CN116723853A

CN116723853A - Combination of STING agonist and complex comprising cell penetrating peptide, cargo and TLR peptide agonist

Info

Publication number: CN116723853A
Application number: CN202180070425.8A
Authority: CN
Inventors: 马泰奥·罗西; 埃洛迪·贝努; 麦迪哈·德鲁阿兹
Original assignee: Boehringer Ingelheim International GmbH
Current assignee: Boehringer Ingelheim International GmbH
Priority date: 2020-10-14
Filing date: 2021-10-14
Publication date: 2023-09-08

Abstract

The present invention provides a combination of an agonist of the interferon response stimulating factor cGAMP interacting factor 1 (STING) and a vaccine comprising a specific antigen or epitope, i.e. a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist. Such a combination is particularly useful in medicine, in particular in the prevention and/or treatment of cancer. Furthermore, the present invention provides compositions, such as pharmaceutical compositions and vaccines, which are useful, for example, in the prevention and/or treatment of cancer.

Description

Combination of STING agonist and complex comprising cell penetrating peptide, cargo and TLR peptide agonist

The present invention relates to the field of vaccination and immunotherapy, in particular cancer immunotherapy.

The immune system recognizes and to some extent eliminates tumor cells, however, such anti-tumor responses are often low-amplitude and inefficient. Enhancement of this weak anti-tumor response by therapeutic vaccination has long been a goal of cancer therapy. Thus, modulating the immune system to enhance immune responses has become a promising therapeutic approach in oncology, as it can be combined with standard of care therapies.

Cancer vaccines can be divided into two main categories: personalized (autologous) and standardized vaccines, and further classified according to the technology platform. Current personalized vaccines include tumor lysate vaccines and dendritic cell-based vaccines (hereinafter cell-based vaccines). For the latter, antigen loading can be achieved by using pulses of tumor lysate or transfection with RNA extracted from the tumor. In this case, the antigen is tumor specific or related, but not specifically defined. Dendritic cells can also be pulsed with peptides or with proteins, e.g.for designThe vaccine is Prostatic Acid Phosphatase (PAP), loaded with defined antigen. However, the manufacturing process of these cell-based therapies is time consuming and labor intensive, while quality standards are difficult to achieve and maintain. Immunomonitoring also causes other complications. Furthermore, unlike established and standardized vaccines, most autologous cancer vaccines do not allow for control over the nature or quantity of antigen used.

Subunit vaccines (proteins or peptides) allow the development of standardized vaccines that are easier to prepare, significantly better reproducible from batch to batch, and can be used in a wide range of patients than cell-based therapies such as Antigen Presenting Cells (APC), T cells, CARs, lysates. Furthermore, the antigen is fully defined, which allows better immune monitoring and reduces the risk of adverse effects of the vaccine components.

Different methods evaluated in preclinical and clinical development include short peptide vaccines (Slingluff CL, jr.the present and future of peptide vaccines for Cancer: single or multiple, long or short, alone or in combinationCancer journal 2011:2011 (5): 343-50), long peptide vaccines (MeliefcJ, van der Burg SH. Immunology of published (pre) malignant disease by synthetic long peptide vaccines. Nature reviews Cancer 2008;8 (5): 351-60) and proteins. Short peptide vaccines have very short half-lives compared to long peptide and protein vaccines and can negatively impact immune responses.

Typically, cancer vaccines are administered to cancer patients in order to enhance their ability of the immune system to recognize and kill tumor cells. The main goal of therapeutic cancer vaccines is to generate killer T cells (also known as cytotoxic T lymphocytes) specific for tumor cells. For this reason, and in order to achieve an effective immune response, vaccines often contain antigens or antigenic epitopes, which are also present in tumors, and need to be delivered to Antigen Presenting Cells (APCs), in particular Dendritic Cells (DCs), to allow initiation of cancer immunity. Dendritic cells process these tumor antigens into small peptides that are presented on MHC class I or MHC class II molecules expressed on the cell surface of T cells. Peptides that are then recognized by T cells and thus induce their stimulation are called epitopes. Presentation by MHC class I and MHC class II molecules allows for activation of both T cells, CD8, respectively ⁺ Cytotoxic T Lymphocytes (CTL) and CD4 ⁺ Helper T (T) _h ) And (3) cells. In addition, in order to become fully activated, T cells require a second signal, the costimulatory signal, in addition to antigen recognition, which is antigen-non-specific, provided by the interaction between the costimulatory molecule expressed on the surface of the APC and the T cell. Thus, two major requirements for an effective therapeutic cancer vaccine are the specificity of tumor antigens and their ability to deliver them efficiently to dendritic cell DCs.

In summary, three main steps are required to induce a tumor-specific immune response: (i) antigen is delivered to dendritic cells, which process the antigen into epitopes, (ii) dendritic cells should receive appropriate activation signals, and (iii) activated tumor antigen-loaded dendritic cells must generate T-cell mediated immune responses in lymphoid organs.

