acids of an antigen and most preferably at least 30 consecutive amino acids of the antigen. Thus, the EpCAM fragment contains at least 10 consecutive EpCAM amino acids (SEQ ID NO: 47), preferably at least 15 consecutive EpCAM amino acids (SEQ ID NO: 47), more preferably at least 20 consecutive EpCAM amino acids (SEQ ID NO: 47). SEQ ID NO: 47), even more preferably at least 25 consecutive amino acids of EpCAM (SEQ ID NO: 47) and most preferably at least 30 consecutive amino acids of EpCAM (SEQ ID NO: 47); the MUC-1 fragment contains at least 10 consecutive amino acids of MUC-1 (SEQ ID NO: 49), preferably at least 15 consecutive amino acids of MUC-1 (SEQ ID NO: 49), more preferably at least 20 consecutive amino acids MUC-1 amino acids (SEQ ID NO: 49), even more preferably at least 25 contiguous amino acids of MUC-1 (SEQ ID NO: 49) and most preferably at least 30 contiguous amino acids of MUC-1 (SEQ ID NO: 49); the survivin fragment contains at least 10 consecutive amino acids of survivin (SEQ ID NO: 52), preferably at least 15 consecutive amino acids of survivin (SEQ ID NO: 52), more preferably at least 20 consecutive amino acids of survivin (SEQ ID NO : 52), even more preferably at least 25 consecutive amino acids of survivin (SEQ ID NO: 52) and most preferably at least 30 consecutive amino acids of survivin (SEQ ID NO: 52); the CEA fragment contains at least 10 consecutive CEA amino acids (SEQ ID NO: 54), preferably at least 15 consecutive CEA amino acids (SEQ ID NO: 54), more preferably at least 20 consecutive CEA amino acids (SEQ ID NO : 54), even more preferably at least 25 consecutive amino acids CEA (SEQ ID NO: 54) and most preferably at least 30 consecutive amino acids CEA (SEQ ID NO: 54); the KRas fragment contains at least 10 consecutive KRas amino acids (SEQ ID NO: 57), preferably at least 15 consecutive KRas amino acids (SEQ ID NO: 57), more preferably at least 20 consecutive KRas amino acids (SEQ ID NO : 57), even more preferably at least 25 consecutive amino acids of KRas (SEQ ID NO: 57) and most preferably at least 30 consecutive amino acids of KRas (SEQ ID NO: 57); the MAGE-A3 fragment contains at least 10 consecutive amino acids of MAGE-A3 (SEQ ID NO: 59), preferably at least 15 consecutive amino acids of MAGE-A3 (SEQ ID NO: 59), more preferably at least 20 consecutive amino acids MAGE-A3 amino acids (SEQ ID NO: 59), even more preferably at least 25 consecutive amino acids of MAGE-A3 (SEQ ID NO: 59) and most preferably at least 30 consecutive amino acids of MAGE-A3 (SEQ ID NO: 59). In addition, the ASCL2 fragment contains at least 10 consecutive amino acids of ASCL2 (SEQ ID NO: 92), preferably at least 15 consecutive amino acids of ASCL2 (SEQ ID NO: 92), more preferably at least 20 consecutive amino acids of ASCL2 ( SEQ ID NO: 92), even more preferably at least 25 consecutive amino acids of ASCL2 (SEQ ID NO: 92) and most preferably at least 30 consecutive amino acids of ASCL2 (SEQ ID NO: 92).

A functional variant sequence of said fragment has an (amino acid) sequence that is at least 70%, at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, most preferably at least 99% identical to the reference sequence, and the epitope function of at least one, preferably all, epitope(s) included in the fragment is retained.

Preferably, said complex contains

XIV) one or more EpCAM epitopes (eg, the epitope having SEQ ID NO: 48) or functional sequence variants thereof;

XV) one or more CEA epitopes (eg, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof; And

XVI) one or more MAGE-A3 epitopes (eg, the epitope having SEQ ID NO: 60) or functional sequence variants thereof.

The complex preferably does not contain any of the following epitopes: HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT, CASP5, COA-1, SART or IL13Ralpha2. More preferably, said complex does not contain any of the following epitopes: ASCL2, HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT, CASP5, COA-1, SART or IL13Ralpha2.

Preferably, said complex also contains

XVII) one or more EpCAM epitopes (eg, the epitope having SEQ ID NO: 48) or functional sequence variants thereof;

XVIII) one or more MUC-1 epitopes (eg, the epitope having SEQ ID NO: 50 and/or the epitope having SEQ ID NO: 51) or functional sequence variants thereof;

XIX) one or more CEA epitopes (eg, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof; And

XX) one or more MAGE-A3 epitopes (eg, the epitope having SEQ ID NO: 60) or functional sequence variants thereof.

The complex preferably does not contain any of the following epitopes: HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT, CASP5, COA-1, SART or IL13Ralpha2. More preferably, said complex does not contain any of the following epitopes: ASCL2, HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT, CASP5, COA-1, SART or IL13Ralpha2.

Preferably, said complex also contains

XXI) one or more EpCAM epitopes (eg, the epitope having SEQ ID NO: 48) or functional sequence variants thereof;

XXII) one or more MUC-1 epitopes (eg, the epitope having SEQ ID NO: 50 and/or the epitope having SEQ ID NO: 51) or functional sequence variants thereof;

XXIII) one or more CEA epitopes (eg, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof; And

XXIV) one or more KRas epitopes (eg, the epitope having SEQ ID NO: 58) or functional sequence variants thereof.

The complex preferably does not contain any of the following epitopes: HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, OGT, CASP5, SOA-1, MAGE, SART or IL13Ralpha2. More preferably, said complex does not contain any of the following epitopes: ASCL2, HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

Preferably, said complex also contains

XXV) one or more EpCAM epitopes (eg, the epitope having SEQ ID NO: 48) or functional sequence variants thereof;

XXVI) one or more survivin epitopes (eg, the epitope having SEQ ID NO: 53) or functional sequence variants thereof;

XXVII) one or more CEA epitopes (eg, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof; And

XXVIII) one or more MAGE-A3 epitopes (eg, the epitope having SEQ ID NO: 60) or functional sequence variants thereof.

The complex preferably does not contain any of the following epitopes: HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, SART or IL13Ralpha2. More preferably, said complex does not contain any of the following epitopes: ASCL2, HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, SART or IL13Ralpha2.

Preferably, said complex also contains

XXIX) one or more MUC-1 epitopes (eg, the epitope having SEQ ID NO: 50 and/or the epitope having SEQ ID NO: 51) or functional sequence variants thereof;

XXX) one or more survivin epitopes (eg, the epitope having SEQ ID NO: 53) or functional sequence variants thereof; And

XXXI) one or more MAGE-A3 epitopes (eg, the epitope having SEQ ID NO: 60) or functional sequence variants thereof.

The complex preferably does not contain any of the following epitopes: EpCAM, HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, CEA, TGFβR2, p53, KRas, OGT, CASP5, COA-1, SART or IL13Ralpha2. More preferably, said complex does not contain any of the following epitopes: ASCL2, EpCAM, HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, CEA, TGFβR2, p53, KRas, OGT, CASP5, COA-1, SART or IL13Ralpha2.

More preferably, said complex contains

XXXII) one or more EpCAM epitopes (for example, the epitope having SEQ ID NO: 48) or functional sequence variants thereof;

XXXIII) one or more MUC-1 epitopes (eg, the epitope having SEQ ID NO: 50 and/or the epitope having SEQ ID NO: 51) or functional sequence variants thereof;

XXXIV) one or more survivin epitopes (eg, the epitope having SEQ ID NO: 53) or functional sequence variants thereof; and/or

XXXV) one or more CEA epitopes (eg, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof.

The complex preferably does not contain any of the following epitopes: HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2. More preferably, said complex does not contain any of the following epitopes: ASCL2, HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

More preferably, said complex contains

XXXVI) one or more EpCAM epitopes (for example, the epitope having SEQ ID NO: 48) or functional sequence variants thereof;

XXXVII) one or more MUC-1 epitopes (eg, the epitope having SEQ ID NO: 50 and/or the epitope having SEQ ID NO: 51) or functional sequence variants thereof;

XXXVIII) one or more survivin epitopes (eg, the epitope having SEQ ID NO: 53) or functional sequence variants thereof;

XXXIX) one or more ASCL2 epitopes (eg, the epitope having SEQ ID NO: 93 and/or the epitope having SEQ ID NO: 94) or functional sequence variants thereof; and/or

XL) one or more CEA epitopes (eg, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof.

The complex preferably does not contain any of the following epitopes: HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

More preferably, said complex contains

XLI) one or more EpCAM epitopes (eg, the epitope having SEQ ID NO: 48) or functional sequence variants thereof;

XLII) one or more survivin epitopes (eg, the epitope having SEQ ID NO: 53) or functional sequence variants thereof;

XLIII) one or more ASCL2 epitopes (eg, the epitope having SEQ ID NO: 93 and/or the epitope having SEQ ID NO: 94) or functional sequence variants thereof; and/or

XLIV) one or more CEA epitopes (eg, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof.

The complex preferably does not contain any of the following epitopes: HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

Particularly preferably, said complex contains

XLV) one or more EpCAM epitopes (eg, the epitope having SEQ ID NO: 48) or functional sequence variants thereof;

XLVI) one or more MUC-1 epitopes (eg, the epitope having SEQ ID NO: 50 and/or the epitope having SEQ ID NO: 51) or functional sequence variants thereof;

XLVII) one or more survivin epitopes (eg, the epitope having SEQ ID NO: 53) or functional sequence variants thereof; And

XLVIII) one or more CEA epitopes (eg, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof.

The complex preferably does not contain any of the following epitopes: HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2. More preferably, said complex does not contain any of the following epitopes: ASCL2, HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

Also particularly preferably, said complex contains

XLIX) one or more CEA epitopes (eg, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof;

L) one or more survivin epitopes (eg, the epitope having SEQ ID NO: 53) or functional sequence variants thereof;

LI) one or more EpCAM epitopes (eg, the epitope having SEQ ID NO: 48) or functional sequence variants thereof; And

LII) one or more ASCL2 epitopes (eg, the epitope having SEQ ID NO: 93 and/or the epitope having SEQ ID NO: 94) or functional sequence variants thereof.

The complex preferably does not contain any of the following epitopes: HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

Also particularly preferably, said complex contains

LIII) one or more EpCAM epitopes (eg, the epitope having SEQ ID NO: 48) or functional sequence variants thereof;

LIV) one or more MUC-1 epitopes (eg, the epitope having SEQ ID NO: 50 and/or the epitope having SEQ ID NO: 51) or functional sequence variants thereof; And

LV) one or more CEA epitopes (eg, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof.

The complex preferably does not contain any of the following epitopes: HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2. More preferably, said complex does not contain any of the following epitopes: ASCL2, HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

Also particularly preferably, said complex contains

LVI) one or more CEA epitopes (eg, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof;

LVII) one or more survivin epitopes (eg, the epitope having SEQ ID NO: 53) or functional sequence variants thereof; And

LVII) one or more ASCL2 epitopes (eg, the epitope having SEQ ID NO: 93 and/or the epitope having SEQ ID NO: 94) or functional sequence variants thereof.

Said complex preferably does not contain any of the following epitopes: HER-2, EpCAM, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2 .

Even more preferably, the complex contains, in the direction from N-terminus to C-terminus:

- one or more CEA epitopes (for example, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof;

- one or more survivin epitopes (for example, the epitope having SEQ ID NO: 53) or functional sequence variants thereof; And

- one or more ASCL2 epitopes (for example, the epitope having SEQ ID NO: 93 and/or the epitope having SEQ ID NO: 94) or functional sequence variants thereof.

Said complex preferably does not contain any of the following epitopes: HER-2, EpCAM, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2

Even more preferably, the complex contains, in the direction from N-terminus to C-terminus:

I) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 54, or a fragment thereof, consisting of at least 10 amino acids (preferably at least 15 amino acids, more preferably at least 20 amino acids, even more preferably at least 25 amino acids and most preferably at least 30 amino acids), or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80%, even more preferably in at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity);

II) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 52, or a fragment thereof, consisting of at least 10 amino acids (preferably at least 15 amino acids, more preferably at least 20 amino acids, even more preferably at least 25 amino acids and most preferably at least 30 amino acids), or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80%, even more preferably in at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity); And

III) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 92, or a fragment thereof, consisting of at least 10 amino acids (preferably at least 15 amino acids, more preferably at least 20 amino acids, even more preferably at least 25 amino acids and most preferably at least 30 amino acids), or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80%, even more preferably in at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity).

The complex preferably does not contain any additional antigen or any additional epitopes of antigens other than CEA, survivin and ASCL2, more preferably the complex does not contain any other (tumor) epitope.

Preferably, in said complex, the C-terminus (I) of a peptide that has an amino acid sequence corresponding to SEQ ID NO: 54, or a fragment or variant thereof, is directly linked to the N-terminus (II) of a peptide that has an amino acid sequence corresponding to SEQ ID NO: : 52, or a fragment or variant thereof; and the C-terminus (II) of a peptide that has an amino acid sequence corresponding to SEQ ID NO: 52, or a fragment or variant thereof, is directly linked to the N-terminus (III) of a peptide that has an amino acid sequence corresponding to SEQ ID NO: 92, or a fragment or variant thereof.

Even more preferably, the complex contains, in the direction from N-terminus to C-terminus:

I) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 96, or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80% , even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity);

ii) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 95 or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity); And

III) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 97, or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80% , even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity).

The complex preferably does not contain any additional antigen or any additional epitopes of antigens other than CEA, survivin and ASCL2, more preferably the complex does not contain any other (tumor) epitope.

Preferably, in said complex, the C-terminus (I) of a peptide that has an amino acid sequence corresponding to SEQ ID NO: 96, or a variant thereof, is directly linked to the N-terminus (II) of a peptide that has an amino acid sequence corresponding to SEQ ID NO: 95 , or its variant; and the C-terminus (II) of the peptide, which has the amino acid sequence corresponding to SEQ ID NO: 95, or a variant thereof, is directly linked to the N-terminus (III) of the peptide, which has the amino acid sequence corresponding to SEQ ID NO: 92, or its option.

Most preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 98, or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80% -, even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity). The complex preferably does not contain any additional antigen or any additional epitopes of antigens other than CEA, survivin and ASCL2, more preferably the complex does not contain any other (tumor) epitope.

Particularly preferably, said complex also contains

- one or more EpCAM epitopes (for example, the epitope having SEQ ID NO: 48) or functional sequence variants thereof; And

- one or more CEA epitopes (for example, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof.

Said complex preferably does not contain any of the following epitopes: HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT, CASP5, SOA-1, MAGE, SART or IL13Ralpha2 . More preferably, said complex does not contain any of the following epitopes: ASCL2, HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

Particularly preferably, said complex also contains

- one or more EpCAM epitopes (for example, the epitope having SEQ ID NO: 48) or functional sequence variants thereof; And

- one or more ASCL2 epitopes (for example, the epitope having SEQ ID NO: 93 and/or the epitope having SEQ ID NO: 94) or functional sequence variants thereof.

Said complex preferably does not contain any of the following epitopes: HER-2, MUC-1, CEA, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

Particularly preferably, said complex also contains

- one or more ASCL2 epitopes (for example, the epitope having SEQ ID NO: 93 and/or the epitope having SEQ ID NO: 94) or functional sequence variants thereof; And

- one or more CEA epitopes (for example, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof.

Said complex preferably does not contain any of the following epitopes: HER-2, EpCAM, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

Particularly preferably, said complex also contains

- one or more survivin epitopes (for example, the epitope having SEQ ID NO: 53) or functional sequence variants thereof; And

- one or more ASCL2 epitopes (for example, the epitope having SEQ ID NO: 93 and/or the epitope having SEQ ID NO: 94) or functional sequence variants thereof.

Said complex preferably does not contain any of the following epitopes: HER-2, MUC-1, EpCAM, CEA, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

Particularly preferably, said complex also contains

- one or more survivin epitopes (for example, the epitope having SEQ ID NO: 53) or functional sequence variants thereof; And

- one or more CEA epitopes (for example, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof.

Said complex preferably does not contain any of the following epitopes: ASCL2, HER-2, EpCAM, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

Particularly preferably, said complex also contains

- one or more EpCAM epitopes (for example, the epitope having SEQ ID NO: 48) or functional sequence variants thereof.

Said complex preferably does not contain any of the following epitopes: HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, CEA, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2. More preferably, said complex does not contain any of the following epitopes: ASCL2, HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, CEA, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

Particularly preferably, said complex also contains

- one or more CEA epitopes (for example, the epitope having SEQ ID NO: 55 and/or the epitope having SEQ ID NO: 56) or functional sequence variants thereof.

Said complex preferably does not contain any of the following epitopes: EpCAM, HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2. More preferably, said complex does not contain any of the following epitopes: ASCL2, EpCAM, HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

Particularly preferably, said complex also contains

- one or more ASCL2 epitopes (for example, the epitope having SEQ ID NO: 93 and/or the epitope having SEQ ID NO: 94) or functional sequence variants thereof.

Said complex preferably does not contain any of the following epitopes: EpCAM, HER-2, MUC-1, CEA, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE , SART or IL13Ralpha2.

Component c) - TLR peptide agonist

In the complex proposed for use according to the present invention, the TLR peptide agonist provides, along with autoimmunogenicity, increased targeting of the vaccine to dendritic cells. The physical association of a TLR peptide agonist with CPP and at least one antigen or antigenic epitope of the present invention, in the complex proposed for use according to the present invention, provides an enhanced immune response by simultaneously stimulating antigen presenting cells, in particular dendritic cells that internalize, metabolize and expose antigen(s).

In the context of the present invention, a “peptide TLR agonist” is a Toll-like receptor (TLR) agonist, i.e. it binds to TLRs and activates TLRs, specifically leading to a biological response. In addition, the TLR peptide agonist is a peptide, polypeptide or protein as described above. Preferably, the TLR peptide agonist contains from 10 to 150 amino acids, more preferably from 15 to 130 amino acids, even more preferably from 20 to 120 amino acids, even more preferably from 25 to 110 amino acids, and most preferably from 30 to 100 amino acids.

Toll-like receptors (TLRs) are transmembrane proteins characterized by the presence of extracellular, transmembrane, and cytosolic domains. Extracellular domains containing horseshoe-shaped leucine-rich repeats (LRRs) are involved in the recognition of standard molecular patterns from a variety of microbes. Toll-like receptors include TLRs 1-10. Compounds having the ability to activate TLR receptors, and modifications and derivatives thereof, are well known in the art. TLR1 can be activated by bacterial lipoproteins and their acetylated forms, TLR2 can in addition be activated by glycolipids from Gram-positive bacteria, LPS, LPA, LTA, fimbriae, outer membrane proteins, heat shock proteins from bacteria or from the host, and mycobacterial lipoarabinomannans. TLR3 can be activated by dsRNA, in particular of viral origin, or by the chemical compound poly(LC). TLR4 can be activated by LPS, LTA of gram-negative bacteria, heat shock proteins of host or bacterial origin, coating or envelope proteins of viruses, taxol or its derivatives, hyaluronan-containing oligosaccharides and fibronectins. TLR5 can be activated by bacterial flagella or flagellin. TLR6 can be activated by mycobacterial lipoproteins and heat-sensitive group B streptococcal factor (GBS-F) or staphylococcal modulins. TLR7 can be activated by imidazoquinolines. TLR9 can be activated by unmethylated CpG-DNA or chromatin-IgG complexes.

Preferably, the TLR peptide agonist included in the complex proposed for use according to the present invention is an agonist of TLR1, 2, 4, 5, 6 and/or 10. TLRs are expressed either on the cell surface (TLR1, 2, 4, 5, 6 and 10 ), or on the membranes of intracellular organelles such as endosomes (TLR3, 4, 7, 8 and 9). Known naturally occurring ligands for endosomal receptors are nucleic acid-based molecules (with the exception of TLR4). Cell surface expressed TLR1, 2, 4, 5, 6 and 10 recognize molecular patterns of extracellular microbes (Monie T.P., Bryant S.E. et al., Activating immunity: Lessons from the TLRs and NLRs. Trends Biochem. Sci 34(11), 2009, pp. 553-561). TLRs are expressed on several cell types, but virtually all TLRs are expressed on DCs, providing these specialized cells with sensitivity to all possible pathogens and danger signals.

In this case, TLR2, 4 and 5 are constitutively expressed on the surface of DCs. Thus, the TLR peptide agonist included in the complex proposed for use according to the present invention is more preferably a TLR2, TLR4 and/or TLR5 peptide agonist. Even more preferably, the TLR peptide agonist is a TLR2 peptide agonist and/or a TLR4 peptide agonist. Most preferably, the TLR peptide agonist is a TLR4 peptide agonist. Most preferably, the TLR agonist peptide is a TLR agonist peptide that is an agonist of both TLR2 and TLR4. TLR2 can detect a wide variety of ligands derived from bacteria, viruses, parasites and fungi. Ligand specificity is often determined by the interaction of TLR2 with other TLRs such as TLR1, 6 or 10, or non-TLR molecules such as Dectin-1, CD14 or CD36. Formation of a heterodimer with TLR1 will allow TLR2 to identify triacyl lipoproteins or lipopeptides of (myco)bacterial origin, such as Pam3CSK4 and peptidoglycan (PGA; Gay N.J. and Gangloff M., Structure and function of Toll receptors and their ligands. Annu. Rev. Biochem. 76, 2007, pp. 141-165; Spohn R., Buwitt-Beckmann U. et al., Synthetic lipopeptide adjuvants and Toll-like receptor 2 - Structure-activity relationships. Vaccine 22(19), 2004, pp. 2494-2499) . Heterodimerization of TLR2 and 6 allows detection of diacyl lipopeptides and zymosan. Lipopolysaccharide (LPS) and its derivatives are ligands for TLR4, and flagellin for TLR5 (Bryant S.E., Spring D.R. et al., The molecular basis of the host response to lipopolysaccharide. Nat. Rev. Microbiol. 8(1), 2010, pp. 8-14).

TLR2 interacts with a wide range of structurally diverse ligands, including molecules expressed by microbes and fungi. A variety of TLR2 agonists have been identified, including naturally occurring and synthetic lipopeptides (e.g., Mycoplasma fermentas macrophage-activating lipopeptide (MALP-2)), peptidoglycans (PG, e.g., from S. aureus), lipopolysaccharides (LPS) from various bacterial strains, polysaccharides (eg, zymosan), glycoside phosphophatidylinositol-anchored structures from gram-positive bacteria (eg, lipoteichoic acid (LTA) and lipoarabinomannan from mycobacteria and lipomannans from M. tuberculosis). Some viral determinants can also be “triggered” through TLR2 (Barbalat R., Lau L., Locksley RM, Barton GM Toll-like receptor 2 on inflammatory monocytes induces type I interferon in response to viral but not bacterial ligands. Nat Immunol. 10( 11), 2009, pp. 1200-1207). Bacterial lipopeptides are structural components of cell membranes. They consist of an acylated s-glycerylcysteine residue to which the peptide can be conjugated via a cysteine residue. Examples of TLR2 agonists that are bacterial lipopeptides include MALP-2 and its synthetic analog dipalmitoyl-S-glycerylcysteine (Pam ₂ Cys) or tripalmitoyl-S-glycerylcysteine (Pam ₃ Cys).

A variety of ligands interact with TLR4, including monophosphoryl lipid A from Salmonella minnesota R595 (MPLA), lipopolysaccharides (LPS), mannans (Candida albicans), glycoinositol phospholipids (Trypanosoma), viral envelope proteins (RSV and MMTV), and endogenous antigens, including fibrinogen and heat shock proteins. These TLR4 agonists are described, for example, in Akira S., Uematsu S., Takeuchi O., Pathogen recognition and innate immunity. Cell. 124(4), 24 February 2006, cc. 783-801 or in Kumar H., Kawai T., Akira S., Toll-like receptors and innate immunity. Biochem Biophys Res Commun. 388(4), 30 October 2009, cc. 621-625. LPS, which is found in the outer membranes of Gram-negative bacteria, is the most widely studied of the TLR4 family of ligands. Suitable LPS-derived TLR4 peptide agonists are described, for example, in WO 2013/120073 (A1).

TLR5 is triggered by a region of the flagellin molecule expressed by virtually all motile bacteria. Thus, flagellin or peptides or proteins derived from flagellin and/or variants or fragments of flagellin are also useful as TLR peptide agonists included in the complex proposed for use according to the present invention.

Thus, examples of TLR peptide agonists include the TLR2 lipopeptide agonists MALP-2, Pam ₂ Cys and Pam ₃ Cys or modifications thereof, various forms of TLR4 LPS agonists, e.g. wild-type N. meningitidis L3-LPS and mutant pentaacylated LpxL1-LPS, and the TLR5 agonist flagellin.

However, it is preferred that the TLR peptide agonist included in the complex proposed for use according to the present invention is neither a lipopeptide, nor a lipoprotein, nor a glycopeptide, nor a glycoprotein, more preferably, the TLR peptide agonist included in the complex proposed for use according to the present invention invention is a classic peptide, polypeptide or protein as defined herein.

A preferred TLR2 agonist is annex II or an immunomodulatory fragment thereof, which is described in detail in WO 2012/048190 A1 and US patent application 13/0331546, particularly preferred is a TLR2 peptide agonist that contains the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 7 described in WO 2012/048190 A1, or fragments or variants thereof.

Thus, a TLR2 peptide agonist containing or consisting of the amino acid sequence of SEQ ID NO: 15 or a variant thereof described above is most preferred as component c), i.e. as at least one TLR peptide agonist included in the complex proposed for use according to the present invention.

SEQ ID NO: 15 (TLR2 peptide agonist Anaxa).

The most preferred functional variant of the TLR agonist peptide sequence having SEQ ID NO: 15 is the TLR agonist peptide sequence shown in SEQ ID NO: 71:

SEQ ID NO: 71

Thus, a TLR2 peptide agonist containing or consisting of the amino acid sequence SEQ ID NO: 71 or a variant thereof described above is most preferred as component c), i.e. as at least one TLR peptide agonist included in the complex. In other words, the TLR agonist peptide complex most preferably contains or consists of a peptide that has an amino acid sequence corresponding to SEQ ID NO: 71, or a functional sequence variant having at least 70% sequence identity (preferably at least 75% -th, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity).

Regarding TLR4, the most preferred peptide TLR agonists are agonists that correspond to motifs that bind to TLR4, in particular (i) peptides that mimic the naturally occurring ligand LPS (RS01: Gln-Glu-Ile-Asn-Ser-Ser- Tyr and RS09: Ala-Pro-Pro-His-Ala-Leu-Ser), and (II) fibronectin-derived peptides. The cellular glycoprotein fibronectin (FN) has multiple isoforms created from a single gene by alternative splicing of three exons. One of these isoforms is extra domain A (EDA), which interacts with TLR4.

Other suitable TLR peptide agonists contain the fibronectin EDA domain or a fragment or variant thereof. Said suitable fibronectin EDA domains or fragments or variants thereof are described in EP 1913954 B1, EP 2476440 A1, US 2009/0220532 A1 and WO 2011/101332 A1. Thus, a TLR4 peptide agonist containing or consisting of the amino acid sequence of SEQ ID NO: 45 or a variant thereof described above is most preferred as component c), i.e. as at least one TLR peptide agonist contained in the complex proposed for use according to the present invention.

SEQ ID NO: 45 (TLR4 EDA peptide agonist).

In addition, high mobility group protein 1 (HMG protein) (HMGB1) and its peptide fragments have been proposed to be agonists for TLR4. Said HMGB1-derived peptides are described, for example, in US 2011/0236406 A1.

In addition, the TLR agonist shown in SEQ ID NO: 15 and the TLR agonist shown in SEQ ID NO: 71 can act as a TLR4 agonist. Thus, a TLR4 peptide agonist containing or consisting of the amino acid sequence of SEQ ID NO: 15 or 71 or a functional variant thereof is most preferred as component c), i.e. as at least one TLR peptide agonist included in the complex.

The complex proposed for use according to the present invention contains at least one TLR peptide agonist, preferably the complex proposed for use according to the present invention contains more than one TLR peptide agonist, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TLR peptide agonists, more preferably the complex proposed for use according to the present invention contains (at least) two or three TLR peptide agonists, even more preferably the complex proposed for use according to the present invention contains ( at least) four or five TLR peptide agonists. If the complex proposed for use according to the present invention contains more than one TLR peptide agonist, then it should be obvious that the specified TLR peptide agonist is also, in particular, covalently linked in the complex proposed for use according to the present invention, for example, to another a TLR peptide agonist and/or with component a), i.e. cell-penetrating peptide, and/or with component b), i.e. antigen or antigenic epitope.

In the most preferred embodiment of the invention, the complex proposed for use according to the present invention contains a single TLR peptide agonist. In particular, in this most preferred embodiment of the invention, the complex proposed for use according to the present invention contains a single TLR agonist peptide and no additional TLR agonist component other than the single TLR peptide agonist specified above is present.

The various TLR peptide agonists included in the complex proposed for use according to the present invention may be the same or different. Preferably, the various TLR peptide agonists included in the complex proposed for use according to the present invention are different from each other.

It is further preferred that more than one antigen or antigenic epitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 antigens or antigenic epitopes, or more than one TLR peptide agonist, in particular 2, 3 4, 5, 6, 7, 8, 9, 10 TLR agonists were arranged sequentially in the complex proposed for use according to the present invention. This means, in particular, that all TLR peptide agonists included in the complex are located in the form of a region that is not interrupted by component a), i.e. cell-penetrating peptide, nor component b), i.e. at least one antigen or antigenic epitope. Preferably, component a) and component b) are located in a complex, for example, before or after the specified region of all TLR peptide agonists. However, TLR peptide agonists arranged in such a sequential manner can be linked to each other, for example, by a spacer or linker described below, which is neither component a), i.e. cell-penetrating peptide, nor component b), i.e. at least one antigen or antigenic epitope.

However, as an alternative to this, the various TLR peptide agonists can also be positioned in any other manner in the complex proposed for use according to the present invention, for example with component a) and/or component b) located between two or more TLR peptide agonists, i.e. e. with one or more TLR peptide agonists located between component a) and component b) (or vice versa) and optionally with one or more TLR peptide agonists located at the corresponding other end of component a) and/or component b).

It will be apparent that it may be appropriate for several different TLR peptide agonists, activating the same or different TLR receptors, to be included in a single complex for use in the present invention. Alternatively, several different peptide TLR agonists activating the same or different TLR receptors can be divided into subsets of different peptide TLR agonists activating the same or different TLR receptors, which are included in different complexes proposed in the present invention, wherein these different complexes containing different subsets are advantageously administered simultaneously, for example as a single vaccine, to an individual who requires it.

The combination of components a), b) and c) in a complex proposed for use according to the present invention

In the complex proposed for use according to the present invention, components a), b) and c) are covalently linked, i.e. the bond between two of the three components a), b) and c) in the complex proposed for use according to the present invention is a covalent bond. Preferably, two of the three components a), b) and c) of the complex proposed for use according to the present invention (i.e., the “first” and “second” components) are covalently linked to each other, and the third component of the three components a) , b) and c) is covalently linked either to the first component of the three components a), b) and c), or to the second component of the three components a), b) and c). A linear molecule is thereby preferably formed. However, it is also possible for each of the three components a), b) and c) to be covalently linked to both of the other three components a), b) and c).

The term “covalent compound” (also covalent bond) in the context of the present invention refers to a chemical bond that involves the overlap (“sharing”) of electron pairs between atoms. A "covalent compound" (also covalent bond) involves, in particular, a stable balance of attractive and repulsive forces between atoms when they share electrons. For many molecules, electron sharing allows each atom to achieve a full outer shell state corresponding to a stable electron configuration. Covalent bonding includes many types of interactions, including, for example, σ-bonding, π-bonding, metal-to-metal bonding, agonistic interactions, and three-center two-electron bonds. Thus, the complex proposed for use according to the present invention may also be referred to as a "compound", in particular, it may be referred to as a "molecule".

Preferably, in the complex proposed for use according to the present invention, components a), b) and c) are covalently linked by chemical coupling by any suitable means known in the art, for example, cross-linking methods. However, one should take into account the fact that many known chemical cross-linking methods are non-specific, i.e. they do not direct the combination point to any particular position on components a), b) and c). Thus, the nonspecific cross-linking agents used may attack functional sites or sterically block active sites, rendering the fused components of the complex biologically inactive. One skilled in the art will know how to block potentially reactive groups by using appropriate protecting groups. Alternatively, techniques can be used based on the strong and universal ligation of oxime and hydrazone, which are chemoselective substances suitable for cross-linking components a), b) and c). This coupling technology is described, for example, in Rose et al., JACS 116, 1994, p. thirty.

The specificity of the combination can be increased by direct chemical binding to a functional group that occurs only once or several times in components a), b) and/or c), while the functional group can be cross-linked with another present in components a), b ) or in). Thus, for example, a cysteine thiol group can be used if only one cysteine residue is present in a particular component a), b) or c) of the complex proposed for use according to the present invention. Thus, for example, if a particular component a), b) or c) does not contain lysine residues, then a cross-linking reagent specific for primary amines may be selective for the amino terminus of the corresponding component. Alternatively, cross-linking can also be carried out through the side chain of a glutamic acid residue located at the N-terminus of the peptide, thereby creating an amide bond through its side chain. Thus, it may be advantageous to link a glutamic acid residue to the N-terminus of a particular component a), b) or c). However, if a cysteine residue is to be introduced into a particular component a), b) or c), then introduction at or near the N- or C-terminus is preferred. Conventional methods are known for making such amino acid sequence changes based on modifications to a particular component a), b) or c), either by adding one or more additional amino acids, such as, but not limited to, a cysteine residue, to translocate the sequence, or by substituting at least at least one residue of the translocated sequence(s) included in the corresponding component. In the case where a cysteine side chain is used for coupling purposes, the particular component a), b) or c) preferably has one cysteine residue. Any second cysteine residue is preferably not required and can optionally be replaced when present in the appropriate component that is included in the complex proposed for use according to the present invention. When a cysteine residue is replaced in the original sequence of a particular component a), b) or c), then, as a rule, it is necessary to minimize the resulting changes in the folding of the peptide of the corresponding component. Changes in folding are minimized when the substitution is a chemically and sterically similar substitution to a cysteine. Thus, series is the preferred replacement for cysteine.

The combination of two of the three components a), b) and c) can be accomplished by a coupling or conjugating agent using standard coupling reagents for peptide synthesis such as HOBt, HBTU, DICI, TBTU. Several intermolecular cross-linking agents are known that can be used, see, for example, Means and Feeney, Chemical Modification of Proteins, Holden-Day, 1974, pp. 39-43. Notable among these reagents are, for example, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) or N,N'-(1,3-phenylene)bismaleimide; N,N'-ethylene-bis(iodoacetamide) or other such reagent having methylene bridges containing 6-11 carbon atoms; and 1,5-difluoro-2,4-dinitrobenzene. Other cross-linking agents that can be used for this purpose include: n,n'-difluoro-m,m'-dinitrodiphenylsulfone; dimethyladipimidate; phenol-1,4-disulfonyl chloride; hexamethylene diisocyanate or diisothiocyanate or azophenyl-n-diisocyanate; glutaraldehyde and disdiazobenzidine. Cross-linking agents can be homobifunctional, i.e. have two functional groups that undergo the same reaction. A preferred homobifunctional cross-linking agent is bismaleimidohexane (BMH). BMN contains two maleimide functional groups that interact specifically with sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7). The two maleimide groups are connected by a hydrocarbon chain. Thus, BMN can be used to irreversibly cross-link proteins (or polypeptides) that contain cysteine residues. Cross-linking agents can also be heterobifunctional. Heterobifunctional cross-linking agents have two different functional groups, for example, an amino-reactive group and a thiol-reactive group, which can cross-link two proteins having free amines and thiols, respectively. Examples of heterobifunctional cross-linkers are succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS) and succinimide-4-(n-maleimidophenyl)butyrate (SMPB) and analogue with extended chain MBS. The succinimidyl group of these cross-linking agents reacts with the primary amine, the thiol-reactive maleimide forms a covalent bond with the thiol of the cysteine residue. Since cross-linking agents often have low water solubility, a hydrophilic moiety, such as a sulfonate group, can be added to the cross-linking agent to increase its solubility in water. Sulfo-MBS and sulfo-SMCC are examples of cross-linking agents modified for water solubility. Many cross-linking agents form a conjugate that is virtually indestructible under cellular conditions. Thus, some cross-linking agents contain a covalent bond, such as a disulfide, which can be cleaved under cellular conditions. For example, Trout's reagent, dithiobis(succinimidylpropionate) (DSP) and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) are well-known cleavable cross-linking agents. The use of a cleavable cross-linking agent allows the cell-penetrating peptide, at least one antigen or antigenic epitope and at least one TLR peptide agonist that form the complex proposed for use according to the present invention to be separated from each other after they are delivered to the target cell. A direct disulfide bond can also be used for this purpose. Chemical cross-linking may also include the use of spacer arms. Spacer arms provide intramolecular flexibility or allow adjustment of intramolecular distances between conjugated moieties and may therefore help maintain biological activity. The spacer arm may be in the form of a protein (or polypeptide) fragment that includes spacer amino acids, for example, proline. Alternatively, the spacer arm may be part of a cross-linking agent such as "long chain SPDP" (Pierce Chem. Co., Rockford, Ill., catalog no. 21651 H). Numerous cross-linking agents, including those described above, are commercially available. Detailed instructions for their use are available from commercial suppliers. More detailed information on the cross-linking of proteins and the preparation of conjugates that can be used in the context of binding components a), b) and c) forming the complex proposed for use according to the present invention can be gleaned from: Wong, Chemistry of Protein Conjugation and Cross-linking Linking, CRC Press, 1991.

Cross-linking agents for cross-linking a peptide or protein include, for example, (i) amine-amine cross-linking agents, for example, homobifunctional amine-specific protein cross-linking reagents based on NHS ester and imido ester reactive groups for selective conjugation of primary amines; available as short, long, cleavable, irreversible, membrane-permeable and cell surface variants; (II) sulfhydryl-carbohydrate cross-linking agents, for example, maleimide and hydrazide reactive group cross-linking agents for conjugation and covalent cross-linking; (III) sulfhydryl-sulfhydryl cross-linking agents, for example, homobifunctional sulfhydryl-specific cross-linking reagents based on maleimide or pyridyl dithiol reactive groups for selective covalent conjugation of protein and peptide thiols (reduced cysteines) to form stable thioether bonds; (IV) photoreactive cross-linkers, for example, arylazide, diazirine and other photoreactive (light-activated) chemical heterobifunctional cross-linkers for conjugation of proteins, nucleic acids and other molecular structures involved in the formation of receptor-ligand complexes through two-step activation; (V) amine-sulfhydryl cross-linking agents, for example, heterobifunctional protein cross-linking agents for conjugation between the primary amine (lysine) and sulfhydryl (cysteine) groups of proteins and other molecules; available with spacer arms of various lengths and types; and (VI) carboxyl-amine cross-linking agents, such as carbodiimide, DCC, and EDC cross-linking reagents (EDAC), to conjugate carboxyl groups (glutamate, aspartate, C-termini) to primary amines (lysine, N -ends), as well as N-hydroxysuccinimide (NHS) to stably activate carboxylates for amine conjugation.

Examples of cross-linking agents that can be used in the complex proposed for use according to the present invention include N-(α-maleimidoacetoxy)succinimidyl ester, N-5-azido-2-nitrobenzyloxysuccinimide, 1,4-bismaleimidobutane, 1,4-bismaleimidyl -2,3-dihydroxybutane, bismaleimidohexane, bismaleimidoethane, N-(β-maleimidopropionic acid)×TPA hydrazide, N-(β-maleimidopropyloxy)succinimidyl ester, 1,8-bismaleimidodiethylene glycol, 1,11-bismaleimidotriethylene glycol, bis(sulfosuccinimidyl) suberate, bis(sulfosuccinimidyl)glutarate-d0, bis(sulfosuccinimidyl)-2,2,4,4-glutarate-d4, bis(sulfosuccinimidyl)suberate-d0, bis(sulfosuccinimidyl)-2,2,7,7-suberate- d4, bis(NHS)PEG5, bis(NHS)PEG9, bis(2-[succinimidoxycarbonyloxy]ethyl)sulfone, N,N-dicyclohexylcarbodiimide, 1-5-difluoro-2,4-dinitrobenzene, dimethyladipimidate×HCI, dimethylpimelimidate×2HCI , dimethyl suberimidate×2HCl, disuccinimidyl glutarate, dithiobis(succimidylpropionate) (Lomant's reagent), disuccinimidylsuberate, disuccinimidyl tartrate, dimethyl-3,3'-dithiobispropionimidate×2HCI, dithiobismaleimidoethane, 3,3'-dithiobis(sulfosuccinimidylpropionate), hydrochloride 1-eth IL-3- (3-dimethylaminopropyl)carbodiimide, ethylene glycol bis(succinimidyl succinate), N-ε-maleimidocaproic acid, N-(ε-maleimidocaproic acid hydrazide), N-(ε-maleimidocaproyloxy)succinimidyl ester, N-(γ-maleimidobutyryloxy)succinimidyl ester, N-(κ-maleimidoundecanoic acid) hydrazide, NHS-LC-diazirine, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy(6-amidocaproate), succinimidyl-6-(3'-[2-pyridylthio ]propionamido)hexanoate, L-photo-leucine, L-photo-methionine, m-maleimidobenzoyl-N-hydroxysuccinimidyl ester, 4-(4-N-maleimidophenyl)butyric acid hydrazide×HCI, 2-[N2-(4- azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-L-lysinyl]ethylmethanethiosulfate, 2-{N2-[N6-(4-azido-2,3,5,6-tetrafluorobenzoyl) -N6-(6-biotinamidocaproyl)-L-lysinyl]}ethylmethanethiosulfate, N-hydroxysuccinimide, ethanazide N-hydroxysuccinimidyl ester, tetraoxapentadecanazide N-hydroxysuccinimidyl ester, dodecaoxanonatriacontanazide N-hydroxysuccinimidyl ester, NHS-phosphine, 3-(2- pyridylthio)propionylhydrazide, 2-pyridyldithioltetraoxatetradecane-N-hydroxysuccinimide, 2-pyridyldithioltetraoxaoctatriacontane-N-hydroxysuccinimide, N-(n-maleimidophenyl)isocyanate, succinimidyl-3-(bromoacetamido)propionate, NHS-diazirine, NHS-SS-diazirine, N- succinimidyl iodoacetate, N-succinimidyl (4-iodoacetyl) aminobenzoate, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, NHS-PEG2-maleimide, NHS-PEG4-maleimide, NHS-PEG6-maleimide, NHS-PEG8-maleimide, NHS-PEG12-maleimide, NHS-PEG24-maleimide, succinimidyl-4-(n-maleimidophenyl)butyrate, succinimidyl-6-(β-maleimidopropionamido)hexanoate, 4-succinimidyloxycarbonylmethyl-α-(2-pyridyldithio)toluene, succinimidyl-( 4-psoralen-8-yloxy)butyrate, N-succinimidyl-3-(2-pyridyldithio)propionate, ethylene glycol bis(sulfosuccinimidyl succinate), N-(ε-maleimidocaproyloxy)sulfosuccinimidyl ester, N-(γ-maleimidobutyloxy)sulfosuccinimidyl ester, N-(κ-maleimidoundecanoyloxy)sulfosuccinimidyl ester, sulfo-NHS-LC-diazirine, sulfosuccinimidyl-6-(3'-[2-pyridyldithio]propionamido)hexanoate, m-maleimidobenzoyl-N-hydroxysulfosuccinimidyl ester, N- hydroxysuccinimide, sulfo-NHS-phosphine, sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate, sulfo-NHS-(2-6-[biotinamido]-2-(n-azidobenzamide), sulfo-NHS- diazirine, sulfo-NHS-SS-diazirine, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, sulfosuccinimidyl-4-(n-maleimidophenyl)butyrate, tris(2-maleimidoethyl) amine (trifunctional) and tris(succinimidylaminotriacetate) (trifunctional).

The connection between two of the three components a), b) and c) in the complex proposed for use according to the present invention can be direct or indirect, i.e. the two components may be linked directly, or they may be linked by an additional component of the complex, such as a spacer or linker.

Preferably, the implementation of the direct linkage is an amide bridge if the components to be linked have reactive amino groups or carboxy groups. More specifically, if the components to be linked are peptides, polypeptides or proteins, then a peptide bond is preferred. Said peptide bond may be created by chemical synthesis involving both components to be linked (the N-terminus of one component and the C-terminus of the other component), or it may be created directly by protein synthesis of the complete peptide sequence of both components, both (protein or peptide) component is preferably synthesized in one step. Said protein synthesis methods include, but are not limited to, for example, liquid phase peptide synthesis methods or solid phase peptide synthesis methods, for example, Merrifield solid phase peptide synthesis methods, solid phase peptide synthesis using t-Boc, solid phase peptide synthesis using Fmoc, solid-phase peptide synthesis based on BOP (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), etc. Alternatively, ester or ether linkages are preferred.

In addition, if, in particular, the components to be linked are peptides, polypeptides or proteins, the linkage can be carried out via side chains, for example by means of a disulfide bridge. Additional components of a different chemical nature, for example, at least one antigen or antigenic epitope, if not of a peptide nature, can also be attached to components of a peptide nature, for example, a cell penetrating peptide, at least one TLR peptide agonist and at least at least one antigen or antigenic epitope, if it is of a peptide nature. The side chain linkage should preferably be based on amino groups, thiol or hydroxyl groups of the side chain, for example an amide or ester or ether linkage. The linkage of a main peptide chain to a side peptide chain of another component may also be an isopeptide bond. An isopeptide bond is an amide bond that is not present in the main chain of a protein. A bond is formed between the carboxyl terminus of one peptide or protein and the amino group of a lysine residue on another peptide or protein (target).