Multi-epitope antigen delivery provides advantages since tumor cells can evade the immune system by down-regulating the expression of individual antigens (passive immune evasion). In fact, protein-based vaccines allow the delivery of multi-epitope antigens to Antigen Presenting Cells (APCs), such as Dendritic Cells (DCs), without restriction by a single MHC allele. Another advantage is the long-lasting epitope presentation in protein-loaded dendritic cells recently described (van Montfoort N, camps MG, khan S, filipcov DV, weterings JJ, griffith JM et al, antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-private capacity. Proceedings of the National Academy of Sciences of the United States of America 2009;106 (16): 6730-5). In addition, proteins need to be taken up and processed by dendritic cell DCs to achieve MHC-restricted presentation of their constituent epitopes. This reduces the risk of inducing peripheral tolerance as shown after inoculation with short peptides that do not have such stringent processing requirements (Toes RE, offringa R, boom RJ, melief CJ, kast WM. Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance introduction of the National Academy of Sciences of the United States of America 1996:1996; 93 (15): 7855-60).

However, most soluble proteins are typically degraded in endolysosomes and are less cross-presented on MHC class I molecules and thus on CD8 ⁺ T cell responses were poorly immunogenic (Rosalia RA, quakkelar 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 2013;43 (10): 2554-65). In addition, although mature treeDendritic cell DCs are more effective than immature dendritic cell DCs in eliciting and eliciting T cell responses (Apetoh L, locher C, ghirengelli F, kroemer G, zitvogel L.Harnessing dendritic cells in cancer.Semin immunol.2011; 23:42-49), but they lose the ability to efficiently absorb exogenous antigens, particularly for MHC class II restricted antigens (Banchereau J, steinman RM.Dendritic cells and the control of immunoy.Nature.1998; 392:245-252). Thus, peptide-pulsed dendritic cell DCs have several limitations as vaccines. For example, peptide degradation, rapid switching of MHC class I and separation of peptide from MHC class I molecules during DC/peptide preparation and injection may result in a shorter half-life of MHC class I/peptide complex on the DC surface, resulting in a weaker T cell response.

To enhance the efficacy of protein-based vaccine delivery, intracellular delivery of cancer peptides into DCs using cell-penetrating peptides has been proposed (Wang RF, wang HY. Enhancement of antitumor immunity by prolonging antigen presentation on dendritic cells. Nat Biotechnol.2002; 20:149-156). Cell Penetrating Peptides (CPP) are peptides that are capable of penetrating the cell membrane and entering most cell types (Copolovici DM, langel K, eriste E, langel U.S. cell-penetrating peptides: design, synchronization, and application. ACS nano 2014;8 (3): 1972-94,Milletti F.Cell-penetrating peptides: classification, origin, and current language cape. Drug discovery 2012). Alternatively, they are also known as Protein Transduction Domains (PTDs), reflecting that they are derived from natural proteins. Several potent CPPs have been identified from proteins, including the Tat protein of human immunodeficiency virus, the VP22 protein of herpes simplex virus and fibroblast growth factor (Berry CC. Intracellulars dehvery of nanoparticles via the HIV-1 Tat peptide.Nanomedicine.2008;3:357-365;Deshayes S,Morris MC,Divita G,Heitz F.Cell-penetrating peptides: tools for intracellular delivery of therapeutics. Cell Mol Life Sci.2005;62:1839-1849;Edenhofer F.Protein transduction revisited:Novel insights into the mechanism underlying intracellular delivery of proteins.Curr Pharm Des.2008;14:3628-3636;Gupta B,Levchenko TS,Torchilin VP.Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides. Adv Drug Deliv Rev.2005;57:637-651;Torchilin VP.Recent approaches to intracellular delivery of drugs and DNA and organelle targeting.Annu Rev Biomed Eng.2006;8:343-375). DC/TAT-TRP 2-induced T cell activity was found to be 3-to 10-fold greater than DC/TRP 2-induced activity (Wang HY, fu T, wang G, gang Z, donna MPL, yang JC, restifo NP, hwu P, wang RF. Instruction of CD4+T cell-dependent antitumor immunity by TAT-mediated tumor antigen delivery into dendritic cells.J Clin invest.2002a; 109:1463-1470).

In order to increase the level of co-stimulatory molecules on dendritic cell DCs and enhance the immune system response to target antigens, adjuvants may be used. Adjuvants can accomplish this task by mimicking conserved microbial components that are recognized naturally by the immune system. They include, for example, lipopolysaccharide (LPS), bacterial cell wall components and nucleic acids, such as double stranded RNA (dsRNA), single stranded DNA (ssDNA) and DNA containing unmethylated CpG dinucleotides. Their presence may increase the innate immune response to the antigen. Furthermore, such adjuvants should promote CTL and type polarization T _h 1, but not a humoral immune response leading to antibody production. Different adjuvants have been evaluated, with a limited number of adjuvants having been approved by regulatory authorities for use in humans. These include alum, MPL (monophosphoryl lipid a) and ASO in the united states ₄ (alum and MPL), MF59 (oil-in-water emulsion), ASO in Europe ₄ Liposomes (Lim, y.t., vaccine adjuvant materials for cancer immunotherapy and control of infectious treatment.clin Exp Vaccine Res,2015.4 (1): p.54-8).

Recently, toll-like receptor (TLR) ligands have emerged as a promising class of adjuvants (Baxevenis, C.N., I.F.Voutsas and O.E.Tstisitronis, TOIL-like receptor agonists: current status and future perspective on their utility as adjuvants in improving anticancer vaccination strategies.immunotherapy,2013.5 (5): p.497-511). Thus, an important development in cancer vaccine research is the inclusion of various TLR agonists into vaccine formulations, including TLR-3 (multimeric I: C), TLR-4 (monophosphoryl lipid A; MPL), TLR-5 (flagellin), TLR-7 (imiquimod) and TLR-9 (CpG) (Duthie MS, windish HP, fox CB, reed SG.use of defined TLR ligands as adiuvants within human vaccines.Immunol Rev.2011; 239:178-196). The signals and cytokine types produced by immune cells upon TLR stimulation control the differentiation of cd4+ T cells into Th1, th2, th17 and Treg cells. Most TLR-based adjuvants stimulate immune cells such as DCs and T cells to produce pro-inflammatory cytokines and promote Th1 and cd8+ T responses (manicassmam S, pulsdran b. Modul of adaptive immunity with Toil-like receptors. Semin immunol.2009; 21:185-193).

Combining a vaccine with a TLR ligand is an attractive approach that provides several advantages over non-combined vaccines, including (i) preferential uptake by TLR-expressing immune cells, (ii) higher immune responses and (iii) reduced risk of inducing peripheral tolerance. Virtually all antigen-loaded antigen-presenting cells will be activated simultaneously. Different groups explored this approach, in which various TLR ligands were predominantly chemically linked to peptide or protein vaccines (Zom GG, khan S, filipply DV, ossendorp F. TLR ligand-peptide conjugate vaccines: topard clinical application. Adv immunol.2012; 114:177-201). Because of the ease of chemical attachment to peptides, the most studied TLR ligands in conjugate vaccines are the TLR2 agonists Pam2Cys and Pam3Cys (Fujita, y. And h.taguchi, overview and outlook of Toll-like receptor hgand-antigen conjugate vaccines. Ter Deliv,2012.3 (6): p.749-60).

Recently, chimeric protein vaccine platforms have been described that provide a complex consisting of three components: cell penetrating peptides, antigen cargo and TLR agonists that confer self-adjuvanticity (belnue, e., et al (2016), "Enhancing Antitumor Immune Responses by Optimized Combinations of Cell-penetrating Peptide-based Vaccines and Adjuvants.," Mol ter 24 (9): 1675-1685). The vaccine platform was shown to induce CD8 and CD4 antigen-specific immune responses in preclinical tumor models, leading to immune memory and high vaccine efficacy as well as increased intratumoral leukocyte infiltration (deruazi, m., et al (2015), "Novel Cell-Penetrating Peptide-Based Vaccine Induces Robust CD4+ and cd8+ T Cell-Mediated Antitumor immunity @" Cancer Res 75 (15): 3020-3031; beioue, e., et al (2019), "Targeting self and neo-epitopes with a modular self-adjuvanting Cancer vaccinee." JCI Insight 5).

Another very promising strategy is to target the interferon gene stimulatory factor/interferon response stimulatory factor cGAMP interacting factor 1 (STING) pathway. STING is an adaptor protein that is activated by binding to a circular GAMP, which is a byproduct of the degradation of viral or bacterial DNA by cytoplasmic DNA sensors (Ishikawa, h. And g.n. barber (2008), "STING is an endoplasmic reticulum adaptor that facilitates innate immune signaling." Nature 455 (7213): 674-678; ablasser, a., et al (2013), "cGAS products a 2'-5' -linked cyclic dinucleotide second messenger that activates STING.," Nature 498 (7454): 380-384), which, upon activation, induces high levels of type I interferon and other pro-inflammatory cytokines such as IL-6 and TNF secretion (Ishikawa, h. And g.n. barber (2008), "STING is an endophsmic reticulum adaptor that facilitates innate immune signaling.," Nature 455 (7213): 674-678; saitoh, t., "2009)," Atg9a control DNA-35delivery "353535 Proc Natl Acad Sci U S A (3892)," 26:378 (37): 6:6:. In addition, STING signaling was shown to enhance NK cell recruitment and activation (Takashima, k., et al (2016), "STING in tumor and host cells cooperatively work for NK cell-mediated tumor growth retrieval," Biochem Biophys Res Commun 478 (4): 1764-1771), and promote chemotaxis of CD4 and CD 8T cells (Parkes, e.e., et al (2017), "Activation of STING-Dependent Innate Immune Signaling By S-Phase-Specific DNA Damage in Breast cancer," J Natl Cancer Inst (1)). Furthermore, STING signaling was found to be inhibited in patient-derived colorectal adenocarcinoma cells, supporting its anti-tumor effect (Xia, x., et al (2015), "Porous silicon microparticle potentiates anti-tumor immunity by enhancing cross-presentation and inducing type I interferon response", "Cell Rep 11 (6): 957-966). Because of these properties, synthetic STING agonists have been tested in preclinical tumor models and clinical studies with the aim of inflaming tumors and eliciting anti-tumor immune responses. Intratumoral injection of STING agonists was shown to induce regression of different murine tumor models, while also inducing a systemic tumor-specific immune response, as highlighted by resistance of tumor-clearing mice to re-challenge (Corrales, l., et al (2015), "Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and immunity," Cell Rep 11 (7): 1018-1030). Furthermore, STING agonists formulated in GM-CSF producing cancer cell vaccines have been shown to delay the progression of several murine tumor models, indicating that intratumoral administration is not the only effective route. At present, a number of phase 1/2 clinical trials have investigated the use of STING agonists in different patients with solid tumors and lymphomas.