The complex proposed for use according to the present invention may optionally contain a spacer or linker, which are non-immunological fragments, which are preferably cleavable and which connect component a) and b) and/or component a) and c), and/or component b) and c), and/or connect sequentially located antigens or antigenic epitopes, and/or connect sequentially located peptide TLR agonists, and/or connect sequentially located cell penetrating peptides, and/or which can be placed in the C-terminal region of components b ) and/or c). The linker or spacer may preferably have additional functionality beyond the ability to link components, preferably being cleavable, more preferably cleavable naturally within the target cell, for example, cleavable by enzymes. However, said other functionality shall not include, in particular, immunological functionality. Examples of additional functionality, particularly regarding linkers in fusion proteins, are presented in Chen X. et al.: Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10), 2013, cc. 1357-1369, which also describe, for example, in vivo cleavable linkers. Also, in Chen X. et al., Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10), 2013, cc. 1357-1369 also describe various linkers, for example, flexible linkers and rigid linkers, and tools and databases for creating linkers that can be used in the complex proposed for use according to the present invention, or for creating a linker that can be used in the complex proposed for use according to the present invention,

Said spacer may be peptide or non-peptide, preferably the spacer is peptide. Preferably, the peptide spacer consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, more preferably about 1, 2, 3, 4 or 5 amino acids. The amino acid sequence of the peptide spacer may be identical to the sequence of the N-terminal or C-terminal flanking region of any of components a), b) or c). Alternatively, the peptide spacer may consist of non-naturally occurring amino acid sequences, such as an amino acid sequence resulting from conservative amino acid substitutions of the naturally occurring flanking regions, or sequences of known protease cleavage sites, such as an enterokinase target site ( amino acid sequence: DDDK, SEQ ID NO: 16), factor Xa target site (amino acid sequence: IEDGR, SEQ ID NO: 17), thrombin target site (amino acid sequence: LVPRGS, SEQ ID NO: 18), site -target site for TEV protease (amino acid sequence: ENLYFQG, SEQ ID NO: 19), target site for PreScission protease (amino acid sequence LEVLFQGP, SEQ ID NO: 20), polycationic amino acids, e.g. target site for polyK, furin (amino acid sequence RX(R/K)R, SEQ ID NO: 21). In a particular embodiment of the invention, the peptide spacer should not contain any Cys (C) residues. In a preferred embodiment of the invention, the linker sequence contains at least 20%, more preferably at least 40%, and even more preferably at least 50% Gly or β-alanine residues, for example, GlyGlyGlyGlyGly (SEQ ID NO: 22), GlyGlyGlyGly ( SEQ ID NO: 23), GlyGlyGly, CysGlyGly or GlyGlyCys, etc. One skilled in the art can easily select and obtain suitable linker sequences. They may consist of D- and/or L-amino acids. Other examples of a peptide spacer include the amino acid sequences EQLE (SEQ ID NO: 24) or TEWT (SEQ ID NO: 25), or any conservative substitutions thereof.

The non-peptide spacer may include or may be an ester, a thioester, and a disulfide.

In particular, the complex proposed for use according to the invention may contain a spacer or linker, in particular a peptide spacer located between components a) and b) and/or between components a) and c), and/or between components b) and V). One skilled in the art may select a peptide spacer such that it can be cleaved by a cell-intrinsic mechanism following internalization of a complex containing a cell penetrating peptide and a cargo molecule.

When the complex contains multiple antigens or antigenic epitopes, or when the complex contains multiple TLR peptide agonists, it will be apparent to one skilled in the art that each of the antigens or antigenic epitopes and/or each of the TLR peptide agonists included in the complex of the invention , can either be directly connected to each other or connected through spacers or linkers, such as a peptide spacer consisting of several amino acids. Alternatively, when the complex proposed for use according to the present invention contains several antigens or antigenic epitopes, or when the complex contains several TLR peptide agonists, it is also possible that some antigens or antigenic epitopes and/or some TLR peptide agonists included in the complex , proposed in the invention, are directly linked to each other, and some other antigens or antigenic epitopes and/or some other TLR peptide agonists are linked through spacers or linkers, such as a peptide spacer consisting of several amino acids.

For example, two sequential antigens or antigenic epitopes or two sequential TLR peptide agonists that form part of the complex of the invention are connected to each other by spacers consisting of naturally occurring flanking regions of said antigens or antigenic epitopes, or TLR peptide agonists, respectively. For example, a spacer used to connect a first antigen/antigenic epitope or a first TLR peptide agonist to a second antigen/antigenic epitope or a second TLR peptide agonist, respectively, may consist of up to about 8 amino acids, corresponding to up to about 4 amino acids N-terminal or A C-terminal flanking region of a first antigen/antigenic epitope or a first TLR peptide agonist, followed by up to about 4 amino acids of an N-terminal or C-terminal flanking region of a second antigen/antigenic epitope or a second TLR peptide agonist. In the present invention, by way of illustration, a spacer used to connect a first antigen/antigenic epitope or a first TLR peptide agonist (“TLR 1 antigen/epitope/peptide agonist”) to a second epitope (“TLR 2 antigen/epitope/peptide agonist” consists of approximately 8 amino acids corresponding to any possible combination of the following: 0 flanking amino acids of the TLR 1 antigen/epitope/peptide agonist and 8 flanking amino acids of the TLR 2 antigen/epitope/peptide agonist, to 8 flanking amino acids of the TLR 1 antigen/epitope/peptide agonist and up to 0 flanking amino acids of the TLR 2 antigen/epitope/peptide agonist, i.e. including 1 flanking amino acid of the TLR 1 antigen/epitope/peptide agonist and 7 flanking amino acids of the TLR 2 antigen/epitope/peptide agonist, 2 flanking amino acids of the TLR 1 antigen/epitope/peptide agonist and 6 flanking amino acids of TLR 2 antigen/epitope/peptide agonist, 3 flanking amino acids of TLR 1 antigen/epitope/peptide agonist and 5 flanking amino acids of TLR 2 antigen/epitope/peptide agonist, 4 flanking amino acids of TLR 1 and 4 antigen/epitope/peptide agonist flanking amino acids of antigen/epitope/peptide agonist TLR 2, 5 flanking amino acids of antigen/epitope/peptide agonist TLR 1 and 3 flanking amino acids of antigen/epitope/peptide agonist TLR 2, 6 flanking amino acids of antigen/epitope/peptide agonist TLR 1 and 2 flanking amino acids antigen/epitope/peptide agonist TLR 2, 7 flanking amino acids of antigen/epitope/peptide agonist TLR 1 and 1 flanking amino acid of antigen/epitope/peptide agonist TLR 2, 8 flanking amino acids of antigen/epitope/peptide agonist TLR 1 and 0 flanking amino acids of antigen/ TLR epitope/peptide agonist 2. It should be obvious that the total of 8 amino acids forming the spacer that connects two consecutive TLR antigens/epitopes/peptide agonists is not an absolute value, and the spacer may consist of a total of, for example, 3 amino acids , 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 9 amino acids or 10 amino acids. Likewise, combinations equivalent to those above may also illustrate the invention in the situation where the spacer consists of less than 8 amino acids or more than 8 amino acids.

According to another specific illustration of the present invention, a spacer used to connect a first antigen/antigenic epitope or a first TLR peptide agonist (“TLR 1 antigen/epitope/peptide agonist”) to a second antigen/antigenic epitope or a second TLR peptide agonist, respectively (“antigen/ epitope/peptide agonist TLR 2"), consists of, for example, 1, 2, 3, 4 or 5 amino acids. More specifically, said spacer amino acid sequence may correspond to the 4 amino acids of the N-terminal or C-terminal flanking region of a TLR 1 antigen/epitope/peptide agonist or a TLR 2 antigen/epitope/peptide agonist. The spacer described above may also be located in the C region the last TLR antigen/epitope/peptide agonist included in the complex proposed for use according to the present invention.

Methods for combining two of the three components a), b) and c) are described in detail in the literature, and they may depend on the nature of at least one antigen or antigenic epitope. For example, the coupling of two of the three components a), b) and c) can be achieved through cleavable disulfide bonds obtained generally by stepwise solid-phase synthesis or combination of solution-phase or solid-phase moieties, stable amide, thiazolidine, oxime and hydrazine linkages , disulfide bond, stable thiomaleimide bond, peptide bond (including peptide bonds between amino acids of the fusion protein), or electrostatic or hydrophobic interactions.

Preferably, at least one antigen or antigenic epitope included in the complex proposed for use according to the present invention, as well as any optional spacer or linker included in the complex proposed for use according to the present invention, are peptide in nature. More preferably, all components of the complex proposed for use according to the present invention, for example, a cell penetrating peptide, at least one antigen or antigenic epitope that is a peptide, polypeptide or protein, at least one peptide TLR agonist and an optional peptide linker or spacer , are connected in the complex proposed for use according to the present invention by a peptide bond. Thus, most preferably, the complex proposed for use according to the present invention is a peptide, polypeptide or protein, such as a fusion protein, for example a recombinant fusion protein.

In this context, a complex that contains or consists of the amino acid sequence SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 46, SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87; or a complex that contains or consists of an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical, at least 75% identical, at least 80% identical 85%, at least 90%, at least 95%, or at least 98% of any of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33 , SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 46, SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO : 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87 is preferred. Additionally, a complex that contains or consists of the amino acid sequence SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 46, SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91; or a complex that contains or consists of an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical, at least 75% identical, at least 80% identical 85%, at least 90%, at least 95%, or at least 98% of any of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33 , SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 46, SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO : 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88 SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91 is preferred. Additionally, a complex that contains or consists of the amino acid sequence SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 46, or SEQ ID NO: 69; or a complex that contains or consists of an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical, at least 75% identical, at least 80% identical 85%, at least 90%, at least 95%, or at least 98% of any of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33 , SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 46 or SEQ ID NO: 69, is preferred. A complex that contains or consists of the amino acid sequence SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69 or a complex that contains or consists of an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical, at least 75% identical, 80%, at least 85%, at least 90%, at least 95%, or at least 98% of any of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33 SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69 is preferred. A complex that contains or consists of the amino acid sequence SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 69 , or a complex that contains or consists of an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85%, at least 90%, at least 95%, or at least 98% of any of the sequences of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69 is preferred. A complex that contains or consists of the amino acid sequence SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 69 , or a complex that contains or consists of an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85%, at least 90%, at least 95%, or at least 98% of any of the sequences of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69 is more preferred. Additionally, a complex that contains or consists of the amino acid sequence SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87, or a complex that contains or consists of an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical, at least 75% identical, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of any of the sequences of SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO : 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82 SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87 is more preferred. Additionally, a complex that contains or consists of the amino acid sequence SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91, or a complex that contains or consists of an amino acid sequence that is at least 50% identical , at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% of any of the sequences of SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78 , SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91 is more preferred. A complex that contains or consists of the amino acid sequence of SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69, or a complex that contains or consists of an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85% identical to at least 90%, at least 95%, or at least 98% of any of the sequences of SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69 is even more preferred. A complex that contains or consists of the amino acid sequence of SEQ ID NO: 28, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69, or a complex that contains or consists of an amino acid sequence, identical by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% of SEQ ID NO: 28, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69 is even more preferred. A complex that contains or consists of the amino acid sequence of SEQ ID NO: 28, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69, or a complex that contains or consists of an amino acid sequence, identical by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% of SEQ ID NO: 28, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69 is particularly preferred. Most preferably, the complex proposed for use according to the present invention contains or consists of the amino acid sequence of SEQ ID NO: 72 or an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical to at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% SEQ ID NO: 72. Most preferably, a complex proposed for use according to the present invention contains or consists of the amino acid sequence of SEQ ID NO: 73 or of an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% SEQ ID NO: 73. Most preferably the complex proposed for use according to of the present invention, contains or consists of an amino acid sequence of SEQ ID NO: 74 or an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical, at least 75% identical, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% SEQ ID NO: 74. Most preferably, the complex proposed for use according to the present invention contains or consists of an amino acid sequence of SEQ ID NO: 75 or an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical, at least 75% identical, or at least 80% identical , at least 85%, at least 90%, at least 95%, or at least 98% SEQ ID NO: 75.

Most preferably, the complex proposed for use according to the present invention contains or consists of the amino acid sequence of SEQ ID NO: 76 or an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical to at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% SEQ ID NO: 76. Most preferably, a complex proposed for use according to the present invention contains or consists of an amino acid sequence of SEQ ID NO: 77 or an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% SEQ ID NO: 77.

Most preferably, the complex proposed for use according to the present invention contains or consists of the amino acid sequence of SEQ ID NO: 78 or an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical to at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% SEQ ID NO: 78. Most preferably, a complex proposed for use according to the present invention contains or consists of an amino acid sequence of SEQ ID NO: 79 or an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% SEQ ID NO: 79.

Most preferably, the complex proposed for use according to the present invention contains or consists of the amino acid sequence of SEQ ID NO: 80 or an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical to at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% SEQ ID NO: 80. Most preferably, a complex proposed for use according to the present invention contains or consists of an amino acid sequence of SEQ ID NO: 81 or an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% SEQ ID NO: 81.

Most preferably, the complex proposed for use according to the present invention contains or consists of the amino acid sequence of SEQ ID NO: 82 or an amino acid sequence that is at least 50% identical, at least 60%, at least 70% identical to at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% SEQ ID NO: 82.

Most preferably, the complex proposed for use according to the present invention contains or consists of the amino acid sequence of SEQ ID NO: 83 or an amino acid sequence that is at least 50% identical, at least 60%, at least 70% identical to at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% SEQ ID NO: 83. Most preferably, a complex proposed for use according to the present invention contains or consists of the amino acid sequence of SEQ ID NO: 84 or of an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% SEQ ID NO: 84.

Most preferably, the complex proposed for use according to the present invention contains or consists of the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical to at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% SEQ ID NO: 85. Most preferably, a complex proposed for use according to the present invention contains or consists of an amino acid sequence of SEQ ID NO: 86 or an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% SEQ ID NO: 86. Most preferably the complex proposed for use according to of the present invention, contains or consists of an amino acid sequence of SEQ ID NO: 87 or an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical, at least 75% identical, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% SEQ ID NO: 87. Most preferably, the complex proposed for use according to the present invention contains or consists of an amino acid sequence of SEQ ID NO: 88 or an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical, at least 75% identical, or at least 80% identical , at least 85%, at least 90%, at least 95%, or at least 98% SEQ ID NO: 88.

Most preferably, the complex proposed for use according to the present invention contains or consists of the amino acid sequence of SEQ ID NO: 89 or an amino acid sequence that is at least 50% identical, at least 60%, at least 70% identical to at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% SEQ ID NO: 89. Most preferably, a complex proposed for use according to the present invention, contains or consists of an amino acid sequence of SEQ ID NO: 90 or an amino acid sequence that is at least 50% identical, at least 60% identical, at least 70% identical, or at least 75 identical %, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% SEQ ID NO: 90.

Most preferably, the complex proposed for use according to the present invention contains or consists of the amino acid sequence of SEQ ID NO: 91 or an amino acid sequence that is at least 50% identical, at least 60%, at least 70% identical to at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% SEQ ID NO: 91.

Most preferably, the complex proposed for use according to the present invention contains or consists of a polypeptide that has an amino acid sequence corresponding to SEQ ID NO: 89, or a functional sequence variant having at least 50% sequence identity (preferably at least 60 %, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, particularly preferably at least 95%, and most preferably at least 98% or 99% sequence identity).

SEQ ID NO: 26:

SEQ ID NO: 27:

SEQ ID NO: 28:

SEQ ID NO: 33:

SEQ ID NO: 34:

SEQ ID NO: 37:

SEQ ID NO: 38:

SEQ ID NO: 39:

SEQ ID NO: 40:

SEQ ID NO: 41:

SEQ ID NO:46:

SEQ ID NO: 69:

SEQ ID NO: 72:

SEQ ID NO: 73:

SEQ ID NO: 74:

SEQ ID NO: 75:

SEQ ID NO: 76:

SEQ ID NO: 77:

SEQ ID NO: 78:

SEQ ID NO: 79:

SEQ ID NO: 80:

SEQ ID NO: 81:

SEQ ID NO: 82:

SEQ ID NO:83:

SEQ ID NO:84:

SEQ ID NO: 85:

SEQ ID NO: 86:

SEQ ID NO: 87:

SEQ ID NO:88:

SEQ ID NO: 89:

SEQ ID NO: 90:

SEQ ID NO:91:

Most preferably, the complex proposed for use according to the present invention contains (or consists of) the following components:

(I) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 6 (CPP3/Z13), or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably to at least 80%, even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity );

(II) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 96, or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80% - identical, even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity);

(III) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 95, or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80% - identical, even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity);

(IV) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 97, or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80% - identical, even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity); And

(V) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 71 or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80% , even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity). Preferably the complex contains components (I) - (V) (described above) in the direction from the N-terminus to the C-terminus. However, the components may be arranged in other ways, as will be described below.

Another object of the present invention is also a complex containing:

cell-penetrating peptide;

at least three antigenic epitopes; And

at least one TLR peptide agonist,

in which components a) - c) are covalently bonded and

wherein at least three antigenic epitopes comprise (I) one or more survivin epitopes or functional sequence variant(s), (II) one or more CEA epitopes or functional sequence variant(s). her), and (III) one or more ASCL2 epitopes or functional sequence variant(s).

Preferred variants of the specified complex proposed in the present invention correspond to the preferred variants of the complex proposed for use according to the present invention, which is described above.

For example, the complex of the present invention preferably contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 52, or a fragment thereof of at least 10 amino acids, or a functional sequence variant having at least 70% sequence identity as above. More preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 53. Even more preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 95, or a functional sequence variant having at least 70% sequence identity as above.

For example, the complex of the present invention preferably contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 54, or a fragment thereof of at least 10 amino acids, or a functional sequence variant having at least 70% sequence identity as above. More preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 55, and/or a peptide that has an amino acid sequence corresponding to SEQ ID NO: 56. Even more preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 96, or a functional sequence variant having at least 70% sequence identity as defined above.

For example, the complex of the present invention preferably contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 92, or a fragment thereof of at least 10 amino acids, or a functional sequence variant having at least 70% sequence identity as above. More preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 93, and/or a peptide that has an amino acid sequence corresponding to SEQ ID NO: 94. Even more preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 97, or a functional sequence variant having at least 70% sequence identity as defined above.

Preferably the complex contains, in the direction from N-terminus to C-terminus:

one or more CEA epitopes or functional sequence variants as defined above;

one or more survivin epitopes or functional sequence variants as defined above; And

one or more ASCL2 epitopes or functional sequence variants as defined above.

More preferably, the complex contains, in the direction from N-terminus to C-terminus:

- a peptide that has an amino acid sequence corresponding to SEQ ID NO: 54, or a fragment thereof consisting of at least 10 amino acids as defined above, or a functional sequence variant having at least 70% sequence identity as defined above ;

- a peptide that has an amino acid sequence corresponding to SEQ ID NO: 52, or a fragment thereof consisting of at least 10 amino acids as defined above, or a functional sequence variant having at least 70% sequence identity as defined above ; And

- a peptide that has an amino acid sequence corresponding to SEQ ID NO: 92, or a fragment thereof consisting of at least 10 amino acids as defined above, or a functional sequence variant having at least 70% sequence identity as defined above ;

Even more preferably, the C-terminus (I) of a peptide that has the amino acid sequence corresponding to SEQ ID NO: 54, or a fragment or variant thereof as defined above, is directly linked to the N-terminus (II) of the peptide specified above that has the amino acid sequence corresponding to SEQ ID NO: 52, or a fragment or variant thereof specified above; and the C-terminus (II) of a peptide that has an amino acid sequence corresponding to SEQ ID NO: 52, or a fragment or variant thereof as defined above, is directly linked to the N-terminus (III) of a peptide that has an amino acid sequence corresponding to SEQ ID NO: : 92, or a fragment or variant thereof indicated above.

Preferably, the complex also contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 96, or a functional sequence variant having at least 70% sequence identity as stated above; a peptide that has an amino acid sequence corresponding to SEQ ID NO: 95, or a functional sequence variant having at least 70% sequence identity as defined above; and a peptide that has an amino acid sequence corresponding to SEQ ID NO: 97, or a functional sequence variant having at least 70% sequence identity as defined above.

Most preferably, the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 98, or a functional sequence variant having at least 70% sequence identity as stated above.

Preferably the complex is a recombinant polypeptide or recombinant protein as described above.

In addition, the cell penetrating peptide in the complex proposed in the present invention preferably corresponds to the cell penetrating peptide in the complex proposed for use according to the present invention mentioned above. This applies in particular to preferred embodiments of the cell penetrating peptide.

In addition, the TLR peptide agonist in the complex proposed in the present invention preferably corresponds to the TLR peptide agonist in the complex proposed for use according to the present invention mentioned above. This applies in particular to preferred TLR agonist peptide embodiments.

For example, the complex proposed in the present invention contains, preferably in the direction from the N-terminus to the C-terminus, the following components as described above:

(I) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 6 (CPP3/Z13), or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably to at least 80%, even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity );

(II) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 96, or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80% - identical, even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity);

(III) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 95, or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80% - identical, even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity);

(IV) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 97, or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80% - identical, even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity); And

(V) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 71, or a functional sequence variant having at least 70% sequence identity (preferably at least 75%, more preferably at least 80% - identical, even more preferably at least 85%, even more preferably at least 90%, especially preferably at least 95%, most preferably at least 99% sequence identity), in which the components (I)-(V) are optionally linked by a linker or spacer.

Most preferably, the complex contains or consists of a polypeptide that has an amino acid sequence corresponding to SEQ ID NO: 89, or a functional variant of this sequence having at least 50% sequence identity (preferably at least 60%, more preferably to at least 70%, more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, particularly preferably at least 95%, most preferably at least 99% sequence identity).

Preferably, the complex proposed in the present invention can be used for the prevention and/or treatment of colorectal cancer specified in the present description.

Location of components a), b) and c) in the complex proposed for use according to the present invention

Components a), b) and c) can be located in the complex proposed for use according to the present invention, in any order.

In particular, if more than one cell penetrating peptide and/or more than one antigen or antigenic epitope, and/or more than one TLR peptide agonist are contained in a complex proposed for use according to the present invention, the multiple cell penetrating peptides may not be arranged in a sequential manner. , i.e. at least one antigen or antigenic epitope (component b)) and/or at least one TLR peptide agonist (component c)) may interrupt a region of sequential cell-penetrating peptides and/or cell-penetrating peptides may alternate with component b ) and/or component c). Likewise, multiple antigens or antigenic epitopes may not be located in a sequential manner, i.e. at least one cell penetrating peptide (component a)) and/or at least one TLR peptide agonist (component b)) may interrupt a region of consecutive antigens or antigenic epitopes and/or antigens or antigenic epitopes may alternate with component a ) and/or component c). Likewise, multiple TLR agonist peptides may not necessarily be arranged in a sequential manner, i.e. at least one cell penetrating peptide (component a)) and/or at least one antigen or antigenic epitope (component b)) may interrupt a stretch of consecutive TLR peptide agonists and/or TLR peptide agonists may alternate with component a) and/or component b).

However, it is preferable that several cell penetrating peptides are located in a complex proposed for use according to the present invention in a sequential manner and/or several antigens or antigenic epitopes are located in a complex proposed for use according to the present invention in a sequential manner and/or several peptide agonists The TLRs were located in the complex proposed for use according to the present invention in a sequential manner. This means, in particular, that all single elements of a certain component, i.e. all cell-penetrating peptides, all antigens or antigenic epitopes, or all TLR peptide agonists that are contained in the complex are arranged in a region that is not interrupted by either of the other two components. More preferably, the other two components are located in a complex, for example, before or after the specified area of all the unit elements of the specified specific component. However, successive units of a particular component may be linked to each other, for example by a spacer or linker as defined herein, in a manner that is not the case for the other two components.

Most preferably, each of components a), b) and c) are arranged in a sequential manner.

Structurally, each component a), b) and c) typically contains one main chain and at least one side chain. The term "main chain" (synonymous with "framework chain") in the context of the present invention refers to the main continuous chain of covalently bonded atoms in a molecule. For example, in peptides, polypeptides and proteins, the main chain (backbone) typically contains alpha carbon atoms and nitrogen atoms, forming amino acids linked by a peptide bond. The framework does not include side chains. The term "side chain" (synonymous with "dangling chain") in the context of the present invention refers to a chemical group attached to the core part of the molecule, called the "main chain" or framework. For example, in peptides, polypeptides and proteins, the side chains are typically the (backbone) amino acid-forming parts that are attached to the alpha carbon atoms of the backbone.

In the complex proposed for use according to the present invention, components a), b) and c) can be covalently linked through a linker or spacer specified in the present description, or they can be directly covalently linked. Regardless of whether a spacer or a linker is used for covalent coupling, there are in principle four possible types of connection to each other of two of the three components in the complex proposed for use according to the present invention, namely:

(I) through the main circuit/main circuit connection,

(ii) through a main chain/side chain bond,

(III) via a side chain/main chain linkage or

(IV) via a side chain/side chain bond.

Preferably, all three components a), b) and c) are connected via a main chain/main chain bond, which allows in particular to obtain a main chain of the complex proposed for use according to the present invention, which contains the main chain of one or more penetrant(s) into the cell of the peptide(s), the main chain of one or more antigen(s) or antigenic epitope(s), and the main chain of one or more TLR agonist(s) peptide(s). In other words, the main chain of one or more cell penetrating peptide(s), the main chain of one or more antigen(s) or antigenic epitope(s), and the main chain of one or more peptide agonist(s) ) TLRs form the main chain of the complex proposed for use according to the present invention, optionally in combination with additional components, for example, linker(s), spacer(s), etc. Thus, the following arrangements of components a), b) and c) are preferred, particularly if the at least one antigen or antigenic epitope is a peptide, polypeptide or protein, wherein said preferred arrangements are presented below in the direction N-terminus → C - the end of the main chain of the complex and where all three components a), b) and c) are connected by a main chain/main chain bond and optionally can be connected by a linker, spacer or other additional component:

(α) component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope) - component c) (at least one TLR peptide agonist);

(β) component c) (at least one TLR peptide agonist) - component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope);

(γ) component a) (cell penetrating peptide) - component c) (at least one TLR peptide agonist) - component b) (at least one antigen or antigenic epitope);

(δ) component c) (at least one TLR peptide agonist) - component b) (at least one antigen or antigenic epitope) - component a) (cell penetrating peptide);

(ε) component b) (at least one antigen or antigenic epitope) - component a) (cell penetrating peptide) - component c) (at least one TLR peptide agonist); or

(ζ) component b) (at least one antigen or antigenic epitope) - component c) (at least one TLR peptide agonist) - component a) (cell penetrating peptide).

In particular, if all three components a), b) and c) are linked through a backbone/backbone linkage, then it is preferred that at least one antigen or antigenic epitope is located at the C-terminus of the cell penetrating peptide, and the cell penetrating peptide the peptide and at least one antigen or antigenic epitope are optionally linked by an additional component, eg, a linker, a spacer, or by at least one TLR peptide agonist. Thus, this corresponds to the arrangement indicated in (α), (β) and (γ) from the arrangement options described above, i.e. Of the arrangement options described above, (α), (β) and (γ) are more preferable.

Even more preferably, the at least one antigen or antigenic epitope is located at the C-terminus of the cell penetrating peptide, wherein the cell penetrating peptide and the at least one antigen or antigenic epitope are optionally linked by an additional component, e.g., a linker, a spacer, but not by at least one TLR peptide agonist. Thus, this corresponds to the arrangement indicated in (α) and (β) from the arrangement options described above, i.e. Of the arrangement options described above, (α) and (β) are even more preferred. Particularly preferably, the complex proposed for use according to the present invention is a recombinant polypeptide or recombinant protein and components a) to c) are located in the direction N-terminus → C-terminus of the main chain of said complex in the following order:

(α) component a) - component b) - component c); or

(β) component c) - component a) - component b),

where the components can be linked using an additional component, such as a linker or spacer.

Particularly preferred is arrangement (α) in which the at least one TLR agonist contains or consists of at least one TLR2 agonist, for example:

(α1) component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope) - one or more TLR2 peptide agonists, for example 1, 2, 3, 4 or 5 peptide agonist(s) (s) TLR2;

(α2) component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope) - one or more TLR2 peptide agonists, for example 1, 2, 3, 4 or 5 peptide agonist(s) (s) TLR2, one or more TLR4 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR4 peptide agonist(s), and one or more TLR5 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR5 peptide agonist(s);

(α3) component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope) - one or more TLR2 peptide agonists, for example 1, 2, 3, 4 or 5 peptide agonist(s) (s) TLR2, and one or more TLR4 peptide agonists, for example, 1, 2, 3, 4 or 5 TLR4 peptide agonist(s); or

(α4) component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope) - one or more TLR2 peptide agonists, for example 1, 2, 3, 4 or 5 peptide agonist(s) (s) TLR2, and one or more TLR5 peptide agonists, for example, 1, 2, 3, 4 or 5 TLR5 peptide agonist(s).

Alternatively, in arrangements containing a TLR2 peptide agonist, additional TLR peptide agonists may be complexed at other positions, for example:

(α5) one or more TLR4 peptide agonists, for example 1, 2, 3, 4 or 5 TLR4 peptide agonist(s) - component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope) - one or more TLR2 peptide agonists, for example, 1, 2, 3, 4 or 5 TLR2 peptide agonist(s);

(α6) one or more TLR5 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR5 peptide agonist(s) - component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope) - one or more TLR2 peptide agonists, for example, 1, 2, 3, 4 or 5 TLR2 peptide agonist(s); or

(α7) one or more TLR4 peptide agonists, for example 1, 2, 3, 4 or 5 TLR4 peptide agonist(s), and one or more TLR5 peptide agonists, for example 1, 2, 3, 4 or 5 TLR5 peptide agonist(s) - component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope) - one or more TLR2 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR2 peptide agonist(s). Most preferred is the arrangement (β) wherein the at least one TLR agonist contains or consists of at least one TLR4 agonist, for example:

(β1) one or more TLR4 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR4 peptide agonist(s) - component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope)

(β2) one or more TLR4 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR4 peptide agonist(s), one or more TLR2 peptide agonists, e.g. 1, 2, 3, 4 or 5 peptide TLR2 agonist(s) and one or more TLR5 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR5 peptide agonist(s) - component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope);

(β3) one or more TLR4 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR4 peptide agonist(s), and one or more TLR2 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR2 peptide agonist(s) - component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope); or

(β4) one or more TLR4 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR4 peptide agonist(s), and one or more TLR5 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR5 peptide agonist(s) - component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope).

Alternatively, in arrangements containing a TLR4 peptide agonist, additional TLR peptide agonists may be complexed at other positions, for example:

(β5) one or more TLR4 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR4 peptide agonist(s) - component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope) - one or more TLR2 peptide agonists, for example, 1, 2, 3, 4 or 5 TLR2 peptide agonist(s);

(β6) one or more TLR4 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR4 peptide agonist(s) - component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope) - one or more TLR5 peptide agonists, for example, 1, 2, 3, 4 or 5 TLR5 peptide agonist(s); or

(β7) one or more TLR4 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR4 peptide agonist(s) - component a) (cell penetrating peptide) - component b) (at least one antigen or antigenic epitope) - one or more TLR2 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR2 peptide agonist(s), and one or more TLR5 peptide agonists, e.g. 1, 2, 3, 4 or 5 TLR5 peptide agonist(s).

Alternatively, only two of the three components a), b) and c) are connected via a main chain/main chain bond in the complex proposed for use according to the present invention.

For example, components a) and b) are connected through a main chain/main chain bond, thereby obtaining the following arrangement of components a) and b) in the complex, in the direction N-terminus → C-terminus of the main chain of the complex, with components a) and b) optionally can be linked using an additional component, for example, a linker, spacer, etc.:

(1) cell-penetrating peptide (a) - antigen/antigenic epitope (b); or

(2) antigen/antigenic epitope (b) - cell-penetrating peptide (a).

In this case, component c), i.e. at least one TLR peptide agonist can then be coupled via a main chain/side chain linkage, via a side chain/main chain linkage, or via a side chain/side chain linkage to either a cell penetrating peptide(s) or an antigen/antigenic epitope (b), or, if present, with an additional component such as a spacer or linker, which may, for example, be located between the cell-penetrating peptide (a) and the antigen/antigenic epitope (b). This provides the following location options:

(i) component c) may be connected - optionally via a spacer or linker - via a backbone/side chain linkage to component a), i.e. the main chain of at least one TLR agonist peptide is covalently linked, optionally through a spacer or linker, to a side chain of the cell penetrating peptide;

(II) component c) may be connected - optionally via a spacer or linker - via a side chain/main chain linkage to component a), i.e. a side chain of at least one TLR agonist peptide is covalently linked, optionally through a spacer or linker, to the main chain of the cell penetrating peptide;

(III) component c) may be connected - optionally via a spacer or linker - via a side chain/side chain linkage to component a), i.e. a side chain of at least one TLR agonist peptide is covalently linked, optionally through a spacer or linker, to a side chain of the cell penetrating peptide;

(IV) component c) may be connected - optionally via a spacer or linker - via a backbone/side chain linkage to component b), i.e. the main chain of at least one TLR agonist peptide is covalently linked, optionally through a spacer or linker, to a side chain of at least one antigen or antigenic epitope;

(V) component c) may be connected - optionally via a spacer or linker - via a side chain/main chain linkage to component b), i.e. a side chain of at least one TLR peptide agonist is covalently linked, optionally through a spacer or linker, to the main chain of at least one antigen or antigenic epitope;

(VI) component c) may be connected - optionally via a spacer or linker - via a side chain/side chain linkage to component b), i.e. a side chain of at least one TLR peptide agonist is covalently linked, optionally through a spacer or linker, to a side chain of at least one antigen or antigenic epitope;

(VII) component c) may be connected - optionally via a spacer or linker - by a backbone/side chain linkage to a linker or spacer located between component a) and component b), i.e. The main chain of at least one TLR agonist peptide is covalently linked, optionally through a spacer or linker, to a side chain of a linker or spacer located between component a) and component b);

(VIII) component c) may be connected - optionally via a spacer or linker - via a side chain/main chain linkage to a linker or spacer located between component a) and component b), i.e. a side chain of at least one TLR agonist peptide is covalently linked, optionally through a spacer or linker, to the main chain of a linker or spacer located between component a) and component b); or

(IX) component c) may be connected - optionally via a spacer or linker - via a side chain/side chain bond to a linker or spacer located between component a) and component b), i.e. a side chain of at least one TLR agonist peptide is covalently linked, optionally through a spacer or linker, to a side chain of a linker or spacer located between component a) and component b).

For example, components b) and c) are connected via a main chain/main chain bond, thereby obtaining the following arrangement of components b) and c) in the complex, in the direction N-terminus → C-terminus of the main chain of the complex, with components b) and c) optionally can be linked using an additional component, for example, a linker, spacer, etc.:

(3) antigen/antigenic epitope (b) - TLR peptide agonist (c); or

(4) peptide TLR agonist (c) - antigen/antigenic epitope (b).

In this case, component a), i.e. the cell-penetrating peptide can then be coupled via a main chain/main chain linkage, a side chain/main chain linkage or a side chain/side chain linkage to either an antigen/antigenic epitope (b) or a TLR peptide agonist (c), or, if present, with an additional component such as a spacer or linker, which may, for example, be between the antigen/antigenic epitope (b) and the TLR peptide agonist (c). This provides the following location options:

(X) component a) may be connected - optionally via a spacer or linker - via a backbone/side chain linkage to component b), i.e. the main chain of the cell-penetrating peptide is covalently linked, optionally through a spacer or linker, to a side chain of at least one antigen or antigenic epitope;

(XI) component a) may be connected - optionally via a spacer or linker - via a side chain/main chain linkage to component b), i.e. a side chain of the cell penetrating peptide is covalently linked, optionally through a spacer or linker, to the main chain of at least one antigen or antigenic epitope;

(XII) component a) may be connected - optionally via a spacer or linker - via a side chain/side chain linkage to component b), i.e. a side chain of the cell penetrating peptide is covalently linked, optionally through a spacer or linker, to a side chain of at least one antigen or antigenic epitope;

(XIII) component a) may be connected - optionally via a spacer or linker - via a backbone/side chain linkage to component c), i.e. the main chain of the cell penetrating peptide is covalently linked, optionally through a spacer or linker, to a side chain of at least one TLR agonist peptide;

(XIV) component a) may be connected - optionally via a spacer or linker - via a side chain/main chain linkage to component c), i.e. a side chain of the cell penetrating peptide is covalently linked, optionally through a spacer or linker, to the main chain of at least one TLR agonist peptide;

(XV) component a) may be connected - optionally via a spacer or linker - via a side chain/side chain linkage to component c), i.e. a side chain of the cell penetrating peptide is covalently linked, optionally through a spacer or linker, to a side chain of at least one TLR agonist peptide;

(XVI) component a) can be connected - optionally through a spacer or linker - through a backbone/side chain linkage to a linker or spacer located between component b) and component c), i.e. The main chain of the cell-penetrating peptide is covalently linked - optionally through a spacer or linker - to the side chain of the linker or spacer located between component a) and component b);

(XVII) component a) may be connected - optionally via a spacer or linker - via a side chain/main chain linkage to a linker or spacer located between component b) and component c), i.e. the side chain of the cell-penetrating peptide is covalently linked - optionally through a spacer or linker - to the main chain of the linker or spacer located between component b) and component c); or

(XVIII) component a) may be connected - optionally via a spacer or linker - via a side chain/side chain linkage to a linker or spacer located between component b) and component c), i.e. the side chain of the cell-penetrating peptide is covalently linked - optionally through a spacer or linker - to the side chain of the linker or spacer located between component b) and component c).

For example, components a) and c) are connected via a main chain/main chain bond, thereby obtaining the following arrangement of components b) and c) in the complex, in the direction N-terminus → C-terminus of the main chain of the complex, with components a) and c) optionally can be linked using an additional component, for example, a linker, spacer, etc.:

(5) cell-penetrating peptide (a) - TLR peptide agonist (c); or

(6) peptide TLR agonist (c) - cell-penetrating peptide (a).

In this case, component b), i.e. at least one antigen or antigenic epitope can then be coupled via a main chain/main chain linkage, a side chain/main chain linkage, or a side chain/side chain linkage to either a cell penetrating peptide(s) or a TLR peptide agonist (c), or, if present, with an additional component such as a spacer or linker, which may, for example, be located between the cell-penetrating peptide (a) and the TLR agonist peptide (c). This provides the following location options:

(XIX) component b) may be connected - optionally via a spacer or linker - via a backbone/side chain linkage to component a), i.e. the main chain of at least one antigen or antigenic epitope is covalently linked, optionally through a spacer or linker, to a side chain of the cell penetrating peptide;

(XX) component b) may be connected - optionally via a spacer or linker - via a side chain/main chain linkage to component a), i.e. a side chain of at least one antigen or antigenic epitope is covalently linked, optionally through a spacer or linker, to the main chain of the cell penetrating peptide;

(XXI) component b) may be connected - optionally via a spacer or linker - via a side chain/side chain linkage to component a), i.e. a side chain of at least one antigen or antigenic epitope is covalently linked, optionally through a spacer or linker, to a side chain of a cell penetrating peptide;

(XXII) component b) may be connected - optionally via a spacer or linker - via a backbone/side chain linkage to component c), i.e. the main chain of at least one antigen or antigenic epitope is covalently linked, optionally through a spacer or linker, to a side chain of at least one TLR agonist peptide;

(XXIII) component b) may be connected - optionally via a spacer or linker - via a side chain/main chain linkage to component c), i.e. a side chain of at least one antigen or antigenic epitope is covalently linked, optionally through a spacer or linker, to the main chain of at least one TLR agonist peptide;

(XXIV) component b) may be connected - optionally via a spacer or linker - via a side chain/side chain linkage to component c), i.e. a side chain of at least one antigen or antigenic epitope is covalently linked, optionally through a spacer or linker, to a side chain of at least one TLR agonist peptide;

(XXV) component b) may be connected - optionally via a spacer or linker - via a backbone/side chain linkage to a linker or spacer located between component a) and component c), i.e. The main chain of at least one antigen or antigenic epitope is covalently linked, optionally through a spacer or linker, to a side chain of a linker or spacer located between component a) and component b);

(XXVI) component b) may be connected - optionally via a spacer or linker - via a side chain/main chain linkage to a linker or spacer located between component a) and component c), i.e. a side chain of at least one antigen or antigenic epitope is covalently linked - optionally through a spacer or linker - to the main chain of a linker or spacer located between component a) and component b); or

(XXVII) component b) may be connected - optionally via a spacer or linker - via a side chain/side chain linkage to a linker or spacer located between component a) and component c), i.e. a side chain of at least one antigen or antigenic epitope is covalently linked, optionally through a spacer or linker, to a side chain c of a linker or spacer located between component a) and component b).

Alternatively, it is also possible, in the complex proposed for use according to the present invention, all three components a), b) and c) are arranged via a main chain/side chain linkage, a side chain/main chain linkage or a side chain/side chain linkage, optionally linked by an additional component, such as a spacer or linker.

Colorectal cancer

The present invention provides the complex described above for use in the prevention and/or treatment of colorectal cancer.

Colorectal cancer (CRC, also called "bowel cancer") is a cancer that includes colon cancers and rectal cancers (RC). Both of these cancers have many common features, but the starting points of the cancers are different. According to Siegel R., S. Desantis and A. Jemal, Colorectal cancer statistics, 2014. CA Cancer J Clin, 64(2), 2014, cc. 104-117, in the USA between 2006 and 2010. The incidence of tumor area was slightly more common in the proximal colon (first and middle colon). Of approximately 19 cases per 100,000 people, they account for 42% of cases. This is followed by rectal cancer, which accounts for 28% of cases, and distal cancer (lower colon), which accounts for 10 cases per 100,000 people.

Anatomically, the concept of “colorectal cancer” includes (I) cancers of the colon, such as cancers of the cecum (including cancers of the ileocecal valve), appendix, ascending colon, hepatic (right) flexure, transverse colon, splenic flexure, descending colon, sigmoid colon (cancers of the sigmoid flexure), as well as overlapping areas of the colon; (II) cancers of the rectosigmoid junction, such as cancers of the colon and rectum and cancers of the rectosigmoid, (III) cancers of the rectum, such as cancers of the rectal ampulla.

Preferably, the colorectal cancer is colon cancer, such as cecal cancer (including ileocecal valve cancer), appendix cancer, ascending colon cancer, hepatic flexure cancer, transverse colon cancer, splenic flexure cancer, descending colon cancer, sigmoid colon cancer intestines (including cancers of the sigmoid flexure), or a combination thereof.

Preferably, the colorectal cancer is also a cancer of the rectosigmoid junction, such as (I) colorectal cancer or (II) cancer of the rectosigmoid region.

Moreover, preferably also the colorectal cancer is rectal cancer, such as rectal ampulla cancer.

Depending on the cell type, colorectal cancers include colorectal adenocarcinoma, colorectal stromal tumors, primary colorectal lymphoma, colorectal leimyosarcoma, colorectal melanoma, colorectal squamous cell carcinoma and colorectal carcinoid tumors, such as carcinoid tumors of the cecum, appendix, ascending colon, transverse colon, descending colon, sigmoid colon and/or rectum. Thus, preferred colorectal cancers include colorectal adenocarcinoma, colorectal stromal tumors, primary colorectal lymphoma, colorectal leimyosarcoma, colorectal melanoma, colorectal squamous cell carcinoma and colorectal carcinoid tumors, such as carcinoid tumors of the cecum, appendix, ascending colon, transverse colon , descending colon, sigmoid colon and/or rectum. More preferably, the colorectal cancer is colorectal adenocarcinoma or colorectal carcinoid carcinoma. Even more preferably, the colorectal cancer is colorectal adenocarcinoma.

More than 95% of CRCs are adenocarcinomas. Colorectal adenocarcinomas typically start from glandular cells that produce mucus to lubricate the colon or rectum. CRC typically begins in the innermost layer and may grow through some or all of the other layers. In rare cases, CRC may form a polyp, making it easier for it to grow into the wall of the original area. In advanced stages (stages III and IV), the cancer has spread to nearby lymph nodes or distant parts of the body through blood vessels.

For example, in colorectal cancer, the system of classification of malignant tumors (TNM) includes the following stages of primary tumors (“T” stages): TX - the primary tumor is not detectable, T0 - no manifestation of the primary tumor, Ta - non-invasive papillary carcinoma, Tis - carcinoma in situ: intraepithelial or invasion into the lamina propria, T1 - invasion of tumors under the mucous membrane, T2 - invasion of tumors into the own muscles, T3 - invasion of tumors through the own muscles into pericolorectal tissues, T4a - penetration of tumors onto the surface of the visceral peritoneum and T4b - direct invasion or attachment of tumors to other organs or structures; the following stages for lymph nodes (“N” stages): NX - the condition of the regional lymph nodes cannot be assessed, N0 - there is no metastasis in the regional lymph nodes, N1 - metastasis in 1-3 regional lymph nodes, where N1a - metastasis in 1 regional lymph node, N1b - metastasis in 2-3 regional lymph nodes and N1c - tumor deposit(s) in subserosal, mesenteric or non-peritonealized peri-intestinal or perirectal tissues without metastasis to regional nodes, N2 - metastasis in 4 or more lymph nodes, where N2a - metastasis in 4-6 regional lymph nodes and N2b - metastasis in 7 or more regional lymph nodes; and the following stages for distant metastasis (“M” stages): M0 - no distant metastasis and M1 - distant metastases, where M1a - metastasis confirmed for 1 organ or area (for example, liver, lung, ovary, non-regional node) and M1b - metastases in more than 1 organ/area or peritoneum.

These stages can be combined using the following numbered classification of colorectal cancer stages: stage 0: Tis, N0, M0; stage I: T1, N0, M0 or T2, N0, M0; stage IIA: T3, N0, M0; stage IIB: T4a, N0, M0; stage IIC: T4b, N0, M0; stage IIIA: T1-T2, N1/N1c, M0 or T1, N2a, M0; stage IIIB: T3-T4a, N1/N1c, M0 or T2-T3, N2a, M0, or T1-T2, N2b, M0; stage IIIC: T4a, N2a, M0 or T3-T4a, N2b, M0, or T4b, N1-N2, M0; stage IVA: any T, any N, M1a and stage IVB: any T, any N, M1b. In general, in stage 0, the cancer does not extend beyond the inner layer of the colon or rectum; in stage I, cancer has spread from the mucosa to the muscle layer; at stage II, the cancer spreads through the muscular layer into the serous layer of nearby organs; in stage III, the cancer has spread to nearby lymph node(s) or cancer cells have spread to tissue near the lymph nodes; and in stage IV, the cancer has spread through the blood and lymph nodes to other parts of the body.