In view of the above, it is an object of the present invention to overcome the above-mentioned drawbacks of the existing cancer vaccines. In particular, it is an object of the present invention to provide a vaccine comprising an antigen or epitope of an antigen in combination with a STING agonist, which provides specificity against a certain tumor. It is another object of the invention to provide vaccines that enhance or prolong the antitumor effect of each of its components (e.g., when administered as an independent therapy). Thus, this combination represents a more effective vaccine for cancer immunotherapy applications, in particular with improved antitumor activity. Accordingly, the present invention relates to combination therapies that initiate, enable, enhance and/or improve an anti-tumor immune response.

This object is achieved by the subject matter set forth below and in the appended claims.

Although the present invention is described in detail below, it is to be understood that the invention is not limited to the particular methodology, protocols, and reagents described herein as these may vary. It is also to be understood that the terminology used herein is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Hereinafter, elements of the present application will be described. These elements are listed with particular embodiments, but it should be understood that they may be combined in any manner and in any number to create additional embodiments. The examples and preferred embodiments described differently should not be construed as limiting the application to only the explicitly described embodiments. The description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of disclosed and/or preferred elements. Furthermore, unless the context indicates otherwise, it is to be considered that in the specification of the application any permutation and/or combination of all of the described elements of the application are disclosed.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated component, integer or step but not the exclusion of any other non-stated component, integer or step. The term "consisting of" is a particular embodiment of the term "comprising" wherein any other unrecited component, integer or step is excluded. In the context of the present application, the term "comprising" encompasses the term "consisting of. Thus, the term "comprising" encompasses "comprising" and "consisting of" for example, "a composition comprising" X may consist of X alone, or may comprise other content, such as x+y.

The use of numerical terms not limited to use in describing the context of the present invention (particularly in the context of the claims) and the like should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The word "substantially" does not exclude "complete", e.g., a composition that is "substantially free" of Y may be completely free of Y. The word "substantially" may be omitted from the definition of the invention, if necessary.

The term "about" with respect to the value x means x±10%.

STING agonists and complexes comprising cell penetrating peptides, at least one antigen or epitope of an antigen and TLR peptide agonists Combination of objects

In a first aspect, the present invention provides a combination of

(i) STING agonists

(ii) A complex comprising:

a) Cell penetrating peptide;

b) At least one antigen or epitope; and

c) A TLR peptide agonist, which is a compound of formula (i),

wherein components a) through c) (i.e., cell penetrating peptide, at least one antigen or epitope, and TLR peptide agonist) are covalently linked.

The present inventors have surprisingly found that the combination of (i) STING agonist and (ii) a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist improves CD4 and CD 8T cell responses, enhances antigen specific CD 8T cells, increases intratumoral immunogenicity, and as a result, a substantial increase in survival and reduced tumor growth. This demonstrates the synergistic effect of STING agonists and complexes acting together, which greatly increases the anti-tumor effect of each of the components administered as independent therapies.

As used herein, the term "combination" refers to any kind of combination of its components, in particular to any kind of combination of (i) STING agonist and (ii) the complex described herein and optionally any other components. In particular, the components of the combination are provided together (i.e., in a combined manner). In some embodiments, the combination may be a kit (e.g., comprising the components in (at least partially) isolated manner). In other embodiments, the combination may be a composition (e.g., the components may be contained in a single composition).

Thus, each component (i) (STING agonist) and (ii) (complex) (and any other optional components) of the combination may be included in a separate composition. In other embodiments, some (but not all) of the components (i) (STING agonist) and (ii) (complex) (and any other optional components) combined may be included in the same composition. Alternatively, all components (i) (STING agonist) and (ii) (complex) (and any other optional components) of the combination may be included in the same composition. Thus, each component (i) (STING agonist) and (ii) (complex) (and any other optional components) of the combination may be contained in a separate container (e.g., syringe). In other embodiments, some (but not all) of the components (i) (STING agonist) and (ii) (complex) (and any other optional components) combined may be contained in the same container (e.g., syringe). Alternatively, all components (i) (STING agonist) and (ii) (complex) (and any other optional components) of the combination may be contained in the same container (e.g., syringe).

More specifically, (i) the STING agonist and (ii) the complex may be contained in the same composition and/or in the same container (e.g., syringe). In some embodiments, (i) the STING agonist and (iii) the optional third component (other than the complex and STING agonist) may be contained in the same composition and/or the same container (e.g., syringe). In some embodiments, (ii) the complex and (iii) optionally a third component (other than the complex and STING agonist) may be contained in the same composition and/or the same container (e.g., syringe). For example, (i) STING agonists; (ii) The complex and (iii) optionally a third component (other than the complex and STING agonist) may be contained in the same composition and/or in the same container (e.g., syringe).

In some embodiments, (i) STING agonist and (ii) complex may be provided in different compositions and/or in different containers (e.g., different syringes). In some embodiments, (i) the STING agonist and (iii) the optional third component (other than the complex and STING agonist) may be provided in different compositions and/or in different containers (e.g., different syringes). In some embodiments, (ii) the complex and (iii) the optional third component (other than the complex and STING agonist) may be provided in different compositions and/or in different containers (e.g., different syringes). For example, (i) STING agonists; (ii) The complex and (iii) optional third component (other than the complex and STING agonist) may be provided in different compositions and/or in different containers (e.g., different syringes).

Hereinafter, components of the combination according to the invention, namely STING agonists and complexes comprising a cell penetrating peptide, at least one antigen or epitope and at least one TLR peptide agonist, and embodiments thereof, are described in detail. It should be understood that preferred embodiments of the combination according to the invention (i) include preferred embodiments of STING agonists; (ii) Preferred embodiments of the combination according to the invention include preferred embodiments of a complex comprising a cell penetrating peptide, at least one antigen or epitope of an antigen, and at least one TLR peptide agonist; and (iii) more preferred embodiments of the combination according to the invention include preferred embodiments of STING agonists and preferred embodiments of complexes comprising a cell penetrating peptide, at least one antigen or epitope of an antigen, and at least one TLR peptide agonist.

In some embodiments, a combination according to the invention, i.e., a STING agonist and a complex comprising a cell penetrating peptide, at least one antigen or epitope, and at least one TLR peptide agonist, can also be administered in combination with other additional active compounds (e.g., in the context of tumor/cancer treatment). In other embodiments, the combination according to the invention, i.e. STING agonist and complex comprising a cell penetrating peptide, at least one antigen or epitope and at least one TLR peptide agonist, is not further administered in combination with other additional active compounds (e.g. in the context of tumor/cancer treatment). In other words, the combination of the invention may also be used as a "stand alone" therapy.

Complexes comprising cell penetrating peptides, at least one antigen or epitope of an antigen

The combination according to the invention comprises a complex comprising:

a) Cell penetrating peptide;

b) At least one antigen or epitope; and

c) At least one TLR peptide agonist, which is selected from the group consisting of,

wherein components a) through c), i.e., the cell penetrating peptide, at least one antigen or epitope and at least one TLR peptide agonist, are covalently linked. Hereinafter, by using the term "complex" or "complex comprised by a combination according to the invention" is also meant such a complex comprising a cell penetrating peptide, at least one antigen or epitope and at least one TLR peptide agonist, which are covalently linked.

Such complexes comprised by the combination according to the invention simultaneously provide (i) multi-epitope cytotoxic T cell mediated immune stimulation, (ii) T _h Induction of cells and (iii) promotion of immune memory. Thus, the complexes comprised by the combination according to the invention provide an effective vaccine, in particular with improved antitumor activity.

Preferably, the complex comprised by the combination according to the invention is a polypeptide or a protein, in particular a recombinant polypeptide or a recombinant protein, preferably a recombinant fusion protein or a recombinant fusion polypeptide.

The term "recombinant" as used herein (i.e., throughout the specification) means that it (herein: polypeptide or protein) is not naturally occurring. Thus, the complex comprised by the combination according to the invention, which is a recombinant polypeptide or recombinant protein, typically comprises components a) to c), wherein components a) to c) may be from different sources, i.e. not naturally occurring in the combination. In some embodiments, the term "recombinant" refers to a peptide, polypeptide, or protein of semisynthetic or synthetic origin. Recombinant peptides, polypeptides or proteins may be produced by the expression of combinations of DNA molecules of different origins, which may be linked using recombinant DNA techniques. In some cases, a recombinant peptide, polypeptide, or protein may not be associated with all or part of the protein with which it is naturally associated, due to its source or manipulation. Furthermore, a recombinant peptide, polypeptide or protein may be linked to a polypeptide other than that to which it is naturally linked. Recombinant peptides, polypeptides or proteins may be produced by any method known in the art, e.g., prokaryotic and eukaryotic expression systems, using a mature protocol (see, e.g., laVallie, current Protocols in Protein Science (1995) 5.1.1-5.1.8; chen et al Current Protocols in Protein Science (1998) 5.10.1-5.10.41). For example, in the complexes comprised in the combinations of the invention, components a) to c) may come from different sources, i.e. components a) to c) of the complex do not normally occur together in the natural state (so that the complex may be "recombinant" due to the combination of components a) to c).