Unlike the term "cancer", colorectal cancer includes all of the above numbered stages, and therefore the preferred stage of colorectal cancer can be selected from the group consisting of stage 0 (Tis, N0, M0), stage I (T1, N0, M0 or T2 , N0, M0), stage IIA (T3, N0, M0), stage IIB (T4a, N0, M0), stage IIC (T4b, N0, M0), stage IIIA (T1-T2, N1/N1c, M0 or T1 , N2a, M0), stage IIIB (T3-T4a, N1/N1c, M0 or T2-T3, N2a, M0, or T1-T2, N2b, M0), stage IIIC (T4a, N2a, M0 or T3-T4a, N2b, M0, or T4b, N1-N2, M0), stage IVA (any T, any N, M1a) and stage IVB (any T, any N, M1b). More preferably, the colorectal cancer is selected from the group consisting of stage I (T1, N0, M0 or T2, N0, M0), stage IIA (T3, N0, M0), stage IIB (T4a, N0, M0), stage IIC (T4b , N0, M0), stage IIIA (T1-T2, N1/N1c, M0 or T1, N2a, M0), stage IIIB (T3-T4a, N1/N1c, M0 or T2-T3, N2a, M0, or T1- T2, N2b, M0), stage IIIC (T4a, N2a, M0 or T3-T4a, N2b, M0, or T4b, N1-N2, M0), stage IVA (any T, any N, M1a) and stage IVB ( any T, any N, M1b). Even more preferably, the colorectal cancer is selected from the group consisting of stage IIA (T3, N0, M0), stage IIB (T4a, N0, M0), stage IIC (T4b, N0, M0), stage IIIA (T1-T2, N1/ N1c, M0 or T1, N2a, M0), stage IIIB (T3-T4a, N1/N1c, M0 or T2-T3, N2a, M0, or T1-T2, N2b, M0), stage IIIC (T4a, N2a, M0 or T3-T4a, N2b, M0, or T4b, N1-N2, M0), stage IVA (any T, any N, M1a) and stage IVB (any T, any N, M1b). Most preferably, the colorectal cancer is (I) stage III colorectal cancer, such as stage IIIA (T1-T2, N1/N1c, M0 or T1, N2a, M0), stage IIIB (T3-T4a, N1/N1c, M0 or T2 -T3, N2a, M0, or T1-T2, N2b, M0) or stage IIIC (T4a, N2a, M0 or T3-T4a, N2b, M0, or T4b, N1-N2, M0), or (II) stage IV colorectal cancer, such as stage IVA (any T, any N, M1a) and stage IVB (any T, any N, M1b).

Nucleic acid encoding peptides and proteins of complexes

Another aspect of the present invention is a nucleic acid, in particular a nucleic acid molecule encoding a complex as defined herein, wherein the complex is a polypeptide or protein for use in the prevention and/or treatment of colorectal cancer. In particular, the present invention provides polynucleotides for use in the prevention and/or treatment of colorectal cancer, said polynucleotides encoding the complex described above. In other words, the present invention provides, in particular, a nucleic acid, in particular a nucleic acid molecule, which contains a polynucleotide, wherein the polynucleotide encodes a complex as described herein, and wherein the complex is a polypeptide or protein for use in preventing and /or treatment of colorectal cancer.

The present invention also provides a nucleic acid, in particular a nucleic acid molecule encoding the complex of the present invention as described above, wherein the complex is a polypeptide or protein. In particular, the present invention provides polynucleotides that encode the complex described above. In other words, the present invention provides, in particular, a nucleic acid, in particular, a nucleic acid molecule comprising a polynucleotide, wherein the polynucleotide encodes a complex of the present invention as described above, and wherein the complex is a polypeptide or protein. Said nucleic acid can be used for the prevention and/or treatment of colorectal cancer.

The nucleic acids preferably comprise single-stranded, double-stranded or partially double-stranded nucleic acids, preferably selected from genomic DNA, cDNA, RNA, siRNA, antisense DNA, antisense RNA, ribozyme, complementary RNA/DNA sequences with minigene expression promoting elements, gene fragments, regulatory elements, with or without promoters, and combinations thereof. Other preferred examples of nucleic acid(s) and/or polynucleotides include, for example, a recombinant polynucleotide, a vector, an oligonucleotide, an RNA molecule such as rRNA, mRNA, miRNA, siRNA or tRNA, or a DNA molecule as described above. Thus, it is preferable that the nucleic acid (molecule) is a DNA molecule or an RNA molecule; preferably selected from genomic DNA; cDNA; siRNA; rRNA; mRNA; antisense DNA; antisense RNA; ribozyme; complementary RNA/DNA sequences; RNA/DNA sequences with or without expression promoting elements, regulatory elements and/or promoters; vector; and their combinations.

Preferably, the invention relates to a nucleic acid (intended for use) proposed in the present invention, where the specified nucleic acid encodes a complex, which is, in particular, a polypeptide or protein, where the complex containing a cell penetrating peptide, at least one antigen or an antigenic epitope that is a polypeptide or protein, and at least one TLR peptide agonist, wherein the cell penetrating peptide, at least one antigen or antigenic epitope, and at least one TLR peptide agonist are linked covalently, optionally by peptide(s) ) spacer(s) or linker(s) specified in the present description. If more than one antigen or antigenic epitope, which is a polypeptide or protein, is contained in the specified complex, then several antigens or antigenic epitopes are also covalently linked, optionally using peptide spacer(s) or linker(s), specified(s) in the present description. Likewise, if more than one TLR peptide agonist is contained in a specified complex, then several TLR peptide agonists are also covalently linked, optionally using a peptide spacer(s) or linker(s) specified herein.

Particularly preferably, the nucleic acid (intended for use) proposed in the present invention encodes a (recombinant) fusion protein complex that contains (a) a cell penetrating peptide described above, (b) at least one, preferably at least two, more preferably at least three, even more preferably at least four, especially preferably at least five, most preferably at least six antigens or antigenic epitopes described above, preferably arranged in a sequential manner as described above, and ( c) at least one TLR agonist described above.

Most preferably, the nucleic acid (molecule) encodes the exemplary complex presented herein, or a functional sequence variant thereof. Thus, the nucleic acid (molecule) most preferably contains a polynucleotide that encodes an amino acid sequence corresponding to any of SEQ ID NO: 26-28, 33-34, 37-41, 46, 69 or 72-87, or an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least at least 95% or at least 98% of any of the sequences of SEQ ID NO: 26-28, 33-34, 37-41, 46, 69 or 72-87. In particular, the nucleic acid (molecule) most preferably contains a polynucleotide that encodes an amino acid sequence corresponding to any of SEQ ID NO: 26-28, 33-34, 37-41, 46, 69 or 72-91, or an amino acid sequence identical at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, according to at least 95% or at least 98% any of SEQ ID NO: 26-28, 33-34, 37-41, 46, 69 or 72-91.

Most preferably, the nucleic acid (molecule) proposed in the present invention contains a polynucleotide that encodes the amino acid sequence corresponding to SEQ ID NO: 89, or a functional variant of this sequence having at least 50% sequence identity (preferably at least 60 %, more preferably at least 70%, more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at at least 90%, particularly preferably at least 95%, most preferably at least 98% or 99% sequence identity).

Preparation and purification of complexes

Another object of the present invention is a vector, preferably intended for the prevention and/or treatment of colorectal cancer, in particular a recombinant vector containing the nucleic acid described above.

The term "vector" in the context of the present invention refers to a nucleic acid molecule, preferably an artificial nucleic acid molecule, i.e. a nucleic acid molecule that does not occur naturally. A vector in the context of the present invention can be used to include or harbor a desired nucleotide sequence. These vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors, etc. A storage vector is a vector that provides convenient storage of a nucleic acid molecule. Thus, the vector may contain a sequence corresponding, for example, to the required complex proposed in the present invention. An expression vector can be used to produce expression products such as RNA, eg mRNA, or peptides, polypeptides or proteins. For example, an expression vector may contain sequences necessary for transcription of a portion of the vector sequence, such as a promoter sequence. A cloning vector is typically a vector that contains a cloning site that can be used to incorporate nucleotide sequences into the vector. The cloning vector may be, for example, a plasmid vector or a bacteriophage-based vector. The transfer vector may be a vector suitable for transferring nucleic acid molecules into cells or organisms, such as viral vectors. The vector in the context of the present invention may be, for example, an RNA vector or a DNA vector. Preferably the vector is a DNA molecule. For example, a vector as used herein comprises a cloning site, a selection marker such as an antibiotic resistance factor, and a sequence useful for propagating the vector, such as an origin of replication. Preferably, as used herein, the vector is a plasmid vector. Preferably, as used herein, the vector is an expression vector.

Also included within the scope of the invention are cells transformed with the vector described above, preferably intended for use in the prevention and/or treatment of colorectal cancer. Examples of such cells include, but are not limited to, bacterial cells, such as E. coli, and eukaryotic cells, such as yeast cells, animal cells, or plant cells. In one embodiment, the cells are mammalian cells, eg human, CHO, HEK293T, PER.C6, NS0, myeloma cells or hybridoma cells. Thus, the present invention also relates to a cell expressing the complex (intended for use) proposed in the present invention; or containing a vector (intended for use) proposed in the present invention.

In particular, the cell can be transfected with a vector according to the present invention, preferably an expression vector. The term "transfection" refers to the introduction of nucleic acid molecules, such as DNA or RNA molecules (eg, mRNA), into cells, preferably eukaryotic cells. In the context of the present invention, the term “transfection” includes any method known to one skilled in the art for introducing nucleic acid molecules into cells, preferably eukaryotic cells, for example mammalian cells. These methods include, for example, electroporation, lipofection, for example based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection or transfection based on cationic polymers such as DEAE-dextran or polyethylenimine, etc. .d. Preferably, the introduction is carried out without the use of viruses.

Numerous expression systems can be used, including (but not limited to) chromosomes, episomes, and viral derivatives. More specifically, the vector of the present invention, in particular a recombinant vector, can be derived from bacterial plasmids, transposons, yeast episomes, insertion elements, yeast chromosome elements, viruses such as baculovirus, papilloma viruses such as SV40, vaccinia viruses smallpox, adenoviruses, varicella-zoster viruses, pseudorabies viruses, retroviruses.

For example, these vectors, in particular recombinant vectors, can be derivatives of both cosmids and phagemids. The nucleotide sequence, in particular the nucleic acid, proposed in the present invention, can be inserted into a recombinant expression vector using methods well known to one skilled in the art, such as, for example, the methods described in MOLECULAR CLONING: A LABORATORY MANUAL, Sambrook, etc. ., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001.

A vector, in particular a recombinant vector, may also include nucleotide sequences that control the regulation of expression, in particular the nucleic acid proposed in the present invention, as well as nucleotide sequences that provide expression, transcription and translation, in particular, a nucleic acid (intended for use ) proposed in the present invention. Typically, these sequences are selected depending on the host cells used.

Thus, for example, an appropriate secretory signal can be integrated into a vector of the present invention, in particular a recombinant vector, such that the polypeptide or protein encoded by the nucleic acid (intended for use) of the present invention is directed, for example, to cavities of the endoplasmic reticulum, to the periplasmic space, to the membrane or to the extracellular environment. Selecting an appropriate sector signal can facilitate subsequent protein purification.

Another aspect of the present invention is a host cell, preferably for use in the prevention and/or treatment of colorectal cancer, where the host cell contains a vector, in particular a recombinant vector as defined herein.

Introduction of a vector, in particular a recombinant vector, into a host cell can be accomplished by methods well known to one skilled in the art, such as those described in BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al., 2nd ed., McGraw-Graw. Hill Professional Publishing, 1995, and MOLECULAR CLONING: A LABORATORY MANUAL, supra, including, for example, the transfection described above, for example, using calcium phosphate, DEAE-dextran or cationic lipids; microinjection, electroporation, transduction or infection.

The host cell may be, for example, bacterial cells such as E. coli, fungal cells such as yeast cells and Aspergillus, Streptomyces cells, insect cells and/or any cell line, such as Chinese hamster ovary (CHO) cells, mouse cell line C127, BHK cell line or Syrian hamster cells, human embryonic kidney 293 (HEK 293) cells. Preferably, the host cell (intended for use) proposed in the present invention is a mammalian cell, for example, human, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells. As the host cell, dendritic cells and dendritic cell lines are most preferred. In general, the choice of culture medium depends, in part, on the choice of cell type and/or cell line, and one skilled in the art will have knowledge of suitable culture media suitable for the selected cell type and/or cell line.

Host cells can be used, for example, to express a polypeptide or protein, in particular a complex (intended for use) according to the present invention, based on a vector and/or nucleic acid according to the present invention. After purification by standard methods of the polypeptide or protein, in particular, the complex (intended for use) proposed in the present invention, it can be used in the method specified in the present description.

Thus, the present invention also relates to a method for producing the complex specified in the present description, in particular when the complex is a polypeptide or protein. This method contains stages in which:

(I) culturing the host cell described above in a culture medium; And

(ii) separating the complex as defined herein from the culture medium or separating the complex as defined herein from the host cell lysate after lysis of the host cells.

Thus, the complex obtained using the specified method proposed in the present invention is preferably the complex (intended for use) proposed in the present invention, which is presented in the present description.

Commercial kits and/or reagents, such as BugBuster™ from Novagen, can be used for protein extraction.

Preferably, the method of obtaining the complex specified in the present description further includes the following step, in which:

(iii) solubilize the complex specified in the present description, for example, by resuspension in solutions containing urea or guanidine hydrochloride (GuHCl),

in this case, stage (III) is carried out after the above-described stage (II).

In addition, it is preferable that the above-described method for obtaining the complex specified in the present description further comprises the following step, in which:

(IV) purify the complex described herein, preferably using a single affinity chromatography step, with step (IV) being carried out after step (II) or, if present, step (III) as described above.

In addition, the complex specified in the present description can also be obtained using chemical synthesis methods, for example, using solid phase peptide synthesis.

Purification of these peptides or proteins can be accomplished by any method known in the art for purifying proteins/peptides. Examples of methods include ion exchange chromatography, hydrophobic interaction chromatography, and immunoaffinity-based methods.

Thus, the present invention also provides a method for preparing the complex described herein, comprising the steps of:

(I) chemically synthesizing said complex; And

(II) purify said complex.

Preferably, in the method for preparing the complex as defined herein, the complex chemically synthesized in step (I) and purified in step (II) contains the amino acid sequences of a cell penetrating peptide as defined herein, the amino acid sequence of a TLR agonist peptide as defined herein, and Optionally, if the at least one antigen and/or antigenic epitope is a peptide or protein, then the amino acid sequence of the antigen or antigenic epitope as specified herein.

Alternatively, the present invention also provides a method for preparing the complex described herein, in which

(i) the cell penetrating peptide, at least one antigen or antigenic fragment, and/or at least one TLR peptide agonist are synthesized separately;

(ii) optionally, the cell penetrating peptide, at least one antigen or antigenic fragment, and/or at least one TLR peptide agonist is purified; And

(III) the cell penetrating peptide, at least one antigen or antigenic fragment and/or at least one TLR peptide agonist are covalently linked as described above, optionally using a spacer or linker or using a cross-linking agent as described above.

Cells loaded with complexes according to the invention

Another object of the present invention is a cell loaded with the complex specified in the present description, preferably intended for use in the prevention and/or treatment of colorectal cancer. For example, the cells loaded with the complex specified herein are cells of the individual being treated, in particular, isolated cells of the individual being treated, i.e. cells isolated from the individual being treated.

In the context of the present invention, the term "individual" refers in particular to mammals. For example, mammals contemplated herein include humans, primates, domesticated animals such as cattle, sheep, pigs, horses, laboratory rodents, and the like. More preferably, the term "individual" refers to a person.

In the context of the present invention, the terms “treating” and “treating” and the like generally mean obtaining the desired pharmacological and physiological effect. The action may be prophylactic, referring to the prevention or partial prevention of a disease, symptom or condition, and/or may be therapeutic, referring to the partial or complete cure of a disease, condition, symptom or adverse event associated with a disease. The term “treatment” as used herein includes any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the onset of the disease in an individual who is susceptible to the disease but in whom the disease has not yet developed, and/or the disease has not yet been diagnosed in this individual, for example, prophylactic early asymptomatic intervention; (b) inhibition of the disease, i.e. cessation or slowdown of its development; or (c) weakening of the disease, i.e. causing at least partial regression of the disease and/or at least one of its symptoms or conditions, such as improvement or cure of the lesion. In particular, the methods, uses, preparations and compositions of the invention are useful for treating various types of cancer or infectious diseases and/or preventing various types of cancer from progressing to an advanced or metastatic stage in individuals with early stage cancer, thereby improving the progression of cancer. When used for various types of cancer, preventing a disease or disorder includes preventing the occurrence or development of cancer in an individual who has been identified as at risk of developing said cancer, for example, associated with the known presence of that cancer in relatives of the individual, and preventing infection by tumor-promoting pathogens such as eg as Epstein-Barr virus (EBV), human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), human herpes virus type 8 (HHV8), human T-cell leukemia virus type 1 (HTLV-1), Merkel cell polyoma virus (MCV) and Helicobacter pylori. The concept of “preventing/treating” cancer also includes stabilizing or slowing down an already diagnosed cancer in an individual. By “stabilization” we mean preventing the development of cancer into an advanced or metastatic stage in individuals with an early stage of cancer.

Preferably, the cell loaded with the complex described herein is an antigen presenting cell (APC). Preferably, the antigen presenting cells are selected from the group consisting of a dendritic cell (DC), a macrophage, and a B cell. Dendritic cells, in particular dendritic cells (conventional and/or plasmacytoid), isolated from the individual to be treated, are more preferred.

Methods for isolating antigen presenting cells, in particular dendritic cells, from an individual are known to those skilled in the art. These involve the collection of monocytes or hematopoietic stem cells from bone marrow, cord blood or peripheral blood. These also include the use of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. Antigen-presenting cells, particularly dendritic cells or their progenitors, can be enriched by methods that include elutriation and magnetic bead-based separation, which can be used to enrich CD14 ⁺ progenitor cells.

Methods for loading the complex described herein into cells, preferably the above-mentioned antigen presenting cells, more preferably dendritic cells, as well as methods for obtaining such cells before administration to an individual are known to one skilled in the art. For example, the production of dendritic cells may involve culturing or differentiating them using cytokines, which may include, for example, GM-CSF and IL-4. Dendritic cell lines can also be used. Loading of the complex of the invention into cells, preferably APCs, more preferably dendritic cells, may involve co-incubation of the complex of the invention with cells in culture, using the properties of the cell penetrating peptide contained in the complex, specified in the present description (ie its ability to be internalized). Additional culture of cells, for example dendritic cells, loaded in this manner may involve the addition of cytokines, including IL-1β, IL-6, TNFα, PEG2, IFNα, and adjuvants, which may include poly-IC, poly-ICLC, to induce effective maturation (i.e., a synthetic complex of carboxymethylcellulose, polyinosinic-polycytidylic acid double-stranded RNA, and poly-L-lysine), as well as TLR agonists and NLR agonists (oligonucleotide-binding oligomerization domain-like receptors).

A method of obtaining cells, in particular antigen presenting cells, loaded with the complex specified in the present description may include the steps of:

(I) transducing or transfecting said cells with the complex proposed in the invention;

(ii) culturing said cells in a culture medium; And

(III) separating said cells from the culture medium.

Preferably, the cells are loaded with a complex as defined herein, wherein the complex is a polypeptide or protein, and is used for the prevention and/or treatment of colorectal cancer.

Preferably, cells loaded with the complex(es) specified herein and which are used for the prevention and/or treatment of colorectal cancer present at least one antigen or antigenic epitope contained in said complex on the cell surface in the context of MHC class I and/or in the context of MHC class II.

Compositions and kits provided by the present invention

Another object of the invention is a composition, preferably intended for use in the prevention and/or treatment of colorectal cancer, where the composition contains at least one component selected from:

(I) the complex, which is described above,

(ii) a nucleic acid as described above,

(III) the vector described above,

(IV) a host cell, which is described above, and

(V) a cell loaded with the complex specified herein, which is described above.

Preferably, the composition proposed in the present invention contains a complex as described above.

The composition (intended for use) proposed in the present invention may also contain more than one of the above components (I)-(V). For example, the composition (intended for use) proposed in the present invention may contain at least two different complexes specified in subparagraph (I), at least two different nucleic acids specified in subparagraph (II), at least two different the vectors specified in subparagraph (III), at least two different host cells specified in subparagraph (IV), and/or at least two different cells specified in subparagraph (V); for example, the composition (intended for use) proposed in the invention may contain at least two different complexes specified in subparagraph (I), and/or at least two different nucleic acids specified in subparagraph (II).

For example, the various complexes specified in subparagraph (I), which are contained in the composition described above, may differ either in component a), i.e. peptides penetrating the cell, or component b), i.e. antigens or antigenic epitopes or subsets of several antigens or antigenic epitopes, or component c), i.e. a TLR peptide agonist or a subset of several TLR peptide agonists; or the various complexes referred to in subparagraph (I), which are contained in the composition described above, may differ in two of the three components a), b) and c); or the various complexes referred to in sub-clause (I), which are contained in the composition described above, may differ in all three components a), b) and c) of the complex. Accordingly, the various nucleic acids specified in subparagraph (II) that are contained in the composition described above may differ in that they encode these various complexes; the various vectors referred to in subparagraph (III), which are contained in the composition described above, may differ in that they contain these different nucleic acids; the various host cells referred to in subparagraph (IV) contained in the composition described above may differ in that they contain the various vectors indicated; and the various complex-loaded cells referred to in sub-clause (V) contained in the composition described above may be distinguished by the fact that they are loaded with said various complexes.

The present invention also provides a vaccine, preferably for use in the prevention and/or treatment of colorectal cancer, wherein the vaccine contains at least one component selected from:

(I) the complex, which is described above,

(ii) a nucleic acid as described above,

(III) the vector described above,

(IV) a host cell, which is described above, and

(V) cell loaded with complex as described above.

Preferably, the vaccine (intended for use) proposed in the present invention contains the complex specified in the present description.

However, the details described above in relation to the composition (intended for use) proposed in the present invention, which relate to the use of more than one component (I)-(V), also apply to the vaccine (intended for use) proposed in the present invention.

In the context of the present invention, the term “vaccine” refers to a biological preparation that confers innate and/or adaptive immunity, typically to a specific disease, preferably cancer. Thus, the vaccine supports the innate and/or adaptive immune response of the immune system of the individual being treated. For example, an antigen or antigenic epitope of a complex as defined herein will generally mediate or support an adaptive immune response in a patient being treated, and a peptide agonist of a TLR complex as defined herein may mediate or support an innate immune response.

The composition according to the invention, in particular the vaccine according to the invention, may also contain the following pharmaceutically acceptable carrier, adjuvant and/or excipient for the pharmaceutical composition according to the invention. According to a particular embodiment of a composition according to the invention, in particular a vaccine according to the invention, the choice of pharmaceutically acceptable carrier is determined in principle by the route in which the composition according to the invention, in particular the vaccine according to the invention, is administered. The composition according to the invention, in particular the vaccine according to the invention, can be used, for example, systemically or locally. Routes of systemic administration generally include, for example, transdermal, oral, parenteral routes, including subcutaneous, intravenous, intramuscular, intra-arterial, intradermal and intraperitoneal injections and/or intranasal route of administration. Routes of topical administration generally include, for example, topical application, but also intradermal, transdermal, subcutaneous or intramuscular injections or intralesional injections, intracranial, intrapulmonary, intracardiac, intranodal (inside lymph nodes) and sublingual injections. More preferably, the composition according to the invention, in particular the vaccine, can be administered by the intradermal, subcutaneous, intranodal or intramuscular route. Even more preferably, the composition according to the invention, in particular the vaccine, can be administered by the subcutaneous, intranodal or intramuscular route. Particularly preferably, the composition according to the invention, in particular the vaccine, can be administered subcutaneously or intranodally. Most preferably, the composition according to the invention, in particular the vaccine, can be administered subcutaneously. Thus, the composition according to the invention, in particular the vaccines according to the invention, is preferably prepared in liquid (or sometimes solid) form.

The appropriate amount of the inventive composition, in particular the inventive vaccine, to be administered can be determined by routine experiments using animal models. Such models include, but are not limited to, those created using rabbits, sheep, mice, rats, dogs and non-human primates. Preferred unit dosage forms for injection include sterile solutions of water, saline, or mixtures thereof. The pH of such solutions should be adjusted to approximately 7.4. Suitable injectable vehicles include hydrogels, controlled or sustained release devices, polylactic acid, and collagen matrices. Suitable pharmaceutically acceptable carriers for topical use include those suitable for use in lotions, creams, gels, and the like. If the composition according to the invention, in particular the vaccine according to the invention, is intended for oral administration, then the preferred unit dosage form is tablets, capsules or the like. Pharmaceutically acceptable carriers for the preparation of unit dosage forms that can be used for oral administration are known in the art. Their choice depends on secondary parameters such as taste, cost and shelf stability, which are not critical for the purposes of the present invention, and the choice can easily be made by one skilled in the art.

The composition proposed in the invention, in particular the vaccine proposed in the invention, may additionally contain one or more auxiliary substances to further increase immunogenicity. This preferably achieves a synergistic effect between the complex according to the invention, as mentioned above, and the auxiliary substance, which may optionally be contained in the vaccine according to the invention, as described above. In this regard, depending on the different types of excipients, different mechanisms can be considered. For example, compounds that promote dendritic cell (DC) maturation, such as lipopolysaccharides, TNF-alpha or CD40 ligand, form a first class of suitable excipients. In general, any agent that affects the immune system in a “danger signal” manner (LPS, GP96, etc.) or cytokines such as GM-CSF that promote enhancement and/or targeting can be used as an excipient. immune response that is produced in response to the immune-stimulating adjuvant proposed in the invention. The most preferred excipients are cytokines, such as monokines, lymphokines, interleukins or chemokines, which further promote the innate immune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL- 20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors such as hGH.

Further additives that can be included in the vaccine according to the invention are emulsifiers, such as ^Tween® ; wetting agents such as sodium lauryl sulfate; dyes; flavoring agents; pharmaceutical carriers; tablet forming agent; stabilizers; antioxidants; preservatives.

The composition proposed in the invention, in particular the vaccine proposed in the invention, may also contain any additional compound that is known to have immunostimulating properties due to its binding affinity (as ligands) to human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or by binding affinity (as ligands) to the murine Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13 .

Another class of compounds that in this context can be added to the composition according to the invention, in particular to the vaccine according to the invention, may be CpG nucleic acids, in particular CpG RNA or CpG DNA. CpG RNA or CpG DNA may be single-stranded CpG DNA (ssCpG DNA), double-stranded CpG DNA (dsDNA), single-stranded CpG RNA (ssCpG RNA), or double-stranded CpG RNA (dsCpG RNA). The CpG nucleic acid is preferably in the form of CpG RNA, more preferably in the form of single-stranded CpG RNA (ssCpG RNA). The CpG nucleic acid preferably contains at least one or more (mitogenic) cytosine/guanine dinucleotide(s) sequence(s) (CpG motif(s)). According to a first preferred alternative, at least one CpG motif in these sequences, in particular the C (cytosine) and G (guanine) CpG motif, is unmethylated. All other cytosines or guanines optionally contained in these sequences may be methylated or unmethylated. However, according to a further preferred alternative, the C (cytosine) and G (guanine) CpG motif may also be present in methylated form.

The present invention also provides a pharmaceutical composition preferably for use in the prevention and/or treatment of colorectal cancer, in particular the vaccine composition described above, and a method of treating an individual, preferably a mammal and most preferably a human, who suffers from colorectal cancer.

In particular, the present invention also provides a pharmaceutical composition (intended for use) for the prevention and/or treatment of colorectal cancer, containing at least one complex specified in the present description, or at least one cell loaded with a complex specified in the present description , and optionally a pharmaceutically acceptable carrier and/or excipient or any excipient, buffer, stabilizer or other materials well known to those skilled in the art, in particular a pharmaceutical composition containing at least one complex as defined herein, or at least one cell loaded with the complex specified herein and a pharmaceutically acceptable carrier.

As an additional ingredient, the pharmaceutical composition according to the invention may, in particular, contain a pharmaceutically acceptable carrier and/or excipient. In the context of the present invention, the pharmaceutically acceptable carrier generally includes a liquid or non-liquid base of the pharmaceutical composition of the invention. If the composition of the invention is in liquid form, the carrier will generally be pyrogen-free water; isotonic saline or buffered (aqueous) solutions, for example, buffered with phosphate, citrate, etc. solutions. In particular, for injection of the pharmaceutical composition according to the invention, water or preferably a buffer, more preferably an aqueous buffer containing a sodium salt, preferably at least 30 mM sodium salt, a calcium salt, preferably at least 0.05 mM calcium salt, and optionally a potassium salt, preferably at least 1 mM potassium salt. According to a preferred embodiment of the invention, the sodium, calcium and optionally potassium salt may be in the form of their halides, for example chlorides, iodides or bromides, in the form of their hydroxides, carbonates, bicarbonates or sulfates, etc. Examples of sodium salts include, but are not limited to, NaCl, NaI, NaBr, Na ₂ CO ₃ , NaHCO ₃ , Na ₂ SO ₄ , examples of optional potassium salts include, but are not limited to, KCl, KI, KBr, K ₂ CO ₃ , KHCO ₃ , K ₂ SO ₄ , and examples of calcium salts include, but are not limited to, CaCl ₂ , CaI ₂ , CaBr ₂ , CaCO ₃ , CaSO ₄ , Ca(OH) ₂ . In addition, the buffer may contain organic anions of the above cations. According to a more preferred embodiment of the invention, the above buffer, which can be used for injection purposes, may contain a salt selected from sodium chloride (NaCl), calcium chloride (CaCl ₂ ) and optionally potassium chloride (KCl), and in addition to chlorides may be present additional anions. CaCl ₂ can also be replaced with another salt such as KCl. Typically, the salts in the injection buffer are present at a concentration of at least 30 mM in the case of sodium chloride (NaCl), at least 1 mM in the case of potassium chloride (KCl), and at least 0.05 mM in the case of calcium chloride (CaCl ₂ ). The injection buffer can be hypertonic, isotonic or hypotonic relative to a specific reference medium, i.e. the buffer may have a higher, identical or lower salt content relative to a particular reference medium, and preferably the above salts may be used at concentrations that do not cause cell damage due to osmosis or other concentration-dependent effects. Reference media are, for example, fluids present in in vivo methods, such as blood, lymph, cytosolic fluids or other body fluids, or, for example, fluids that can be used as reference media in in vitro methods. , such as conventional buffers or liquids. Such conventional buffers or liquids are known to those skilled in the art. Preferred liquid bases are saline (0.9% NaCl) and lactated Ringer's solution.

Preferably, the (pharmaceutical) composition which contains the complex specified in the present description further contains arginine, for example L-arginine.

However, the pharmaceutical composition of the invention may also include one or more compatible solid or liquid excipients or diluents or encapsulating compounds that can be used for administration to the individual being treated. The term “compatible” in the context of the present description means that these components of the pharmaceutical composition proposed in the invention can be mixed with the above complex proposed in the present invention, in such a way that there is no interaction that could lead to a significant decrease in the pharmaceutical effectiveness of the pharmaceutical composition proposed in the invention compositions under normal conditions of use. Pharmaceutically acceptable carriers, excipients and diluents will naturally be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the individual being treated. Some examples of compounds that can be used as pharmaceutically acceptable carriers, excipients or constituents are sugars, such as lactose, glucose and sucrose; starches, such as corn starch or potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt, gelatin; fat; solid glidants such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and theobroma (chocolate tree) oil; polyols such as, for example, polypropylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid.

The pharmaceutical composition of the invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intraarticular, synovial, epigastric, intrathecal, intrahepatic, intralesional, intracranial, percutaneous, intradermal, intrapulmonary, intraperitoneal, intracardiac, intraarterial, intranodal, and sublingual injection or infusion. Preferably, the pharmaceutical composition according to the invention can be administered intradermally, intramuscularly, intranodally or subcutaneously. More preferably, the pharmaceutical composition according to the invention can be administered intramuscularly, intranodally or subcutaneously. Even more preferably, the pharmaceutical composition according to the invention can be administered intranodally or subcutaneously. Most preferably, the pharmaceutical composition according to the invention can be administered subcutaneously. Also particularly preferably, the pharmaceutical composition according to the invention can be administered subcutaneously or intradermally.

Preferably, the pharmaceutical composition of the invention can be administered by parenteral injection, more preferably subcutaneous, intravenous, intraarticular, intrasynovial, epigastric, intrathecal, intrahepatic, intralesional, intracranial percutaneous, intradermal, intrapulmonary, intraperitoneal, intracardiac, intraarterial, intranodal and sublingual injections or infusions. Sterile injectable forms of the pharmaceutical compositions proposed in the invention can be an aqueous or oily suspension. These suspensions can be prepared using methods known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or injectable suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable excipients and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile non-volatile oils are usually used as a solvent or suspending medium. For this purpose, soft fixed oils, including synthetic mono- or diglycerides, can be used. Fatty acids such as oleic acid and its glycerides can be used in injection preparations in the form of naturally occurring pharmaceutically acceptable oils such as olive oil or castor oil, primarily in the form of their polyoxyethylated derivatives. These oily solutions or suspensions may also contain a long chain alcohol as a diluent or dispersing agent, such as carboxymethylcellulose or similar dispersing agents that are commonly used to prepare pharmaceutically acceptable dosage forms, including emulsions and suspensions. Conventional surfactants, such as Tweens, Spans and other emulsifying agents or bioavailable enhancers, which are usually used for the production of pharmaceutically acceptable solid, liquid or other dosage forms, can also be used to prepare the pharmaceutical composition according to the invention.

For intravenous, dermal or subcutaneous injection or injection into the affected area, the active substance should preferably be in the form of a parenterally acceptable aqueous solution, free of pyrogens and having an acceptable pH value, isotonicity and stability. Those skilled in the art can prepare suitable solutions using, for example, isotonicizing excipients such as sodium chloride injection, Ringer's solution for injection, lactated Ringer's solution for injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as necessary. Regardless of whether a polypeptide, a peptide or a nucleic acid molecule, another pharmaceutically acceptable compound of the invention to be administered to an individual is used, the administration is preferably carried out in a "prophylactically effective amount" or a "therapeutically effective amount" (as appropriate) , which is sufficient to provide a beneficial effect on the individual. The actual amount administered and the rate and duration of administration should depend on the nature and severity of the condition being treated.

The inventive pharmaceutical composition set forth above can also be administered orally in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral administration, common carriers are lactose and corn starch. Typically, lubricants such as magnesium stearate are also added. For oral administration in capsule form, acceptable diluents include lactose and anhydrous corn starch. When aqueous suspensions are required for oral administration, the active substance, i.e. the above proposed in the invention, the transporter of the conjugated cargo molecule mentioned above is combined with emulsifying and suspending agents. If necessary, certain sweeteners, flavoring agents or coloring agents can be added.

The pharmaceutical composition according to the invention can also be applied topically, especially if the target of treatment includes areas or organs that are easily accessible for topical application, for example, including diseases of the skin or any other accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs. For topical use, the pharmaceutical composition of the invention can be formulated as a suitable ointment containing the immunostimulating composition of the invention, in particular the components described above, suspended or dissolved in one or more vehicles. Carriers for topical use include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifiable wax and water. Alternatively, the pharmaceutical composition of the invention may be formulated as a suitable lotion or cream. In the context of the present invention, acceptable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, wax cetyl esters, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

In this context, the prescription of treatment, such as the choice of doses, etc. the use of the pharmaceutical composition described above is within the skill of ordinary practitioners and other physicians, and will typically take into account the disorder being treated, the condition of the individual patient, the site of administration, the method of treatment, and other factors known to the practitioner. Examples of the techniques and protocols mentioned above can be gleaned from REMINGTON'S PHARMACEUTICAL SCIENCES, 160th ed., ed. Osol A., 1980.

Thus, the pharmaceutical composition proposed in the invention, as a rule, contains in a “safe and effective” amount the components of the pharmaceutical composition proposed in the invention, in particular the above-mentioned complex presented in the present description, and/or cells loaded with the specified complex. As used herein, a “safe and effective amount” means an amount of a complex as defined herein that is sufficient to significantly induce beneficial changes in the disease or disorder, i.e. an amount of a complex as defined herein, or cells loaded with said complex, that produces a biological or medical response in the tissue, system, animal or human in question. An effective amount may be a “therapeutically effective amount” for alleviating symptoms of a disease or condition being treated, and/or a “prophylactically effective amount” for preventing symptoms of a disease or condition being prevented. The concept also includes an amount of the active complex sufficient to slow the progression of the disease, in particular to reduce or inhibit the growth of a tumor or infection, and thereby produce an expected response, in particular, said response may be an immune response directed against antigens or antigenic epitopes, which are contained in the complex (i.e., the “inhibitory effective amount”). However, at the same time, the “safe and effective amount” is small enough to avoid serious side effects, i.e. provides a reasonable balance between benefit and risk. These limits are generally determined by reasonable medical judgment. The "safe and effective" amount of the components of the pharmaceutical composition proposed in the invention, in particular, the above-mentioned complex presented in the present description, should also vary depending on the specific condition being treated, as well as the age and physical condition of the patient being treated, body weight , general health, gender, diet, treatment time, excretion rate, drug combination, activity of specific components a), b) and c) of the complex described above presented herein, severity of the condition, duration of treatment, nature of concomitant therapy, specific the pharmaceutically acceptable carrier used and similar factors which are known and within the control of the appropriate physician. The pharmaceutical composition proposed in the invention can be used for humans, as well as in veterinary medicine, preferably in human medicine, as a pharmaceutical composition in general, or as a vaccine.

Pharmaceutical compositions, in particular vaccine compositions, or formulations proposed in the invention, can be administered in the form of a pharmaceutical formulation, which may contain the complex presented in the present description, in any form specified in the present description.

The terms “pharmaceutical formulation” and “pharmaceutical composition” in the context of the present invention refer, in particular, to preparations that are in a form that provides the biological activity of the active substance(s), such that it(ies) clearly exhibit (i) effectiveness, and which does not contain an additional component that may be toxic to the individuals to whom the formulation is to be administered.

In the context of the present invention, the "effectiveness" of treatment can be measured based on changes in the course of the disease in response to the use proposed in the present invention or to the method proposed in the present invention. For example, the effectiveness of cancer treatment can be measured by reduction in tumor volume and/or prolongation of progression-free survival time, and/or reduction in the risk of recurrence after resection of the primary cancer. More specifically, for cancers treated with immunotherapy, assessment of efficacy may include the spectrum of clinical patterns of antitumor response to immunotherapeutic agents using the new Immunotherapy Response Criteria (irRC), which are an adaptation of the Response Evaluation Criteria in Solid Tumors (RECIST), and the World Health Organization (RECIST) criteria. health organizations (WHO) (J. Natl. Cancer Inst. 102(18), 2010, pp. 1388-1397). The effectiveness of infectious disease prevention is ultimately assessed through epidemiological studies of human populations, which are often correlated with serum neutralizing antibody titers, and the induction of multifunctional pathogen-specific T-cell responses. Preclinical evaluation may include resistance to infection following challenge with an infectious pathogen. Treatment of an infectious disease can be assessed by inhibition of pathogen growth or elimination of the pathogen (and the resulting absence of detectable pathogen), which correlates with pathogen-specific antibodies and/or T-cell immune responses.

Pharmaceutical compositions, in particular vaccine compositions, or formulations proposed in the invention, can also be administered in the form of a pharmaceutical formulation, which can contain antigen-presenting cells loaded with the complex proposed in the invention, in any form specified in the present description.

The vaccine and/or composition (intended for use) proposed in the present invention can also be prepared in the form of pharmaceutical compositions and their unit doses, in particular in combination with conventional adjuvants, immunomodulatory agent, carrier, diluent or excipient, which are described above and will be described below, in such form they can be administered in the form of solid preparations, such as tablets or filled capsules, or in the form of liquids, such as solutions, suspensions, emulsions, elixirs, or capsules filled therewith, all for oral administration, or in the form sterile injectable solutions for parenteral (including subcutaneous and intradermal administration), which are administered by injection or continuous infusion.

In the context of the present invention, particularly in the context of the pharmaceutical composition and vaccines proposed in the present invention, the base of the injectable compositions is typically injectable sterile saline or phosphate buffered saline, or other injectable carriers known in the art. Said pharmaceutical compositions and unit dosage forms thereof may contain the ingredients in conventional proportions, with or without the addition of additional active ingredients or ingredients, and such unit dosages may contain any suitable effective amount of the active ingredient, consistent with the intended daily dosage range at application.

Examples of suitable adjuvants and/or immunomodulatory agents in the context of the present invention include MPL® (Corixa), aluminum-based minerals including aluminum derivatives (generally called alum), ASO1-4, MF59, calcium phosphate, liposomes, Iscom, polyinosinic -polycytidylic acid (polyIC), including its stabilized form poly-ICLC (Hiltonol), CpG oligodeoxynucleotides, granulocyte-macrophage colony-stimulating factor (GM-CSF), lipopolysaccharide (LPS), montanide, polylactide-polyglycolide copolymer (PLG), flagellin , soapbark saponins (QS21), aminoalkylglucosamides (e.g. RC529), two components of antibacterial peptides with synthetic oligodeoxynucleotides (e.g. IC31), imiquimod, resiquimod, immunostimulatory sequences (ISS), monophosphoryl lipid A (MPLA), fibroblast-stimulating lipopeptide (FSL1) and antibodies to CD40.

The compositions, in particular pharmaceutical compositions and vaccines (intended for use), proposed in the present invention may be liquid formulations, including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups and elixirs. The compositions may also be formulated as an anhydrous product for reconstitution with water or other suitable excipient before use. These liquid preparations may contain additives including, but not limited to, suspending agents, emulsifying agents, non-aqueous excipients and preservatives. Suspending agents include, but are not limited to, sorbitol syrup, methylcellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate and gum arabic. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Dispersing or wetting agents include, but are not limited to, poly(ethylene glycol), glycerin, bovine serum albumin, Tween®, Span®.

The compositions, in particular pharmaceutical compositions and vaccines (intended for use), proposed in the present invention, can also be prepared in the form of a depot preparation, which can be administered by implantation or intramuscular injection.

The compositions, in particular the pharmaceutical compositions and vaccines (intended for use), proposed in the present invention may be in the form of solid compositions, which may be in the form of tablets or lozenges, which are prepared in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients, including, but not limited to, binding agents, fillers, lubricants, disintegrants and wetting agents. Binding agents include, but are not limited to, syrup, gum arabic, gelatin, sorbitol, tragacanth, starch mucilage, and polyvinylpyrrodidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, corn starch, calcium phosphate and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycolate. Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets can be coated using methods known in the art.

The compositions, in particular the pharmaceutical compositions and vaccines (intended for use), proposed in the present invention, can also be administered in the form of sustained release forms or delivery systems for sustained release dosage forms.

In addition, the compositions, in particular the pharmaceutical compositions and vaccines (intended for use), proposed in the present invention, can be adapted for delivery by repeated administration.

Additional materials, as well as methods of processing of formulations, etc., which can be used in the context of compositions, in particular, pharmaceutical compositions and vaccines (intended for use) or in the context of their preparation, are set out in “Part 5 of Remington's “The Science and Practice of Pharmacy", 22nd ed., University of the Sciences in Philadelphia, Lippincott Williams & Wilkins, 2012.

A further object of the present invention is also kits consisting of parts, preferably intended for use in the prevention and/or treatment of colorectal cancer, which contain at least one of the following components:

(I) the complex as described above,

(ii) a nucleic acid as described above,

(III) vector, which is described above,

(IV) a host cell as described above, and

(V) a cell loaded with the complex as described above.

In particular, the multipart kit according to the invention may contain more than one component (I)-(V). For example, the multipart kit of the present invention may contain at least two different complexes as described in (I), at least two different nucleic acids as described in (II), at least two different vectors, specified in subparagraph (III), at least two different host cells specified in subparagraph (IV), and/or at least two different cells specified in subparagraph (V); for example, the multipart kit of the invention may contain at least two different complexes referred to in (I) and/or at least two different nucleic acids referred to in (II).

For example, the various complexes specified in sub-clause (I), which are included in the above-described set of parts, may differ either in component a), i.e. peptides penetrating the cell, or component b), i.e. antigens or antigenic epitopes or subsets of several antigens or antigenic epitopes, or component c), i.e. a TLR peptide agonist or subsets of several TLR peptide agonists; or the various complexes referred to in subparagraph (I), which are included in the above-described multi-part set, may differ in two of the three components a), b) and c); The various complexes referred to in sub-clause (I), which are included in the above-described set of parts, may differ in all three components a), b) and c) of the complex. Accordingly, the various nucleic acids specified in subparagraph (II) that are included in the above-described multipart set may differ in that they encode these various complexes; the various vectors referred to in sub-clause (III), which are included in the above-described multipart kit, may differ in that they contain these different nucleic acids; the various host cells referred to in sub-clause (IV), which are included in the above-described multi-part set, may differ in that they contain these different vectors; and the various complex-loaded cells referred to in sub-clause (V) that are included in the above-described multipart kit may be distinguished by the fact that they are loaded with said various complexes.

The various components of a multi-part kit may be packaged in one or more containers. The above components may be in lyophilized or anhydrous form, or may be dissolved in a suitable buffer. The kit may also contain additional reagents, including, for example, preservatives, growth media and/or buffers for storage and/or reconstitution of the above components, wash solutions, and the like. In addition, the kit of parts provided by the present invention may optionally contain instructions for use.

In addition, the present invention also provides a vaccination kit for the treatment, prevention and/or stabilization of colorectal cancer, which contains a pharmaceutical composition specified in the present description or a vaccine specified in the present description, and instructions for use of the specified pharmaceutical composition or specified vaccine for the prevention and/or treatment of colorectal cancer.

Thus, the present invention also provides a kit that contains a complex as specified herein, a cell as specified herein, a composition as specified herein, a vaccine as specified herein, and/or a pharmaceutical composition as specified herein.

Preferably, the kit also contains a package insert or leaflet with instructions for use for the treatment of colorectal cancer using the complex specified in the present description (intended for use) proposed in the present invention, specified in the present description of the cell specified in the present description of the composition, the vaccine specified in the present description, and/or the pharmaceutical composition specified in the present description.

It is also preferred that, in addition to any components (I) to (V) above, the kit contains a chemotherapeutic agent, a targeted drug and/or an immunotherapeutic agent, such as an immune checkpoint modulator, which are specified herein in the context of a combination intended for the use specified in this specification.

Application and methods proposed in the invention

Another object of the present invention is the use of one of the following components: (I) the complex specified in the present description, and/or (II) cells, such as antigen presenting cells, loaded with the complex specified in the present description (for the preparation of a drug), for the prevention , treatment or stabilization of colorectal cancer. Thus, the present invention provides any of the following components: (i) the complex specified in the present description, and/or (ii) cells, such as antigen presenting cells, loaded with the complex specified in the present description, for use in the prevention, treatment or stabilization of colorectal cancer.

The present invention also provides a complex proposed for use according to the present invention, which ensures the transport and presentation of at least one antigen or antigenic epitope contained in the complex on the cell surface of antigen-presenting cells in the context of MHC class I and/or MHC class II, for applications for vaccination and/or immunotherapy.

Thus, another aspect of the present invention is a method of preventing, treating or suppressing colorectal cancer, wherein said method comprises administering to said individual any one of the following components: (i) the complex of the invention, (ii) cells, such as antigen presenting cells, loaded the complex proposed in the invention, or (III) a pharmaceutical formulation containing the components specified in subparagraphs (I)-(II).