In the context of the present application, i.e. throughout the present application, the terms "peptide", "polypeptide", "protein" and variants of these terms refer to peptides, oligopeptides, oligomers or proteins comprising fusion proteins, respectively, comprising at least two amino acids linked to each other, preferably by normal peptide bonds, or alternatively by modified peptide bonds, e.g. in the case of isostere peptides. The peptide, polypeptide or protein may consist of L-amino acids and/or D-amino acids. Preferably, such a peptide, polypeptide or protein is either (entirely) composed of L-amino acids or (entirely) composed of D-amino acids, thereby forming a "reverse-reverse peptide sequence". The term "reverse-inverted (peptide) sequence" 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, e.g., jameson et al, nature,368, 744-746 (1994); brady et al, nature,368, 692-693 (1994)). In particular, the terms "peptide", "polypeptide", "protein" also include "peptidomimetic", which is defined as a peptide analogue comprising non-peptide structural elements that are capable of mimicking or antagonizing the biological effects of a native parent peptide. Peptide mimetics lack classical peptide properties such as enzymatically cleavable peptide bonds. In particular, the peptide, polypeptide or protein may comprise or may consist of amino acids other than the 20 amino acids defined by the genetic code in addition to these amino acids. In particular, a peptide, polypeptide or protein in the context of the present application may likewise consist of amino acids which are well known to the person skilled in the art to be modified by natural processes, such as posttranslational maturation processes or chemical processes. These modifications are described in detail in the literature. These modifications may occur at any position in the polypeptide: in the peptide backbone, in the amino acid chain or even at the carboxyl or amino terminus. In particular, the peptide or polypeptide may be branched after ubiquitination or cyclic with or without branching. Such modifications may be the result of natural or synthetic post-translational processes well known to those skilled in the art. In the context of the present application, the terms "peptide", "polypeptide", "protein" in particular also include modified peptides, polypeptides and proteins. For example, modifications of peptides, polypeptides or proteins may include acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of a nucleotide or nucleotide derivative, covalent fixation of a lipid or lipid derivative, covalent fixation of phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including polyethylene glycol, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, selenoylation, sulfation, amino acid addition such as arginylation or ubiquitination. Such modifications are described in detail in the literature (Seffier et al (1990) Analysis for protein modifications and nonprotein cofactors, meth. Enzymol.182:626-646 and Rattan et al (1992) Protein Synthesis:post-translational Modifications and Aging, ann NY Acad Sci, 663:48-62). Thus, the terms "peptide", "polypeptide", "protein" preferably include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, and the like.

However, in particularly preferred embodiments, the complexes described herein are "classical" peptides, polypeptides or proteins, wherein a "classical" peptide, polypeptide or protein typically consists of amino acids selected from the group consisting of 20 amino acids linked by the genetic code, which are linked to each other by normal peptide bonds.

If the complex comprised by the combination according to the application is a polypeptide or a protein, it preferably comprises at least 50, at least 60, at least 70, preferably at least 80, at least 90, more preferably at least 100, at least 110, even more preferably at least 120, at least 130, particularly preferably at least 140 or most preferably at least 150 amino acid residues.

As used herein (i.e., throughout the present application), the term "sequence variant" refers to any change in a 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 "sequence and SEQ ID No." (sequence listing), i.e., SEQ ID NO:1 to SEQ ID NO:55. in particular, the sequence variant (over the entire length of the sequence) has at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity to the reference sequence. Sequence identity can be calculated as follows. In particular, sequence variants retain the specific function of the reference sequence. In particular, amino acid sequence variants have altered sequences in which one or more amino acids in a reference sequence are deleted or replaced, or one or more amino acids are inserted into the sequence of a reference amino acid sequence. As a result of the change, the amino acid sequence variant has a sequence which is at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% identical to the reference sequence. For example, a variant sequence having at least 90% identity has no more than 10 changes, i.e., any combination of deletions, insertions, or substitutions, to every 100 amino acids of the reference sequence.

In the context of the present invention, a "shared sequence identity" with a query amino acid sequence of the present invention, e.g., an amino acid sequence of at least 95% means that the subject amino acid sequence is identical to the query sequence except that the subject amino acid sequence may contain up to 5 amino acid changes per 100 amino acids of the query amino acid sequence. In other words, in order to obtain an amino acid sequence having at least 95% identity to the query amino acid sequence, up to 5% (5 out of 100) of the amino acid residues in the subject sequence may be inserted or replaced by another amino acid or deleted, preferably within the above definition of variant or fragment. Of course, the same applies to nucleic acid sequences.

For sequences that do not correspond exactly (amino acids or nucleic acids), the "% identity" of the first sequence relative to the second sequence may be determined. In general, the two sequences to be compared can be aligned to give the greatest correlation between the sequences. This may include the insertion of a "gap" in one or both sequences to increase the extent of alignment. The% identity can then be determined over the full length of each sequence being compared (so-called global alignment), which is particularly applicable to sequences of identical or similar length, or to similar sequences of shorter defined length (so-called local alignment), which is more applicable to sequences of unequal length.

The method of comparing identity and homology of two or more sequences is well known in the art and the percentage of identity of two sequences can be determined, for example, using a mathematical algorithm. A preferred but non-limiting example of a mathematical algorithm that may be used is Karlin et al (1993), PNAS USA,90: 5873-5877. Such algorithms are integrated into BLAST series programs, such as BLAST or NBLAST programs (see Altschul et al, 1990, J. Mol. Biol.215, 403-410 or Altschul et al (1997), nucleic Acids Res, 25:3389-3402), accessible through the homepage of NCBI with the website NCBI. Nl. Nih. Gov) and FASTA (Pearson (1990), methods enzymol.183, 63-98; pearson and Lipman (1988), proc.Natl.Acad.Sci.U.S. A85, 2444-2448. Sequences that are to some extent identical to other sequences can be identified by these procedures. In addition, programs available in Wisconsin sequence analysis software package, version 9.1 (Devereux et al, 1984,Nucleic Acids Res, 387-395), such as programs BESTFIT and GAP, can be used to determine% identity between two polynucleotides and% identity and% homology or identity between two polypeptide sequences. The "local homology" algorithm used by BESTFIT (Smith and Waterman (1981), J.mol. Biol.147, 195-197.) and found the best single region of similarity between the two sequences.