In addition, the present invention provides a method of inducing or enhancing in an individual an immune response against one or more epitopes that is dependent on CD4 ⁺ T helper cells and/or cytotoxic CD8 ⁺ T cells, wherein the method comprises administering to the individual any one of the following components: (I) the complex proposed in the present invention, (II) cells, such as antigen presenting cells loaded with the complex, or (III) a pharmaceutical formulation containing the components specified in subparagraphs (I)-(II) .

The immune response, which is dependent on the CD4 ⁺ and/or CD8 ⁺ response, can be determined by the inflammatory response, a pro-inflammatory cytokine response, including an increase in the expression of one or more of the mRNAs or proteins of IFN-γ, TNF-α and IL-2 relative to the level before the introduction of the compounds proposed in the invention. It can also be measured by the increase in frequency or absolute number of antigen-specific T cells following administration of compounds of the invention, as measured by HLA peptide multimer staining, by ELISPOT assays, and in delayed-type hypersensitivity assays. It can be indirectly assessed by increases in serum antigen-specific antibody titers, which are dependent on antigen-specific helper T cells.

The present invention provides a method of inducing or enhancing in an individual an immune response against one or more epitopes that is restricted to multiple MHC class I molecules and/or multiple MHC class II molecules, wherein said method comprises administering to said individual one of the following components: (I) the complex proposed in the present invention, (II) cells, such as antigen presenting cells loaded with the complex, or (III) a pharmaceutical formulation containing the components specified in subparagraphs (I)-(II).

A method of inducing or enhancing in an individual an immune response against one or more epitopes set forth herein that is restricted to multiple MHC class I molecules and/or multiple MHC class II molecules can be assessed by a cytokine response involving an increase in the expression of one/one or more from mRNA or proteins of IFN-γ, TNF-α and IL-2 relative to the level before administration of the compounds proposed in the invention, after stimulation in vitro of T cells with individual peptides that bind discretely to MHC class I and class II molecules on antigen-presenting cells. Restriction for different MHC molecules can be confirmed by using antigen presenting cells that express different MHC molecules or by using MHC blocking antibodies. This can also be measured by the increase in frequency or absolute number of antigen-specific T cells following administration of the compounds of the invention, as measured by HLA peptide multimer staining, which uses multimers combined with discrete MHC molecules.

Preferably, in the methods of inducing or enhancing an immune response against one or more antigens or antigenic epitopes proposed in the present invention, the immune response is directed against one or more epitopes of a tumor associated antigen or tumor-specific antigen, such as a combination of epitopes specified in the present description .

Alternatively or additionally, the immune response may be directed against multiple epitopes of the pathogen's antigenic protein.

The methods provided herein can be used to induce or enhance an immune response in an individual against one or more epitopes that are restricted by MHC class I molecules and/or MHC class II molecules.

In particular, the present invention provides a method of preventing and/or treating colorectal cancer or initiating, enhancing or prolonging an antitumor response in an individual in need thereof, which comprises administering to the individual a complex comprising:

cell-penetrating peptide;

at least one antigen or antigenic epitope; And

at least one TLR peptide agonist,

in which components a)-c) are covalently bonded.

In this method, the individual is preferably administered the complex (intended for use) proposed in the present invention, the nucleic acid specified in the present description, the cell specified in the present description, the composition specified in the present description, the vaccine specified in the present description and/or the specified in the present description, a pharmaceutical composition.

Preferably, the individual has colorectal cancer and/or has been diagnosed with colorectal cancer.

Another object of the present invention is the use of any one of the following components: (I) the complex specified in the present description, and/or (II) cells, such as antigen presenting cells, loaded with the complex specified in the present description, for the preparation of an imaging composition intended for use in imaging techniques in the context of (diagnosing) colorectal cancer or for the preparation of diagnostic compositions (“diagnostic compositions”) intended for diagnosing colorectal cancer. The diagnostic composition proposed in the present invention for diagnosing colorectal cancer contains at least one component selected from:

(I) the complex, which is described above,

(II) a nucleic acid as described above,

(III) the vector described above,

(IV) a host cell, which is described above, and

(V) cell loaded with complex as described above.

Preferably, the diagnostic composition proposed in the present invention contains the complex described above.

In particular, the complex (intended for use) proposed in the present invention, the nucleic acid specified in the present description, a cell, such as an antigen presenting cell, loaded with the complex (intended for use) proposed in the present invention, the composition proposed in the invention In accordance with the invention, a pharmaceutical composition or a vaccine according to the invention, or most preferably a diagnostic composition according to the invention, can be used in diagnostics as a diagnostic tool, for example in assays (in vivo or in vitro), for example in immunoassays, for detection, prognosis, diagnosis or monitoring colorectal cancer.

For example, in vitro assays can be performed by delivering a complex of the invention to a cell, such as an antigen presenting cell, loaded with a complex of the invention, a composition of the invention, a composition of the invention. inventing a pharmaceutical composition or an inventive vaccine or most preferably an inventive diagnostic composition, to target cells typically selected, for example, from cultured animal cells, human cells or microorganisms, and monitoring the cellular response using biophysical methods, typically , known to specialists in this field. The target cells used for this purpose may typically be cultured cells (in vitro), for example cells isolated from a human or animal body, for example blood cells isolated from a human or animal body, or cells in vivo, i.e. e. cells contained in organs or tissues of living animals or humans, or microorganisms present in living animals or humans. Particularly preferred in this context are so-called markers or tags, which can be included in the complex (intended for use) proposed in the present invention, and, in particular, in the diagnostic composition proposed in the present invention.

Another aspect of the invention is a method for diagnosing colorectal cancer in an individual, the method comprising administering any of: (i) a complex of the invention, (ii) cells, such as antigen presenting cells loaded with the complex of the invention, or (iii) a pharmaceutical formulation containing the components specified in subparagraphs (I)-(II), to a specified individual or ex vivo into a sample obtained from the body of a specified individual.

Preferably, the uses and methods proposed in the present invention include the administration of the complex (intended for use) proposed in the invention.

In addition, the uses and methods proposed in the present invention include the administration of several complexes, cells or pharmaceutical formulations proposed in the invention. For example, in the uses and methods of the present invention, at least two different complexes are used or administered, each complex containing at least one antigen or antigenic epitope, and said antigen or antigenic epitope or (if said complex contains multiple antigens or antigenic epitopes) a specified subset of antigens or antigenic epitopes differs between the two complexes.

For example, the various complexes (I) contained in the composition described above may differ either in component a), i.e. peptides penetrating the cell, or component b), i.e. antigens or antigenic epitopes, or subsets of several antigens or antigenic epitopes, or component c), i.e. a TLR peptide agonist or a subset of several TLR peptide agonists; or the various complexes (I) contained in the composition described above may differ in two of the three components a), b) and c); or the various complexes (I) contained in the composition described above may differ in all three components a), b) and c) of the complex. Accordingly, the various nucleic acids (II) contained in the composition described above may differ in that they encode these various complexes; the various vectors (III) contained in the composition described above may differ in that they contain said different nucleic acids; the various host cells (IV) contained in the composition described above may differ in that they contain these different vectors; and the various complex (V)-loaded cells contained in the composition described above may differ in that they are loaded with said various complexes.

Additionally, in the uses and methods of the present invention, the cells of the present invention may be antigen presenting cells, particularly dendritic cells, more preferably dendritic cells from the individual being treated.

Route of administration

The complex (intended for use) proposed in the present invention; nucleic acid (intended for use) proposed in the present invention; a cell, such as an antigen presenting cell, loaded with the complex (intended for use) proposed in the present invention; the composition according to the invention; The pharmaceutical composition of the invention or the vaccine of the invention can be administered by any of the routes described above, including orally, parenterally, intravenously, rectally, or a combination of these routes. Parenteral administration includes, but is not limited to, intravenous, intraarterial, intraperitoneal, subcutaneous, intradermal, and intramuscular administration. Preferably the complex proposed in the present invention; a cell, such as an antigen presenting cell, loaded with the complex of the present invention; the composition according to the invention; The pharmaceutical composition according to the invention or the vaccine according to the invention is preferably administered by the enteral route, such as the oral, sublingual and rectal route. The complex (intended for use) proposed in the present invention; nucleic acid (intended for use) proposed in the present invention; a cell, such as an antigen presenting cell, loaded with the complex (intended for use) proposed in the present invention; the composition according to the invention; The pharmaceutical composition proposed in the invention or the vaccine proposed in the invention can also be applied locally, intratumorally, intradermally, subcutaneously, intramuscularly, intranasally or intranodally. The complex (intended for use) proposed in the present invention; nucleic acid (intended for use) proposed in the present invention; a cell, such as an antigen presenting cell, loaded with the complex (intended for use) proposed in the present invention; the composition according to the invention; The pharmaceutical composition proposed in the invention or the vaccine proposed in the invention can also be used in the form of an implant, which provides a slow release of the compositions, as well as using a slow controlled i.v. infusion. For example, the complex (intended for use) proposed in the present invention; nucleic acid (intended for use) proposed in the present invention; a cell, such as an antigen presenting cell, loaded with the complex (intended for use) proposed in the present invention; the composition according to the invention; The pharmaceutical composition according to the invention or the vaccine according to the invention can be administered subcutaneously or intradermally.

When administering the complex (intended for use) proposed in the present invention; nucleic acid (intended for use) proposed in the present invention; cells, such as an antigen presenting cell, loaded with the complex (intended for use) proposed in the present invention; the composition proposed in the invention; The pharmaceutical composition or vaccine of the invention may require several sequential injections. Thus, administration can be repeated at least twice, for example once as primary immunization injections and then as booster injections/administrations.

In particular, the complex (intended for use) proposed in the present invention; nucleic acid (intended for use) proposed in the present invention; a cell, such as an antigen presenting cell, loaded with the complex (intended for use) proposed in the present invention; the composition according to the invention; The pharmaceutical composition proposed in the invention or the vaccine proposed in the invention can be administered repeatedly or continuously. The complex (intended for use) proposed in the present invention; nucleic acid (intended for use) proposed in the present invention; a cell, such as an antigen presenting cell, loaded with the complex (intended for use) proposed in the present invention; the composition according to the invention; the pharmaceutical composition of the invention or the vaccine of the invention can be administered repeatedly or continuously over a period of at least 1, 2, 3 or 4 weeks; 2, 3, 4, 5, 6, 8, 10 or 12 months; or 2, 3, 4 or 5 years. For example, the complex (intended for use) proposed in the present invention can be administered twice a day, once a day, every two days, every three days, once a week, every two weeks, every three weeks, once a month or every two months. Preferably, the complex (intended for use) proposed in the present invention can be administered repeatedly, for example, once a week or (once) every two weeks.

In addition, the cell-penetrating peptide, components a), b) and c), i.e. at least one antigen or antigenic epitope and at least one TLR peptide agonist included in the complex (intended for use) proposed in the present invention may be contained in various compositions that are mixed before administration or that are administered simultaneously to an individual in need thereof .

According to one approach, the complex (intended for use) proposed in the present invention; nucleic acid (intended for use) proposed in the present invention; a cell, such as an antigen presenting cell, loaded with the complex (intended for use) proposed in the present invention; the composition according to the invention; The pharmaceutical composition according to the invention or the vaccine according to the invention can be administered directly to the patient using the routes of administration described above, in particular for pharmaceutical compositions. Alternatively, the complex (intended for use) proposed in the present invention; nucleic acid (intended for use) proposed in the present invention; a cell, such as an antigen presenting cell, loaded with the complex (intended for use) proposed in the present invention; the composition according to the invention; The inventive pharmaceutical composition or the inventive vaccine can be administered to a patient using an ex vivo approach, for example by introducing the pharmaceutical composition, vaccine or the inventive conjugated cargo molecule transporter mentioned above into cells, preferably autologous cells, i.e. . cells obtained from the body of the patient to be treated, and transplanting these cells into the area to be treated in the patient, optionally, by sorting and/or culturing these cells prior to treatment.

The dose administered to an individual, whether as a single dose or multiple doses, will vary significantly depending on various factors, including pharmacokinetic properties, the condition and characteristics of the individual (gender, age, body weight, health status, constitution), severity of symptoms, concomitant therapy, frequency of treatments and the required action.

Typically, for the treatment of cancer, a therapeutically effective dose of the complex (intended for use) proposed in the present invention is from about 0.01 to 5 mg per injection, in particular from about 0.1 to 2 mg per injection, or from about 0. 01 nmol to 1 mmol per injection, in particular 1 nmol to 1 mmol per injection, preferably 1 µmol to 1 mmol per injection.

Typically, for the treatment of cancer, a therapeutically effective dose of the complex (intended for use) proposed in the present invention is from about 0.2 million cells to 2 million cells per injection.

Combination therapy

Introduction of the complex (intended for use) proposed in the present invention; nucleic acid (intended for use) proposed in the present invention; cells, such as an antigen presenting cell, loaded with a complex (intended for use) proposed in the present invention; the composition proposed in the invention; The pharmaceutical composition proposed in the invention or the vaccine proposed in the invention according to the methods and applications proposed in the invention can be carried out individually or in combination with a co-agent suitable for the treatment and/or stabilization of colorectal cancer.

For example, in the case of treatment, prevention or stabilization of colorectal cancer, the administration of pharmaceutical compositions according to the methods and uses proposed in the invention can be carried out in combination with substances used for conventional chemotherapy, which are directed against solid colorectal tumors, and for the control of the occurrence of metastases, or any other molecule whose effect is to initiate programmed cell death, for example, with a co-agent selected from members of the tumor necrosis factor family, including (but not limited to) Fas ligand and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). According to another embodiment of the invention, administration of the complex (intended for use) proposed in the present invention; nucleic acid (intended for use) proposed in the present invention; cells, such as an antigen presenting cell, loaded with a complex (intended for use) proposed in the present invention; the composition proposed in the invention; The pharmaceutical composition proposed in the invention or the vaccine proposed in the invention according to the methods and applications proposed in the invention can be carried out in parallel with radiation therapy.

The scope of the invention includes the administration of the complex (intended for use) proposed in the present invention; nucleic acid (intended for use) proposed in the present invention; cells, such as an antigen presenting cell, loaded with a complex (intended for use) proposed in the present invention; the composition proposed in the invention; a pharmaceutical composition of the invention or a vaccine of the invention, wherein the administration to the individual is carried out prior to, simultaneously or sequentially with other therapeutic regimens or co-agents useful for treating and/or stabilizing colorectal cancer and/or preventing recurrence of colorectal cancer (for example, multiple regimens) drugs) in a therapeutically effective amount. The specified complex, specified cell, composition, vaccine or pharmaceutical composition, which is administered simultaneously with the specified co-agents, can be administered in the same or different compositions and using the same or different routes of administration.

These other therapeutic regimens or co-agents can be selected from the group consisting of radiation therapy, chemotherapy, surgery, targeted therapy (including the use of small molecules, peptides and monoclonal antibodies) and antiangiogenic therapy. As used herein, anti-angiogenic therapy refers to the administration of an agent that directly or indirectly targets tumor-associated vasculature.

Thus, the present invention also provides a combination of (I) the complex specified in the present description; And

(II) a chemotherapeutic agent, a targeted drug and/or an immunotherapeutic agent such as an immune checkpoint modulator.

Preferably for use in the prevention and/or treatment of colorectal cancer.

Traditional chemotherapeutic agents are cytotoxic, i.e. they work by killing cells that are dividing rapidly, which is one of the main properties of most cancer cells. Preferred chemotherapeutic agents for use in combination with the complex described herein are chemotherapeutic agents known to one skilled in the art for the treatment of colorectal cancer. Preferred chemotherapeutic agents for use in combination include 5-fluorouracil (5-FU), capecitabine (Xeloda®), irinotecan (Camptosar®), and oxaliplatin (Eloxatin®). It is also preferable that the complex specified herein be combined with a combination chemotherapy, preferably selected from (I) FOLFOX (5-FU, leucovorin and oxaliplatin); (II) Capecox (capecitabine and oxaliplatin); (III) 5-FU and leucovorin; (IV) FOLFOXIRI (leucovorin, 5-FU, oxaliplatin and irinotecan); and (V) FOLFIRI (5-FU, leucovorin and irinotecan). For cancer that has not spread, the preferred combination is with (I) FOLFOX (5-FU, leucovorin and oxaliplatin); (II) Capecox (capecitabine and oxaliplatin); or (III) 5-FU and leucovorin. In cases of advanced cancer, the preferred combination is with (IV) FOLFOXIRI (leucovorin, 5-FU, oxaliplatin and irinotecan); (I) FOLFOX (5-FU, leucovorin and oxaliplatin); or (V) FOLFIRI (5-FU, leucovorin and irinotecan).

Targeted drugs for use in combination with the complex specified herein for the treatment of colorectal cancer include drugs that target VEGF and drugs that target EGFR. Preferred examples of drugs that target VEGF include bevacizumab (Avastin®), ramucirumab (Cyramza®) or ziv-aflibercept (Zaltrap®). Preferred examples of drugs that target EGFR include cetuximab (Erbitux®), panitumumab (Vectibix®) or regorafenib (Stivarga®).

Immunotherapeutic agents for use in combination with the complex described herein for the treatment of colorectal cancer include vaccines, chimeric antigen receptors (CARs), checkpoint modulators and oncolytic viral therapies.

Preferred vaccines for use in combination with the complex described herein for the treatment of colorectal cancer include TroVax, OncoVax, IMA910, ETBX-011, MicOryx, EP-2101, MKC1106-PP, CDX-1307, V934/V935, MelCancerVac, imprimPGG , FANG, tecemotide, AlloStim, DCVax, GI-6301, AVX701, OCV-C02.

Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immunoreceptors, chimeric antigen receptors (CARs)) are engineered receptors that impart arbitrary specificity to an immune effector cell. Artificial T cell receptors (CARs) are preferred in the context of adoptive cell transfer. For this purpose, T cells are isolated from the patient and modified to express receptors specific for colorectal cancer. T cells, which can then recognize and destroy cancer cells, are reintroduced into the patient's body.

As used herein, the term “immune checkpoint modulator” (also referred to as “checkpoint modulator”) refers to a molecule or compound that modulates (e.g., fully or partially reduces, inhibits, interacts with, activates, stimulates, increases, enhances or maintains) the function of one or more checkpoint molecules. Thus, the immune checkpoint modulator may be an “immune checkpoint inhibitor” (also referred to as a “checkpoint inhibitor” or “inhibitor”) or an “immune checkpoint activator” (also referred to as a “checkpoint activator” or “ activator"). An “immune checkpoint inhibitor” (also referred to as a “checkpoint inhibitor” or “inhibitor”) completely or partially reduces, inhibits, interacts with, or negatively modulates the function of one or more checkpoint molecules. An “immune checkpoint activator” (also referred to as “checkpoint activator” or “activator”) fully or partially activates, stimulates, enhances, enhances, maintains or positively modulates the function of one or more checkpoint molecules.

Immune checkpoint modulators generally have the ability to modulate (i) autotolerance and/or (ii) the amplitude and/or duration of the immune response. Preferably, the immune checkpoint modulator used according to the present invention modulates the function of one or more human checkpoint molecules and is thus a “human checkpoint modulator.”

Checkpoint molecules are molecules, such as proteins, typically involved in immune response pathways and, for example, regulate T cell activation, T cell proliferation, and/or T cell function. Thus, the function of checkpoint molecules that is modulated (e.g., fully or partially reduced, inhibited, interacted with, activated, stimulated, increased, enhanced, or maintained) by checkpoint modulators typically represents (regulation of) T activation -cells, T cell proliferation and/or T cell function. Thus, immune checkpoint molecules regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. Many immune checkpoint molecules belong to the B7:CD28 family or the tumor necrosis factor receptor (TNFR) superfamily and, by binding to specific ligands, activate signaling molecules that are characterized by recruitment to the cytoplasmic domain (see Susumu Suzuki et al., Current status of immunotherapy Japanese Journal of Clinical Oncology, 2016: doi: 10.1093/jjco/hyv201 [Epub ahead of print]; see in particular Table 1).

Preferably, the immune checkpoint modulator for use in combination with the complex described herein for the treatment of colorectal cancer is an activator or inhibitor of one or more immune checkpoint molecules selected from CD27, CD28, CD40, CD122, CD137, OX40, GITR , ICOS, A2AR, В7-Н3, В7-Н4, BTLA, CD40, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, SEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO , GITR, TNFR and/or FasR/DcR3; or an activator or inhibitor of one or more of their ligands.

More preferably, the immune checkpoint modulator is an activator of a (co)stimulatory checkpoint molecule or an inhibitor of an inhibitory checkpoint molecule, or a combination thereof. Thus, the immune checkpoint modulator is more preferably (I) an activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR and/or ICOS or (II) an inhibitor of A2AR, B7-H3, B7-H4, BTLA, CD40 , CTLA-4, IDO, KIR, LAG3, PD-1, PDL-1, PD-L2, TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR and/or FasR/DcR3 .

Even more preferably, the immune checkpoint modulator is an inhibitory checkpoint molecule inhibitor (but preferably not a stimulatory checkpoint molecule inhibitor). Thus, even more preferably, the immune checkpoint modulator is an inhibitor of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PDL-1, PD-L2, TIM-3 , VISTA, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR and/or DcR3 of their ligand.

Preferably, the immune checkpoint modulator is also an activator of a stimulatory or costimulatory checkpoint molecule (but preferably not an activator of an inhibitory checkpoint molecule). Thus, even more preferably, the immune checkpoint modulator is an activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR and/or ICOS or a ligand thereof.

Even more preferably, the immune checkpoint modulator is a modulator of the CD40 pathway, the IDO pathway, the CTLA-4 pathway and/or the PD-1 pathway. In particular, the immune checkpoint modulator is preferably a CD40, CTLA-4, PD-L1, PD-L2, PD-1 and/or IDO modulator, more preferably the immune checkpoint modulator is a CTLA-4, PD-L1 inhibitor, PD-L2, PD-1 and/or IDO or a CD40 activator, even more preferably the immune checkpoint modulator is an inhibitor of CTLA-4, PD-L1, PD-1 and/or IDO and most preferably the immune checkpoint modulator is an inhibitor CTLA-4 and/or PD-1.

Even more preferably, the immune checkpoint modulator is a modulator of the CD40 pathway, the IDO pathway, the LAG3 pathway, the CTLA-4 pathway and/or the PD-1 pathway. In particular, the immune checkpoint modulator is preferably a modulator of CD40, LAG3, CTLA-4, PD-L1, PD-L2, PD-1 and/or IDO, more preferably the immune checkpoint modulator is an inhibitor of CTLA-4, PD- L1, PD-L2, PD-1, LAG3, and/or IDO or a CD40 activator, even more preferably the immune checkpoint modulator is an inhibitor of CTLA-4, PD-L1, PD-1, LAG3 and/or IDO, even more preferably the immune checkpoint modulator is an inhibitor of LAG3, CTLA-4 and/or PD-1 and most preferably the immune checkpoint modulator is an inhibitor of CTLA-4 and/or PD-1.

Thus, the checkpoint modulator for use in combination with the complex described herein for the treatment of colorectal cancer can be selected from known modulators of the CD40 pathway, the CTLA-4 pathway or the PD-1 pathway. Preferably, the checkpoint modulator for use in combination with the complex specified herein for the treatment of colorectal cancer can be selected from known modulators of the CD40 pathway, LAG3 pathway, CTLA-4 pathway or PD-1 pathway. Particularly preferably, the immune checkpoint modulator is a PD-1 inhibitor. Preferred CTLA-4 pathway and PD-1 pathway inhibitors include the monoclonal antibodies Yervoy ^® (ipilimumab; Bristol Myers Squibb) and tremelimumab (Pfizer/MedImmune), as well as Opdivo ^® (nivolumab; Bristol Myers Squibb), Keytruda ^® (pembrolizumab ; Merck), durvalumab (MedImmune/AstraZeneca), MEDI4736 (AstraZeneca; see WO 2011/066389 A1), MPDL3280A (Roche/Genentech; see US 8217149 B2), pidilizumab (CT-011; CureTech ), Medi0680 (AMR-514; ASTRAZENECA company), MSB-0010718C (Merck), MiH1 (AFFYMETRIX) and LambroLizumab (for example, described as HPD109A and its humanized derivatives H409A11, H409A16 and H409A17 in W O2008/156712; Hamid and al., N. Engl. J. Med. 369, 2013, pp. 134-144). More preferred checkpoint inhibitors include the CTLA-4 inhibitors Yervoy ^® (ipilimumab; Bristol Myers Squibb) and tremelimumab (Pfizer/MedImmune), as well as the PD-1 inhibitors Opdivo ^® (nivolumab; Bristol Myers Squibb), Keytruda ^® (pembrolizumab ; Merck), pidilizumab (CT-011; CureTech), MEDI0680 (AMP-514; AstraZeneca), AMP-224 and lambrolizumab (for example, described as hPD109A and its humanized derivatives h409A11, h409A16 and h409A17 in WO 2008/156712 ; in Hamid et al., N. Engl. J. Med. 369, 2013, pp. 134-144). As stated above, a preferred example of a LAG3 inhibitor is the anti-LAG3 monoclonal antibody BMS-986016 (Bristol-Myers Squibb). Other preferred examples of a LAG3 inhibitor include LAG525 (Novartis), IMP321 (Immutep) and LAG3-Ig described in WO 2009/044273 A2 and Brignon et al., Clin. Cancer Res. 15, 2009, cc. 6225-6231, as well as murine or humanized antibodies blocking human LAG3 (for example, IMP701 described in WO 2008/132601 A1), or fully human antibodies blocking human LAG3 (for example, described in EP 2320940 A2).

Preferably, the immune checkpoint modulator is not a CD40 modulator. In particular, the preferred immune checkpoint modulator is not a CD40 ligand. Preferably, the immune checkpoint modulator is also not an anti-CD40 antibody.

Also preferably, the checkpoint modulator for use in combination with the complex specified herein for the treatment of colorectal cancer is selected from the group consisting of pembrolizumab, ipilimumab, nivolumab, MPDL3280A, MEDI4736, tremelimumab, avelumab, PDR001, LAG525, INCB24360, varlimumab, urelumab, AMP-224 and CM-24.

Oncolytic viruses are engineered to cause cell lysis as a result of their replication in the tumor, thereby inactivating the anticancer immune response. The oncolytic virus-based therapy for use in combination with the complex described herein for the treatment of colorectal cancer is preferably selected from the group consisting of JX594 (thymidine kinase-deactivating variola virus), ColoAd1 (adenovirus), NV1020 (HSV-derived), ADXS11 -001 (attenuated listeria vaccine), Reolysin® (specific form of human reovirus), PANVAC (recombinant smallpox virus CEA-MUC-1-TRICOM), Ad5-hGCC-PADRE (recombinant adenovirus vaccine) and vvDD-CDSR ( smallpox virus).

Preferably, (I) the complex and (II) the chemotherapeutic agent, targeted drug, and/or immunotherapeutic agent, such as an immune checkpoint modulator, are administered at approximately the same time.

As used herein, the term “at about the same time” means, in particular, simultaneous administration or the option where immediately after administration of (I) a chemotherapeutic agent, a targeted drug and/or an immunotherapeutic agent, such as an immune checkpoint modulator, (II) complex is administered, or immediately after administration of (I) complex, (II) a chemotherapeutic agent, a targeted drug, and/or an immunotherapeutic agent, such as an immune checkpoint modulator, is administered. It will be apparent to one skilled in the art that the term "immediately thereafter" includes the time required to prepare the second administration - in particular, the time required to expose and disinfect the second administration site, as well as, if necessary, prepare the "administration device" (e.g. syringe, pump, etc.). Co-administration also includes the option where the periods of administration of (I) the complex and (II) the chemotherapeutic agent, targeted drug and/or immunotherapy agent, such as an immune checkpoint modulator, overlap, or, for example, the option where one component is administered over over a longer period of time, for example, over 30 minutes, 1 hour, 2 hours or more, for example, by infusion, and the other component is administered at a certain point in time over a specified long period. Administration of (I) the complex and (II) the chemotherapeutic agent, targeted drug, and/or immunotherapeutic agent, such as an immune checkpoint modulator, at approximately the same time is particularly preferred if different routes of administration are used and/or administration at different sites. areas.

Preferably also (I) the complex and (II) the chemotherapeutic agent, targeted drug and/or immunotherapeutic agent, such as an immune checkpoint modulator, are administered sequentially. This means that (I) the complex is administered before or after (II) a chemotherapeutic agent, a targeted drug and/or an immunotherapeutic agent such as an immune checkpoint modulator. When administered sequentially, the time interval between administration of the first component and administration of the second component preferably does not exceed one week, more preferably does not exceed 3 days, even more preferably does not exceed 2 days, and most preferably does not exceed 24 hours. Most preferably (I) complex and (II) ) a chemotherapeutic agent, a targeted drug, and/or an immunotherapeutic agent, such as an immune checkpoint modulator, are administered on the same day, with a time interval between the administration of the first component (checkpoint modulator or complex) and the administration of the second component (another modulator control points and complex) preferably does not exceed 6 hours, more preferably does not exceed 3 hours, even more preferably does not exceed 2 hours and most preferably does not exceed 1 hour.

Preferably, (I) the complex and (II) the chemotherapeutic agent, targeted drug, and/or immunotherapeutic agent, such as an immune checkpoint modulator, are administered via the same route of administration. Preferably also (I) the complex and (II) the chemotherapeutic agent, targeted drug and/or immunotherapeutic agent, such as an immune checkpoint modulator, are administered via different routes of administration.

In addition, (I) the complex and (II) a chemotherapeutic agent, a targeted drug and/or an immunotherapeutic agent such as an immune checkpoint modulator are preferably in different compositions. Alternatively, (I) the complex and (II) the chemotherapeutic agent, targeted drug and/or immunotherapeutic agent such as an immune checkpoint modulator are preferably in the same composition.

Thus, the present invention provides a pharmaceutical formulation containing a complex (intended for use) according to the invention or a cell (intended for use) according to the invention, especially an antigen presenting cell (intended for use) according to the invention, in combination with at least one co-agent, which can be used to treat and/or stabilize cancer, and/or prevent recurrence of colorectal cancer, and at least one pharmaceutically acceptable carrier.

In addition, the complex (intended for use) proposed in the present invention; a cell, such as an antigen presenting cell, loaded with the complex (intended for use) proposed in the present invention; the composition according to the invention; The pharmaceutical composition according to the invention or the vaccine according to the invention can be administered after surgery in which solid tumors are removed as a preventative measure for relapse and/or metastasis.

Additionally, administration of the imaging or diagnostic composition according to the methods and uses of the invention may be performed alone or in combination with a coagent useful for imaging and/or diagnosing colorectal cancer.

Individuals

The present invention can be used for any individual suffering from colorectal cancer or at risk of developing colorectal cancer. In particular, the therapeutic effect of said complex may manifest itself in the form of an immune response directed against said antigens or antigenic epitopes, in particular a response dependent on CD4 ⁺ T helper cells and/or cytotoxic CD8 ⁺ T cells and/or restricted to MHC class I molecules and/or MHC class II molecules.

According to the present invention, individuals preferably undergo surgical removal of the tumor.

The scope of the present invention is not limited to the specific embodiments of the invention presented in the present description. In fact, those skilled in the art upon reading the above description and the following drawings will recognize various modifications of the invention that can be made in addition to those set forth herein. These modifications fall within the scope of the appended claims.

All references cited herein are incorporated herein by reference.

Unless otherwise stated, all technical and scientific terms used herein have the same meaning as is known to one of ordinary skill in the art to which the present invention relates. Although methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention, acceptable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of disagreement, the present specification, including definitions, shall be relied upon. Moreover, materials, methods and examples are presented for purposes of illustration only and are not intended to limit the scope of the invention.

Brief description of drawings

Below is a brief description of the accompanying drawings. The drawings are intended to illustrate the present invention in more detail. However, they are not intended to limit the scope of the invention in any way.

The drawings show:

in fig. 1 - data obtained in example 1 on the expression of the activation marker CD40 in human blood monocyte-derived dendritic cells (DC) from one buffy coat. DCs were stimulated with 300 nM EDAZ13Mad5, Z13Mad5, Mad5, or 25 ng/ml LPS for 48 h. Isotype staining was also performed for each condition (isotype data not shown in Fig. 1) (one experiment);

in fig. 2 - data obtained in example 1 on the expression of the activation marker CD86 in human blood monocyte-derived dendritic cells (DC) from one buffy coat. DCs were stimulated with 300 nM EDAZ13Mad5, Z13Mad5, Mad5 or ng/ml LPS for 48 hours. Isotype staining was also performed for each condition (isotype data not shown in Fig. 2) (one experiment);

in fig. 3 - data obtained in example 1 on the expression of the activation marker HLADR in human blood monocyte-derived dendritic cells (DC) from one buffy coat. DCs were stimulated with 300 nM EDAZ13Mad5, Z13Mad5, Mad5 or ng/ml LPS for 48 hours. Isotype staining was also performed for each condition (isotype data not shown in Fig. 3) (one experiment);

in fig. 4 - data obtained in example 1 on the expression of the activation marker CD83 in human blood monocyte-derived dendritic cells (DC) from one buffy coat. DCs were stimulated with 300 nM EDAZ13Mad5, Z13Mad5, Mad5 or ng/ml LPS for 48 hours. Isotype staining was also performed for each condition (isotype data not shown in Fig. 4) (one experiment);

in fig. 5 - Data obtained in Example 2 for functional MHC class I-restricted cross-presentation in a murine in vitro system using bone marrow-derived dendritic cells (BMDCs) and splenocytes from various TCR transgenic mice. For this purpose, BMDCs were loaded overnight with 300 nM EDAZ13Mad5, EDAMad5, or Mad5. The efficiency of MHC class I-restricted presentation of the OVACD8 epitope and the gp 100 epitope was monitored after 4 days using CFSE-labeled OT1 cells and P-Mel cells, respectively. The effectiveness of MHC class II-restricted OVACD4 epitope presentation was monitored after 4 days using CFSE-labeled OT2 cells. As a control, BMDCs were pulsed for 1 h with 5 μM peptide (one experiment, representative of 2 separate experiments);

in fig. 6 - results obtained in example 3 for groups treated with compounds at a dose of 2 nmol. C57BL/6 mice were vaccinated twice (Wk0 and Wk2) using 2 nmol EDAMad5 or EDAZ13Mad5. The positive control group was vaccinated with Mad5 and MPLA (equimolar dose of EDA). Mice were bled 7 days after the last vaccination and stained with pentamer (3-4 mice per group, one experiment);

in fig. 7 - results obtained in example 3 for groups treated with compounds at a dose of 10 nmol. C57BL/6 mice were vaccinated twice (Wk0 and Wk2) with EDAMad5 or EDAZ13Mad5 at a dose of 10 nmol. The positive control group was vaccinated with Mad5 and MPLA (equimolar dose of EDA). Mice were bled 7 days after the last vaccination and stained with pentamer (3-4 mice per group, one experiment);

in fig. 8 - data obtained in example 3 on the percentage of pentamer-positive CD8 ⁺ -T cells in all tested groups. C57BL/6 mice were vaccinated twice (Wk0 and Wk2) using 10 nmol of EDAMad5 or EDAZ13Mad5. The positive control group was vaccinated with Mad5 and MPLA (equimolar dose of EDA). Mice were bled 7 days after the last vaccination and stained with pentamer (3-4 mice per group, one experiment);

in fig. 9 - data obtained in example 4 on the growth of tumors in 7 mice per group (average ± SD); *, p<0.05 EDAZ13Mad5 relative to the control group (2-way analysis of variance). C57BL/6 mice were implanted with sc 3×10 ⁵ EG7-OVA tumor cells in the left flank region and vaccinated twice (d5 and d13) by subcutaneous injection (sc) with EDAZ13Mad5, EDAMad5, Mad5 or Mad5 and MPLA at a dose of 10 nmol (dose, equimolar EDA) to the right lateral region. Tumor size was determined using calipers;

in fig. 10 - growth curves of individual tumors obtained in example 4 (for each of 7 mice per group). C57BL/6 mice were implanted with sc 3×10 ⁵ EG7-OVA tumor cells in the left flank region and vaccinated twice (d5 and d13) by subcutaneous (sc) injection at a dose of 10 nmol EDAZ13Mad5, EDAMad5, Mad5 or Mad5 and MPLA (dose, equimolar EDA) to the right lateral region. Tumor size was determined using calipers;

in fig. 11 - survival curve obtained in example 4 (A) for 7 mice per group; *, p<0.05 EDAZ13Mad5 relative to the control group (log-rank test) and (B) curve of the period without tumor progression for 7 mice per group; *, p<0.05 EDAZ13Mad5 relative to the control group (log-rank test);

in fig. 12 - data obtained in example 5 on the number of metastases in each experimental group. C57BL/6 mice were implanted with iv 1×10 ⁵ B16-OVA melanoma tumor cells and vaccinated twice (d0 and d9) by subcutaneous (sc) injection with a dose of 2 nmol EDAZ13Mad5, EDAMad5 or Z13Mad5 + MPLA (dose equimolar to EDA) or only MPLA to the right side area. Mice were sacrificed on day 13 and lungs were removed. The number of metastatic foci was counted in each lung. **, p<0.01; ****, p<0.0001 (unpaired t-test);

in fig. 13 - data obtained in example 6 on the number of metastases in each experimental group. C57BL/6 mice were vaccinated twice (d-21 and d-7) by subcutaneous (sc) injection of 2 nmol EDAZ13Mad5, EDAMad5, or Z13Mad5 + MPLA (dose equimolar to EDA) in the right flank. On day 0, mice were implanted with iv 1×10 ⁵ B16-OVA melanoma tumor cells. Mice were sacrificed on day 14 and lungs were removed. The number of metastatic foci was counted in each lung. *, p<0.05. ***, p<0.001 (unpaired t-test);

in fig. 14 - Results obtained in Example 8. HEK-hTLR2 cell lines were seeded in a flat-bottomed 96-well plate in culture medium, stimulated with 0.3 µM, 1 µM or 3 µM AnaxaZ13Mad5 or Z13Mad5Anaxa and incubated at 37°C for 24 hours. To obtain a positive control, 500 ng/ml Pam3CSK4 was used. (A) Add 20 µl of supernatant to QuantiBlue® detection medium and incubate at 37°C for 1 hour before determining the OD (620 nm). (B) IL-8 secretion was assessed (by ELISA) in the supernatant;

in fig. 15 - results obtained in example 9. C57BL/6 mice were vaccinated twice (Wk0 and Wk2), using a dose of 2 nmol Z13Mad5Anaxa or AnaxaZ13Mad5. Mice were bled 7 days after the last vaccination and stained with pentamer (one experiment);

in fig. 16 - results obtained in example 9. C57BL/6 mice were vaccinated twice (Wk0 and Wk2), using a dose of 2 nmol Z13Mad5Anaxa or AnaxaZ13Mad5. Mice were bled 7 days after the last vaccination and stained with pentamer (one experiment with 4 mice per group). *, p<0.05;

in fig. 17 - data obtained in example 10 on the growth of tumors in 7 mice per group (average ± SD). C57BL/6 mice were implanted with sc 3×10 ⁵ EG7-OVA tumor cells in the left flank region and vaccinated twice (d5 and d13) either by subcutaneous injection at a dose of 10 nmol AnaxZ13Mad5, Z13Mad5Anaxa, or by co-injection of sc-injection Z13Mad5 + Pam3CSK4 ( dose equimolar to the Anaxa dose) to the right lateral area. Tumor size was determined using calipers. *, p<0.05; ***, p<0.001, ****, p<0.0001;

in fig. 18 - growth curves of individual tumors obtained in example 10 (7 mice per group). C57BL/6 mice were implanted with sc 3×10 ⁵ EG7-OVA tumor cells in the left flank region and vaccinated twice (d5 and d 13) either by subcutaneous injection at a dose of 10 nmol AnaxZ13Mad5, Z13Mad5Anaxa, or by joint sc-injection of Z13Mad5 + Pam3CSK4 (dose equimolar to Anaxa) to the right lateral area. Tumor size was determined using calipers;

in fig. 19 - survival curves obtained in example 10 for 7 mice per group. C57BL/6 mice were implanted with sc 3×10 ⁵ EG7-OVA tumor cells in the left flank region and vaccinated twice (d5 and d13) either by subcutaneous injection at a dose of 10 nmol AnaxZ13Mad5, Z13Mad5Anaxa, or by co-injection of sc-injection Z13Mad5 + Pam3CSK4 ( dose equimolar to Anaxa) to the right lateral region. Tumor size was determined using calipers. *, p<0.05, **, p<0.01, ****, p<0.0001 (log-rank test);

in fig. 20 - tumor growth curves obtained in example 11 in 7 mice per group (mean ± SD). C57BL/6 mice were implanted with sc 3×10 ⁵ EG7-OVA tumor cells in the left flank and vaccinated twice (d5 and d13) at a dose of 2 nmol by subcutaneous injection of Hp91Z13Mad5,

EDAZ13Mad5, Z13Mad5 Anaxa, Z13Mad5EDA or Z13Mad5 and MPLA (dose equimolar to EDA) to the right lateral area. *, p<0.05; **, p<0.01; ***, p<0.001; p<0.0001 (2-way ANOVA on day 23);

in fig. 21 - growth curves of individual tumors obtained in example 11 (7 mice per group). C57BL/6 mice were implanted with sc 3×10 ⁵ EG7-OVA tumor cells in the left flank region and vaccinated twice (d5 and d 13) by subcutaneous injection at a dose of 2 nmol Hp91Z13Mad5, EDAZ13Mad5, Z13Mad5 Anaxa, Z13Mad5EDA or Z13Mad5 and MPLA (dose , equimolar EDA) to the right lateral region;

in fig. 22 - survival curves obtained in example 11 for all 7 mice per group. The graph shows median survival (m.s.). *, p<0.05; **, p<0.01 (log-rank test);

in fig. 23 - data obtained in example 12 on the growth of tumors in 7 mice per group (average ± SD); ****, p<0.0001 (log-rank test). C57BL/6 mice were implanted with sc 3×10 ⁵ EG7-OVA tumor cells in the left flank and vaccinated twice (d5 and d13) by subcutaneous injection into the right flank of Z13Mad5Anaxa at a dose of either 0.5 nmol, 2 nmol, or 10 nmoles. Tumor size was determined using calipers;

in fig. 24 - data obtained in example 13 on SIINFEKL-specific CD8 T-cell responses detected in the blood of C57BL/6 mice vaccinated three times (once in Wk0, once in Wk2 and once in Wk4) s.c, i.d. or i.m. using Z13Mad5Anaxa at a dose of 0.5 nmol (A) or 2 nmol (B). Blood was collected from mice 7 days after the 2nd and 3rd vaccinations and stained with multimer (one experiment with 4 mice per group). *, p<0.05;

in fig. 25 - data obtained in example 13 on the expression of KLRG1 (A) and the expression of PD-1 (B), for the analysis of which multimer-positive CD8 T cells were used (one experiment with 4 mice per group). In general, the method was as follows: C57BL/6 mice were vaccinated three times (once in Wk0, once in Wk2 and once in Wk4) s.c, i.d. or i.m. using Z13Mad5 Anaxa at a dose of 2 nmol. Blood was taken from mice 7 days after the 2nd and 3rd vaccinations and FACS staining was performed;

in fig. 26 - Data from Example 14 on SIINFEKL-specific CD8 T-cell responses detected in C57BL/6 mice vaccinated twice (once in Wk0 and once in Wk2) intranodally with Z13Mad5Anaxa at a dose of 0.5 nmol . Blood was collected from mice 7 days after the 2nd vaccination and stained with multimer (3 mice per group);

in fig. 27 - data obtained in example 15 on the percentage of pentamer-positive cells among CD8 T cells (A and B; *, p < 0.05) and geometric mean values for (expression of) KLRG1 in pentamer-positive CD8 T cells (B and D). In general, the method was as follows: C57BL/6 mice were vaccinated 3 times (A and B: Wk0, Wk2 and Wk4; B and D: Wk0, Wk2 and Wk8) s.c. using Z13Mad5Anaxa at a dose of 2 nmol. Mice were bled 7 days after the last vaccination and stained with pentamer (one experiment, 4 mice per group);

in fig. 28 - data obtained in example 15 on the percentage of multimer-positive cells among CD8 T cells (A and D); geometric means for KLRG1 in multimer-positive CD8 T cells (B and E) and geometric means for PD1 in multimer-positive CD8 T cells (B and E). A-B: C57BL/6 mice were vaccinated 3 times on day 0, day 3 and day 7 and blood was collected on day 7 and day 14. D-E, C57BL/6 mice were vaccinated 3 times on day 0, day 7 and day 14 and blood was drawn on day 14 and day 21. Vaccination was carried out s.c. using Z13Mad5Anaxa at a dose of 0.5 nmol. Multimer staining was performed on blood samples (one experiment, 4 mice per group);

in fig. 29 - data obtained in example 16 on the secretion of IL-6, indicating the activation of APC after incubation of BMDC with various constructs, which are indicated in the drawing. The overall method was as follows: BMDCs were plated in a flat-bottomed 96-well plate in culture medium, stimulated with 1 μM Z13Mad5Anaxa, Mad5Anaxa, Z13Mad5, EDAZ13Mad5, or EDAMad5, and incubated for 24 h at 37°C. IL-6 secretion was quantified in the supernatant using ELISA. Mean values ± SEM for 2–3 separate experiments are presented;

in fig. 30 - data obtained in example 16 on the secretion of TNF-α, indicating the activation of APC after incubation of Raw 264.7 cells with various constructs indicated in the drawing. In general, the method was as follows: Raw 264.7 cells were seeded in a flat-bottomed 96-well plate in culture medium, stimulated with 1 μM Z13Mad5Anaxa, Mad5Anaxa or Z13Mad5 and incubated for 24 h at 37°C. TNF-α secretion was quantified in the supernatant using ELISA. Mean values ± SEM for 2–3 separate experiments are presented;

in fig. 31 - data obtained in example 17 on the secretion of IL-8, indicating the binding of TLR4 after incubation of HEK-hTLR4 cells with the various constructs indicated in the drawing. In general, the method was as follows: HEK-hTLR4 cells were seeded in a flat-bottomed 96-well plate in culture medium, stimulated with 1 μM Z13Mad5Anaxa, Mad5Anaxa, Z13Mad5, EDAZ13Mad5 or EDAMad5 and incubated for 24 h at 37°C. IL-8 secretion was quantified in the supernatant using ELISA. Means ± SEM for 2 separate experiments are presented;

in fig. 32 - data obtained in example 18 on the number of metastases in a model of lung metastasis using a semi-therapeutic regimen. In general, the method was as follows: C57BL/6 mice were implanted with iv 1 × 10 ⁵ B16-OVA melanoma tumor cells and vaccinated twice (d0 and d9) by subcutaneous injection at a dose of 2 nmol EDAZ13Mad5, Z13Mad5 + MPLA (dose equimolar EDA) or MPLA only to the right side area. Mice were sacrificed on day 13 and lungs were removed. The number of metastatic foci was counted in each lung. **, p<0.01 (one-way analysis of variance with Tukey's test for multiple comparisons);

in fig. 33 - data obtained in example 19 on the number of metastases in a model of lung metastasis using a semi-therapeutic regimen. In general, the method was as follows: C57BL/6 mice were implanted with iv 1×10 ⁵ B16-OVA melanoma tumor cells and vaccinated twice (d0 and d9) by subcutaneous injection (sc) at a dose of 0.5 nmol Z13Mad5Anaxa, Mad5Anaxa or Z13Mad5 + Pam3CSK4 (dose equimolar to Anaxa) to the right lateral area. Mice were sacrificed on day 21 and lungs were removed. The number of metastatic foci was counted in each lung. *, p<0.05; **, p<0.01 (unpaired t-test);

in fig. 34 - data obtained in example 20 for quantitative assessment of SIINFEKL-specific CD8 T cells in the Quad-Gl261 glioblastoma model. In general, the method was as follows: C57BL/6 mice were implanted with ie 5×10 ⁵ Gl261-Quad tumor cells and vaccinated twice (d7 and 21) by sc injection at a dose of 2 nmol Z13Mad5Anaxa or 2 nmol Z13Mad5 and 2 nmol Anaxa. SIINFEKL-specific CDS-T cells were quantified in blood and BIL (brain-infiltrating leukocytes) at d28 by multimer staining (5-8 mice per group);

in fig. 35 - data on the secretion of cytokines obtained in example 20. In general, the method was as follows: C57BL/6 mice were implanted with ie 5×10 ⁵ Gl261-Quad tumor cells and vaccinated twice (d7 and 21) by sc injection at a dose of 2 nmol Z13Mad5Anaxa or 2 nmol Z13Mad5 and 2 nmol Anaxa. BIL were isolated and cultured for 6 h with mature BMDCs loaded or unloaded with SllNFEKL peptide in the presence of brefeldin A before intracellular cytokine staining. Data are presented as % CD8 T cells secreting cytokine (5-8 mice per group);

in fig. 36 - data obtained in Example 21 on the effect of Z13Mad5Anaxa on survival in the Quad-Gl261 glioblastoma model. In general, the method was as follows: C57BL/6 mice were implanted with ie 5x10 ⁵ Gl261-Quad tumor cells and vaccinated three times (d7, d21 and d35) by sc-injection of Z13Mad5Anaxa at a dose of 2 nmol. Mice were weighed daily and sacrificed when weight loss reached more than 15%;

in fig. 37 - Data obtained in Example 22 on the effects of Z13Mad5Anaxa on tumor growth and survival in the subcutaneous EG7-OVA tumor model using a prophylactic regimen. In general, the method was as follows: C57BL/6 mice were vaccinated twice (d-21 and d-7) by sc injection of Z13Mad5Anaxa at a dose of 0.5 nmol in the right flank, and then implanted on day 0 sc 3×10 ⁵ EG7-OVA tumor cells in the left lateral area. Tumor size was determined using calipers. (A) Tumor growth in 7 mice per group (mean ± SD); ****, p<0.0001 (2-way ANOVA at day 30). (B) Survival curve for 7 mice per group. The graph shows median survival (ms). ***, p<0.001 (log-rank test);

in fig. 38 - Data obtained in Example 23 on the effects of Z13Mad5Anaxa on tumor growth and survival in the subcutaneous B16-OVA tumor model using a therapeutic regimen on mature tumors. In general, the method was as follows: C57BL/6 mice were implanted with sc 1×10 ⁵ B16-OVA tumor cells in the left flank and vaccinated twice (d14 and d21) by sc-injecting Z13Mad5Anaxa at a dose of 0.5 nmol in the right flank region. (A) Tumor growth in 7 mice per group (mean ± SD); *, p < 0.05 (2-way ANOVA on day 32). (B) Survival curve for 7 mice per group. The graph shows median survival (ms); **, p<0.01; ***, p<0.001;

in fig. 39 - data obtained in example 24 on the effect of CPP as part of the Z13Mad5Anaxa construct on tumor growth and survival in the subcutaneous EG7-OVA tumor model. In general, the method was as follows: C57BL/6 mice were implanted on day 0 sc with 3×10 ⁵ EG7-OVA tumor cells in the left flank region, and then vaccinated twice (d5 and d13) by sc injection of Z13Mad5Anaxa or Mad5Anaxa at a dose of 0.5 nmol to the right side region. Tumor size was determined using calipers. (A) Tumor growth in 7 mice per group (mean ± SD); ****, p<0.0001. (B) Survival curve for 7 mice per group. The graph shows median survival (ms). **, p<0.01; ***, p<0.001;

in fig. 40 - data obtained in example 25 on the effect of complexes containing various CPPs on the immune response. C57BL/6 mice were vaccinated five times (Wk0, Wk2, Wk4, Wk6 and Wk8) s.c. using a dose of either 2 nmol (A) or 0.5 nmol (B) Z13Mad5Anaxa, Z14Mad5Anaxa or Z18Mad5Anaxa. Mice were bled 7 days after the 2nd, 3rd, 4th and 5th vaccinations and multimer staining was performed (one experiment, 4 mice per group). *, p < 0.05 when comparing vaccinated and naive mice at each time point except the Vac2 time point for Z18Mad5Anaxa vaccinated mice;

in fig. 41 - data obtained in example 26 on the effects of complexes containing various CPPs on CD8 T cells in the spleen (A), draining lymph nodes (B) and bone marrow (C). C57BL/6 mice were vaccinated five times (Wk0, Wk2, Wk4, Wk6 and Wk8) s.c using 2 nmol Z13Mad5Anaxa or Z14Mad5Anaxa. Nine days after the 5th vaccination, mice were sacrificed, organs were removed, and multimer staining was performed;

in fig. 42 - data obtained in example 26 on the effects of complexes containing various CPPs on T cells in the spleen (CD8 T-cell response (A) and CD4 T-cell response (B)). C57BL/6 mice were vaccinated five times (Wk0, Wk2, Wk4, Wk6 and Wk8) s.c using 2 nmol Z13Mad5Anaxa or Z14Mad5Anaxa. (A) 9 days after the 5th vaccination, an Elispot assay was performed on spleen cells stimulated with the SIINFEKL OVACD8 peptide. (B) 9 days after the 5th vaccination, an Elispot assay was performed on spleen cells stimulated with the OVACD4 peptide;

in fig. 43 - data obtained in example 26 on the effect of complexes containing various CPPs on the effector function of CDS-T cells. C57BL/6 mice were vaccinated five times (Wk0, Wk2, Wk4, Wk6 and Wk8) s.c using 2 nmol Z13Mad5Anaxa or Z14Mad5Anaxa. 9 days after the 5th vaccination, intracellular staining of spleen cells stimulated with the SIINFEKL OVACD8 peptide was carried out;

in fig. 44 - data obtained in example 27 on the effect of complexes containing various CPPs on tumor growth (A) and survival rates (B). C57BL/6 mice were implanted with sc 3×10 ⁵ EG7-OVA tumor cells in the left flank and vaccinated twice (d5 and d13) with sc injection of Z13Mad5Anaxa or Z14Mad5Anaxa at a dose of 0.5 nmol in the right flank. (A) Tumor growth in 7 mice per group (mean ± SD); *, p<0.05; ****, p<0.0001 (2-way ANOVA, tested on day 28). (B) Survival curve for 7 mice per group. The graph shows median survival (ms). *, p<0.05; **, p<0.01; ***, p<0.001 (log-rank test);

in fig. 45 - data obtained in example 28 on the effect of complexes containing various CPPs on the immune response. C57BL/6 mice were vaccinated three times (Wk0, Wk2 and Wk4) s.c using 2 nmol (A) or 0.5 nmol (B) EDAZ13Mad5, EDAZ14Mad5 or EDAZ18Mad5. Mice were bled 7 days after the 3rd vaccination and stained with multimer (one experiment, 4 mice per group). *, p<0.05;

in fig. 46 - data obtained in example 29 on the effect of EDAZ14Mad5 on tumor growth (A) and survival rates (B). C57BL/6 mice were implanted with sc 3×10 ⁵ EG7-OVA tumor cells in the left flank and vaccinated twice (d5 and d 13) with sc injection of EDAZ14Mad5 at a dose of 2 nmol in the right flank. Left panel: tumor growth in 7 mice per group (mean ± SD); **, p<0.01 (2-way ANOVA, tested on day 27). Right panel: survival curve for 7 mice per group. The graph shows median survival (ms);

in fig. 47 - results of quantitative assessment of SIINFEKL-specific CD8 T cells obtained in example 30 in the Quad-Gl261 glioblastoma model. In general, the method was as follows: C57BL/6 mice were implanted with ie 5×10 ⁵ Gl261-Quad tumor cells and vaccinated twice (d7 and 21) by sc injection of Z13Mad5Anaxa at a dose of 2 nmol or Z13Mad5 at a dose of 2 nmol and Anaxa at a dose of 2 nmol. SIINFEKL-specific CD8 T cells were quantified in blood and BIL at d28 by multimer staining (7–16 mice per group);

in fig. 48 - data on the secretion of cytokines obtained in example 30. In general, the method was as follows: C57BL/6 mice were implanted with ie 5×10 ⁵ Gl261-Quad tumor cells and vaccinated twice (d7 and 21) by sc injection of Z13Mad5Anaxa at a dose of 2 nmol or Z13Mad5 at a dose of 2 nmol and Apakha at a dose of 2 nmol. BIL were isolated and cultured for 6 h with mature BMDCs loaded or unloaded with SllNFEKL peptide in the presence of brefeldin A before intracellular cytokine staining. Data are presented as % CDS-T cells secreting cytokine (7-16 mice per group);

in fig. 49 - data obtained in example 31 on the effect of Z13Mad8Anaxa on T cells in the spleen (CD8 T cell response (A) and CD4 T cell response (B)). C57BL/6 mice were vaccinated four times (Wk0, Wk2, Wk4 and Wk6) s.c. using Z13Mad8Anaxa at a dose of 2 nmol. (A) One week after the 4th vaccination, Elispot assay was performed on spleen cells stimulated with gp70CD8 peptide. (B) One week after the 4th vaccination, Elispot assay was performed on spleen cells stimulated with gp70CD4 peptide;

in fig. 50 - data obtained in example 32 on the effect of Z13Mad11Anaxa on the number of metastases in the B16 lung metastasis model (A) and on the T-cell response in the spleen (B). C57BL/6 mice were vaccinated twice (day 0, day 10) s.c. using Z13Mad11Anaxa at a dose of 1 nmol;

in fig. 51 - data obtained in example 33 on the effect of Z13Mad9Anaxa on T cells in the spleen (CD8 T cell response). C57BL/6 mice were vaccinated four times (Wk0, Wk2, Wk4 and Wk6) s.c. using Z13Mad9Anaxa at a dose of 2 nmol. One week after the 4th vaccination, an Elispot assay was performed on spleen cells stimulated with adpgk peptide;

in fig. 52 - data obtained in example 34 on the effect of complexes containing various CPPs on the immune response. C57BL/6 mice were vaccinated twice (Wk0 and Wk2) s.c. using either Z13Mad5Anaxa or TatFMad5Anaxa at a dose of 2 nmol. Mice were bled 7 days after the 2nd vaccination and stained with multimer (one experiment, 8 mice per group):

in fig. 53 shows the results of quantitative assessment of SIINFEKL-specific CD8 T cells in naive mice obtained in example 35. In general, the method was as follows: C57BL/6 mice were vaccinated once (day 0) by sc injection with Z13Mad5Anaxa at a dose of 2 nmol (Z13Mad5Anaxa group), or with Z13Mad5 at a dose of 2 nmol and Anaxa at a dose of 2 nmol (group “Z13Mad5+Anaxa”). SIINFEKL-specific CD8 T cells were quantified in blood at d7 by multimer staining (4–8 mice per group);

in fig. 54 - data obtained in example 36 on the effect of Z13Mad12Anaxa on T cells in the blood (CD8 T cell response). C57BL/6 mice were vaccinated twice (Wk0 and Wk2) s.c. using Z13Mad12Anaxa at a dose of 2 nmol. 1 week after the 2nd vaccination, multimer staining for the reps1 neoantigen was performed on blood cells;

in fig. 55 - data obtained in example 37 on the expression of activation markers HLA-DR, CD83, CD80 and CD86 (from left to right) in dendritic cells (DC) isolated from human blood monocytes from one buffy coat. DCs were stimulated with 300 nM Z13Mad5Anaxa (lower panels) or Z13Mad5 (upper panels) for 48 h. Isotype staining was also performed for each condition.

in fig. 56 - data obtained in example 38 on the percentage of multimer-positive cells (% of CD8 T cells) in the process of repeated vaccination. In general, the method was as follows: C57BL/6 mice were vaccinated subcutaneously 6 times (weeks 0, 2, 4, 8, 12, 16) using Z13Mad5Anaxa at a dose of 2 nmol. Mice were bled 7 days after each vaccination or before vaccination and stained with pentamer (two experiments, 4 mice per group). The arrows below the time axis indicate the time points of vaccination;

in fig. 57 - data obtained in example 39 on tumor volumes (A) and survival levels (B) of mice vaccinated with Z13Mad11Anaxa and control mice on the MS-38 tumor model. In general, the method was as follows: C57BL/6 mice were implanted with sc 2×10 ⁵ MC38 tumor cells in the left lateral region and vaccinated twice (-d21 and -d7 before tumor implantation) by sc-injection of Z13Mad11Anaxa at a dose of 2 nmol in the right side area. (A) - tumor growth data and (B) - survival curve for 7 mice per group. The graph shows median survival (ms). *, p<0.05; **, p<0.01 (log-rank test);

in fig. 58 - data obtained in example 40 on the release of selected cytokines after administration of the complex proposed in the present invention in general, the method consisted of the following: C57BL/6 mice were injected i.v. Z13Mad5Anaxa at a dose of 10 nmol. At 0.5, 1 and 3 hours after administration, blood samples were obtained to monitor serum IL-6, TNFα, IFNY, IL1-β and IL12 using a Luminex multiplex (n=4);

in fig. 59 - data obtained in example 41 on the adpgk neoantigen-specific immune response in the tumor region (tumor infiltrating cells, TILs) in control (column (A)) and Z13Mad12Anaxa vaccinated mice (column (B)). FACS-derived scatter plots for TILs are presented. Each dot plot shows data for multimer-positive cells (% of CD8 T cells);

in fig. 60 - data obtained in example 41 on the reps1 neoantigen-specific immune response in the tumor region (tumor infiltrating cells, TILs) in control (column (A)) and Z13Mad12Anaxa vaccinated mice (column (B)). FACS-derived dot plots for blood cells are presented. Each dot plot shows data for multimer-positive cells (% of CD8 T cells);

in fig. 61 - data obtained in example 41 on neoantigen-specific immune response in the tumor region (tumor infiltrating cells, TILs) in control and Z13Mad12Anaxa vaccinated mice. Data on multimer-positive cells (% of CD8 T cells) for each epitope (adpgk (A) and reps1 (B)) are presented;

in fig. 62 - data obtained in example 42 on the expression of activation markers HLA-DR, CD83, CD80 and CD86 in dendritic cells (DCs) isolated from human blood monocytes from one buffy coat. DCs were stimulated with 300 nM ATP110 overnight. Isotype staining was also performed for each condition;

in fig. 63 - data obtained in example 42 on the expression of activation markers HLA-DR, CD83, CD80 and CD86 in human blood monocyte-derived dendritic cells (DC) from one buffy coat. DCs were stimulated with 300 nM ATP112 overnight. Isotype staining was also performed for each condition;

in fig. 64 - data obtained in example 42 on the expression of activation markers HLA-DR, CD83, CD80 and CD86 in dendritic cells (DCs) isolated from human blood monocytes from one buffy coat. DCs were stimulated with 300 nM ATP115 overnight. For each condition, the indicated isotype staining was also performed;

in fig. 65 - data obtained in example 42 on the expression of activation markers HLA-DR, CD83, CD80 and CD86 in human blood monocyte-derived dendritic cells (DC) from one buffy coat. DCs were stimulated with 300 nM ATP117 overnight. Isotype staining was also performed for each condition;

in fig. 66 - data obtained in example 42 on the expression of activation markers HLA-DR, CD83, CD80 and CD86 in human blood monocyte-derived dendritic cells (DC) from one buffy coat. DCs were stimulated with 300 nM ATP118 overnight. Isotype staining was also performed for each condition;

in fig. 67 - data obtained in example 42 on the expression of activation markers HLA-DR, CD83, CD80 and CD86 in human blood monocyte-derived dendritic cells (DC) from one buffy coat. DCs were stimulated with 300 nM ATP119 overnight. Isotype staining was also performed for each condition;

in fig. 68 - data obtained in example 42 on the expression of activation markers HLA-DR, CD83, CD80 and CD86 in human blood monocyte-derived dendritic cells (DC) from one buffy coat. DCs were stimulated with 300 nM ATP120 overnight. Isotype staining was also performed for each condition;

in fig. 69 - data obtained in example 42 on the expression of activation markers HLA-DR, CD83, CD80 and CD86 in human blood monocyte-derived dendritic cells (DC) from one buffy coat. DCs were stimulated with 300 nM ATP122 overnight. Isotype staining was also performed for each condition;

in fig. 70 - data obtained in example 42 on the expression of activation markers HLA-DR, CD83, CD80 and CD86 in dendritic cells (DCs) isolated from human blood monocytes from one buffy coat. DCs were stimulated with 300 nM ATP123 overnight. Isotype staining was also performed for each condition;

in fig. 71 - data obtained in example 42 on the expression of activation markers HLA-DR, CD83, CD80 and CD86 in human blood monocyte-derived dendritic cells (DC) from one buffy coat. DCs were stimulated with 300 nM ATP125 overnight. Isotype staining was also performed for each condition;

in fig. 72 - data obtained in example 43 on tumor growth (A) and survival levels (B) for 13-14 mice per group (mean ± SD). C57BL/6 mice were implanted with sc 2×10 ⁵ MC-38 tumor cells in the back area. Mice from the “Z13Mad12Anaxa” and “Z13Mad 12Anaxa + anti-PD1” groups were vaccinated 3 times (d3, d 10 and d 17) by subcutaneously injecting Z13Mad12Anaxa at a dose of 2 nmol into the base of the tail. 200 μg of anti-PD1 antibody was administered IP on each of days 6, 10, 13, 17, 20, 24 and 27 to mice from the anti-PD1 and Z13Mad12Anaxa + anti-PD1 groups. Tumor size was determined using calipers. Each tumor growth curve indicates the number of tumor-free (tumor-free) mice in each group. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001;

in fig. 73 - growth curves of individual tumors obtained in example 43 for 13-14 mice per group. C57BL/6 mice were implanted with sc 2×10 ⁵ MC-38 tumor cells in the back area. Mice from the Z13Mad12Anaxa and Z13Mad12Anaxa + anti-PD1 groups were vaccinated 3 times (d3, d10 and d17) by subcutaneously injecting Z13Mad12Anaxa at a dose of 2 nmol into the base of the tail. 200 μg of anti-PD1 antibody was administered IP on each of days 6, 10, 13, 17, 20, 24 and 27 to mice from the anti-PD1 and Z13Mad12Anaxa + anti-PD1 groups. Tumor size was determined using calipers;

in fig. 74 - results obtained in example 44. C57BL/6 mice were vaccinated once using ATP128 at a dose of 4 nmol. Mice were bled after 7 days and Elispot assays were performed using blood cells stimulated with ATP128-loaded dendritic cells (one experiment);

in fig. 75 - activation index data obtained in example 45 for human blood monocyte-derived dendritic cells (DC) from ten different buffy coats. DCs were stimulated with 300 nM ATP128 overnight. To obtain negative and positive controls, cells were incubated with buffer and MPLA, respectively;

in fig. 76 - data obtained in example 46 on regions of ATP128 containing peptides that are found to be presented by MHC class I (A) and class II (B) on the surface of human dendritic cells from two different donors, donor 9 (solid line) and donor 10 (dashed line), after loading overnight with ATP128, and the corresponding number of peptides that are presented on MHC class I and class II (B). The underlined numbers correspond to peptides that have been previously described in the literature as immunogenic peptides.

Examples

Below are specific examples illustrating various embodiments and objects of the invention. However, the scope of the present invention is not limited to the specific embodiments described herein. The preparations and examples presented below are given for the purpose of better understanding of the invention by those skilled in the art and the implementation of the present invention in practice. However, the scope of the present invention is not limited to the exemplary embodiments of the invention, which are presented only for the purpose of illustrating specific aspects of the invention, and functionally equivalent methods fall within the scope of the invention. Thus, various modifications of the invention in addition to those set forth in the present description will be apparent to those skilled in the art from the above description, the accompanying drawings and the examples described below. These modifications fall within the scope of the appended claims.

Example 1: In Vitro Maturation of Human Dendritic Cells

The basis of the present study was to study the ability of the complex proposed for use according to the present invention to induce the maturation of dendritic cells. In the present study, the complex proposed for use according to the present invention was a fusion protein containing the cell penetrating peptide "Z13", the protein "MAD5", consisting of various CD8 ⁺ - and CD4 ⁺ epitopes from various antigens, and a TLR4 agonist peptide "EDA". Thus, a fusion protein was created with the EDA peptide at the N-terminal position and various control conjugated proteins without Z13 or EDA, or without both.

Thus, the following constructs were created in which the amino acid sequence of the cell-penetrating peptide “Z13” is underlined, and the TLR peptide agonist “EDA” is indicated in italics:

EDAZ13Mad5

Subsequence:

Molecular weight: 25057 Yes.

Characteristics:

- The Mad5 cargo molecule contains epitopes OVACD4, gp100CD8, EalphaCD4 and OVACD8.

- Contains the TLR agonist EDA (Lasarte J.J. et al., The extra domain A from fibronectin targets antigens to TLR4-expressing cells and induces cytotoxic T cell responses in vivo. J Immunol, 178(2), 2007, cc. 748-756) .

- Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, 2 mM DTT, 1 M L-arginine, pH 8.

- Endotoxin level: <0.01 endotoxin units (EU)/µg.

Z13Mad5

Subsequence:

Molecular weight: 15196 Yes

Characteristics:

- The Mad5 cargo molecule contains epitopes OVACD4, gp100CD8, EalphaCD4 and OVACD8.

- Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, 2 mM DTT, 1 M L-arginine, pH 9.

- Endotoxin level:

- batch 1: 0.32 EU/mg

- batch 2: 0.44 EU/mg.

Mad5

Subsequence:

Molecular weight: 10154.6 Yes

Characteristics:

- The Mad5 cargo molecule contains epitopes OVACD4, gp100CD8, EalphaCD4 and OVACD8.

- Storage buffer: 50 mM Tris-Cl, 150 mM NaCl, 10% glycerol, 2 mM DTT, 0.5 M L-arginine, pH 8.

- Endotoxin level: 0.069 EU/mg.

The proteins EDAZ13Mad5, Z13Mad5 and Mad5 were examined for their ability to induce maturation of human dendritic cells (DC). After incubation for 48 h with 300 nM protein, the expression of activation markers (CD86, CD40, CD83 and HLA-DR) on human DCs was assessed by FACS (Fig. 1-4). Buffers specific for each protein were used as a negative control.

Results for CD40 are presented in FIG. 1, for CD86 in FIG. 2, for HLADR in FIG. 3 and for CD83 in FIG. 4. While the EDAZ13Mad5 construct induced maturation of human DCs, as evidenced by up-regulation of CD86, HLADR, and CD83, the Z13Mad5 and Mad5 proteins did not have the ability to activate human DCs. These results suggest that the EDA component of the protein is responsible for the upregulation of activation markers on human DCs.

Example 2: In vitro presentation of epitopes (MHC I)

The present study was designed to examine functional MHC class I-restricted cross-presentation in a murine in vitro system using bone marrow-derived dendritic cells (BMDCs) and splenocytes from various TCR transgenic mice. For this purpose, the EDAZ13Mad5 and Mad5 constructs (described above in example 1) and the EDAMad5 construct were used:

EDAMad5

Subsequence

Molecular weight: 20017 Yes.

Characteristics:

- The Mad5 cargo molecule contains epitopes OVACD4, gp100CD8, EalphaCD4 and OVACD8.

- Contains the TLR agonist EDA.

- Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, 2 mM DTT, 0.5 M L-arginine, pH 8.

- Endotoxin level: 1.8 EU/mg.

BMDCs were loaded overnight with 300 nM EDAMad5, EDAZ13Mad5 and Mad5 proteins containing the OVACD8, OVACD4 and gp 100 epitopes. Processing and presentation of these MHC I-restricted OVACD8 and gp 100 epitopes was monitored by measuring in vitro proliferation of naive OVA _257-264 -specific CD8 ⁺ -T cells from OT-1 T cell receptor (TCR) transgenic mice and gp100-specific CD8 ⁺ -T cells from P-mel T cell TCR transgenic mice, respectively. Thus, the efficiency of MHC class I-restricted presentation of the OVACD8 epitope and the gp 100 epitope was monitored after 4 days using CFSE-labeled OT1 cells and P-Mel cells, respectively. Processing and presentation of the MHC II-restricted OVACD4 epitope was monitored by measuring in vitro proliferation of naïve OVA _323-339 -specific CD4 ⁺ T cells from OT-2 T cell receptor (TCR) transgenic mice. Thus, the efficiency of MHC class II-restricted OVACD4 epitope presentation was monitored after 4 days using CFSE-labeled OT2 cells. As a control, BMDC were pulsed for 1 h with 5 μM peptide (one experiment, representative of 2 separate experiments).

The results are presented in Fig. 5. Similar cross-presentation and processing ability of all Mad5-based proteins studied were found.

Example 3: CD8 T-cell immune response

To study the effectiveness of EDA-conjugated proteins in inducing CD8 ⁺ T-cell responses, C57BL/6 mice were vaccinated twice (Wk0 and Wk2) by subcutaneous injection of the EDAZ13Mad5 or EDAMad5 constructs (described in Examples 1 and 2) at a dose of either 2 nmol, or 10 nmol. The positive control group was vaccinated with Mad5 and the TLR4 agonist MPLA (at an equimolar dose of EDA). Two doses of the constructs (2 nmol) (Fig. 6) and (10 nmol) (Fig. 7) were studied. 3-4 mice were used in each group.

7 days after the last vaccination, mice were bled and pentamer stained to monitor the OVA-specific immune response in the blood. In fig. Figure 8 shows the percentage of pentamer-positive CD8 ⁺ -T cells in all groups and for both doses tested.

These data suggest that this immune response was lower at the 10 nmol dose than at the 2 nmol dose. At both doses, 2 nmol and 10 nmol, the vaccine-mediated immune response was more robust in the EDAZ13Mad5-treated group than in the EDAMad5-treated group. Additionally, an increased immune response was found when the TLR4 agonist was conjugated to the vaccine.

Example 4: Vaccine Efficacy on Tumor Growth in the Standard Tumor Model EG.7-OVA

To evaluate the effect of EDA-containing protein constructs on tumor growth control, the sc model of EG.7-OVA thymoma cells was chosen. C57BL/6 mice were implanted with sc 3×10 ⁵ EG7-OVA tumor cells in the left lateral region. After tumor implantation, mice were vaccinated on days 5 and 13 using a dose of 10 nmol of one of the following constructs (for a description of the constructs, see Examples 1 and 2): EDAZ13Mad5, EDAMad5, Mad5 or Mad5 and MPLA (at a dose equimolar to EDA) sc in right side area. Tumor size was determined using calipers.

In fig. Figure 9 shows data on tumor growth in 7 mice per group (mean ± SD); *, p<0.05 EDAZ13Mad5 relative to the control group (2-way analysis of variance). In fig. Figure 10 shows the growth curves of individual tumors (for each of 7 mice per group). In fig. 11A shows the survival curve of 7 mice per group; *, p<0.05 EDAZ13Mad5 relative to the control group (log-rank test). In fig. 11B shows the tumor-free period curve for 7 mice per group; *, p<0.05 EDAZ13Mad5 relative to the control group (log-rank test).

The results demonstrated that when used in a therapeutic regimen, the EDAZ13Mad5 construct was the only protein vaccine that significantly controlled tumor growth compared to controls, resulting in significant improvements in tumor progression-free and survival curves.

Thus, the results suggest that the EDAZ13Mad5 protein construct is a highly effective vaccine for controlling tumor growth when used in a therapeutic regimen.

Example 5: Vaccine Efficacy on Tumor Growth in a Metastatic Melanoma Model

To evaluate the effectiveness in a model of lung metastases using B16-OVA tumor cells, various protein constructs were used in a semi-therapeutic mode: EDAMad5, EDAZ13Mad5, Z13Mad5 + MPLA (see examples 1 and 2 for a description of the constructs) and individually MPLA. C57BL/6 mice were implanted with iv 1×10 ⁵ B16-OVA melanoma tumor cells and simultaneously (d0) a vaccine (EDAMad5, EDAZ13Mad5, Z13Mad5 + MPLA, MPLA individually) was administered by subcutaneous injection into the right lateral region at a dose of 2 nmol. After 9 days, mice were vaccinated a second time with the same dose. Additionally, control groups were vaccinated with 2 nmol Z13Mad5 and the TLR4 agonist MPLA (at an equimolar dose of EDA) or MPLA alone. Mice were sacrificed on day 13 and lungs were removed. The number of metastatic foci in each lung was counted. The results are presented in Fig. 12.

The results demonstrated that the EDAZ13Mad5 conjugate had the same efficacy as Z13Mad5 + MPLA in inhibiting tumor metastasis in the lung. In addition, EDA-Mad5 was found to have lower efficacy than EDAZ13Mad5, suggesting a critical role for Z13 in vaccine efficacy.

Example 6: Vaccine Efficacy on Tumor Growth in a Metastatic Melanoma Model - Prophylactic Regime

In addition, the effectiveness of various protein constructs EDAMad5, EDAZ13Mad5 and Z13Mad5 + MPLA (see examples 1 and 2 for a description of the constructs) was assessed in a model of lung metastases in a prophylactic mode. C57BL/6 mice were vaccinated 21 and 7 days before tumor cell implantation (d-21 and d-7) by subcutaneous injection (sc) of 2 nmol EDAZ13Mad5, EDAMad5 or Z13Mad5 + MPLA (at a dose equimolar to EDA) in the right side area. On day 0, mice were implanted with iv 1×10 ⁵ B16-OVA melanoma tumor cells. Mice were sacrificed on day 14 and lungs were removed. The results are presented in Fig. 13.

Example 7: Generation of Additional Constructs Containing a TLR2 Peptide Agonist

In this case, the complex proposed for use according to the present invention was a fusion protein that contained the cell-penetrating peptide "Z13", the protein "MAD5", consisting of various CD8 ⁺ - and CD4 ⁺ epitopes of various antigens, and a TLR2 agonist peptide "Anaxa". Thus, fusion proteins were created with the Anaxa peptide at the C-terminal or N-terminal position.

Thus, the following constructs were created in which the amino acid sequence of the cell-penetrating peptide “Z13” is underlined, and the TLR peptide agonist “Anaxa” is indicated in italics:

AnaxaZ13Mad5

Subsequence:

Molecular weight: 18973 Yes.

Characteristics:

- The Mad5 cargo molecule contains epitopes OVACD4, gp100CD8, EalphaCD4 and OVACD8.

- Contains 35-mer annexin peptide.

- Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, 2 mM DTT, 0.5 M L-arginine, pH 8.

- Endotoxin level: 5.17 EU/mg.

Z13Mad5Anaxa

Subsequence:

Molecular weight: 18973 Yes

Characteristics:

- The Mad5 cargo molecule contains epitopes OVACD4, gp100CD8, EalphaCD4 and OVACD8.

- Contains 35-mer annexin peptide.

- Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, 2 mM DTT, 0.5 M L-arginine, pH 8.

- Endotoxin level: 3.1 EU/mg.

Example 8: Assessment of TLR2 Binding (HEK-hTLR2 Cell Lines)

The basis of this study was to evaluate whether the protein constructs Z13Mad5Anaxa and AnaxaZ13Mad5 (see example 7 for a description of the constructs of these proteins) can bind to TLR2 as an agonist. HEK-Blue™ hTLR2 cells were seeded in a flat-bottomed 96-well plate in culture medium stimulated with 0.3, 1 or 3 µM AnaxaZ13Mad5 or Z13Mad5Anaxa and incubated at 37°C for 24 hours. Pam3CSK4, a TLR2 agonist, was used as a positive control , at a concentration of 500 ng/ml.

To monitor NF-κB/AP1 activation, 20 μl of supernatant was added to QuantiBlue® detection medium and incubated at 37°C 1 h before OD determination (620 nm). The results are presented in Fig. 14A.

Secretion of IL-8 in the supernatant was quantified by ELISA. The results are presented in Fig. 14B.

The results (Fig. 14A, B) indicate that Z13Mad5Anaxa and AnaxaZ13Mad5 had the same ability to bind to TLR2 in a dose-dependent manner.

Example 9: In Vivo Induction of Specific CD8 ⁺ T Cells

To study the effectiveness of the Anaxa-conjugated proteins described in Example 7 in inducing CD8 ⁺ T-cell responses, C57BL/6 mice were vaccinated twice (Wk0 and Wk2) by subcutaneous injection of AnaxaZ13Mad5 at a dose of 2 nmol or Z13Mad5Anaxa at a dose of 2 nmol. Mice were bled 7 days after the last vaccination and pentamer staining was performed to monitor the OVA-specific immune response in the blood (one experiment with 4 mice per group). The results are presented in Fig. 15 and 16.

These data indicate that both the Z13Mad5Anaxa vaccine and the AnaxaZ13Mad5 construct induced a strong immune response.

Example 10: Therapeutic Effects on Tumor Growth

To evaluate the effect of the Anaxa-conjugated protein constructs described in Example 7 on tumor growth control, a standard tumor model was used, namely, created by sc-implantation of EG.7-OVA thymoma cells.

C57BL/6 mice were implanted with sc 3×10 ⁵ EG7-OVA tumor cells in the left lateral region. After tumor implantation, each of three groups of 7 mice each was vaccinated with sc in the right flank region on days 5 and 13 by subcutaneous injection at a dose of 10 nmol of either AnaxZ13Mad5 (group 1), or Z13Mad5Anaxa (group 2), or Z13Mad5 and Pam3CSK4 (in dose equimolar to Anaxa; group 3). To compare the effects of protein mixed with another adjuvant, a control group was vaccinated with Z13Mad5 and Pam3CSK4 (at an equimolar dose of Anaxa). Tumor size was determined using calipers. The results are presented in Fig. 17-19.

When used therapeutically, the Z13Mad5Anaxa and AnaxaZ13Mad5 protein vaccines were more effective in controlling tumor growth compared to the control group, i.e. co-injection of Z13Mad5 and Pam3CSK provided a significantly improved survival curve. In particular, Z13Mad5Anaxa and AnaxaZ13Mad5 showed significantly higher efficacy than Z13Mad5 when administered separately from Pam3CSK4. Thus, the results suggest that the Z13Mad5Anaxa and AnaxaZ13Mad5 protein constructs are promising conjugate vaccines for controlling tumor growth when used in a therapeutic regimen.

Example 11: Therapeutic Effects on Tumor Growth - Comparison of Constructs Containing Different TLR Agonists

The objective of this study was to compare the effectiveness of various protein vaccine designs conjugated to various TLR agonists, namely EDAZ13Mad5 and Z13Mad5Anaxa, which are described in examples 1 and 7, in controlling tumor growth. For this purpose, C57BL/6 mice were implanted with sc 3×10 ⁵ EG.7-OVA thymoma cells in the left flank region according to the method described in example 10. Mice (each of 7 mice in the group) were vaccinated with sc in the right flank region on days 5 and 13, using a 2 nmol dose of either EDAZ13Mad5, Z13Mad5Anaxa, or using a co-injection of Z13Mad5+MPLA (at a dose equimolar to EDA).

The results are presented in Fig. 20, 21, and 22. In this experimental design, Z13Mad5Anaxa, EDAZ13Mad5, and Z13Mad5+MPLA had similar abilities to provide significant control of tumor growth. In addition, these data indicate that Z13Mad5Anaxa is the most effective design in terms of tumor growth control, and the effectiveness of EDAZ13Mad5 was slightly superior to that of Z13Mad5+MPLA when studied using this experimental design.

Example 12: Dose Effect of Z13Mad5Anaxa on Tumor Growth Control

To determine the optimal dose of the conjugate vaccine, the ability of Z13Mad5Anaxa (see Example 7) at three different doses (0.5, 2 and 10 nmol) to control tumor growth was assessed. The dose dependence of the Z13Mad5Anaxa construct was assessed on the sc model created using EG.7-OVA thymoma cells, according to the method described above in example 10. After tumor implantation, mice were vaccinated twice (on day 5 and on day 13 after tumor implantation ) in a therapeutic regimen using Z13Mad5Anaxa at a dose of 0.5, 2 or 10 nmol.

C57BL/6 mice were implanted with sc 3x10 ⁵ EG7-OVA tumor cells in the left flank and vaccinated twice (d5 and d13) by subcutaneous injection of either 0.5, 2, or 10 nmol Z13Mad5Anaxa in the right flank. Tumor size was determined using calipers.

Data on tumor growth in a group of 7 mice are presented in Fig. 23 These data suggest that the 0.5 and 2 nmol doses were at least as effective as the 10 nmol dose in controlling tumor growth.

Example 13: Effect of different routes of administration of Z13Mad5Anaxa

This study was based on the results obtained in the examples described above, demonstrating the effectiveness of the Z13Mad5Anaxa conjugate vaccine (see Example 7), which had the ability to induce specific immune responses and was effective in controlling tumor growth in the subcutaneous EG7 tumor model described above.

To study the effects of subcutaneous, intramuscular, and intradermal routes of administration, immune responses elicited by subcutaneous, intramuscular, and intradermal injections were compared. Intradermal injections were performed using the PLEASE ^® device from Pantec Biosolutions.

Mice were vaccinated three times every two weeks (Wk0, Wk2 and Wk4) using Z13Mad5Anaxa at a dose of 0.5 or 2 nmol (see example 7). In order to have a targeted effect on several lymph nodes, the 1st and 3rd vaccinations were carried out in the right lateral area, and the 2nd were carried out in the left lateral area. The response of SIINFEKL-specific CD8 ⁺ T cells was analyzed 1 week after the 2nd and 3rd vaccinations in the blood. In fig. Figure 24 shows SIINFEKL-specific CD8 T cell responses after each vaccination detected in the blood of C57BL/6 mice vaccinated three times (Wk0, Wk2 and Wk4) with sc, id or im using 0.5 nmol (Fig. 24 A) or 2 nmol (Fig. 24 B) Z13Mad5Anaxa. Blood was collected from mice 7 days after the 2nd and 3rd vaccinations and multimer staining was performed (one experiment with 4 mice per group).

The results demonstrated that at the two doses studied (0.5 and 2 nmol) (I) administration by all routes studied resulted in a SIINFEKL-specific CD8 T-cell immune response and (II) subcutaneous vaccination resulted in the strongest SIINFEKL-specific CD8 -T-cell immune response. When administered subcutaneously, the maximum response was achieved after the 3rd vaccination and was still maintained after the 3rd vaccination. The SIINFEKL-specific CD8 T-cell immune response after the 2nd vaccination, caused by intradermal and intramuscular vaccinations, was lower than with subcutaneous vaccination, and did not increase after the 3rd vaccination.

Next, the effector function and exhaustion status of SIINFEKL-specific CD8 T cells were assessed by analyzing KLRG 1 (killer cell receptor lectin-like receptor member G 1) and PD-1, respectively.

For this purpose, C57BL/6 mice were vaccinated three times (Wk0, Wk2 and Wk4) s.c., i.d. or i.m. Z13Mad5Anaxa at a dose of 2 nmol (see example 7). Blood was taken from mice 7 days after the 2nd and 3rd vaccinations and FACS staining was performed. KLRG1 and PD-1 expression was analyzed on multimer-positive CD8 T cells (one experiment with 4 mice per group). The results are presented in Fig. 25.

These data indicate that KLRG 1 expression was strongly increased on SIINFEKL-specific CD8 T cells after subcutaneous vaccination. After i.d. - or i.m. - the effect of vaccination was found to be lower. The percentage of KLRG 1-positive cells among SIINFEKL-specific CD8 T cells also increased after sc vaccination (data not shown).

In contrast to KLRG 1, PD-1 expression decreased over time and with number of vaccinations using the subcutaneous and intramuscular routes of vaccination. This suggests that depletion of SIINFEKL-specific CD8 T cells did not occur. The percentage of PD1-positive cells among SIINFEKL-specific CD8 T cells also decreased after s.c. and i.m. vaccinations (data not shown). Importantly, PD-1 expression was found to be higher after the 2nd vaccination when mice were vaccinated subcutaneously, reflecting the earlier activation status of specific T cells (Keir M.E. et al., PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol, 26, 2008, pp. 677-704).

The late depletion marker Tim-3 was also analyzed. A very low level of its expression was found in all groups.

Taken together, the results suggest that subcutaneous vaccination elicits the most potent CD8 T cell-specific immune response compared with intramuscular or intradermal injections.

Example 14: Intranodal route of administration

Taking into account the results of previous experiments (example 13), an additional study of the intranodal route of administration was carried out. To do this, we examined the immune response caused by intranodal injection of Z13Mad5Anaxa (see example 7).

For this purpose, mice were first injected subcutaneously with Evans blue, which allows easy visualization of the injected lymph nodes and intranodal injection without invasive surgery, for example, according to the method described by Jewell C.M., S.C. Lopez and D.J. Irvine, In situ engineering of the lymph node microenvironment via intranodal injection adjuvant-releasing polymer particles. Proc Nati Acad Sci USA, 108(38), 2011, pp. 15745-15750.

C57BL/6 mice were vaccinated twice every two weeks (Wk0 and Wk2) intranodally using 0.5 nmol Z13Mad5Anaxa (see Example 7). The first vaccination was carried out in the right inguinal lymph node, and the second vaccination was carried out in the left inguinal lymph node. Blood was taken from mice 7 days after the 2nd vaccination and multimer staining was performed (3 mice per group). In other words, the SIINFEKL-specific response of CD8 ⁺ T cells in the blood was analyzed 1 week after the 2nd vaccination. In fig. 26 shows SIINFEKL-specific CD8 T cell responses. These data suggest that intranodal injection can also induce a SIINFEKL-specific CD8 T cell response.

Example 15: Vaccination schedule

Work on studying the vaccination schedule began with identifying the effect of the third vaccination when using the Z13Mad5Anaxa design described above (see example 7). Based on previous results, the subcutaneous route of administration was chosen.

In this experiment, the first two vaccinations were carried out in wk0 and wk2, and the 3rd vaccination was carried out in either wk4 (Fig. 27A) or wk8 (Fig. 27B). Thus, C57BL/6 mice were vaccinated three times (Fig. 27 A and B: Wk0, Wk2 and Wk4 and Fig. 27 B and D: Wk0, Wk2 and Wk8) sc using Z13Mad5Anaxa at a dose of 2 nmol. Blood was collected from mice 7 days after the last vaccination and staining with pentamer was performed (one experiment with 4 mice per group). Accordingly, the SIINFEKL-specific CD8 ⁺ T-cell response was analyzed 1 week after the 2nd and 3rd vaccinations (Fig. 27 A and B). Additionally, the effector function of SIINFEKL-specific T cells was assessed by analyzing the expression of KLRG 1 on specific CD8 T cells (Fig. 27 B and D).

The data indicate that, compared with controls, the percentage of SIINFEKL-specific CD8 T cells was significantly increased at all time points examined (Vac2 and Vac3), as well as with both vaccination regimens (Fig. 27 A and B).

It is important to note that the third vaccination at Wk4 provided the most significant increase in the percentage of SIINFEKL-specific CD8 T cells (Fig. 27 A). These same cells showed increased effector function due to higher levels of KLRG 1 expression (Fig. 27 B). In contrast, when performing the third vaccination in Wk8, no improvement was found after the third vaccination relative to the second vaccination in terms of SIINFEKL-specific immune response and KLRG 1 expression.

Taken together, these results suggest that CD8 T-cell immune responses can be enhanced by shortening the time interval between the second and third vaccinations.

Given that an earlier third vaccination was likely to enhance the immune response, two short vaccination schedules were tested in the following experiment:

I) three vaccinations on day 0, day 3 and day 7 and

ii) three vaccinations on day 0, day 7 and day 14.

And in this case, C57BL/6 mice were also used and vaccination was carried out s.c. using Z13Mad5Anaxa at a dose of 0.5 nmol (see example 7). Multimer staining was performed on blood samples obtained 1 week after the 2nd and 3rd vaccinations (one experiment with 4 mice per group).

Thus, the SIINFEKL-specific CD8 ⁺ T cell response was analyzed 1 week after the 2nd and 3rd vaccinations (Fig. 28 A and D). In addition, effector function of SIINFEKL-specific T cells was assessed by analyzing KLRG 1 expression on specific CD8 T cells (FIGS. 28B and 28E) and exhaustion status by analyzing PD-1 expression on specific T cells (FIGS. 28B and 28E). 28E).

The data indicate that - similar to the results obtained in the first experiment, regardless of the vaccination schedule described above - compared with the control, the percentage of SIINFEKL-specific CD8 T cells increased at all time points studied (Vac2 and Vac3), as well as in both vaccination schemes (Fig. 28 A and B).

However, compared with the wk0-wk2-wk4 regimen, the day 0, day 3, and day 7 vaccination regimens did not elicit as strong a SIINFEKL-specific CD8 T-cell immune response. Regarding the schedule with vaccinations on day 0, day 7 and day 14, the SIINFEKL-specific CD8 T-cell immune response was stronger compared with the previously described schedule (d0-d3-d7), but it was not maintained after the 3rd vaccinations.

Taken together, the results obtained with these vaccination regimens indicate that the Wk0-Wk2-Wk4 vaccination regimen is the best vaccination regimen for eliciting an effective OVA-specific CD8 T cell immune response with high effector function.

Example 16: Ability of TLR-CPP Agonist Conjugate Constructs to Activate Murine Antigen Presenting Cells (APCs)

To study the effects of both the CPP component and the TLR agonist component of the complex of the present invention, the fusion proteins described above were again used (see Examples 1, 2 and 7).

In addition, an additional “control peptide” was created, which was also a fusion protein and which contained the “MAD5” protein, consisting of various CD8 ⁺ and CD4 ⁺ epitopes from various antigens, and the TLR2 agonist peptide “Anaxa” (i.e. without a cell-penetrating peptide). Thus, the following control structures were additionally created:

Mad5Anaxa

Subsequence:

Molecular weight: 13933 Yes.

Characteristics:

- The Mad5 cargo molecule contains epitopes OVACD4, gp100CD8, EalphaCD4 and OVACD8.

- Contains a 35-mer annexin peptide at the C-terminal position.

- Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, 2 mM DTT, 0.5 M L-arginine, pH 8.

- Endotoxin level: batch 1-12, 15 EU/mg.