In general, substitutions of one or more amino acids present in the reference amino acid sequence are preferably made conservatively. Examples of conservative substitutions include the replacement of one aliphatic residue for another, such as Ile, vaI, leu or Ala for each other, or the replacement of one polar residue for another, such as between Lys and Arg; between Glu and Asp or between Gln and Asn. Other such conservative substitutions, for example substitutions of the entire region with similar hydrophobicity, are well known (Kyte and Doolittle,1982, J.mol. Biol.157 (1): 105-132). In the context of the present invention, one or more L-amino acid substitutions by one or more D-amino acid substitutions are considered conservative substitutions. Exemplary amino acid substitutions are listed in table 1 below:

(Table 1)

Component a) -cell penetrating peptide

Cell Penetrating Peptides (CPPs) allow for efficient delivery, i.e. transport and loading, of in particular at least one antigen or epitope of an antigen into Antigen Presenting Cells (APCs), in particular into Dendritic Cells (DCs), and thus into the antigen processing mechanism of dendritic cells.

The term "cell penetrating peptide" ("CPP") is generally used to denote a short peptide that is capable of transporting different types of cargo molecules across the plasma membrane, thereby facilitating cellular uptake of various molecular cargo (from nano-sized particles to small chemical molecules and large DNA fragments). "cellular internalization" of a cargo molecule linked to a cell penetrating peptide generally means that the cargo molecule is transported across the plasma membrane, such that the cargo molecule enters the cell. Depending on the circumstances, the cargo molecule may be released in the cytoplasm, directed to an intracellular organelle, or further presented on the cell surface. The cell penetrating ability or internalization of a cell penetrating peptide or complex comprising said cell penetrating peptide comprised by a combination according to the invention can be examined by standard methods known to those skilled in the art, including flow cytometry or fluorescence microscopy of living and fixed cells, immunocytochemistry of cells transduced with said peptide or complex, and western blotting.

Cell penetrating peptides typically have an amino acid composition comprising a high relative abundance of positively charged amino acids, such as lysine or arginine, or a sequence comprising alternating patterns of polar/charged amino acids and non-polar hydrophobic amino acids. These two types of structures are referred to as polycationic or amphiphilic, respectively. Cell penetrating peptides have different sizes, amino acid sequences, and charges, but all CPPs share the common feature of being able to translocate the plasma membrane and facilitate delivery of various molecular cargo to the cytoplasm or organelle. Currently, the theory of CPP translocation distinguishes three major entry mechanisms: direct membrane penetration, endocytosis-mediated entry, and translocation through the formation of transitional structures. CPP transduction is an ongoing area of research. Cell penetrating peptides have found many applications in medicine as drug delivery agents for the treatment of different diseases, including cancer and viral inhibitors, as well as contrast agents for cell labeling and imaging.

In general, cell Penetrating Peptides (CPPs) are peptides having 8 to 50 residues with the ability to penetrate cell membranes and enter most cell types. Alternatively, they are also known as Protein Transduction Domains (PTDs), reflecting their origin in the natural protein. Frankel and Pabo describe the ability of transactivating transcriptional activators from human immunodeficiency Virus 1 (HIV-TAT) to penetrate into cells simultaneously to Green and Lowenstein (Frankel, A.D. and C.O. Pabo, cellular uptake of the TAT protein from human immunodeficiency viruses. Cell,1988.55 (6): p.1189-93). In 1991, transduction of neural cells from the antennapedia homology domain (DNA binding domain) of Drosophila melanogaster was described (Joliot, A. Et al, antennapedia homeobox peptide regulates neural morphism. Proc Natl Acad Sci U S A,1991.88 (5): p.1864-8). In 1994, the first 16-mer peptide CPP, termed the transmembrane peptide, was identified from the third helix of the antennapedia homeodomain, having the amino acid sequence RQIKIYFQNRRMKWKK (SEQ ID NO: 1) (Derossi, D. Et al, the third helix of the Antennapedia homeodomain translocates through biological membranes.J Biol Chem,1994.269 (14): p.10444-50), followed by the identification of the minimum domain of TAT in 1998, which has the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 2) required for protein transduction (vitamins, 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,1997.272 (25): p.16010-7). Over the last two decades, tens of peptides from different sources have been described, including viral proteins such as VP22 (Elliott, g. And P.O' Hare, intercellular trafficking and protein delivery by a herpesvirus structural protein. Cell,1997.88 (2): p.223-33) and ZEBRA (Rothe, R.et al, characterization of the cell-penetrating properties of the Epstein-Barr virus ZEBRA trans-activator.J Biol Chem,2010.285 (26): p.20224-33), or from venom, e.g., melittin (Dempsey, C.E., the actions of melittin on membrane.Biochim biophysia Acta,1990.1031 (2): p.143-61), wasp toxin (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,2000.38 (11): p.1505-15), scorpion dark peptide toxoid (es teve, E.et al, transduction of the scorpion toxin maurocalcine into cells.Evence that the toxin crosses the plasma membrane, J Biol Chem,2005.280 (13): p.33-9), tail 128snake amine (Nascintimo, F.D, et al, crotamine mediates gene delivery into cells through the binding to heparan sulfate protein 86 (29.J. 62) or (Konno.213-15) (e.213, 29-49) of the whole organism Chem (e.e.26). Synthetic CPPs have also been designed, including poly-Arginine (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,2001.276 (8): p.5836-40) or transporters (Pooga, M.et al, cell penetration by transportan. FASEB J,1998.12 (1): p.67-77). Any of the above CPPs may be used as cell penetrating peptides, i.e. as component a), present in a complex comprised in a combination according to the invention. In particular, component a), i.e. the CPP, of the complex comprised in the combination according to the invention may comprise the minimal domain of TAT, having the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 2). In some embodiments, component a), i.e., CPP, in a complex comprised in a combination according to the invention may comprise a transmembrane peptide having the amino acid sequence RQIKIYFQNRRMKWKK (SEQ ID NO: 1).