The basis of this study was to evaluate the ability of two exemplary complexes proposed in the present invention, namely: EDAZ13Mad5 (see example 1) and Z13Mad5Anaxa (see example 7), to increase the activation of antigen presenting cells compared to reference complexes , which lacked either the cell-penetrating peptide Z13 component (Mad5Anaxa, see above; EDAMad5, see example 2) or the TLR agonist (Z13Mad5, see example 1).

For this purpose, the ability of the above constructs to enhance the activation of antigen presenting cells (APCs) on bone marrow-derived dendritic cells (BMDCs) that express all TLRs except TLR7 was assessed.

BMDCs were seeded in a flat-bottomed 96-well plate in culture medium stimulated with 1 μM of either Z13Mad5Anaxa (see example 7), or Mad5Anaxa (see above), or Z13Mad5 (see example 1), EDAZ13Mad5 (see example 1), or EDAMad5 (see example 2), and incubated for 24 hours at 37°C.

APC activation was studied by monitoring the secretion of IL-6 into the BMDC culture supernatant. IL-6 secretion was quantified in the supernatant using ELISA.

The results are presented in Fig. 29 These data clearly demonstrate that the Z13Mad5Anaxa construct had the ability to activate BMDCs, but no activation was detected when cells were cultured in the presence of Z13Mad5 or Mad5Anaxa. This suggests that not only is the TLR agonist (Anaxa or EDA) critical for macrophage and dendritic cell activation, but CPP is also required. In addition, it has been established that the presence of CPP without a TLR agonist is not sufficient, thus, in fact, both CPP and the TLR agonist are critical for the activation of macrophages and dendritic cells.

These results were confirmed using another cell line, such as the murine macrophage cell line Raw 264.7, which expresses all TLRs except TLR5 (Applequist S.E., R.P. Wallin, and H.G. Ljunggren, Variable expression of Toll-like receptor in murine innate and adaptive immune cell lines Int Immunol, 14(9), 2002, pp. 1065-1074).

Raw 264.7 cells were seeded in a flat-bottomed 96-well plate in culture medium stimulated with 1 μM of either Z13Mad5Anaxa (see example 7), Mad5Anaxa (see above), or Z13Mad5 (see example 1), and incubated for 24 hours at 37°C.

APC activation in Raw 264.7 cells was studied by monitoring TNF-α secretion in the Raw 264.7 culture supernatant. TNF-α secretion was quantified by ELISA in the supernatant. The results are presented in Fig. thirty.

It is believed that CPP may facilitate the entry of the molecule into cells, allowing for improved targeting of intracellular TLRs.

Taken together, the data confirmed the critical importance of both CPP and TLR agonist in the conjugated constructs for APC activation. This action may result from facilitating the entry of the construct into the cell, resulting in optimal targeting of intracellular TLRs.

Example 17: Ability of Conjugated Constructs to Bind Human TLR4

Anaxa peptide has now been shown to have adjuvant activity through signaling through TLR2 (WO 2012/048190 A1), while EDA peptide is a naturally occurring ligand for TLR4 (Okamura Y. et al., The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem, 276(13), 2001, pp. 10229-10233).

Moreover, as demonstrated above in Example 8 and FIG. 14, the complex proposed for use according to the present invention, which contains the Anaxa peptide as a TLR agonist, for example Z13Mad5Anaxa, has the ability to bind to human TLR2 and enhance the secretion of IL-8 by HEK-hTLR2 cells (see example 8, Fig. 14) .

The present invention assessed the ability of the complexes of the present invention, which contained either Anaxa peptide as a TLR agonist or EDA peptide as a TLR agonist, to bind to human TLR4. For this purpose, HEK cells transfected with human TLR4 (HEK-hTLR4) were seeded in a flat-bottomed 96-well plate in culture medium stimulated with 1 μM of either Z13Mad5Anaxa (see example 7), Mad5Anaxa (see above), Z13Mad5 (see example 1), EDAZ13Mad5 (see example 1), or EDAMad5 (see example 2), and incubated for 24 hours at 37°C. Secretion of IL-8 in the supernatant was quantified by ELISA.

The results are presented in Fig. 31 As expected, incubation of HEK-hTLR4 with EDAZ13Mad5 resulted in significant IL-8 secretion, indicating binding of EDAZ13Mad5 to TLR4. Consistent with the results obtained in Example 16, IL-8 secretion using EDAMad5 (without CPP) was significantly lower compared to EDAZ13Mad5, demonstrating the effect of the presence of CPP. It was found that the Z13Mad5 construct, which lacks the TLR agonist, did not induce IL-8 secretion, indicating - as expected - a lack of binding to TLR4.

Importantly, incubation of HEK-hTLR4 with the Z13Mad5Anaxa construct resulted in the most pronounced IL-8 secretion, indicating the binding of Z13Mad5Anaxa to TLR4. This is unexpected since Anaxa was previously hypothesized to be a TLR2 agonist. Again, a similar design but without CPP (Mad5Anaxa) resulted in significantly lower IL-8 secretion, confirming the results obtained in Example 16.

Taken together, these data (I) confirm the results obtained in Example 16, (II) confirm that EDA is indeed a TLR4 agonist, and (III) and suggest, surprisingly, that the Anaxa peptide is also a TLR4 agonist ( in addition to being a TLR2 agonist, see Example 8 and Figure 14).

Example 18: Vaccine Efficacy on Tumor Growth in a Lung Metastasis Model - Semi-Therapeutic Regime: As a TLR EDA Agonist

This study was based on the results obtained in Example 6 demonstrating the effectiveness of the complex proposed for use according to the present invention, such as EDAZ13Mad5, when studied in a model of melanoma lung metastases in a prophylactic regimen (see FIG. 13).

In the present study, we used the same model of lung metastases, as well as the EDAZ13Mad5 and Z13Mad5 + MPLA protein constructs (see examples 1 and 2 for a description of the constructs). However, using the semi-therapeutic regimen, C57BL/6 mice were vaccinated simultaneously with tumor cell implantation (d0) and a second time 9 days after implantation (d9). Vaccination was carried out by subcutaneous injection (sc) of 2 nmol EDAZ13Mad5, Z13Mad5 + MPLA (at an equimolar dose of EDA) or MPLA into the right flank. On day 0, mice were implanted iv with 1x10 ⁵ B16-OVA melanoma tumor cells and vaccinated twice (d0 and d9) by subcutaneous injection (sc) with 2 nmol EDAZ13Mad5, Z13Mad5 + MPLA (at a dose equimolar to EDA), or MPLA individually at right side area. Mice were sacrificed on day 13 and lungs were removed. The results are presented in Fig. 32.

The results demonstrated that the EDAZ13Mad5 construct was slightly more effective than Z13Mad5 + MPLA in inhibiting the growth of melanoma metastases. In addition, no adjuvant effect was observed when mice were injected with MPLA alone.

Both EDAZ13Mad5 and Z13Mad5 + MPLA significantly inhibited melanoma lung metastasis when administered in both prophylactic and semitherapeutic regimens.

Example 19: Vaccine Efficacy on Tumor Growth in a Lung Metastasis Model - Semi-Therapeutic Regime: As a TLR Agonist Anaxa

This study was based on the results obtained in Example 18 and used the same model (in semi-therapeutic mode) and experimental design. However, the effects of the complexes of the present invention were studied that contained the Anaxa peptide as a TLR agonist instead of the TLR agonist EDA, which was used in Example 18.

For this purpose, C57BL/6 mice were implanted with iv 1×10 ⁵ B16-OVA melanoma tumor cells and vaccinated twice (d0 and d9) by subcutaneous injection (sc) at a dose of 0.5 nmol Z13Mad5Anaxa, Mad5Anaxa or Z13Mad5 + Pam3CSK4 (at a dose , equimolar Anaxa) to the right lateral region. Mice were sacrificed on day 21 and lungs were removed. The number of metastatic foci was counted in each lung. The results are presented in Fig. 33.

The results demonstrated that the Z13Mad5Anaxa construct was significantly more effective than Z13Mad5 + Pam3CSK4 in inhibiting the growth of melanoma metastases. In contrast, Mad5Anaxa had no ability to control the growth of lung metastases, again confirming the importance of CPP.

Overall, the experiment conducted in the B16-OVA lung metastasis model demonstrated that the Z13Mad5Anaxa construct was highly effective in inhibiting the growth of melanoma lung metastases.

Example 20: Vaccine efficacy in a glioblastoma model

This study used a different cancer model, namely the glioblastoma model. Glioma is the most common form of primary brain tumor in adults, with glioblastoma multiforme (GBM) being the most lethal. This tumor is notorious for its high invasiveness and aggressive behavior. Currently, the best treatment for GBM is a regimen that includes a combination of surgery, chemotherapy, and radiation therapy, which provides a median survival of only 14.6 months. There is an urgent, unmet need for new treatment options that improve the prognosis of glioma patients. T cell-mediated immunotherapy is speculatively an attractive treatment option when used in combination with existing treatment pathways for glioma, particularly highly invasive GBM.

Gl261 glioma is a carcinogen-induced murine glioma model. This model is one of a very small number of brain tumor models generated in immunocompromised animals that has growth characteristics similar to human GBM (Newcomb E. and D. Zagzag, The murine GL261 glioma experimental model to assess novel brain tumor treatments, in: CNS Cancer Models, Markers, Prognostic, Factors, Targets, and Therapeutic Approaches, edited by E. G. Van Meir, Humana Press: Atlanta, 2009, pp. 227-241; Jacobs V. L. et al., Current review of in vivo GBM rodent models: emphasis on the CNS-1 tumor model. ASN Neuro, 3(3): 2011, e00063). Small numbers of intracranially transplanted G1261 cells form intracranial tumors in C57BL/6 mice (Zhu X. et al., Poly-/CLC promotes the infiltration of effector T cells into intracranial gliomas via induction of CXCL10 in IFN-alpha and IFN-gamma dependent manners, Cancer Immunol Immunother, 59(9), 2010, pp. 1401-1409; Zhu X. et al., Toll like receptor-3 ligand poly-/CLC promotes the efficacy of peripheral vaccinations with tumor antigen-derived peptide epitopes in murine CNS tumor models. J Transl Med, 5, 2007, p. 10). The cells are moderately immunogenic: they have the ability to induce a tumor-specific immune response in the tumor area. However, tumor-specific immune cells do not ensure complete tumor clearance.

Currently, M. Ollin has created a new model Gl261 (Ohlfest JR, et al., Vaccine injection site matters: qualitative and quantitative defects in CDB T cells primed as a function of proximity to the tumor in a murine glioma model. J Immunol, 190 (2), 2013, pp. 613-620) by transfecting Gl261 cells with a “Quad cassette” expressing four peptides presented by class I or II H-2b molecules: human gp100 _25-33 , chicken OVA _257-264 , chicken OVA _323-339 and mouse I-Eα _52-68 . The Quad-Gl261 cell line also stably expresses luciferase, allowing tumor growth to be monitored by bioluminescence.

The purpose of this study was to evaluate the effectiveness of the complex proposed for use according to the present invention in the Quad-Gl261 glioblastoma model.

The effect of the complex proposed for use according to the present invention, namely: Z13Mad5Anaxa (see example 7) was assessed using the glioblastoma model described above. At the same time, T-cell homing in the tumor area was analyzed in mice bearing the Gl261-Quad tumor, which were vaccinated twice (Wk1 and Wk3) with the Z13Mad5Anaxa vaccine. A group vaccinated with Z13Mad5 and Anaxa (at an equimolar dose of Z13Mad5Anaxa), administered individually, was used as a control. In general, the method was as follows: C57BL/6 mice were implanted ie (inside the skull) with 5x10 ⁵ Gl261-Quad tumor cells and vaccinated twice (d7 and d21 after implantation) by sc injection at a dose of 2 nmol Z13Mad5Anaxa (group 1) or 2 nmol Z13Mad5 and 2 nmol Anaxa (group 2). Blood and brain-infiltrating leukocytes (BIL) were analyzed in Wk4, with SIINFEKL-specific CD8 T cells in blood and BIL at d28 quantified by multimer staining (5-8 mice per group). The results are presented in Fig. 34.

Overall, a low frequency of SIINFEKL-specific CD8 T cells was found in the blood. However, a higher percentage of SIINFEKL-specific CD8 T cells was detected in the blood of Z13Mad5Anaxa-vaccinated mice. All groups showed significantly greater accumulation of SIINFEKL-specific CD8 T cells in the BIL.

After two vaccinations with Z13Mad5Anaxa, the frequency of SIINFEKL-specific CD8 ⁺ T cells in BIL was 2 times higher (24%) than with Z13Mad5 + Anaxa (12%).

Cytokine secretion was then assessed. To do this, C57BL/6 mice were implanted with ie 5×10 ⁵ Gl261-Quad tumor cells and vaccinated twice (d7 and 21) by sc injection using a dose of 2 nmol Z13Mad5Anaxa or 2 nmol Z13Mad5 and 2 nmol Anaxa. BIL were isolated and cultured for 6 h with mature BMDCs loaded or unloaded with SllNFEKL peptide in the presence of brefeldin A before intracellular cytokine staining. The results are presented in Fig. 35.

Despite the heterogeneity, high levels of cytokine secretion were detected for brain-infiltrating CD8 T cells from mice vaccinated with Z13Mad5Anaxa. These results demonstrated that the Z13Mad5Anaxa vaccine had the ability to induce a stronger SIINFEKL-specific CD8 T cell immune response in the brains of tumor-bearing mice with strong effector function.

Our results indicate that Z13Mad5Anaxa is effective in eliciting an immune response from brain-infiltrating SIINFEKL-specific CD8 T cells.

Example 21: Vaccine Efficacy on Survival in the Gl261-Quad Glioblastoma Model

In an independent experiment, the survival of mice vaccinated with control and Z13Mad5Anaxa was monitored. The therapeutic regimen included three sequential vaccinations using a dose of 2 nmol Z13Mad5Anaxa on days 7, 21 and 35 after i.e. implantation of the tumor.

C57BL/6 mice were implanted with ie 5×10 ⁵ Gl261-Quad tumor cells and vaccinated three times (d7, d21 and d35) by sc injection of Z13Mad5Anaxa at a dose of 2 nmol. Mice were weighed daily and sacrificed when weight loss reached more than 15%. The results are presented in Fig. 36.

The results demonstrated that therapeutic vaccination with Z13Mad5Anaxa was more effective than the control group, extending median survival by 10 days.

Example 22: Vaccine Efficacy in a Subcutaneous Tumor Model - Prophylactic Regime

This experiment was based on the results obtained in Example 10, which are presented in FIG. 17-19.

To evaluate the effects of the Anaxa-conjugated protein constructs described in Example 7 on tumor growth control, a standard tumor model was used, namely, sc-implantation of EG.7-OVA thymoma cells. In contrast to Example 10, in which vaccination was carried out on days 5 and 13, the present study used a prophylactic regimen in which mice were vaccinated 21 and 7 days before tumor implantation.

C57BL/6 mice were vaccinated twice (d-21 and d-7) by sc injection using 0.5 nmol Z13Mad5Anaxa in the right flank and then implanted on day 0 sc with 3×10 ⁵ EG7- tumor cells OVA to the left side area. Tumor size was determined using calipers.

The results presented in Fig. 37 relate to tumor volume (FIG. 37A) and survival rate (FIG. 37B). Data demonstrate that prophylactic vaccination with Z13Mad5Anaxa was highly effective in controlling tumor growth and survival rates. Tumor volume was significantly reduced in mice treated with Z13Mad5Anaxa compared to control mice. The survival rate was significantly increased in mice treated with Z13Mad5Anaxa compared to control mice.

Example 23: Vaccine Efficacy in a Subcutaneous Tumor Model - Therapeutic Regime in Advanced Tumors

This experiment was based on the results obtained in Example 10, which are presented in FIG. 17-19, and on the results obtained in example 22, which are presented in Fig. 37. The purpose of this study was to evaluate the effect of Z13Mad5Anaxa (see Example 7) on an advanced tumor.

For this purpose, an s.c. model was used, obtained using B16-OVA melanoma cells. In this model, tumor cells proliferated slowly, allowing for a larger window of vaccination duration.

The first vaccination using Z13Mad5Anaxa at a low dose of 0.5 nmol was carried out in the presence of a developed tumor visible to the naked eye, i.e. on day 14 after tumor cell implantation. The second vaccination was carried out on day 21.

Thus, C57BL/6 mice were implanted with sc 1×10 ⁵ B16-OVA tumor cells in the left flank and vaccinated twice (d14 and d21) by sc-injecting 0.5 nmol Z13Mad5Anaxa into the right flank. Tumor growth was monitored and survival curves were plotted. The results are presented in Fig. 38.

The results demonstrated that Z13Mad5Anaxa effectively controlled the growth of advanced and visible to the naked eye tumors. Moreover, despite the presence of advanced and visible tumors to the naked eye, survival rates were increased in mice treated with Z13Mad5Anaxa compared to controls.

Example 24: Vaccine Efficacy in a Subcutaneous Tumor Model - Therapeutic Regime: Role of CPP

The protocol for this study was the same as that described in Example 10, with the only difference being that an additional Mad5Anaxa group was evaluated (see Example 16).

In general, the method consisted of the following: a standard tumor model was used, namely, obtained as a result of sc-implantation of EG.7-OVA thymoma cells. C57BL/6 mice were implanted with sc 3×10 ⁵ EG7-OVA tumor cells in the left lateral region. After tumor implantation, groups of 7 mice each were sc vaccinated in the right flank region on days 5 and 13 by subcutaneous injection using 0.5 nmol of either Z13Mad5Anaxa (group 1) or Mad5Anaxa (group 2) and compared with the control group . Tumor size was determined using calipers. The results are presented in Fig. 39.

The results demonstrated that mice treated with Z13Mad5Anaxa showed a significant reduction in tumor volume and a significantly increased survival rate compared to both control and Mad5Anaxa-treated mice, i.e. design without CPP. These results indicate that the presence of CPP leads to a significant reduction in tumor volume and a significant increase in survival rates, i.e. increased effectiveness of vaccination. Thus, the results demonstrated - in conjunction with the results obtained in example 10 - that the presence of CPP and a TLR agonist leads to a synergistic effect on tumor growth and survival rates.

Example 25: Comparison of the kinetics of immune responses when using complexes containing various cell-penetrating peptides

To study the effects of various CPPs in the complex proposed for use according to the present invention, the Z13Mad5Anaxa fusion protein described above was used (see Example 7).

Additionally, fusion proteins were constructed containing CPPs other than Z13, namely Z14 (SEQ ID NO: 7) or Z18 (SEQ ID NO: 11). These fusion proteins also contained the MAD5 protein, which consists of various CD8 ⁺ and CD4 ⁺ epitopes from various antigens, and the TLR2 agonist peptide Anaxa. Thus, the following additional structures were created:

Z14Mad5Anaxa

Subsequence:

Z18Mad5Anaxa

Subsequence:

C57BL/6 mice were divided into 8 different groups (4 mice per group): three groups treated with either Z13Mad5Anaxa, Z14Mad5Anaxa or Z18Mad5Anaxa at a dose of 2 nmol and their corresponding control, and three groups treated with Z13Mad5Anaxa, Z14Mad5Anaxa or Z18Mad5Anaxa at a dose of 0.05 nmol, and the corresponding control. Mice were vaccinated five times (Week0, Week2, Week4, Week6 and Week8) s.c. Mice were bled 7 days after the 2nd, 3rd, 4th and 5th vaccinations and multimer staining was performed (one experiment, 4 mice per group).

The results are presented in Fig. 40. All groups vaccinated with Z13Mad5Anaxa, Z14Mad5Anaxa or Z18Mad5Anaxa showed an increased percentage of multimer-positive cells compared to the control group (with the exception of the second vaccination with Z18Mad5Anaxa). These results indicate that the complexes of the present invention, which contain various cell penetrating peptides, have the ability to induce an immune response when administered at varying doses.

Example 26: Comparison of T Cell Immune Responses Using Complexes Containing Various Cell Penetrating Peptides

To study CD8 T-cell immune responses in more detail, C57BL/6 mice were divided into three different groups (3-4 mice per group): naïve Z13Mad5Anaxa or Z14Mad5Anaxa.

C57BL/6 mice from the Z13Mad5Anaxa group and the Z14Mad5Anaxa group were vaccinated 5 times (Week0, Week2, Week4, Week6 and Week8) s.c. using either Z13Mad5Anaxa (see Example 7) or Z14Mad5Anaxa (see Example 25) at a dose of 2 nmol ). Nine days after the 5th vaccination, mice were sacrificed, organs were removed, and multimer staining was performed to determine the percentage of SIINFEKL-specific CD8 T cells in the spleen, bone marrow, and draining lymph nodes (inguinal and axillary).

The results are presented in Fig. 41. Mice vaccinated with Z13Mad5Anaxa or Z14Mad5Anaxa showed a similar increase in multimer-positive cells, particularly in the spleen and bone marrow, as well as a slight increase in draining lymph nodes.

To further study the effector function of CD8 T cells after vaccination with complexes with various CPPs, Elispot analysis of spleen cells stimulated with the SIINFEKL-OVACD8 peptide (SEQ ID NO: 35) was performed in the same groups of mice as described above after 9 days after the 5th vaccination to quantify IFN-γ-producing cells.

The results are presented in Fig. 42A. Mice vaccinated with Z13Mad5Anaxa showed a significant increase in IFN-γ-producing cells compared to naïve mice. Mice vaccinated with Z14Mad5Anaxa also showed an increase in IFN-γ-producing cells compared to naïve mice, but the increase was not significant, which may be a consequence of the small number of mice (3 mice in the Z14Mad5Anaxa-treated group).

To study CD4 T cell responses after vaccination with complexes containing different CPPs, Elispot analysis of spleen cells stimulated with the SIINFEKL-OVACD4 peptide (SEQ ID NO: 36) was performed in the same groups of mice as described above, 9 days after 5th vaccination to quantify IFN-γ-producing cells.

The results are presented in Fig. 42B. Mice vaccinated with Z13Mad5Anaxa showed a very significant increase in the number of INF-γ-producing cells compared to naïve mice. Mice vaccinated with Z14Mad5Anaxa also showed an increase in INF-γ-producing cells compared to naïve mice, but the increase was not significant, which may be a consequence of the small number of mice (3 mice in the Z14Mad5Anaxa-treated group).

In addition, intracellular staining of spleen cells stimulated with the SIINFEKL OVACD8 peptide (SEQ ID NO: 35) was performed in the above groups of mice to identify CD107a ⁺ IFN-γ ⁺ TNF-α ⁺ cells. The results are presented in Fig. 43. Mice vaccinated with Z13Mad5Anaxa or Z14Mad5Anaxa showed a similar increase in CD107a ⁺ IFN-γ ⁺ TNF-α ⁺ -cells.

Example 27: Comparison of the effect of complexes containing various cell-penetrating peptides on tumor growth and survival in the s.c. model EG.7-OVA

An sc model created using EG.7-OVA cells was used to study the effects of complexes containing various cell-penetrating peptides on tumor growth and survival. At d0, C57BL/6 mice were implanted with sc 3×10 ⁵ EG7-OVA tumor cells in the left flank region and divided into three different groups (naïve, Z13Mad5Anaxa and Z14Mad5Anaxa). Mice were vaccinated twice on d5 and d13 after tumor implantation by sc injection using 0.5 nmol Z13Mad5Anaxa or Z14Mad5Anaxa in the right flank.

The results are presented in Fig. 44. Vaccination with Z13Mad5Anaxa or Z14Mad5Anaxa resulted in a significant reduction in tumor volumes compared to control mice (Figure 44A) as well as significantly increased survival rates compared to control mice (Figure 44B). These results demonstrated that both complexes, Z13Mad5Anaxa and Z14Mad5Anaxa, had the ability to significantly reduce tumor growth and significantly prolong survival.

Example 28: Comparison of Immune Responses Following Vaccination with Complexes Containing Various Cell Penetrating Peptides

In this experiment, the effects of various CPPs in the complex proposed for use according to the present invention were studied using a complex with the TLR agonist "EDA". For this purpose, the EDAZ13Mad5 fusion protein described above was used (see example 1).

In addition, additional fusion proteins were created that contained CPPs other than Z13, namely Z14 (SEQ ID NO: 7) or Z18 (SEQ ID NO: 11). These fusion proteins also contained the "MAD5" protein, consisting of various CD8 ⁺ - and CD4 ⁺ -epitopes from various antigens, and the TLR4 agonist peptide "EDA". Thus, the following structures were additionally created:

EDAZ14Mad5

Subsequence:

EDAZ18Mad5

Subsequence:

C57BL/6 mice were divided into 8 different groups (4 mice per group): three groups that were treated with either EDAZ13Mad5, EDAZ14Mad5, or EDAZ18Mad5 at a dose of 2 nmol and their corresponding control, and three groups that were treated with either EDAZ13Mad5, EDAZ14Mad5 or EDAZ18Mad5 at a dose of 5 nmol, and their corresponding control group. Mice were vaccinated three times (week 0, week 2 and week 4) s.c. Mice were bled 7 days after the 2nd and 3rd vaccinations and multimer staining was performed (one experiment with 4 mice per group).

The results are presented in Fig. 45. All groups vaccinated with EDAZ13Mad5, EDAZ14Mad5 or EDAZ18Mad5 showed an increased percentage of multimer-positive cells compared to the control group. These results demonstrated that the complexes of the present invention, which contained various cell penetrating peptides, had the ability to induce an immune response at varying doses.

Example 29: Effect of EDAZ14Mad5 on tumor growth and survival in the sc model EG.7-OVA

To study the effect of EDAZ14Mad5 on tumor growth and survival, the sc model EG.7-OVA was used (see example 4 and Fig. 9-11 for the results of the EDAZ13Mad5 study on the same model).

At d0, C57BL/6 mice were implanted with sc 3×10 ⁵ EG7-OVA tumor cells in the left flank region and divided into 2 groups (naïve and EDAZ14Mad5). Mice were vaccinated twice on d5 and d13 after tumor implantation by sc-injecting 0.5 nmol EDAZ14Mad5 into the right flank.

The results are presented in Fig. 46. Similar to EDAZ13Mad5 (see Example 4, FIGS. 9-11), vaccination with EDAZ14Mad5 resulted in a significant reduction in tumor volumes compared to control mice (FIG. 46 A) as well as significantly increased survival rates (FIG. 46 B). These results demonstrated that the EDAZ14Mad5 construct had the ability to significantly reduce tumor growth and significantly prolong survival similar to EDAZ13Mad5 (see Example 4, Figs. 9-11).

Example 30: Increased Efficacy of Z13Mad5Anaxa Fusion Construct Compared to Z13Mad5 and Anaxa in a Glioblastoma Model

To study the effectiveness of the complex proposed in the present invention, a glioblastoma model was chosen (see example 20). Thus, Z13Mad5Anaxa (see Example 7; SEQ ID NO: 28) was administered to one group of mice, and Z13Mad5 (SEQ ID NO: 29) and Anaxa (SEQ ID NO: 15) were administered (together) to another group of mice.

T-cell homing in the tumor region was analyzed in Gl261-Quad tumor-bearing mice (7-16 mice per group), which were vaccinated twice, namely: day 7 and day 21 after tumor implantation (day 0), using the Z13Mad5Anaxa vaccine at a dose of 2 nmol. A group vaccinated with both Z13Mad5 and Anaxa (at an equimolar dose of Z13Mad5Anaxa) was used as a control. In general, the method was as follows: C57BL/6 mice were implanted ie (intraskull) with 5x10 ⁵ Gl261-Quad tumor cells and vaccinated twice (at d7 and d21 after implantation) by sc-injection of Z13Mad5Anaxa at a dose of 2 nmol (group 1) or Z13Mad5 at a dose of 2 nmol and Anaxa at a dose of 2 nmol (group 2). On day 28, blood and brain-infiltrating leukocytes (BIL) were analyzed, with SIINFEKL-specific CD8 T cells in blood and BIL quantified at d28 by multimer staining (7-16 mice per group).

The results are presented in Fig. 47. A significantly higher percentage of SIINFEKL-specific CD8 T cells was detected in the blood of Z13Mad5Anaxa-vaccinated mice compared to mice vaccinated with both Z13Mad5 and Anaxa (FIG. 47A). Likewise, there was a greater accumulation of SIINFEKL-specific CD8 T cells in the BIL of Z13Mad5Anaxa-vaccinated mice compared to mice vaccinated with Z13Mad5 and Anaxa alone (FIG. 47B, p=0.0539).

Next, cytokine secretion was assessed. For this purpose, C57BL/6 mice were implanted with ie 5×10 ⁵ Gl261-Quad tumor cells and vaccinated twice (d7 and 21) by sc injection of Z13Mad5Anaxa at a dose of 2 nmol or Z13Mad5 at a dose of 2 nmol and Anaxa at a dose of 2 nmol . BIL were isolated and cultured for 6 h with mature BMDCs loaded or not with SllNFEKL peptide (SEQ ID NO: 35) in the presence of Brefeldin A before intracellular cytokine staining.

The results are presented in Fig. 48. Overall, higher levels of cytokine secretion were found for brain-infiltrating CD8 T cells from mice vaccinated with Z13Mad5Anaxa. In particular, significantly higher secretion levels of total IFN-γ and IFN-γ and TNF-α together were found for brain-infiltrating CD8 T cells from mice vaccinated with Z13Mad5Anaxa compared to mice vaccinated with Z13Mad5 and Anaxa alone.

Taken together, these results demonstrated that the Z13Mad5Anaxa vaccine (compared to Z13Mad5 and Anaxa alone) was able to induce a stronger SIINFEKL-specific CD8-T cell immune response in the brains of tumor-bearing mice with robust effector function.

The results indicate that Z13Mad5Anaxa is effective in inducing a high immune response of brain-infiltrating SIINFEKL-specific CD8 T cells. Z13Mad5Anaxa also has the ability to enhance cytokine secretion by antigen-specific CD8 T cells in the brain.

Example 31: Effect of other antigenic cargo molecules in the complex proposed in the present invention

To study the effects of various antigenic cargo molecules (“Mad8”), another complex was created containing a cell-penetrating peptide, various antigens, and a TLR peptide agonist (“Z13Mad8Anaxa”). Z13Mad8Anaxa differed from Z13Mad5Anaxa (described in example 7) in antigenic cargo molecules. Specifically, "Z13Mad8Anaxa" is a fusion protein containing the cell-penetrating peptide "Z13", the antigenic cargo molecule "MAD8", which contains the CD8 and CD4 epitopes of glycoprotein 70, and the TLR agonist peptide "Anaxa". Below is the amino acid sequence of Z13Mad8Anaxa, with the cell penetrating peptide "Z13" underlined and the TLR agonist peptide "Anaxa" in italics:

Naive Balb/c mice (4 mice per group) were vaccinated 4 times with s.c. (week 0, week 2, week 4 and week 6) Z13Mad8Anaxa at a dose of 2 nmol.

To study CD4 T-cell responses after vaccination, mice were sacrificed 1 week after the 4th vaccination; organs were removed and an ex vivo Elispot assay was performed on spleen cells stimulated with gp70CD4 peptide (SEQ ID NO: 64) or gp70CD8 peptide (SEQ ID NO: 65) to quantify IFN-γ producing epitope-specific CD4 and CD8 T cells .

The results are presented in Fig. 49. Mice vaccinated with Z13Mad8Anaxa showed a significant increase in IFN-γ-producing cells compared to naïve mice. These data demonstrate that the Z13Mad8Anaxa vaccine had the ability to induce an effective immune response of epitope-specific CD8 and CD4 T cells, and therefore the complex proposed in the present invention can induce an immune response to the self-antigen.

Example 32: Effect of other antigenic cargo molecules in the complex proposed in the present invention

To study the effects of additional other antigenic cargo molecules (“Mad 11”), another complex was created containing a cell-penetrating peptide, other antigens, and a TLR peptide agonist (“Z13Mad11Anaxa”). Z13Mad11Anaxa differed from Z13Mad5Anaxa (described in example 7) in antigenic cargo molecules. In particular, “Z13Mad11Anaxa” is a fusion protein containing the cell-penetrating peptide “Z13”, the antigenic cargo molecule “MAD11” containing two CD8 survivin epitopes, described in detail by Derouazi M., Wang Y., Marlu R. and etc. Optimal epitope composition after antigen screening using a live bacterial delivery vector: Application to TRP-2. Bioengineered Bugs. 1(1), 2010, pp. 51-60, doi:10.4161/bbug.1.1.9482, and the peptide TLR agonist Anaxa. Below is the amino acid sequence of Z13Mad11Anaxa, with the cell penetrating peptide "Z13" underlined and the TLR agonist peptide "Anaxa" in italics:

Naïve C57BL/6 mice (5 mice per group) were implanted with iv 1×10 ⁵ B16 melanoma tumor cells and vaccinated twice (d0 and d10) by subcutaneous injection of Z13Mad11Anaxa at a dose of 1 nmol. On day 18, mice were sacrificed, organs were removed, and ex vivo Elispot analysis was performed on spleen cells stimulated with the survivin peptides survivin 20-28 (SEQ ID NO: 67) and survivin 97-104: (SEQ ID NO: 68) to quantify the production IFN-γ survivin-specific T cells.

The results are presented in Fig. 50. Mice vaccinated with Z13Mad11Anaxa showed fewer metastases compared to naïve mice (Figure 50A). In addition, the number of IFN-γ-producing survivin-specific T cells was significantly increased in the spleen of mice vaccinated with Z13Mad11Anaxa (Fig. 49B).

The results demonstrated that Z13Mad11Anaxa was effective in reducing the number of metastases, and Z13Mad11Anaxa could enhance the secretion of cytokines by antigen-specific CD8 T cells in the spleen.

Example 33: Effect of other antigenic cargo molecules in the complex proposed in the present invention

To study the effects of additional other antigenic cargo molecules (“Mad9”), another complex was created containing a cell-penetrating peptide, another antigen, and a TLR peptide agonist (“Z13Mad9Anaxa”). Z13Mad9Anaxa differed from Z13Mad5Anaxa (described in example 7) by an antigenic cargo molecule. Specifically, "Z13Mad9Anaxa" is a fusion protein containing the cell-penetrating peptide "Z13", an antigenic cargo molecule "Mad9" containing a neoantigen described in Yadav et al. Nature. 515(7528), November 27, 2014, cc. 572-576, from the MC-38 tumor cell line, and the peptide TLR agonist Anaxa. Below is the amino acid sequence of Z13Mad9Anaxa, with the cell penetrating peptide "Z13" underlined and the TLR agonist peptide "Anaxa" in italics:

Naive C57BL/6 mice (4 mice per group) were vaccinated 4 times s.c. (week 0, week 2, week 4 and week 6) using Z13Mad9Anaxa at a dose of 2 nmol. To study CD8 T cell responses after vaccination, 1 week after the 4th vaccination, mice were sacrificed, organs were harvested and Elispot analysis was performed on spleen cells after in vitro restimulation on day 7 using adpgk peptide stimulation (SEQ ID NO: 66). to quantify IFN-γ-producing epitope-specific CD8 T cells.

The results are presented in Fig. 51. Mice vaccinated with Z13Mad9Anaxa showed a significant increase in effector neoantigen-specific CD8 T cells compared to naive mice

Example 34: Comparison of immune responses after vaccination with complexes having different cell-penetrating peptides

This experiment examined the effects of an additional other CPP in the complex of the present invention using a complex containing the TLR agonist "Anaxa". For this purpose, the Z13Mad5Anaxa fusion protein described above was used (see Example 7, SEQ ID NO: 28).

In addition, an additional fusion protein was created that contained TAT-CPP combined with furin linkers, described in Lu et al., Multiepitope trojan antigen peptide vaccines for the induction of antitumor CTL and Th immune responses J. Immunol., 172, 2004, cc. 4575-4582. This fusion protein also contained the "MAD5" protein, consisting of various CD8 ⁺ - and CD4 ⁺ - epitopes from various antigens, and the TLR4 agonist peptide "Anaxa". Thus, the following construction was additionally created:

TatFMad5Anaxa

Subsequence:

C57BL/6 mice were divided into three different groups (8 mice per group): one group that was treated with Z13Mad5Anaxa at a dose of 2 nmol, one group that was treated with TatFMad5Anaxa at a dose of 2 nmol, and a corresponding control. Mice were vaccinated twice (week 0 and week 2) s.c. using either Z13Mad5Anaxa at 2 nmol or TatFMad5Anaxa at 2 nmol. Mice were bled 7 days after the 2nd vaccination and multimer staining was performed (8 mice per group).

The results are presented in Fig. 52. Mice vaccinated with Z13Mad5Anaxa or TatFMad5Anaxa showed an increased percentage of multimer-positive cells compared to controls. These results indicate that the complexes of the present invention, which have different cell penetrating peptides, can induce an immune response at different doses. However, CPP derived from ZEBRA (Z13) was superior to TAT-CPP.

Example 35: Increased efficacy of the Z13Mad5Anaxa fusion construct compared to Z13Mad5 and Anaxa in naïve mice

Next, the effectiveness of the complex proposed in the present invention was studied in naive mice. To do this, Z13Mad5Anaxa (see Example 7; SEQ ID NO: 28) was administered to one group of mice, and Z13Mad5 (SEQ ID NO: 29) and Anaxa (SEQ ID NO: 15) were administered (together) to another group of mice.

C57BL/6 mice from the Z13Mad5Anaxa group and Z13Mad5 + Anaxa group were vaccinated once (week 0) by sc injection at a dose of 2 nmol Z13Mad5Anaxa (group 1) or 2 nmol Z13Mad5 and 2 nmol Anaxa (group 2). On day 14, blood was analyzed by quantifying SIINFEKL-specific CD8 T cells in the blood by multimer staining (4-8 mice per group).

The results are presented in Fig. 53. A significantly higher percentage of SIINFEKL-specific CD8 T cells was found in the blood of mice vaccinated with Z13Mad5Anaxa compared to mice vaccinated with Z13Mad5 and Anaxa alone (FIG. 53).

Taken together, these results demonstrated that the Z13Mad5Anaxa vaccine (compared to Z13Mad5 and Anaxa alone) had the ability to induce a stronger SIINFEKL-specific CD8 T cell peripheral immune response.

Example 36: Effect of another antigenic cargo molecule in the complex proposed in the present invention

To study the effects of an additional different antigenic cargo molecule (“Mad12”), another complex was created containing a cell-penetrating peptide, another antigen, and a TLR peptide agonist (“Z13Mad12Anaxa”). Z13Mad12Anaxa differed from Z13Mad5Anaxa (described in example 7) by an antigenic cargo molecule. Specifically, "Z13Mad12Anaxa" is a fusion protein containing the cell-penetrating peptide "Z13", the antigenic cargo molecule "MAD12", which contains three neoantigens described in Yadav et al. Nature. 515(7528), November 27, 2014, cc. 572-576, from the MC-38 tumor cell line, and the peptide TLR agonist “Anaxa”. Below is the amino acid sequence of Z13Mad12Anaxa:

Naïve C57BL/6 mice (4 mice per group) were vaccinated twice with s.c. (week 0, week 2) using Z13Mad12Anaxa at a dose of 2 nmol. To study CD8 T cell responses after vaccination, blood was analyzed 1 week after the 2nd vaccination and reps1 neoantigen-specific CD8 T cells in the blood were quantified by multimer staining (4 mice per group).

The results are presented in Fig. 54. Mice vaccinated with Z13Mad12Anaxa showed a significant increase in effector neoantigen-specific CD8 T cells compared to naïve mice.

Example 37: In Vitro Maturation of Human Dendritic Cells

The basis of this study was to examine the ability of the complex proposed for use according to the present invention (Z13Mad5Anaxa, SEQ ID NO: 28, see Example 7) to induce the maturation of dendritic cells in comparison with a complex lacking the TLR agonist peptide ( "Z13Mad5", SEQ ID NO: 29, see example 1).

The Z13Mad5Anaxa polypeptide and the Z13Mad5 polypeptide were examined for their ability to induce maturation of human dendritic cells (DC). After overnight incubation with 300 nM protein, expression of activation markers (CD86, CD80, CD83 and HLA-DR) on human DCs was assessed by FACS (FIG. 55). Buffer was used in the same volumes as a negative control for each protein.

The results are presented in Fig. 55. While the Z13Mad5Anaxa complex induced the maturation of human DCs, as evidenced by upregulation of CD86, HLADR, and CD83, Z13Mad5 did not have the ability to activate human DCs. These results suggest that the Anaxa component of the protein is responsible for the upregulation of activation markers on human DCs.

Example 38: CD8 T-cell immune response during booster vaccination

To address the question of whether the pool of effector T cells can be maintained for several months, repeated vaccination was carried out. For this purpose, C57BL/6 mice were vaccinated subcutaneously six times (weeks 0, 2, 4, 8, 12, 16) using the Z13Mad5Anaxa construct (SEQ ID NO: 28) at a dose of 2 nmol. Seven days after each vaccination (and before some vaccinations), mice were bled and stained with pentamer to monitor the OVA-specific immune response in the blood (two experiments with 4 mice per group). In fig. 56 shows the percentage of pentamer-positive CD8 ⁺ T cells in mice vaccinated with Z13Mad5Anaxa and control mice.

These data demonstrate that the effector T cell pool can be maintained for 4 months (17 weeks) using 6 vaccinations.

Example 39: Evaluation of the complex proposed in the present invention in the MC38 murine colorectal cancer model

To evaluate the effects of the complex of the present invention in a murine colorectal cancer model, the MC38 murine colorectal cancer model was selected. MC38 is a colon carcinoma cell line.

Naïve C57BL/6 mice were implanted with sc 2×10 ⁵ MC38 tumor cells in the left flank region and vaccinated twice (on day -21 and day -7 before tumor implantation) by subcutaneous injection of the Z13Mad11Anaxa construct at a dose of 2 nmol (see example 32, SEQ ID NO: 40) to the right side area. The antigenic cargo molecule “Mad11” contains two survivin epitopes, the expression of which took place in the MC38 mouse model of colorectal cancer.

The results are presented in Fig. 57. Mice vaccinated with Z13Mad11Anaxa showed significantly less tumor volume compared to naive mice (Fig. 57A). In addition, mice vaccinated with Z13Mad11Anaxa showed a significantly higher survival rate (Fig. 57B).

The results demonstrate that Z13Mad11Anaxa can significantly reduce and slow tumor growth compared to controls. In addition, mice vaccinated with Z13Mad11Anaxa had increased survival rates.

Example 40: Preliminary Toxicity Test in Mice

To evaluate the toxicity of the complex of the present invention in mice, Z13Mad5Anaxa (SEQ ID NO: 28) was injected at a dose of 2 nmol s.c. and i.v. naive mice of the C57BL/6 line. Blood samples were collected at 0.5, 1.5 and 3 hours after administration. A commercial multiplex detection kit (Luminex) was used to detect and quantify blood cytokines, and blood samples from 4 mice were obtained at each time point. However, no expression of proinflammatory cytokines could be detected. It should be noted that when comparing data obtained using the multiplex detection kit with data obtained using classical ELISA, the results were similar (data not shown).

Next, the dose of Z13Mad5Anaxa (SEQ ID NO: 28) was increased to 10 nmol. However, in this case, no cytokines were detected after s.c. administration. In contrast to s.c. administration, a transient increase in IL-6 and TNFα was found 1.5 hours after i.v. administration. However, this slight increase disappeared after 3 hours (Fig. 58). Subcutaneous injection of 10 nmol Z13Mad5Anaxa mice did not induce any cytokine release until 6 h after treatment (data not shown).

Example 41: Effects of a complex containing a cell-penetrating peptide, various antigens and a TLR peptide agonist on T cell homing to the MS-38 tumor area

To evaluate the effects of a complex containing a cell-penetrating peptide, various antigens, and a TLR peptide agonist on T cell homing to the tumor site, mice were vaccinated with the cell-penetrating peptide, various antigens, and a TLR peptide agonist using the MC-38 tumor model. On day 27, mice were sacrificed and FACS staining was performed to monitor neoantigen-specific immune response in TILs (tumor infiltrating lymphocytes).

C57BL/6 mice (4 mice per group, female, 7 weeks old) were implanted with sc 2×10 ⁵ tumor cells of the MC-38 line in the left flank region (day 0). After implantation of tumors, Z13Mad12Anaxa group mice were vaccinated on days 3, 10 and 17 subcutaneously with a dose of 2 nmol Z13Mad12Anaxa (see Example 36; SEQ ID NO: 69) at the base of the tail.

As shown in FIG. 59, 60, and 61, neoantigen-specific T cells accumulated at the tumor site in vaccinated mice. The percentage of multimer-positive cells increased significantly in mice vaccinated with Z13Mad12Anaxa. Control mice showed a lower percentage of multimer-positive cells.

Example 42: Activation of Human Dendritic Cells by Constructs Containing Human Antigens

The purpose of the present study was to study the various complexes proposed in the present invention, which contain various human antigens as cargo molecules, to induce the maturation of human dendritic cells. For this purpose, each of the constructs ATP110 (SEQ ID NO: 72), ATP112 (SEQ ID NO: 74), ATP115 (SEQ ID NO: 77), ATP117 (SEQ ID NO: 79), ATP118 (SEQ ID NO: 80) was tested ), ATP119 (SEQ ID NO: 81), ATP120 (SEQ ID NO: 82), ATP122 (SEQ ID NO: 84), ATP123 (SEQ ID NO: 85) and ATP125 (SEQ ID NO: 87). The biological indicator used to assess DC activation was the activation index, which indicates the percentage of activation based on the intensity of expression of four membrane antigens: HLA-DR, CD80, CD83 and CD86.

After overnight incubation using 300 nM or 600 nM of each of the above constructs (ATP110 (SEQ ID NO: 72), ATP 112 (SEQ ID NO: 74), ATP 115 (SEQ ID NO: 77), ATP 117 (SEQ ID NO: 79), ATP118 (SEQ ID NO: 80), ATP119 (SEQ ID NO: 81), ATP120 (SEQ ID NO: 82), ATP122 (SEQ ID NO: 84), ATP123 (SEQ ID NO: 85) and ATP125 (SEQ ID NO: 87)), the expression of activation markers (CD86, CD80, CD83 and HLA-DR) on human dendritic cells was assessed using FACS (Fig. 62-71). The same volumes of buffer were used as negative controls for each construct.

The results presented in Fig. 62-71 demonstrate that dendritic cell maturation occurred with all constructs tested, as evidenced by up-regulation of CD86, HLADR and CD83.

Example 43: Effects of a combination of a PD1 inhibitor and a complex containing a cell penetrating peptide, various antigens and a TLR agonist peptide. on tumor growth and survival rate in a colon carcinoma model

To evaluate the effectiveness of the combination of a PD1 inhibitor and a complex containing a cell-penetrating peptide, various antigens and a TLR peptide agonist in the treatment of colorectal cancer, the MS-38 tumor model was used. MS-38 is a colon carcinoma cell line.

For this purpose, C57BL/6 mice (13-14 mice per group, females, 7 weeks old) were implanted with sc 2×10 ⁵ MC-38 tumor cells in the left flank region (day 0). After tumor implantation, mice from the Z13Mad12Anaxa and Z13Mad12Anaxa + anti-PD1 groups were vaccinated on day 3, day 10 and 17 by subcutaneous tail base administration of Z13Mad12Anaxa (see Example 36; SEQ ID NO: 69) at a dose of 2 nmol. 200 μg of anti-PD1 antibody RMP1-14 (BioXcell, West Lebanon, NH, USA) was administered ip on each of days 6, 10, 13, 17, 20, 24 and 27 to mice in the anti-PD1 groups. and “Z13Mad12Anaxa + anti-PD1.” On days 10 and 17, when both Z13Mad12Anaxa and anti-PD1 antibody were co-administered in the “Z13Mad12Anaxa + anti-PD1” group, the antibody was administered ip directly after sc-treatment with Z13Mad12Anaxa. The size of tumors was determined using calipers.

As shown in FIG. 72 and 73, treatment with PD1 inhibitor alone or Z13Mad12Anaxa alone resulted in significantly reduced tumor volumes (FIG. 72A) and increased survival (FIG. 72B) compared to the control group. However, a combination of both of them, i.e. PD1 inhibitor and Z13Mad12Anaxa, led to the most pronounced improvement, namely, a strong reduction in tumor volumes and a strong increase in survival rates. It should be noted that in the Z13Mad12Anaxa + aPD1 group, only three mice developed tumors (while the remaining 10 mice had no tumors (tumor-free mice)), while in the aPD1 group and in the Z13Mad12Anaxa group "8 and 10 mice, respectively, developed tumors. In the control group, all mice developed tumors. These data suggest that the combination of anti-PD1 antibody therapy and Z13Mad12Anaxa vaccination was superior to anti-PD1 therapy alone or Z13Mad12Anaxa vaccination alone.

As shown in FIG. 73, the number of tumor-free mice in the “Z13Mad12Anaxa + aPD1” group (10 mice out of 13 mice) turned out to be greater than the sum of the numbers of tumor-free mice in the “aPD1” group (6 mice out of 14 mice) and in the “Z13Mad12Anaxa” group (3 mice from 13 mice). These results suggest a synergistic effect of anti-PD1 antibody therapy and Z13Mad12Anaxa vaccination.

Example 44: Immune response induced in mice after vaccination with ATP128

Another human-derived construct, ATP128 (SEQ ID NO: 89), was created comprising a cell penetrating peptide, epitopes of three antigens (survivin, CEA and ASCL2) and a TLR agonist peptide. Specifically, "ATP128" (SEQ ID NO: 89) is a fusion protein that contains the cell-penetrating peptide "Z13" (SEQ ID NO: 6), epitopes of the three survivin antigens, CEA and ASCL2, and the sequence of a TLR agonist variant peptide " Anaxa" (namely, a TLR peptide agonist having SEQ ID NO: 71). Below is the amino acid sequence of ATP128:

Naive C57BL/6 mice (5 mice per group, female, 7 weeks old) were vaccinated by subcutaneous injection of ATP128 (SEQ ID NO: 89) at a dose of 4 nmol. Control mice were treated with vehicle (vaccine buffer). 7 days after treatment, mice were bled and an ex vivo Elispot assay was performed on blood cells stimulated with ATP128-loaded mouse dendritic cells to determine the number of vaccine-specific IFN-γ-producing T cells.

The results are presented in Fig. 74. Mice vaccinated with ATP128 exhibited a higher ATP128-specific immune response compared to naïve mice.

The results indicate that ATP128 is effective in inducing an immune response in mice.

Example 45: Activation of Human Dendritic Cells by ATP128 Construct

The purpose of this study was to study the ability of the ATP128 construct (SEQ ID NO: 89), containing a human antigen as a cargo molecule, to induce the maturation of human dendritic cells (human DC). ATP128 (SEQ ID NO: 89) was tested for this purpose. The biological metric used to assess DC activation was the activation index (FIG. 75), which indicates the percentage of activation based on the intensity of expression of four membrane antigens: HLA-DR, CD80, CD83 and CD86.

After overnight incubation with ATP128 (SEQ ID NO: 89) at a dose of 300 nM, the expression of activation markers (CD86, CD80, CD83 and HLA-DR) on human dendritic cells was assessed using FACS (Fig. 75). The same volume of buffer was used as a negative control, and MPLA was used as a positive control.

The results are presented in Fig. 75 The results demonstrate that ATP128 can effectively activate human DCs.

Example 46: Activation of Human Dendritic Cells by ATP128 Construct

To confirm that human cells can process and present the epitope peptides included in ATP128 (SEQ ID NO: 89), human dendritic cells (human DCs) from two different donors (namely, donor "9" and "10") were loaded overnight with ATP128 and processed for purification of MHC class I and class II peptides presented by MHC molecules, peptide elution and characterization by mass spectroscopy. The overall method was as follows: peptide pools from shock-frozen human DC samples were prepared by immunoprecipitation using HLA-specific antibodies, acid treatment, and ultrafiltration. Pools of HLA peptides were separated based on their hydrophobicity using reverse phase chromatography and the eluted peptides were analyzed using a mass spectrometer (Thermo Fisher Scientific). The data was then acquired and automatically processed by analyzing signals corresponding to the masses of unfragmented peptides, as well as information about the spectra of fragments containing the peptide sequence. False discovery rate (FDR) was determined using the Percolator algorithm ( L., Canterbury JD, Weston J., Noble WS, MacCoss MJ Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat Methods 4(11), 2007, cc. 923-925)) based on the processing of a “decoy” database (a “decoy” database), consisting of a shuffled target database. For MHC class I molecules, peptides are limited to 8–12 amino acids in length. For MHC class II molecules, peptides are limited to 12–25 amino acids in length. GCSG annotation was performed using SYFPEITHI (www.syfpeithi.de).

The data demonstrated that human DCs loaded with ATP128 could process and present both class I and class II epitope peptides derived from the polyantigen domain (Fig. 76). In the class I pool, peptides derived from all antigens were identified. A greater number of class II-related peptides were identified in all antigenic regions of the vaccine.

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LIST OF SEQUENCES

<110> AMAL THERAPYUTICS SA

<120> FUSION CONSTRUCTION CONTAINING A CELL PENETRATING PEPTIDE,

POLYEPITOPE AND TLR PEPTIDE AGONIST, INTENDED

FOR CANCER TREATMENT

<130>AM05P013WO

<150> PCT/EP2016/072475

<151> 2016-09-21

<160> 98

<170> Patent In version 3.5

<210> 1

<211> 16

<212>PRT

<213> Artificial Sequence

<220>

<223> CPP: Penetratin

<400> 1

Arg Gln Ile Lys Ile Tyr Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

<210> 2

<211> 11

<212>PRT

<213> Artificial Sequence

<220>

<223> CPP: TAT minimal domain

<400> 2

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5 10

<210> 3

<211> 245

<212>PRT

<213> Artificial Sequence

<220>

<223> ZEBRA amino acid sequence (natural sequence from Epstein -

Barr

virus (EBV)) (YP_401673)

<400> 3

Met Met Asp Pro Asn Ser Thr Ser Glu Asp Val Lys Phe Thr Pro Asp

1 5 10 15

Pro Tyr Gln Val Pro Phe Val Gln Ala Phe Asp Gln Ala Thr Arg Val

20 25 30

Tyr Gln Asp Leu Gly Gly Pro Ser Gln Ala Pro Leu Pro Cys Val Leu

35 40 45

Trp Pro Val Leu Pro Glu Pro Leu Pro Gln Gly Gln Leu Thr Ala Tyr

50 55 60

His Val Ser Thr Ala Pro Thr Gly Ser Trp Phe Ser Ala Pro Gln Pro

65 70 75 80

Ala Pro Glu Asn Ala Tyr Gln Ala Tyr Ala Ala Pro Gln Leu Phe Pro

85 90 95

Val Ser Asp Ile Thr Gln Asn Gln Gln Thr Asn Gln Ala Gly Gly Glu

100 105 110

Ala Pro Gln Pro Gly Asp Asn Ser Thr Val Gln Thr Ala Ala Ala Val

115 120 125

Val Phe Ala Cys Pro Gly Ala Asn Gln Gly Gln Gln Leu Ala Asp Ile

130 135 140

Gly Val Pro Gln Pro Ala Pro Val Ala Ala Pro Ala Arg Arg Thr Arg

145 150 155 160

Lys Pro Gln Gln Pro Glu Ser Leu Glu Glu Cys Asp Ser Glu Leu Glu

165 170 175

Ile Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Cys Arg Ala Lys

180 185 190

Phe Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser

195 200 205

Ser Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Gln Met Cys Pro Ser

210 215 220

Leu Asp Val Asp Ser Ile Ile Pro Arg Thr Pro Asp Val Leu His Glu

225 230 235 240

Asp Leu Leu Asn Phe

245

<210> 4

<211> 45

<212>PRT

<213> Artificial Sequence

<220>

<223> CPP1 (Z11)

<400> 4

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Cys Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Gln Met Cys

35 40 45

<210> 5

<211> 42

<212>PRT

<213> Artificial Sequence

<220>

<223> CPP2 (Z12)

<400> 5

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Cys Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys

35 40

<210> 6

<211> 42

<212>PRT

<213> Artificial Sequence

<220>

<223> CPP3 (Z13)

<400> 6

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys

35 40

<210> 7

<211> 30

<212>PRT

<213> Artificial Sequence

<220>

<223> CPP4 (Z14)

<400> 7

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys

20 25 30

<210> 8

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223> CPP5 (Z15)

<400> 8

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys

<210> 9

<211> 15

<212>PRT

<213> Artificial Sequence

<220>

<223> CPP6 (Z16)

<400> 9

Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser Glu Asn Asp

1 5 10 15

<210> 10

<211> 13

<212>PRT

<213> Artificial Sequence

<220>

<223> CPP7 (Z17)

<400> 10

Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys

1 5 10

<210> 11

<211> 19

<212>PRT

<213> Artificial Sequence

<220>

<223> CPP8 (Z18)

<400> 11

Arg Glu Val Ala Ala Ala Lys Ser Ser Glu Asn Asp Arg Leu Arg Leu

1 5 10 15

Leu Leu Lys

<210> 12

<211> 8

<212>PRT

<213> Artificial Sequence

<220>

<223> CPP9 (Z19)

<400> 12

Lys Arg Tyr Lys Asn Arg Val Ala

15

<210> 13

<211> 11

<212>PRT

<213> Artificial Sequence

<220>

<223> CPP10 (Z20)

<400> 13

Val Ala Ser Arg Lys Ser Arg Ala Lys Phe Lys

1 5 10

<210> 14

<211> 85

<212>PRT

<213> Artificial Sequence

<220>

<223> MAD5 cargo

<400> 14

Glu Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile

1 5 10 15

Asn Glu Ala Gly Arg Glu Val Val Gly Val Gly Ala Leu Lys Val Pro

20 25 30

Arg Asn Gln Asp Trp Leu Gly Val Pro Arg Phe Ala Lys Phe Ala Ser

35 40 45

Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala Val Asp Lys Ala Asn

50 55 60

Leu Asp Val Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr

65 70 75 80

Glu Trp Thr Gly Ser

85

<210> 15

<211> 35

<212>PRT

<213> Artificial Sequence

<220>

<223> TLR2 peptide agonist Anaxa

<400> 15

Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser Leu Glu Gly Asp His

1 5 10 15

Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe

20 25 30

Asp Ala Glu

35

<210> 16

<211> 4

<212>PRT

<213> Artificial Sequence

<220>

<223> enterokinase target site

<400> 16

Asp Asp Asp Lys

1

<210> 17

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> factor Xa target site

<400> 17

Ile Glu Asp Gly Arg

15

<210> 18

<211> 6

<212>PRT

<213> Artificial Sequence

<220>

<223> thrombin target site

<400> 18

Leu Val Pro Arg Gly Ser

15

<210> 19

<211> 7

<212>PRT

<213> Artificial Sequence

<220>

<223> protease TEV target site

<400> 19

Glu Asn Leu Tyr Phe Gln Gly

15

<210> 20

<211> 8

<212>PRT

<213> Artificial Sequence

<220>

<223> PreScission protease target site

<400> 20

Leu Glu Val Leu Phe Gln Gly Pro

15

<210> 21

<211> 4

<212>PRT

<213> Artificial Sequence

<220>

<223> furin target site

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X may be any amino acid

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X is R or K

<400> 21

Arg Xaa Xaa Arg

1

<210> 22

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> peptidic linker

<400> 22

Gly Gly Gly Gly Gly

15

<210> 23

<211> 4

<212>PRT

<213> Artificial Sequence

<220>

<223> peptidic linker

<400> 23

Gly Gly Gly Gly

1

<210> 24

<211> 4

<212>PRT

<213> Artificial Sequence

<220>

<223> peptidic linker

<400> 24

Glu Gln Leu Glu

1

<210> 25

<211> 4

<212>PRT

<213> Artificial Sequence

<220>

<223> peptidic linker

<400> 25

Thr Glu Trp Thr

1

<210> 26

<211> 224

<212>PRT

<213> Artificial Sequence

<220>

<223>EDAZ13Mad5

<400> 26

Met His His His His His Asn Ile Asp Arg Pro Lys Gly Leu Ala

1 5 10 15

Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile Ala Trp Glu Ser Pro

20 25 30

Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser Ser Pro Glu Asp

35 40 45

Gly Ile Arg Glu Leu Phe Pro Ala Pro Asp Gly Glu Asp Asp Thr Ala

50 55 60

Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu Tyr Thr Val Ser Val Val

65 70 75 80

Ala Leu His Asp Asp Met Glu Ser Gln Pro Leu Ile Gly Ile Gln Ser

85 90 95

Thr Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys

100 105 110

Phe Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser

115 120 125

Ser Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Glu Ser Leu Lys Ile

130 135 140

Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly Arg

145 150 155 160

Glu Val Val Gly Val Gly Ala Leu Lys Val Pro Arg Asn Gln Asp Trp

165 170 175

Leu Gly Val Pro Arg Phe Ala Lys Phe Ala Ser Phe Glu Ala Gln Gly

180 185 190

Ala Leu Ala Asn Ile Ala Val Asp Lys Ala Asn Leu Asp Val Glu Gln

195 200 205

Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr Gly Ser

210 215 220

<210> 27

<211> 169

<212>PRT

<213> Artificial Sequence

<220>

<223> AnaxaZ13Mad5

<400> 27

Met His His His His His Ser Thr Val His Glu Ile Leu Cys Lys

1 5 10 15

Leu Ser Leu Glu Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser

20 25 30

Val Lys Pro Tyr Thr Asn Phe Asp Ala Glu Lys Arg Tyr Lys Asn Arg

35 40 45

Val Ala Ser Arg Lys Ser Arg Ala Lys Phe Lys Gln Leu Leu Gln His

50 55 60

Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser Glu Asn Asp Arg Leu Arg

65 70 75 80

Leu Leu Leu Lys Glu Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala

85 90 95

His Ala Glu Ile Asn Glu Ala Gly Arg Glu Val Val Gly Val Gly Ala

100 105 110

Leu Lys Val Pro Arg Asn Gln Asp Trp Leu Gly Val Pro Arg Phe Ala

115 120 125

Lys Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala Val

130 135 140

Asp Lys Ala Asn Leu Asp Val Glu Gln Leu Glu Ser Ile Ile Asn Phe

145 150 155 160

Glu Lys Leu Thr Glu Trp Thr Gly Ser

165

<210> 28

<211> 169

<212>PRT

<213> Artificial Sequence

<220>

<223> Z13Mad5Anaxa

<400> 28

Met His His His His His Lys Arg Tyr Lys Asn Arg Val Ala Ser

1 5 10 15

Arg Lys Ser Arg Ala Lys Phe Lys Gln Leu Leu Gln His Tyr Arg Glu

20 25 30

Val Ala Ala Ala Lys Ser Ser Glu Asn Asp Arg Leu Arg Leu Leu Leu

35 40 45

Lys Glu Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu

50 55 60

Ile Asn Glu Ala Gly Arg Glu Val Val Gly Val Gly Ala Leu Lys Val

65 70 75 80

Pro Arg Asn Gln Asp Trp Leu Gly Val Pro Arg Phe Ala Lys Phe Ala

85 90 95

Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala Val Asp Lys Ala

100 105 110

Asn Leu Asp Val Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu

115 120 125

Thr Glu Trp Thr Gly Ser Ser Thr Val His Glu Ile Leu Cys Lys Leu

130 135 140

Ser Leu Glu Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val

145 150 155 160

Lys Pro Tyr Thr Asn Phe Asp Ala Glu

165

<210> 29

<211> 134

<212>PRT

<213> Artificial Sequence

<220>

<223>Z13Mad5

<400> 29

Met His His His His His Lys Arg Tyr Lys Asn Arg Val Ala Ser

1 5 10 15

Arg Lys Ser Arg Ala Lys Phe Lys Gln Leu Leu Gln His Tyr Arg Glu

20 25 30

Val Ala Ala Ala Lys Ser Ser Glu Asn Asp Arg Leu Arg Leu Leu Leu

35 40 45

Lys Glu Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu

50 55 60

Ile Asn Glu Ala Gly Arg Glu Val Val Gly Val Gly Ala Leu Lys Val

65 70 75 80

Pro Arg Asn Gln Asp Trp Leu Gly Val Pro Arg Phe Ala Lys Phe Ala

85 90 95

Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala Val Asp Lys Ala

100 105 110

Asn Leu Asp Val Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu

115 120 125

Thr Glu Trp Thr Gly Ser

130

<210> 30

<211> 92

<212>PRT

<213> Artificial Sequence

<220>

<223> Mad5

<400> 30

Met His His His His His Glu Ser Leu Lys Ile Ser Gln Ala Val

1 5 10 15

His Ala Ala His Ala Glu Ile Asn Glu Ala Gly Arg Glu Val Val Gly

20 25 30

Val Gly Ala Leu Lys Val Pro Arg Asn Gln Asp Trp Leu Gly Val Pro

35 40 45

Arg Phe Ala Lys Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn

50 55 60

Ile Ala Val Asp Lys Ala Asn Leu Asp Val Glu Gln Leu Glu Ser Ile

65 70 75 80

Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr Gly Ser

85 90

<210> 31

<211> 182

<212>PRT

<213> Artificial Sequence

<220>

<223> EdaMad5

<400> 31

Met His His His His His Asn Ile Asp Arg Pro Lys Gly Leu Ala

1 5 10 15

Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile Ala Trp Glu Ser Pro

20 25 30

Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser Ser Pro Glu Asp

35 40 45

Gly Ile Arg Glu Leu Phe Pro Ala Pro Asp Gly Glu Asp Asp Thr Ala

50 55 60

Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu Tyr Thr Val Ser Val Val

65 70 75 80

Ala Leu His Asp Asp Met Glu Ser Gln Pro Leu Ile Gly Ile Gln Ser

85 90 95

Thr Glu Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu

100 105 110

Ile Asn Glu Ala Gly Arg Glu Val Val Gly Val Gly Ala Leu Lys Val

115 120 125

Pro Arg Asn Gln Asp Trp Leu Gly Val Pro Arg Phe Ala Lys Phe Ala

130 135 140

Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala Val Asp Lys Ala

145 150 155 160

Asn Leu Asp Val Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu

165 170 175

Thr Glu Trp Thr Gly Ser

180

<210> 32

<211> 127

<212>PRT

<213> Artificial Sequence

<220>

<223> Mad5Anaxa

<400> 32

Met His His His His His Glu Ser Leu Lys Ile Ser Gln Ala Val

1 5 10 15

His Ala Ala His Ala Glu Ile Asn Glu Ala Gly Arg Glu Val Val Gly

20 25 30

Val Gly Ala Leu Lys Val Pro Arg Asn Gln Asp Trp Leu Gly Val Pro

35 40 45

Arg Phe Ala Lys Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn

50 55 60

Ile Ala Val Asp Lys Ala Asn Leu Asp Val Glu Gln Leu Glu Ser Ile

65 70 75 80

Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr Gly Ser Ser Thr Val His

85 90 95

Glu Ile Leu Cys Lys Leu Ser Leu Glu Gly Asp His Ser Thr Pro Pro

100 105 110

Ser Ala Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe Asp Ala Glu

115 120 125

<210> 33

<211> 157

<212>PRT

<213> Artificial Sequence

<220>

<223> Z14Mad5Anaxa

<400> 33

Met His His His His His Lys Arg Tyr Lys Asn Arg Val Ala Ser

1 5 10 15

Arg Lys Ser Arg Ala Lys Phe Lys Gln Leu Leu Gln His Tyr Arg Glu

20 25 30

Val Ala Ala Ala Lys Glu Ser Leu Lys Ile Ser Gln Ala Val His Ala

35 40 45

Ala His Ala Glu Ile Asn Glu Ala Gly Arg Glu Val Val Gly Val Gly

50 55 60

Ala Leu Lys Val Pro Arg Asn Gln Asp Trp Leu Gly Val Pro Arg Phe

65 70 75 80

Ala Lys Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala

85 90 95

Val Asp Lys Ala Asn Leu Asp Val Glu Gln Leu Glu Ser Ile Ile Asn

100 105 110

Phe Glu Lys Leu Thr Glu Trp Thr Gly Ser Ser Thr Val His Glu Ile

115 120 125

Leu Cys Lys Leu Ser Leu Glu Gly Asp His Ser Thr Pro Pro Ser Ala

130 135 140

Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe Asp Ala Glu

145 150 155

<210> 34

<211> 146

<212>PRT

<213> Artificial Sequence

<220>

<223> Z18Mad5Anaxa

<400> 34

Met His His His His His Arg Glu Val Ala Ala Ala Lys Ser Ser

1 5 10 15

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Glu Ser Leu Lys Ile Ser

20 25 30

Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly Arg Glu

35 40 45

Val Val Gly Val Gly Ala Leu Lys Val Pro Arg Asn Gln Asp Trp Leu

50 55 60

Gly Val Pro Arg Phe Ala Lys Phe Ala Ser Phe Glu Ala Gln Gly Ala

65 70 75 80

Leu Ala Asn Ile Ala Val Asp Lys Ala Asn Leu Asp Val Glu Gln Leu

85 90 95

Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr Gly Ser Ser

100 105 110

Thr Val His Glu Ile Leu Cys Lys Leu Ser Leu Glu Gly Asp His Ser

115 120 125

Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe Asp

130 135 140

Ala Glu

145

<210> 35

<211> 8

<212>PRT

<213> Artificial Sequence

<220>

<223>SIINFEKL OVACD8

<400> 35

Ser Ile Ile Asn Phe Glu Lys Leu

15

<210> 36

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223>OVACD4 peptide

<400> 36

Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly

1 5 10 15

Arg

<210> 37

<211> 212

<212>PRT

<213> Artificial Sequence

<220>

<223>EDAZ14Mad5

<400> 37

Met His His His His His Asn Ile Asp Arg Pro Lys Gly Leu Ala

1 5 10 15

Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile Ala Trp Glu Ser Pro

20 25 30

Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser Ser Pro Glu Asp

35 40 45

Gly Ile Arg Glu Leu Phe Pro Ala Pro Asp Gly Glu Asp Asp Thr Ala

50 55 60

Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu Tyr Thr Val Ser Val Val

65 70 75 80

Ala Leu His Asp Asp Met Glu Ser Gln Pro Leu Ile Gly Ile Gln Ser

85 90 95

Thr Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys

100 105 110

Phe Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Glu

115 120 125

Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn

130 135 140

Glu Ala Gly Arg Glu Val Val Gly Val Gly Ala Leu Lys Val Pro Arg

145 150 155 160

Asn Gln Asp Trp Leu Gly Val Pro Arg Phe Ala Lys Phe Ala Ser Phe

165 170 175

Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala Val Asp Lys Ala Asn Leu

180 185 190

Asp Val Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu

195 200 205

Trp Thr Gly Ser

210

<210> 38

<211> 201

<212>PRT

<213> Artificial Sequence

<220>

<223>EDAZ18Mad5

<400> 38

Met His His His His His Asn Ile Asp Arg Pro Lys Gly Leu Ala

1 5 10 15

Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile Ala Trp Glu Ser Pro

20 25 30

Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser Ser Pro Glu Asp

35 40 45

Gly Ile Arg Glu Leu Phe Pro Ala Pro Asp Gly Glu Asp Asp Thr Ala

50 55 60

Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu Tyr Thr Val Ser Val Val

65 70 75 80

Ala Leu His Asp Asp Met Glu Ser Gln Pro Leu Ile Gly Ile Gln Ser

85 90 95

Thr Arg Glu Val Ala Ala Ala Lys Ser Ser Glu Asn Asp Arg Leu Arg

100 105 110

Leu Leu Leu Lys Glu Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala

115 120 125

His Ala Glu Ile Asn Glu Ala Gly Arg Glu Val Val Gly Val Gly Ala

130 135 140

Leu Lys Val Pro Arg Asn Gln Asp Trp Leu Gly Val Pro Arg Phe Ala

145 150 155 160

Lys Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala Val

165 170 175

Asp Lys Ala Asn Leu Asp Val Glu Gln Leu Glu Ser Ile Ile Asn Phe

180 185 190

Glu Lys Leu Thr Glu Trp Thr Gly Ser

195 200

<210> 39

<211> 116

<212>PRT

<213> Artificial Sequence

<220>

<223> Z13Mad8Anaxa

<400> 39

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Val Thr Tyr His Ser Pro

35 40 45

Ser Tyr Ala Tyr His Gln Phe Glu Arg Arg Ala Ile Leu Asn Arg Leu

50 55 60

Val Gln Phe Ile Lys Asp Arg Ile Ser Val Val Gln Ala Leu Val Leu

65 70 75 80

Thr Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser Leu Glu Gly Asp

85 90 95

His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Pro Tyr Thr Asn

100 105 110

Phe Asp Ala Glu

115

<210> 40

<211> 110

<212>PRT

<213> Artificial Sequence

<220>

<223> Z13Mad11Anaxa

<400> 40

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Asn Tyr Arg Ile Ala Thr

35 40 45

Phe Lys Asn Trp Pro Phe Leu Glu Asp Cys Ala Met Glu Glu Leu Thr

50 55 60

Val Ser Glu Phe Leu Lys Leu Asp Arg Gln Arg Ser Thr Val His Glu

65 70 75 80

Ile Leu Cys Lys Leu Ser Leu Glu Gly Asp His Ser Thr Pro Pro Ser

85 90 95

Ala Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe Asp Ala Glu

100 105 110

<210> 41

<211> 94

<212>PRT

<213> Artificial Sequence

<220>

<223> Z13Mad9Anaxa

<400> 41

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys His Leu Glu Leu Ala Ser

35 40 45

Met Thr Asn Met Glu Leu Met Ser Ser Ile Val Ser Thr Val His Glu

50 55 60

Ile Leu Cys Lys Leu Ser Leu Glu Gly Asp His Ser Thr Pro Pro Ser

65 70 75 80

Ala Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe Asp Ala Glu

85 90

<210> 42

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223> Mad9

<400> 42

His Leu Glu Leu Ala Ser Met Thr Asn Met Glu Leu Met Ser Ser Ile

1 5 10 15

Val

<210> 43

<211> 20

<212>PRT

<213> Artificial Sequence

<220>

<223> Mad8

<400> 43

Val Thr Tyr His Ser Pro Ser Tyr Ala Tyr His Gln Phe Glu Arg Arg

1 5 10 15

Ala Ile Leu Asn

20

<210> 44

<211> 33

<212>PRT

<213> Artificial Sequence

<220>

<223> Mad11

<400> 44

Asn Tyr Arg Ile Ala Thr Phe Lys Asn Trp Pro Phe Leu Glu Asp Cys

1 5 10 15

Ala Met Glu Glu Leu Thr Val Ser Glu Phe Leu Lys Leu Asp Arg Gln

20 25 30

Arg

<210> 45

<211> 90

<212>PRT

<213> Artificial Sequence

<220>

<223>EDA

<400> 45

Asn Ile Asp Arg Pro Lys Gly Leu Ala Phe Thr Asp Val Asp Val Asp

1 5 10 15

Ser Ile Lys Ile Ala Trp Glu Ser Pro Gln Gly Gln Val Ser Arg Tyr

20 25 30

Arg Val Thr Tyr Ser Ser Pro Glu Asp Gly Ile Arg Glu Leu Phe Pro

35 40 45

Ala Pro Asp Gly Glu Asp Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro

50 55 60

Gly Ser Glu Tyr Thr Val Ser Val Val Ala Leu His Asp Asp Met Glu

65 70 75 80

Ser Gln Pro Leu Ile Gly Ile Gln Ser Thr

85 90

<210> 46

<211> 115

<212>PRT

<213> Artificial Sequence

<220>

<223> TatFMad5Anaxa

<400> 46

Arg Lys Lys Arg Arg Gln Arg Arg Arg Arg Val Lys Arg Ile Ser Gln

1 5 10 15

Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly Arg Arg Val

20 25 30

Lys Arg Lys Val Pro Arg Asn Gln Asp Trp Leu Arg Val Lys Arg Ala

35 40 45

Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala Val Asp Lys Ala

50 55 60

Arg Val Lys Arg Ser Ile Ile Asn Phe Glu Lys Leu Arg Val Lys Arg

65 70 75 80

Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser Leu Glu Gly Asp His

85 90 95

Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe

100 105 110

Asp Ala Glu

115

<210> 47

<211> 314

<212>PRT

<213> Artificial Sequence

<220>

<223> EpCAM

<400> 47

Met Ala Pro Pro Gln Val Leu Ala Phe Gly Leu Leu Leu Ala Ala Ala

1 5 10 15

Thr Ala Thr Phe Ala Ala Ala Gln Glu Glu Cys Val Cys Glu Asn Tyr

20 25 30

Lys Leu Ala Val Asn Cys Phe Val Asn Asn Asn Arg Gln Cys Gln Cys

35 40 45

Thr Ser Val Gly Ala Gln Asn Thr Val Ile Cys Ser Lys Leu Ala Ala

50 55 60

Lys Cys Leu Val Met Lys Ala Glu Met Asn Gly Ser Lys Leu Gly Arg

65 70 75 80

Arg Ala Lys Pro Glu Gly Ala Leu Gln Asn Asn Asp Gly Leu Tyr Asp

85 90 95

Pro Asp Cys Asp Glu Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly

100 105 110

Thr Ser Met Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp

115 120 125

Lys Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile

130 135 140

Ile Ile Glu Leu Lys His Lys Ala Arg Glu Lys Pro Tyr Asp Ser Lys

145 150 155 160

Ser Leu Arg Thr Ala Leu Gln Lys Glu Ile Thr Thr Arg Tyr Gln Leu

165 170 175

Asp Pro Lys Phe Ile Thr Ser Ile Leu Tyr Glu Asn Asn Val Ile Thr

180 185 190

Ile Asp Leu Val Gln Asn Ser Ser Gln Lys Thr Gln Asn Asp Val Asp

195 200 205

Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser

210 215 220

Leu Phe His Ser Lys Lys Met Asp Leu Thr Val Asn Gly Glu Gln Leu

225 230 235 240

Asp Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala

245 250 255

Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile Ala Val Ile

260 265 270

Val Val Val Val Ile Ala Val Val Ala Gly Ile Val Val Leu Val Ile

275 280 285

Ser Arg Lys Lys Arg Met Ala Lys Tyr Glu Lys Ala Glu Ile Lys Glu

290 295 300

Met Gly Glu Met His Arg Glu Leu Asn Ala

305 310

<210> 48

<211> 9

<212>PRT

<213> Artificial Sequence

<220>

<223> EpCAM epitope

<400> 48

Gly Leu Lys Ala Gly Val Ile Ala Val

15

<210> 49

<211> 1255

<212>PRT

<213> Artificial Sequence

<220>

<223> MUC-1

<400> 49

Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr

1 5 10 15

Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly

20 25 30

Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser

35 40 45

Thr Glu Lys Asn Ala Val Ser Met Thr Ser Ser Val Leu Ser Ser His

50 55 60

Ser Pro Gly Ser Gly Ser Ser Thr Thr Gln Gly Gln Asp Val Thr Leu

65 70 75 80

Ala Pro Ala Thr Glu Pro Ala Ser Gly Ser Ala Ala Thr Trp Gly Gln

85 90 95

Asp Val Thr Ser Val Pro Val Thr Arg Pro Ala Leu Gly Ser Thr Thr

100 105 110

Pro Pro Ala His Asp Val Thr Ser Ala Pro Asp Asn Lys Pro Ala Pro

115 120 125

Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr

130 135 140

Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser

145 150 155 160

Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His

165 170 175

Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala

180 185 190

Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro

195 200 205

Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr

210 215 220

Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser

225 230 235 240

Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His

245 250 255

Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala

260 265 270

Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro

275 280 285

Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr

290 295 300

Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser

305 310 315 320

Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His

325 330 335

Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala

340 345 350

Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro

355 360 365

Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr

370 375 380

Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser

385 390 395 400

Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His

405 410 415

Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala

420 425 430

Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro

435 440 445

Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr

450 455 460

Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser

465 470 475 480

Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His

485 490 495

Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala

500 505 510

Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro

515 520 525

Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr

530 535 540

Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser

545 550 555 560

Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His

565 570 575

Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala

580 585 590

Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro

595 600 605

Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr

610 615 620

Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser

625 630 635 640

Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His

645 650 655

Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala

660 665 670

Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro

675 680 685

Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr

690 695 700

Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser

705 710 715 720

Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His

725 730 735

Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala

740 745 750

Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro

755 760 765

Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr

770 775 780

Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser

785 790 795 800

Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His

805 810 815

Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala

820 825 830

Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro

835 840 845

Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr

850 855 860

Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser

865 870 875 880

Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His

885 890 895

Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala

900 905 910

Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro

915 920 925

Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Asn

930 935 940

Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr Ser

945 950 955 960

Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn Gly

965 970 975

Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro Phe

980 985 990

Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser His

995 1000 1005

Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Ser Val Pro

1010 1015 1020

Pro Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr

1025 1030 1035

Gly Val Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln

1040 1045 1050

Phe Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu

1055 1060 1065

Leu Gln Arg Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln

1070 1075 1080

Gly Gly Phe Leu Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser

1085 1090 1095

Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn

1100 1105 1110

Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala

1115 1120 1125

Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser Asp

1130 1135 1140

Val Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly Val Pro Gly

1145 1150 1155

Trp Gly Ile Ala Leu Leu Val Leu Val Cys Val Leu Val Ala Leu

1160 1165 1170

Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys Gln Cys Arg Arg

1175 1180 1185

Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr

1190 1195 1200

His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg Tyr

1205 1210 1215

Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser

1220 1225 1230

Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val

1235 1240 1245

Ala Ala Thr Ser Ala Asn Leu

1250 1255

<210> 50

<211> 9

<212>PRT

<213> Artificial Sequence

<220>

<223> MUC-1 epitope

<400> 50

Gly Ser Thr Ala Pro Pro Val His Asn

15

<210> 51

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223> MUC-1 epitope

<400> 51

Thr Ala Pro Pro Ala His Gly Val Thr Ser

1 5 10

<210> 52

<211> 142

<212>PRT

<213> Artificial Sequence

<220>

<223> survivin

<400> 52

Met Gly Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp

1 5 10 15

His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Cys Ala

20 25 30

Cys Thr Pro Glu Arg Met Ala Glu Ala Gly Phe Ile His Cys Pro Thr

35 40 45

Glu Asn Glu Pro Asp Leu Ala Gln Cys Phe Phe Cys Phe Lys Glu Leu

50 55 60

Glu Gly Trp Glu Pro Asp Asp Asp Pro Ile Glu Glu His Lys Lys His

65 70 75 80

Ser Ser Gly Cys Ala Phe Leu Ser Val Lys Lys Gln Phe Glu Glu Leu

85 90 95

Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn Lys

100 105 110

Ile Ala Lys Glu Thr Asn Asn Lys Lys Lys Glu Phe Glu Glu Thr Ala

115 120 125

Lys Lys Val Arg Arg Ala Ile Glu Gln Leu Ala Ala Met Asp

130 135 140

<210> 53

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223> survivin epitope

<400> 53

Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe

1 5 10

<210> 54

<211> 701

<212>PRT

<213> Artificial Sequence

<220>

<223>CEA

<400> 54

Met Glu Ser Pro Ser Ala Pro Pro His Arg Trp Cys Ile Pro Trp Gln

1 5 10 15

Arg Leu Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr

20 25 30

Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly

35 40 45

Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly

50 55 60

Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Ile

65 70 75 80

Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser

85 90 95

Gly Arg Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile

100 105 110

Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp

115 120 125

Leu Val Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu

130 135 140

Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys

145 150 155 160

Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr

165 170 175

Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

180 185 190

Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn

195 200 205

Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg

210 215 220

Arg Ser Asp Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro

225 230 235 240

Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn

245 250 255

Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe

260 265 270

Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn

275 280 285

Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln Ala His Asn Ser

290 295 300

Asp Thr Gly Leu Asn Arg Thr Thr Val Thr Thr Ile Thr Val Tyr Ala

305 310 315 320

Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu

325 330 335

Asp Glu Asp Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr

340 345 350

Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg

355 360 365

Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr

370 375 380

Arg Asn Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Lys Leu Ser

385 390 395 400

Val Asp His Ser Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp

405 410 415

Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn

420 425 430

Leu Ser Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser

435 440 445

Trp Leu Ile Asp Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile

450 455 460

Ser Asn Ile Thr Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn

465 470 475 480

Asn Ser Ala Ser Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val

485 490 495

Ser Ala Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro

500 505 510

Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Ala Gln

515 520 525

Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser

530 535 540

Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn

545 550 555 560

Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Cys Gly Ile Gln Asn Ser

565 570 575

Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly

580 585 590

Pro Asp Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly

595 600 605

Ala Asn Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln

610 615 620

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

625 630 635 640

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

645 650 655

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

660 665 670

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Gly Ala Thr

675 680 685

Val Gly Ile Met Ile Gly Val Leu Val Gly Val Ala Leu

690 695 700

<210> 55

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223> CEA epitope

<400> 55

Tyr Leu Ser Gly Ala Asn Leu Asn Leu Ser

1 5 10

<210> 56

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223> CEA epitope

<400> 56

Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln

1 5 10

<210> 57

<211> 189

<212>PRT

<213> Artificial Sequence

<220>

<223>Kirsten Ras

<400> 57

Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr

20 25 30

Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly

35 40 45

Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr

50 55 60

Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys

65 70 75 80

Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His Tyr

85 90 95

Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp Val Pro Met Val

100 105 110

Leu Val Gly Asn Lys Cys Asp Leu Pro Ser Arg Thr Val Asp Thr Lys

115 120 125

Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr

130 135 140

Ser Ala Lys Thr Arg Gln Arg Val Glu Asp Ala Phe Tyr Thr Leu Val

145 150 155 160

Arg Glu Ile Arg Gln Tyr Arg Leu Lys Lys Ile Ser Lys Glu Glu Lys

165 170 175

Thr Pro Gly Cys Val Lys Ile Lys Lys Cys Ile Ile Met

180 185

<210> 58

<211> 9

<212>PRT

<213> Artificial Sequence

<220>

<223> Kirsten Ras epitope

<400> 58

Val Val Val Gly Ala Gly Gly Val Gly

15

<210> 59

<211> 314

<212>PRT

<213> Artificial Sequence

<220>

<223> MAGE-A3

<400> 59

Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu

1 5 10 15

Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala

20 25 30

Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val

35 40 45

Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser

50 55 60

Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp

65 70 75 80

Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser

85 90 95

Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys

100 105 110

Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu

115 120 125

Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln

130 135 140

Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Phe Ser Ser Leu Gln Leu

145 150 155 160

Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr

165 170 175

Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp

180 185 190

Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile

195 200 205

Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu

210 215 220

Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly

225 230 235 240

Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu

245 250 255

Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu

260 265 270

Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His

275 280 285

His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu

290 295 300

His Glu Trp Val Leu Arg Glu Gly Glu Glu

305 310

<210> 60

<211> 9

<212>PRT

<213> Artificial Sequence

<220>

<223> MAGE-A3 epitope

<400> 60

Lys Val Ala Glu Leu Val His Phe Leu

15

<210> 61

<211> 380

<212>PRT

<213> Artificial Sequence

<220>

<223> IL13Ralpha2

<400> 61

Met Ala Phe Val Cys Leu Ala Ile Gly Cys Leu Tyr Thr Phe Leu Ile

1 5 10 15

Ser Thr Thr Phe Gly Cys Thr Ser Ser Ser Asp Thr Glu Ile Lys Val

20 25 30

Asn Pro Pro Gln Asp Phe Glu Ile Val Asp Pro Gly Tyr Leu Gly Tyr

35 40 45

Leu Tyr Leu Gln Trp Gln Pro Pro Leu Ser Leu Asp His Phe Lys Glu

50 55 60

Cys Thr Val Glu Tyr Glu Leu Lys Tyr Arg Asn Ile Gly Ser Glu Thr

65 70 75 80

Trp Lys Thr Ile Ile Thr Lys Asn Leu His Tyr Lys Asp Gly Phe Asp

85 90 95

Leu Asn Lys Gly Ile Glu Ala Lys Ile His Thr Leu Leu Pro Trp Gln

100 105 110

Cys Thr Asn Gly Ser Glu Val Gln Ser Ser Trp Ala Glu Thr Thr Tyr

115 120 125

Trp Ile Ser Pro Gln Gly Ile Pro Glu Thr Lys Val Gln Asp Met Asp

130 135 140

Cys Val Tyr Tyr Asn Trp Gln Tyr Leu Leu Cys Ser Trp Lys Pro Gly

145 150 155 160

Ile Gly Val Leu Leu Asp Thr Asn Tyr Asn Leu Phe Tyr Trp Tyr Glu

165 170 175

Gly Leu Asp His Ala Leu Gln Cys Val Asp Tyr Ile Lys Ala Asp Gly

180 185 190

Gln Asn Ile Gly Cys Arg Phe Pro Tyr Leu Glu Ala Ser Asp Tyr Lys

195 200 205

Asp Phe Tyr Ile Cys Val Asn Gly Ser Ser Glu Asn Lys Pro Ile Arg

210 215 220

Ser Ser Tyr Phe Thr Phe Gln Leu Gln Asn Ile Val Lys Pro Leu Pro

225 230 235 240

Pro Val Tyr Leu Thr Phe Thr Arg Glu Ser Ser Cys Glu Ile Lys Leu

245 250 255

Lys Trp Ser Ile Pro Leu Gly Pro Ile Pro Ala Arg Cys Phe Asp Tyr

260 265 270

Glu Ile Glu Ile Arg Glu Asp Asp Thr Thr Leu Val Thr Ala Thr Val

275 280 285

Glu Asn Glu Thr Tyr Thr Leu Lys Thr Thr Asn Glu Thr Arg Gln Leu

290 295 300

Cys Phe Val Val Arg Ser Lys Val Asn Ile Tyr Cys Ser Asp Asp Gly

305 310 315 320

Ile Trp Ser Glu Trp Ser Asp Lys Gln Cys Trp Glu Gly Glu Asp Leu

325 330 335

Ser Lys Lys Thr Leu Leu Arg Phe Trp Leu Pro Phe Gly Phe Ile Leu

340 345 350

Ile Leu Val Ile Phe Val Thr Gly Leu Leu Leu Arg Lys Pro Asn Thr

355 360 365

Tyr Pro Lys Met Ile Pro Glu Phe Phe Cys Asp Thr

370 375 380

<210> 62

<211> 7

<212>PRT

<213> Artificial Sequence

<220>

<223> IL13Ralpha2 epitope

<400> 62

Leu Pro Phe Gly Phe Ile Leu

15

<210> 63

<211> 50

<212>PRT

<213> Artificial Sequence

<220>

<223> Mad12

<400> 63

Leu Phe Arg Ala Ala Gln Leu Ala Asn Asp Val Val Leu Gln Ile Met

1 5 10 15

Glu His Leu Glu Leu Ala Ser Met Thr Asn Met Glu Leu Met Ser Ser

20 25 30

Ile Val Val Ile Ser Ala Ser Ile Ile Val Phe Asn Leu Leu Glu Leu

35 40 45

Glu Gly

50

<210> 64

<211> 14

<212>PRT

<213> Artificial Sequence

<220>

<223> gp70CD4 peptide

<400> 64

Leu Val Gln Phe Ile Lys Asp Arg Ile Ser Val Val Gln Ala

1 5 10

<210> 65

<211> 9

<212>PRT

<213> Artificial Sequence

<220>

<223> gp70CD8 peptide

<400> 65

Ser Pro Ser Tyr Val Tyr His Gln Phe

15

<210> 66

<211> 9

<212>PRT

<213> Artificial Sequence

<220>

<223> adpgk peptide

<400> 66

Ala Ser Met Thr Asn Met Glu Leu Met

15

<210> 67

<211> 8

<212>PRT

<213> Artificial Sequence

<220>

<223> survivin20-28

<400> 67

Ala Thr Lys Asn Trp Pro Phe Leu

15

<210> 68

<211> 8

<212>PRT

<213> Artificial Sequence

<220>

<223> survivin97-104

<400> 68

Thr Val Ser Glu Phe Leu Lys Leu

15

<210> 69

<211> 127

<212>PRT

<213> Artificial Sequence

<220>

<223> Z13Mad12Anaxa

<400> 69

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Leu Phe Arg Ala Ala Gln

35 40 45

Leu Ala Asn Asp Val Val Leu Gln Ile Met Glu His Leu Glu Leu Ala

50 55 60

Ser Met Thr Asn Met Glu Leu Met Ser Ser Ile Val Val Ile Ser Ala

65 70 75 80

Ser Ile Ile Val Phe Asn Leu Leu Glu Leu Glu Gly Ser Thr Val His

85 90 95

Glu Ile Leu Cys Lys Leu Ser Leu Glu Gly Asp His Ser Thr Pro Pro

100 105 110

Ser Ala Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe Asp Ala Glu

115 120 125

<210> 70

<211> 1255

<212>PRT

<213> Artificial Sequence

<220>

<223> Her2/neu

<400> 70

Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu

1 5 10 15

Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys

20 25 30

Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His

35 40 45

Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr

50 55 60

Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val

65 70 75 80

Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu

85 90 95

Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr

100 105 110

Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro

115 120 125

Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser

130 135 140

Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln

145 150 155 160

Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn

165 170 175

Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys

180 185 190

His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser

195 200 205

Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys

210 215 220

Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys

225 230 235 240

Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu

245 250 255

His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val

260 265 270

Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg

275 280 285

Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu

290 295 300

Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln

305 310 315 320

Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys

325 330 335

Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu

340 345 350

Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys

355 360 365

Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp

370 375 380

Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe

385 390 395 400

Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro

405 410 415

Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg

420 425 430

Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu

435 440 445

Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly

450 455 460

Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val

465 470 475 480

Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr

485 490 495

Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His

500 505 510

Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys

515 520 525

Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys

530 535 540

Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys

545 550 555 560

Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys

565 570 575

Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp

580 585 590

Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu

595 600 605

Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln

610 615 620

Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys

625 630 635 640

Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser

645 650 655

Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly

660 665 670

Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg

675 680 685

Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly

690 695 700

Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu

705 710 715 720

Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys

725 730 735

Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile

740 745 750

Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu

755 760 765

Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg

770 775 780

Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu

785 790 795 800

Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg

805 810 815

Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly

820 825 830

Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala

835 840 845

Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe

850 855 860

Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp

865 870 875 880

Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg

885 890 895

Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val

900 905 910

Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala

915 920 925

Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro

930 935 940

Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met

945 950 955 960

Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe

965 970 975

Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu

980 985 990

Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu

995 1000 1005

Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr

1010 1015 1020

Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly

1025 1030 1035

Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg

1040 1045 1050

Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu

1055 1060 1065

Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser

1070 1075 1080

Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu

1085 1090 1095

Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser

1100 1105 1110

Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val

1115 1120 1125

Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln Pro

1130 1135 1140

Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro

1145 1150 1155

Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu

1160 1165 1170

Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly

1175 1180 1185

Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala

1190 1195 1200

Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp

1205 1210 1215

Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro

1220 1225 1230

Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr

1235 1240 1245

Leu Gly Leu Asp Val Pro Val

1250 1255

<210> 71

<211> 35

<212>PRT

<213> Artificial Sequence

<220>

<223> TLR peptide agonist "Anaxa" sequence variant

<400> 71

Ser Thr Val His Glu Ile Leu Ser Lys Leu Ser Leu Glu Gly Asp His

1 5 10 15

Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe

20 25 30

Asp Ala Glu

35

<210> 72

<211> 118

<212>PRT

<213> Artificial Sequence

<220>

<223>ATP110

<400> 72

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Ala Pro Pro Gln Val Leu

35 40 45

Ala Phe Gly Leu Leu Leu Ala Ala Ala Thr Ala Tyr Val Asp Glu Lys

50 55 60

Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile Ala Val

65 70 75 80

Ile Val Val Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser Leu Glu

85 90 95

Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Pro Tyr

100 105 110

Thr Asn Phe Asp Ala Glu

115

<210> 73

<211> 331

<212>PRT

<213> Artificial Sequence

<220>

<223> ATP111

<400> 73

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Arg Thr Leu Thr Leu Phe

35 40 45

Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln Asn

50 55 60

Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Pro Asp Ser Ser Tyr

65 70 75 80

Leu Ser Gly Ala Asn Leu Asn Leu Ser Ser His Ser Ala Ser Pro Gln

85 90 95

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

100 105 110

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

115 120 125

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

130 135 140

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Gly Ala Thr

145 150 155 160

Val Gly Ile Met Ile Gly Val Leu Val Gly Val Ala Leu Ile Leu Gly

165 170 175

Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu

180 185 190

Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Ser Tyr Glu Phe Leu

195 200 205

Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Ala Leu

210 215 220

Ser Arg Lys Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg

225 230 235 240

Ala Arg Glu Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Ala

245 250 255

Pro Pro Gln Val Leu Ala Phe Gly Leu Leu Leu Ala Ala Ala Thr Ala

260 265 270

Tyr Val Asp Glu Lys Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Ala

275 280 285

Gly Val Ile Ala Val Ile Val Val Ser Thr Val His Glu Ile Leu Cys

290 295 300

Lys Leu Ser Leu Glu Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly

305 310 315 320

Ser Val Lys Pro Tyr Thr Asn Phe Asp Ala Glu

325 330

<210> 74

<211> 313

<212>PRT

<213> Artificial Sequence

<220>

<223> ATP112

<400> 74

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Arg Thr Leu Thr Leu Phe

35 40 45

Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln Asn

50 55 60

Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Pro Asp Ser Ser Tyr

65 70 75 80

Leu Ser Gly Ala Asn Leu Asn Leu Ser Ser His Ser Ala Ser Pro Gln

85 90 95

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

100 105 110

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

115 120 125

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

130 135 140

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Leu Gly Asp Pro

145 150 155 160

Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu Glu Tyr

165 170 175

Arg Gln Val Pro Gly Ser Asp Pro Ala Ser Tyr Glu Phe Leu Trp Gly

180 185 190

Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Ala Leu Ser Arg

195 200 205

Lys Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg

210 215 220

Glu Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Ala Pro Pro

225 230 235 240

Gln Val Leu Ala Phe Gly Leu Leu Leu Ala Ala Ala Thr Ala Tyr Val

245 250 255

Asp Glu Lys Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val

260 265 270

Ile Ala Val Ile Val Val Ser Thr Val His Glu Ile Leu Cys Lys Leu

275 280 285

Ser Leu Glu Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val

290 295 300

Lys Pro Tyr Thr Asn Phe Asp Ala Glu

305 310

<210> 75

<211> 366

<212>PRT

<213> Artificial Sequence

<220>

<223>ATP113

<400> 75

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Arg Thr Leu Thr Leu Phe

35 40 45

Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln Asn

50 55 60

Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Pro Asp Ser Ser Tyr

65 70 75 80

Leu Ser Gly Ala Asn Leu Asn Leu Ser Ser His Ser Ala Ser Pro Gln

85 90 95

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

100 105 110

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

115 120 125

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

130 135 140

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Leu Gly Asp Pro

145 150 155 160

Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu Glu Tyr

165 170 175

Arg Gln Val Pro Gly Ser Asp Pro Ala Ser Tyr Glu Phe Leu Trp Gly

180 185 190

Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Ala Leu Ser Arg

195 200 205

Lys Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg

210 215 220

Glu Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Ala Pro Pro

225 230 235 240

Gln Val Leu Ala Phe Gly Leu Leu Leu Ala Ala Ala Thr Ala Tyr Val

245 250 255

Asp Glu Lys Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val

260 265 270

Ile Ala Val Ile Val Val Ala Pro Gly Ser Thr Ala Pro Pro Ala His

275 280 285

Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala

290 295 300

Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Arg Pro Ala Leu Gly

305 310 315 320

Ser Thr Ala Pro Pro Val His Asn Val Thr Ser Ser Thr Val His Glu

325 330 335

Ile Leu Cys Lys Leu Ser Leu Glu Gly Asp His Ser Thr Pro Pro Ser

340 345 350

Ala Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe Asp Ala Glu

355 360 365

<210> 76

<211> 335

<212>PRT

<213> Artificial Sequence

<220>

<223>ATP114

<400> 76

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Arg Thr Leu Thr Leu Phe

35 40 45

Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln Asn

50 55 60

Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Pro Asp Ser Ser Tyr

65 70 75 80

Leu Ser Gly Ala Asn Leu Asn Leu Ser Ser His Ser Ala Ser Pro Gln

85 90 95

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

100 105 110

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

115 120 125

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

130 135 140

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Pro Thr Leu

145 150 155 160

Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp His Arg Ile Ser Thr Phe

165 170 175

Lys Asn Trp Pro Phe Leu Glu Gly Ser Ala Val Lys Lys Gln Phe Glu

180 185 190

Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Pro

195 200 205

Pro Gln Val Leu Ala Phe Gly Leu Leu Leu Ala Ala Ala Thr Ala Tyr

210 215 220

Val Asp Glu Lys Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly

225 230 235 240

Val Ile Ala Val Ile Val Val Ala Pro Gly Ser Thr Ala Pro Pro Ala

245 250 255

His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr

260 265 270

Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Arg Pro Ala Leu

275 280 285

Gly Ser Thr Ala Pro Pro Val His Asn Val Thr Ser Ser Thr Val His

290 295 300

Glu Ile Leu Cys Lys Leu Ser Leu Glu Gly Asp His Ser Thr Pro Pro

305 310 315 320

Ser Ala Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe Asp Ala Glu

325 330 335

<210> 77

<211> 303

<212>PRT

<213> Artificial Sequence

<220>

<223>ATP115

<400> 77

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Arg Thr Leu Thr Leu Phe

35 40 45

Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln Asn

50 55 60

Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Pro Asp Ser Ser Tyr

65 70 75 80

Leu Ser Gly Ala Asn Leu Asn Leu Ser Ser His Ser Ala Ser Pro Gln

85 90 95

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

100 105 110

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

115 120 125

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

130 135 140

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Glu Tyr Lys Leu

145 150 155 160

Val Val Val Gly Ala Val Gly Val Gly Lys Ser Ala Leu Thr Ala Pro

165 170 175

Pro Gln Val Leu Ala Phe Gly Leu Leu Leu Ala Ala Ala Thr Ala Tyr

180 185 190

Val Asp Glu Lys Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly

195 200 205

Val Ile Ala Val Ile Val Val Ala Pro Gly Ser Thr Ala Pro Pro Ala

210 215 220

His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr

225 230 235 240

Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Arg Pro Ala Leu

245 250 255

Gly Ser Thr Ala Pro Pro Val His Asn Val Thr Ser Ser Thr Val His

260 265 270

Glu Ile Leu Cys Lys Leu Ser Leu Glu Gly Asp His Ser Thr Pro Pro

275 280 285

Ser Ala Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe Asp Ala Glu

290 295 300

<210> 78

<211> 363

<212>PRT

<213> Artificial Sequence

<220>

<223> ATP116

<400> 78

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Arg Thr Leu Thr Leu Phe

35 40 45

Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln Asn

50 55 60

Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Pro Asp Ser Ser Tyr

65 70 75 80

Leu Ser Gly Ala Asn Leu Asn Leu Ser Ser His Ser Ala Ser Pro Gln

85 90 95

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

100 105 110

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

115 120 125

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

130 135 140

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Pro Thr Leu

145 150 155 160

Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp His Arg Ile Ser Thr Phe

165 170 175

Lys Asn Trp Pro Phe Leu Glu Gly Ser Ala Val Lys Lys Gln Phe Glu

180 185 190

Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Pro

195 200 205

Pro Gln Val Leu Ala Phe Gly Leu Leu Leu Ala Ala Ala Thr Ala Tyr

210 215 220

Val Asp Glu Lys Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly

225 230 235 240

Val Ile Ala Val Ile Val Val Leu Gly Asp Pro Lys Lys Leu Leu Thr

245 250 255

Gln His Phe Val Gln Glu Asn Tyr Leu Glu Tyr Arg Gln Val Pro Gly

260 265 270

Ser Asp Pro Ala Ser Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Val

275 280 285

Glu Thr Ser Tyr Val Lys Val Ala Leu Ser Arg Lys Val Ala Glu Leu

290 295 300

Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val Thr Lys

305 310 315 320

Ala Glu Met Leu Gly Ser Val Val Ser Thr Val His Glu Ile Leu Cys

325 330 335

Lys Leu Ser Leu Glu Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly

340 345 350

Ser Val Lys Pro Tyr Thr Asn Phe Asp Ala Glu

355 360

<210> 79

<211> 285

<212>PRT

<213> Artificial Sequence

<220>

<223>ATP117

<400> 79

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Arg Thr Leu Thr Leu Phe

35 40 45

Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln Asn

50 55 60

Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Pro Asp Ser Ser Tyr

65 70 75 80

Leu Ser Gly Ala Asn Leu Asn Leu Ser Ser His Ser Ala Ser Pro Gln

85 90 95

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

100 105 110

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

115 120 125

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

130 135 140

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Pro Pro Gln

145 150 155 160

Val Leu Ala Phe Gly Leu Leu Leu Ala Ala Ala Thr Ala Tyr Val Asp

165 170 175

Glu Lys Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile

180 185 190

Ala Val Ile Val Val Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly

195 200 205

Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro

210 215 220

Pro Ala His Gly Val Thr Ser Ala Pro Asp Arg Pro Ala Leu Gly Ser

225 230 235 240

Thr Ala Pro Pro Val His Asn Val Thr Ser Ser Thr Val His Glu Ile

245 250 255

Leu Cys Lys Leu Ser Leu Glu Gly Asp His Ser Thr Pro Pro Ser Ala

260 265 270

Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe Asp Ala Glu

275 280 285

<210> 80

<211> 232

<212>PRT

<213> Artificial Sequence

<220>

<223>ATP118

<400> 80

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Arg Thr Leu Thr Leu Phe

35 40 45

Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln Asn

50 55 60

Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Pro Asp Ser Ser Tyr

65 70 75 80

Leu Ser Gly Ala Asn Leu Asn Leu Ser Ser His Ser Ala Ser Pro Gln

85 90 95

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

100 105 110

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

115 120 125

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

130 135 140

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Pro Pro Gln

145 150 155 160

Val Leu Ala Phe Gly Leu Leu Leu Ala Ala Ala Thr Ala Tyr Val Asp

165 170 175

Glu Lys Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile

180 185 190

Ala Val Ile Val Val Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser

195 200 205

Leu Glu Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys

210 215 220

Pro Tyr Thr Asn Phe Asp Ala Glu

225 230

<210> 81

<211> 261

<212>PRT

<213> Artificial Sequence

<220>

<223>ATP119

<400> 81

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Leu Gly Asp Pro Lys Lys

35 40 45

Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu Glu Tyr Arg Gln

50 55 60

Val Pro Gly Ser Asp Pro Ala Ser Tyr Glu Phe Leu Trp Gly Pro Arg

65 70 75 80

Ala Leu Val Glu Thr Ser Tyr Val Lys Val Ala Leu Ser Arg Lys Val

85 90 95

Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro

100 105 110

Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Ala Pro Thr Leu Pro

115 120 125

Pro Ala Trp Gln Pro Phe Leu Lys Asp His Arg Ile Ser Thr Phe Lys

130 135 140

Asn Trp Pro Phe Leu Glu Gly Ser Ala Val Lys Lys Gln Phe Glu Glu

145 150 155 160

Leu Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Pro Gly

165 170 175

Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg

180 185 190

Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala

195 200 205

Pro Asp Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val

210 215 220

Thr Ser Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser Leu Glu Gly

225 230 235 240

Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Pro Tyr Thr

245 250 255

Asn Phe Asp Ala Glu

260

<210> 82

<211> 191

<212>PRT

<213> Artificial Sequence

<220>

<223>ATP120

<400> 82

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Arg Thr Leu Thr Leu Phe

35 40 45

Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln Asn

50 55 60

Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Pro Asp Ser Ser Tyr

65 70 75 80

Leu Ser Gly Ala Asn Leu Asn Leu Ser Ser His Ser Ala Ser Pro Gln

85 90 95

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

100 105 110

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

115 120 125

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

130 135 140

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ser Thr Val His

145 150 155 160

Glu Ile Leu Cys Lys Leu Ser Leu Glu Gly Asp His Ser Thr Pro Pro

165 170 175

Ser Ala Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe Asp Ala Glu

180 185 190

<210> 83

<211> 311

<212>PRT

<213> Artificial Sequence

<220>

<223>ATP121

<400> 83

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Asn Arg Thr Leu Thr Leu

35 40 45

Phe Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln

50 55 60

Asn Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu

65 70 75 80

Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn Leu Asn Leu Ser Ser His

85 90 95

Ser Ala Ser Pro Gln Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln

100 105 110

His Thr Gln Val Leu Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly

115 120 125

Thr Tyr Ala Cys Phe Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser

130 135 140

Ile Val Lys Ser Ile Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu

145 150 155 160

Ser Ala Gly Ala Thr Val Gly Ile Met Ile Gly Val Leu Val Gly Val

165 170 175

Ala Leu Ile Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr

180 185 190

Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala

195 200 205

His Gly Val Thr Ser Ala Pro Asp Arg Pro Ala Leu Gly Ser Thr Ala

210 215 220

Pro Pro Val His Asn Val Thr Ser Ala Pro Pro Gln Val Leu Ala Phe

225 230 235 240

Gly Leu Leu Leu Ala Ala Ala Thr Ala Leu Ile Tyr Tyr Val Asp Glu

245 250 255

Lys Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile Ala

260 265 270

Val Ile Val Val Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser Leu

275 280 285

Glu Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Pro

290 295 300

Tyr Thr Asn Phe Asp Ala Glu

305 310

<210> 84

<211> 294

<212>PRT

<213> Artificial Sequence

<220>

<223>ATP122

<400> 84

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Asn Arg Thr Leu Thr Leu

35 40 45

Phe Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln

50 55 60

Asn Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu

65 70 75 80

Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn Leu Asn Leu Ser Ser His

85 90 95

Ser Ala Ser Pro Gln Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln

100 105 110

His Thr Gln Val Leu Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly

115 120 125

Thr Tyr Ala Cys Phe Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser

130 135 140

Ile Val Lys Ser Ile Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu

145 150 155 160

Ser Ala Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser

165 170 175

Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His

180 185 190

Gly Val Thr Ser Ala Pro Asp Arg Pro Ala Leu Gly Ser Thr Ala Pro

195 200 205

Pro Val His Asn Val Thr Ser Ala Pro Pro Gln Val Leu Ala Phe Gly

210 215 220

Leu Leu Leu Ala Ala Ala Thr Ala Leu Ile Tyr Tyr Val Asp Glu Lys

225 230 235 240

Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile Ala Val

245 250 255

Ile Val Val Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser Leu Glu

260 265 270

Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Pro Tyr

275 280 285

Thr Asn Phe Asp Ala Glu

290

<210> 85

<211> 294

<212>PRT

<213> Artificial Sequence

<220>

<223> ATP123

<400> 85

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Asn Arg Thr Leu Thr Leu

35 40 45

Phe Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln

50 55 60

Asn Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu

65 70 75 80

Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn Leu Asn Leu Ser Cys His

85 90 95

Ser Ala Ser Pro Gln Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln

100 105 110

His Thr Gln Val Leu Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly

115 120 125

Thr Tyr Ala Cys Phe Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser

130 135 140

Ile Val Lys Ser Ile Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu

145 150 155 160

Ser Ala Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser

165 170 175

Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His

180 185 190

Gly Val Thr Ser Ala Pro Asp Arg Pro Ala Leu Gly Ser Thr Ala Pro

195 200 205

Pro Val His Asn Val Thr Ser Ala Pro Pro Gln Val Leu Ala Phe Gly

210 215 220

Leu Leu Leu Ala Ala Ala Thr Ala Leu Ile Tyr Tyr Val Asp Glu Lys

225 230 235 240

Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile Ala Val

245 250 255

Ile Val Val Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser Leu Glu

260 265 270

Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Pro Tyr

275 280 285

Thr Asn Phe Asp Ala Glu

290

<210> 86

<211> 344

<212>PRT

<213> Artificial Sequence

<220>

<223>ATP124

<400> 86

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Asn Arg Thr Leu Thr Leu

35 40 45

Phe Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln

50 55 60

Asn Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu

65 70 75 80

Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn Leu Asn Leu Ser Cys His

85 90 95

Ser Ala Ser Pro Gln Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln

100 105 110

His Thr Gln Val Leu Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly

115 120 125

Thr Tyr Ala Cys Phe Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser

130 135 140

Ile Val Lys Ser Ile Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu

145 150 155 160

Ser Ala Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp

165 170 175

His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Ser Ala

180 185 190

Val Lys Lys Gln Phe Glu Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu

195 200 205

Asp Arg Glu Arg Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val

210 215 220

Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro

225 230 235 240

Ala His Gly Val Thr Ser Ala Pro Asp Arg Pro Ala Leu Gly Ser Thr

245 250 255

Ala Pro Pro Val His Asn Val Thr Ser Ala Pro Pro Gln Val Leu Ala

260 265 270

Phe Gly Leu Leu Leu Ala Ala Ala Thr Ala Leu Ile Tyr Tyr Val Asp

275 280 285

Glu Lys Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile

290 295 300

Ala Val Ile Val Val Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser

305 310 315 320

Leu Glu Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys

325 330 335

Pro Tyr Thr Asn Phe Asp Ala Glu

340

<210> 87

<211> 344

<212>PRT

<213> Artificial Sequence

<220>

<223>ATP125

<400> 87

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Asn Arg Thr Leu Thr Leu

35 40 45

Phe Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln

50 55 60

Asn Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu

65 70 75 80

Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn Leu Asn Leu Ser Cys His

85 90 95

Ser Ala Ser Pro Gln Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln

100 105 110

His Thr Gln Val Leu Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly

115 120 125

Thr Tyr Ala Cys Phe Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser

130 135 140

Ile Val Lys Ser Ile Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu

145 150 155 160

Ser Ala Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp

165 170 175

His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Ser Ala

180 185 190

Val Lys Lys Gln Phe Glu Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu

195 200 205

Asp Arg Glu Arg Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val

210 215 220

Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro

225 230 235 240

Ala His Gly Val Thr Ser Ala Pro Asp Arg Pro Ala Leu Gly Ser Thr

245 250 255

Ala Pro Pro Val His Asn Val Thr Ser Ala Pro Pro Gln Val Leu Ala

260 265 270

Phe Gly Leu Leu Leu Ala Ala Ala Thr Ala Leu Ile Tyr Tyr Val Asp

275 280 285

Glu Lys Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile

290 295 300

Ala Val Ile Val Val Ser Thr Val His Glu Ile Leu Ser Lys Leu Ser

305 310 315 320

Leu Glu Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys

325 330 335

Pro Tyr Thr Asn Phe Asp Ala Glu

340

<210> 88

<211> 397

<212>PRT

<213> Artificial Sequence

<220>

<223>ATP127

<400> 88

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Asn Arg Thr Leu Thr Leu

35 40 45

Phe Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln

50 55 60

Asn Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu

65 70 75 80

Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn Leu Asn Leu Ser Cys His

85 90 95

Ser Ala Ser Pro Gln Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln

100 105 110

His Thr Gln Val Leu Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly

115 120 125

Thr Tyr Ala Cys Phe Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser

130 135 140

Ile Val Lys Ser Ile Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu

145 150 155 160

Ser Ala Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp

165 170 175

His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Ser Ala

180 185 190

Val Lys Lys Gln Phe Glu Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu

195 200 205

Asp Arg Glu Arg Ala Pro Pro Gln Val Leu Ala Phe Gly Leu Leu Leu

210 215 220

Ala Ala Ala Thr Ala Leu Ile Tyr Tyr Val Asp Glu Lys Ala Pro Glu

225 230 235 240

Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile Ala Val Ile Val Val

245 250 255

Ala Ala Val Ala Arg Arg Asn Glu Arg Glu Arg Asn Arg Val Lys Leu

260 265 270

Val Asn Leu Gly Phe Gln Ala Leu Arg Gln His Val Pro His Gly Gly

275 280 285

Ala Ser Lys Lys Leu Ser Lys Val Glu Thr Leu Arg Ser Ala Val Glu

290 295 300

Tyr Ile Arg Ala Leu Gln Arg Leu Leu Ala Glu His Asp Ala Val Arg

305 310 315 320

Asn Ala Leu Ala Gly Gly Leu Arg Pro Gln Ala Val Arg Pro Ser Ala

325 330 335

Pro Arg Gly Pro Ser Glu Gly Ala Leu Ser Pro Ala Glu Arg Glu Leu

340 345 350

Leu Asp Phe Ser Ser Trp Leu Gly Gly Tyr Ser Thr Val His Glu Ile

355 360 365

Leu Ser Lys Leu Ser Leu Glu Gly Asp His Ser Thr Pro Pro Ser Ala

370 375 380

Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe Asp Ala Glu

385 390 395

<210> 89

<211> 353

<212>PRT

<213> Artificial Sequence

<220>

<223>ATP128

<400> 89

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Asn Arg Thr Leu Thr Leu

35 40 45

Phe Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln

50 55 60

Asn Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu

65 70 75 80

Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn Leu Asn Leu Ser Cys His

85 90 95

Ser Ala Ser Pro Gln Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln

100 105 110

His Thr Gln Val Leu Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly

115 120 125

Thr Tyr Ala Cys Phe Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser

130 135 140

Ile Val Lys Ser Ile Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu

145 150 155 160

Ser Ala Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp

165 170 175

His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Ser Ala

180 185 190

Val Lys Lys Gln Phe Glu Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu

195 200 205

Asp Arg Glu Arg Ala Ala Val Ala Arg Arg Asn Glu Arg Glu Arg Asn

210 215 220

Arg Val Lys Leu Val Asn Leu Gly Phe Gln Ala Leu Arg Gln His Val

225 230 235 240

Pro His Gly Gly Ala Ser Lys Lys Leu Ser Lys Val Glu Thr Leu Arg

245 250 255

Ser Ala Val Glu Tyr Ile Arg Ala Leu Gln Arg Leu Leu Ala Glu His

260 265 270

Asp Ala Val Arg Asn Ala Leu Ala Gly Gly Leu Arg Pro Gln Ala Val

275 280 285

Arg Pro Ser Ala Pro Arg Gly Pro Ser Glu Gly Ala Leu Ser Pro Ala

290 295 300

Glu Arg Glu Leu Leu Asp Phe Ser Ser Trp Leu Gly Gly Tyr Ser Thr

305 310 315 320

Val His Glu Ile Leu Ser Lys Leu Ser Leu Glu Gly Asp His Ser Thr

325 330 335

Pro Pro Ser Ala Tyr Gly Ser Val Lys Pro Tyr Thr Asn Phe Asp Ala

340 345 350

Glu

<210> 90

<211> 358

<212>PRT

<213> Artificial Sequence

<220>

<223> ATP129

<400> 90

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Asn Arg Thr Leu Thr Leu

35 40 45

Phe Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln

50 55 60

Asn Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu

65 70 75 80

Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn Leu Asn Leu Ser Cys His

85 90 95

Ser Ala Ser Pro Gln Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln

100 105 110

His Thr Gln Val Leu Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly

115 120 125

Thr Tyr Ala Cys Phe Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser

130 135 140

Ile Val Lys Ser Ile Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu

145 150 155 160

Ser Ala Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp

165 170 175

His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Ser Ala

180 185 190

Val Lys Lys Gln Phe Glu Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu

195 200 205

Asp Arg Glu Arg Ala Lys Asn Lys Ile Ala Ala Val Ala Arg Arg Asn

210 215 220

Glu Arg Glu Arg Asn Arg Val Lys Leu Val Asn Leu Gly Phe Gln Ala

225 230 235 240

Leu Arg Gln His Val Pro His Gly Gly Ala Ser Lys Lys Leu Ser Lys

245 250 255

Val Glu Thr Leu Arg Ser Ala Val Glu Tyr Ile Arg Ala Leu Gln Arg

260 265 270

Leu Leu Ala Glu His Asp Ala Val Arg Asn Ala Leu Ala Gly Gly Leu

275 280 285

Arg Pro Gln Ala Val Arg Pro Ser Ala Pro Arg Gly Pro Ser Glu Gly

290 295 300

Ala Leu Ser Pro Ala Glu Arg Glu Leu Leu Asp Phe Ser Ser Trp Leu

305 310 315 320

Gly Gly Tyr Ser Thr Val His Glu Ile Leu Ser Lys Leu Ser Leu Glu

325 330 335

Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Pro Tyr

340 345 350

Thr Asn Phe Asp Ala Glu

355

<210> 91

<211> 396

<212>PRT

<213> Artificial Sequence

<220>

<223> ATP130

<400> 91

Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg Lys Ser Arg Ala Lys Phe

1 5 10 15

Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser Ser

20 25 30

Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Asn Arg Thr Leu Thr Leu

35 40 45

Phe Asn Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Ser Gly Ile Gln

50 55 60

Asn Ser Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu

65 70 75 80

Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn Leu Asn Leu Ser Cys His

85 90 95

Ser Ala Ser Pro Gln Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln

100 105 110

His Thr Gln Val Leu Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly

115 120 125

Thr Tyr Ala Cys Phe Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser

130 135 140

Ile Val Lys Ser Ile Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu

145 150 155 160

Ser Ala Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp

165 170 175

His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Ser Ala

180 185 190

Val Lys Lys Gln Phe Glu Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu

195 200 205

Asp Arg Glu Arg Ala Lys Asn Lys Ile Ala Ala Val Ala Arg Arg Asn

210 215 220

Glu Arg Glu Arg Asn Arg Val Lys Leu Val Asn Leu Gly Phe Gln Ala

225 230 235 240

Leu Arg Gln His Val Pro His Gly Gly Ala Ser Lys Lys Leu Ser Lys

245 250 255

Val Glu Thr Leu Arg Ser Ala Val Glu Tyr Ile Arg Ala Leu Gln Arg

260 265 270

Leu Leu Ala Glu His Asp Ala Val Arg Asn Ala Leu Ala Gly Gly Leu

275 280 285

Arg Pro Gln Ala Val Arg Pro Ser Ala Pro Arg Gly Pro Pro Gly Thr

290 295 300

Thr Pro Val Ala Ala Ser Pro Ser Arg Ala Ser Ser Ser Pro Gly Arg

305 310 315 320

Gly Gly Ser Ser Glu Pro Gly Ser Pro Arg Ser Ala Tyr Ser Ser Asp

325 330 335

Asp Ser Gly Ser Glu Gly Ala Leu Ser Pro Ala Glu Arg Glu Leu Leu

340 345 350

Asp Phe Ser Ser Trp Leu Gly Gly Tyr Ser Thr Val His Glu Ile Leu

355 360 365

Ser Lys Leu Ser Leu Glu Gly Asp His Ser Thr Pro Pro Ser Ala Tyr

370 375 380

Gly Ser Val Lys Pro Tyr Thr Asn Phe Asp Ala Glu

385 390 395

<210> 92

<211> 193

<212>PRT

<213> Artificial Sequence

<220>

<223> ASCL2

<400> 92

Met Asp Gly Gly Thr Leu Pro Arg Ser Ala Pro Pro Ala Pro Pro Val

1 5 10 15

Pro Val Gly Cys Ala Ala Arg Arg Arg Pro Ala Ser Pro Glu Leu Leu

20 25 30

Arg Cys Ser Arg Arg Arg Arg Pro Ala Thr Ala Glu Thr Gly Gly Gly

35 40 45

Ala Ala Ala Val Ala Arg Arg Asn Glu Arg Glu Arg Asn Arg Val Lys

50 55 60

Leu Val Asn Leu Gly Phe Gln Ala Leu Arg Gln His Val Pro His Gly

65 70 75 80

Gly Ala Ser Lys Lys Leu Ser Lys Val Glu Thr Leu Arg Ser Ala Val

85 90 95

Glu Tyr Ile Arg Ala Leu Gln Arg Leu Leu Ala Glu His Asp Ala Val

100 105 110

Arg Asn Ala Leu Ala Gly Gly Leu Arg Pro Gln Ala Val Arg Pro Ser

115 120 125

Ala Pro Arg Gly Pro Pro Gly Thr Thr Pro Val Ala Ala Ser Pro Ser

130 135 140

Arg Ala Ser Ser Ser Pro Gly Arg Gly Gly Ser Ser Glu Pro Gly Ser

145 150 155 160

Pro Arg Ser Ala Tyr Ser Ser Asp Asp Ser Gly Cys Glu Gly Ala Leu

165 170 175

Ser Pro Ala Glu Arg Glu Leu Leu Asp Phe Ser Ser Trp Leu Gly Gly

180 185 190

Tyr

<210> 93

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223> ASCL2 epitope

<400> 93

Ser Ala Val Glu Tyr Ile Arg Ala Leu Gln

1 5 10

<210> 94

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223> ASCL2 epitope

<400> 94

Glu Arg Glu Leu Leu Asp Phe Ser Ser Trp

1 5 10

<210> 95

<211> 50

<212>PRT

<213> Artificial Sequence

<220>

<223> Survivin fragment

<400> 95

Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp His Arg

1 5 10 15

Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Ser Ala Val Lys

20 25 30

Lys Gln Phe Glu Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg

35 40 45

Glu Arg

50

<210> 96

<211> 120

<212>PRT

<213> Artificial Sequence

<220>

<223> CEA fragment

<400> 96

Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn Asp Ala Arg Ala

1 5 10 15

Tyr Val Ser Gly Ile Gln Asn Ser Val Ser Ala Asn Arg Ser Asp Pro

20 25 30

Val Thr Leu Asp Val Leu Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn

35 40 45

Leu Asn Leu Ser Cys His Ser Ala Ser Pro Gln Tyr Ser Trp Arg Ile

50 55 60

Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu Phe Ile Ala Lys Ile

65 70 75 80

Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe Val Ser Asn Leu Ala

85 90 95

Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile Thr Val Ser Ala Ser

100 105 110

Gly Thr Ser Pro Gly Leu Ser Ala

115 120

<210> 97

<211> 106

<212>PRT

<213> Artificial Sequence

<220>

<223> ASCL2 fragment

<400> 97

Ala Ala Val Ala Arg Arg Asn Glu Arg Glu Arg Asn Arg Val Lys Leu

1 5 10 15

Val Asn Leu Gly Phe Gln Ala Leu Arg Gln His Val Pro His Gly Gly

20 25 30

Ala Ser Lys Lys Leu Ser Lys Val Glu Thr Leu Arg Ser Ala Val Glu

35 40 45

Tyr Ile Arg Ala Leu Gln Arg Leu Leu Ala Glu His Asp Ala Val Arg

50 55 60

Asn Ala Leu Ala Gly Gly Leu Arg Pro Gln Ala Val Arg Pro Ser Ala

65 70 75 80

Pro Arg Gly Pro Ser Glu Gly Ala Leu Ser Pro Ala Glu Arg Glu Leu

85 90 95

Leu Asp Phe Ser Ser Trp Leu Gly Gly Tyr

100 105

<210> 98

<211> 276

<212>PRT

<213> Artificial Sequence

<220>

<223> antigenic cargo of ATP128

<400> 98

Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn Asp Ala Arg Ala

1 5 10 15

Tyr Val Ser Gly Ile Gln Asn Ser Val Ser Ala Asn Arg Ser Asp Pro

20 25 30

Val Thr Leu Asp Val Leu Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn

35 40 45

Leu Asn Leu Ser Cys His Ser Ala Ser Pro Gln Tyr Ser Trp Arg Ile

50 55 60

Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu Phe Ile Ala Lys Ile

65 70 75 80

Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe Val Ser Asn Leu Ala

85 90 95

Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile Thr Val Ser Ala Ser

100 105 110

Gly Thr Ser Pro Gly Leu Ser Ala Ala Pro Thr Leu Pro Pro Ala Trp

115 120 125

Gln Pro Phe Leu Lys Asp His Arg Ile Ser Thr Phe Lys Asn Trp Pro

130 135 140

Phe Leu Glu Gly Ser Ala Val Lys Lys Gln Phe Glu Glu Leu Thr Leu

145 150 155 160

Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Ala Val Ala Arg Arg

165 170 175

Asn Glu Arg Glu Arg Asn Arg Val Lys Leu Val Asn Leu Gly Phe Gln

180 185 190

Ala Leu Arg Gln His Val Pro His Gly Gly Ala Ser Lys Lys Leu Ser

195 200 205

Lys Val Glu Thr Leu Arg Ser Ala Val Glu Tyr Ile Arg Ala Leu Gln

210 215 220

Arg Leu Leu Ala Glu His Asp Ala Val Arg Asn Ala Leu Ala Gly Gly

225 230 235 240

Leu Arg Pro Gln Ala Val Arg Pro Ser Ala Pro Arg Gly Pro Ser Glu

245 250 255

Gly Ala Leu Ser Pro Ala Glu Arg Glu Leu Leu Asp Phe Ser Ser Trp

260 265 270

Leu Gly Gly Tyr

275

<---

Claims (86)

1. Complex for the treatment of colorectal cancer, containing: a) cell-penetrating peptide; b) at least three antigenic epitopes; And c) at least one TLR peptide agonist, in which components a)-c) are covalently linked and in which at least three antigenic epitopes contain (I) one or more epitopes of survivin or functional variant(s) of sequence(s) thereof, (II) one or more epitopes carcinoembryonic antigen (CEA) or functional variant(s) of sequence(s) thereof and (III) one or more epitopes of Achaete-scute complex homologue 2 (ASCL2) or functional variant(s) of sequence(s) thereof . 2. The complex of claim 1, wherein the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 52, or a fragment thereof of at least 10 amino acids in length, or a functional variant of this sequence that is at least 70% identical to the sequence. 3. The complex according to claim 2, where the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 53. 4. The complex of claim 2, wherein the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 95, or a functional variant of this sequence that is at least 70% identical to the sequence. 5. The complex of claim 1, wherein the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 54, or a fragment thereof of at least 10 amino acids in length, or a functional variant of this sequence that is at least 70% identical to the sequence. 6. The complex according to claim 5, where the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 55, and/or a peptide that has an amino acid sequence corresponding to SEQ ID NO: 56. 7. The complex of claim 5, wherein the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 96, or a functional variant of this sequence that is at least 70% identical to the sequence. 8. The complex of claim 1, wherein the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 92, or a fragment thereof of at least 10 amino acids in length, or a functional variant of this sequence that is at least 70% identical to the sequence. 9. The complex of claim 8, wherein the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 93, and/or a peptide that has an amino acid sequence corresponding to SEQ ID NO: 94. 10. The complex of claim 8, wherein the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 97, or a functional variant of this sequence that is at least 70% identical to the sequence. 11. The complex according to claim 1, where the complex contains, in the direction from the N-terminus to the C-terminus: (I) one or more CEA epitopes or functional sequence variants thereof; (II) one or more survivin epitopes or functional sequence variants thereof; And (III) one or more ASCL2 epitopes or functional sequence variants thereof. 12. The complex according to claim 11, where the complex contains, in the direction from the N-terminus to the C-terminus: (I) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 54, or a fragment thereof of at least 10 amino acids in length, or a functional variant of this sequence that is at least 70% identical to the sequence; (II) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 52, or a fragment thereof of at least 10 amino acids in length, or a functional variant of this sequence that is at least 70% identical to the sequence; And (III) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 92, or a fragment thereof of at least 10 amino acids in length, or a functional variant of this sequence that is at least 70% identical to the sequence. 13. The complex according to claim 12, wherein the C-terminus (I) of the peptide, which has the amino acid sequence corresponding to SEQ ID NO: 54, or a fragment or variant thereof, is directly linked to the N-terminus (II) of the peptide, which has the amino acid sequence corresponding to SEQ ID NO: 52, or a fragment or variant thereof; and the C-terminus (II) of a peptide that has an amino acid sequence corresponding to SEQ ID NO: 52, or a fragment or variant thereof, is directly linked to the N-terminus (III) of a peptide that has an amino acid sequence corresponding to SEQ ID NO: 92 , or its fragment, or variant. 14. The complex according to claim 1, where the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 96, or a functional variant of this sequence that is at least 70% identical to the sequence; a peptide that has an amino acid sequence corresponding to SEQ ID NO: 95, or a functional variant of this sequence that is at least 70% identical to the sequence; and a peptide that has an amino acid sequence corresponding to SEQ ID NO: 97, or a functional variant of this sequence that is at least 70% identical to the sequence. 15. The complex of claim 1 or 14, wherein the complex contains a peptide that has an amino acid sequence corresponding to SEQ ID NO: 98, or a functional variant of this sequence that is at least 70% identical to the sequence. 16. The complex according to claim 1, where the complex is a recombinant polypeptide or a recombinant protein. 17. Complex according to claim 1, where the cell-penetrating peptide: (i) has an amino acid sequence length of said peptide of only 5 to 50 amino acids, preferably only 10 to 45 amino acids, more preferably only 15 to 45 amino acids; and/or (ii) has an amino acid sequence containing a fragment of a ZEBRA minimal domain, said minimal domain located from residue 170 to residue 220 of the ZEBRA amino acid sequence corresponding to SEQ ID NO: 3, where optionally 1, 2, 3, 4 or 5 amino acids have been replaced, deleted and/or added without interfering with the permeability of said peptide or variant thereof. 18. The complex of claim 17, wherein the cell penetrating peptide has an amino acid sequence containing Ser (S) at position equivalent to 189 of the ZEBRA amino acid sequence according to SEQ ID NO: 3. 19. Complex according to claim 17 or 18, where the peptide penetrating the cell has an amino acid sequence containing: (i) sequence according to the following general formula (I): X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ SX ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ with 0, 1, 2, 3, 4 or 5 amino acids that are replaced, deleted and/or added without interfering with the cell penetration ability of said peptide, wherein X ₁ represents K, R or H, preferably X ₁ represents K or R; X ₂ represents R, K or H, preferably X ₂ represents R or K; X ₃ is Y, W or F, preferably X ₃ is Y, W or F; X ₄ represents K, R or H, preferably X ₄ represents K or R; X ₅ represents N or Q; X ₆ represents R, K or H, preferably X ₆ represents R or K; X ₇ is V, I, M, L, F or A, preferably X ₇ is V, I, M or L; X ₈ is A, V, L, I or G, preferably X ₈ is A or G; X ₉ represents S or T; X ₁₀ is R, K or H, preferably X ₁₀ is R or K; X ₁₁ represents K, R or H, preferably X ₁₁ represents K or R; X ₁₃ is R, K or H, preferably X ₁₃ is R or K; X ₁₄ is A, V, L, I or G, preferably X ₁₄ is A or G; X ₁₅ is K, R or H, preferably X ₁₅ is K or R; X ₁₆ is F, L, V, I, Y, W or M, preferably X ₁₆ is F, Y or W; And X ₁₇ is K, R or H, preferably X ₁₇ is K or R. 20. The complex of claim 17 or 18, wherein the cell penetrating peptide has an amino acid sequence comprising an amino acid sequence selected from the group consisting of amino acid sequences corresponding to SEQ ID NO: 4-13, or a variant of this sequence without disrupting the penetrating the ability of said peptide to enter a cell, in particular sequence variants thereof, that are at least 70% sequence identical, preferably at least 80% sequence identical, and more preferably at least 90% sequence identical, without impairing the ability of said peptide to enter cells . 21. The complex according to claim 20, where the cell-penetrating peptide has an amino acid sequence containing or consisting of an amino acid sequence corresponding to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14), SEQ ID NO: 8 (CPP5/Z15) or SEQ ID NO: 11 (CPP8/Z18), or a variant thereof without interfering with the cell penetrability of said peptide, in particular sequence variants thereof having at least 70% sequence identity, preferably at least 80% sequence identical, and more preferably at least 90% sequence identical, without impairing the ability of said peptide to enter cells. 22. The complex of claim 21, wherein the cell penetrating peptide comprises or consists of a peptide having an amino acid sequence corresponding to SEQ ID NO: 6, or a functional variant of that sequence having at least 70% sequence identity. 23. The complex according to claim 1, wherein the at least one TLR peptide agonist is TLR2, TLR4 and/or TLR5, preferably a TLR2 peptide agonist and/or a TLR4 peptide agonist. 24. The complex according to claim 23, wherein at least one TLR peptide agonist contains or consists of an amino acid sequence corresponding to SEQ ID NO: 15 or 71; or a variant of that sequence, in particular a variant of that sequence that is at least 70% identical to the sequence, preferably at least 80% identical to the sequence, and more preferably at least 90% identical to the sequence, without interfering with the ability of said peptide to be a TLR agonist. 25. The complex of claim 24, wherein the at least one TLR peptide agonist includes or consists of a peptide having the amino acid sequence corresponding to SEQ ID NO: 71; or a functional variant of that sequence that is at least 70% sequence identical, preferably at least 80% sequence identical, and more preferably at least 90% sequence identical, without interfering with the ability of said peptide to be a TLR agonist. 26. The complex according to claim 1, preferably containing in the direction from the N-terminus to the C-terminus the following components: (I) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 6, or a functional variant of this sequence that is at least 70% identical to the sequence; (II) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 96, or a functional variant of this sequence that is at least 70% identical to the sequence; (III) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 95, or a functional variant of this sequence that is at least 70% identical to the sequence; (IV) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 97, or a functional variant of this sequence that is at least 70% identical to the sequence; And (V) a peptide that has an amino acid sequence corresponding to SEQ ID NO: 71, or a functional variant of this sequence that is at least 70% identical to the sequence, where components (I)-(V) are optionally linked to each other using a linker or spacer. 27. The complex of claim 1 or 26, wherein said complex contains or consists of a polypeptide that has an amino acid sequence corresponding to SEQ ID NO: 89, or a functional variant of that sequence that is at least 90% identical to the sequence. 28. Method for obtaining the complex according to any one of paragraphs. 1-27, involving cultivation in a culture medium of a host cell transformed with an expression vector containing a nucleotide sequence encoding said complex, followed by isolation of the complex from the cell or culture medium and its purification, where the complex is a polypeptide or protein. 29. Antigen-presenting cell loaded with the complex according to any one of paragraphs. 1-27. 30. The cell of claim 29, wherein said cell is a dendritic cell. 31. A composition for the preparation of a pharmaceutical composition or vaccine for the treatment of colorectal cancer, containing an effective amount of at least one of the complex, as defined in any one of paragraphs. 1-27. 32. A vaccine for the treatment of colorectal cancer, comprising an effective amount of at least one of the complex as defined in any one of paragraphs. 1-27, and an excipient to increase its immunogenicity. 33. A pharmaceutical composition for the treatment of colorectal cancer, containing an effective amount of at least one complex, as defined in any one of paragraphs. 1-27, and a pharmaceutically acceptable carrier. 34. The pharmaceutical composition according to claim 33, wherein said composition contains an effective amount of at least two different complexes. 35. Combination for the treatment of colorectal cancer containing an effective amount of: (i) the complex according to any of paragraphs. 1-27; And (ii) a chemotherapeutic agent, a targeted drug and/or an immunotherapeutic agent such as an immune checkpoint modulator. 36. The combination of claim 35, wherein (I) the complex and (II) the chemotherapeutic agent, targeted drug, and/or immunotherapeutic agent, such as an immune checkpoint modulator, are administered at approximately the same time. 37. Combination according to any one of paragraphs. 35, 36, wherein (I) the complex and (II) a chemotherapeutic agent, a targeted drug and/or an immunotherapeutic agent such as an immune checkpoint modulator are administered sequentially. 38. Combination according to any one of paragraphs. 35-37, wherein (I) the complex and (II) the chemotherapeutic agent, targeted drug, and/or immunotherapeutic agent, such as an immune checkpoint modulator, are administered using the same route of administration. 39. Combination according to any one of paragraphs. 35-37, wherein (I) the complex and (II) a chemotherapeutic agent, a targeted drug, and/or an immunotherapeutic agent, such as an immune checkpoint modulator, are administered using different routes of administration. 40. Combination according to any one of paragraphs. 35-39, wherein (I) the complex and (II) a chemotherapeutic agent, a targeted drug and/or an immunotherapeutic agent such as an immune checkpoint modulator are in different compositions. 41. Combination according to any one of paragraphs. 35-38, wherein (I) the complex and (II) the chemotherapeutic agent, targeted drug, and/or immunotherapeutic agent, such as an immune checkpoint modulator, are in the same composition. 42. Combination according to any one of paragraphs. 35-41, wherein the chemotherapeutic agent, targeted drug and/or immunotherapeutic agent is an immune checkpoint modulator. 43. The combination of claim 42, wherein the immune checkpoint modulator is a PD-1 inhibitor. 44. A kit for the treatment of colorectal cancer, comprising (1) a complex as defined in any one of paragraphs. 1-27, and (2) instructions for use. 45. The kit according to claim 44, where the kit further contains a package insert or a leaflet with instructions for use for the treatment of colorectal cancer using the complex, as defined in any of paragraphs. 1-27. 46. The kit of claim 44 or 45, wherein the kit further comprises a chemotherapeutic agent, a targeted drug, and/or an immunotherapeutic agent, such as an immune checkpoint modulator. 47. A method of treating colorectal cancer in an individual in need thereof, comprising administering to the individual a complex as defined in any one of claims. 1-27.