In the complexes comprised in the composition according to the invention, various CPPs that can be used as cell penetrating peptides, i.e. as component a), are also disclosed in the review: milletti, f., cell-penetrating peptides: class, origin, and current land slope. Drug discovery Today 17 (15-16): 850-60, 2012. In other words, disclosed in Milletti, f.,2012, cell-penetrating peptides: class, origin, and current land slope. Drug discovery Today 17 (15-16): 850-60 can be used as cell penetrating peptide, i.e. as component a), in a complex comprised in a combination according to the invention. This includes, inter alia, cationic CPPs, amphiphilic CPPs and hydrophobic CPPs as well as CPPs derived from heparinoids, RNA-and DNA-binding proteins (see table 2 of Milletti, f., cell-penetrating peptides: seals, origin, and current lands tape. Drug discovery Today 17 (15-16): 850-60, 2012), CPPs derived from signal peptides (see table 1 of Milletti, f., cell-penetrating peptides: seals, origin, and current lands tape. Drug discovery 17 (15-16): 850-60, 2012), CPPs derived from antimicrobial peptides (see table 2 of Milletti, f., cell-penetrating peptides: seals, origin, and current lands tape. Drug discovery 17 (15-16): 850-60, 2012), CPPs derived from signal peptides (see table 3 of Milletti, f., cell-penetrating peptides: seals, 2012), CPPs derived from signal peptides (see table 6, 37-60, and tape. 37-16): 1) and CPPs derived from Cell-60, 2012.

Preferably, the cell penetrating peptide is derived from the "ZEBRA" protein of Epstein Barr Virus (EBV). "ZEBRA" (also known as Zta, Z, EB1 or BZLF 1) generally refers to the basic-leucine zipper (bZIP) transcriptional activator of EBV (EBV). The minimal domain of ZEBRA exhibits cell penetration and has been identified as from residue 170 to residue 220 of ZEBRA. The amino acid sequence of ZEBRA is disclosed in NCBI accession number yp_401673, comprising SEQ ID NO: 245 amino acids shown in 3:

( SEQ ID NO:3-ZEBRA amino acid sequence (native sequence from Epstein-Barr virus (EBV)) (YP_ 401673) )

Preferably, the cell penetrating peptide

i) The length of the amino acid sequence of the peptide having a total of 5 to 50 amino acids, preferably a total of 10 to 45 amino acids, more preferably a total of 15 to 45 amino acids; and/or

ii) an amino acid sequence having a fragment comprising the minimal domain of ZEBRA from the amino acid sequence according to SEQ ID NO:3 to residue 220, wherein, optionally, 1, 2, 3, 4 or 5 amino acids are substituted, deleted and/or added without losing the cell penetrating ability of the peptide.

Therefore, it is preferable that the cell penetrating peptide

i) The length of the amino acid sequence of the peptide having a total of 5 to 50 amino acids, preferably a total of 10 to 45 amino acids, more preferably a total of 15 to 45 amino acids; and

Such a preferred CPP is disclosed in, for example, WO 2014/04505.

More recently, CPP from the viral protein ZEBRA has been described as transducing protein cargo across biological membranes by (i) direct translocation and (ii) lipid raft mediated endocytosis (Roth R, liguori L, viilegas-Mendez A, marques B, 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 2010;285 (26): 20224-33). The inventors hypothesize that these two entry mechanisms should promote the antigen of cargo to CD8, respectively ⁺ And CD4 ⁺ MHC class I and class II restricted presentation of T cells. Thus, such CPPs can deliver polyepitopic peptides to Dendritic Cells (DCs) and subsequently promote CTL and Th cell activation and anti-tumor function. Thus, such CPPs can effectively deliver complexes comprised by a combination according to the invention to Antigen Presenting Cells (APCs) and cause multi-epitope MHC class I and class II MHC-restricted presentation.

In the context of the present invention, the term "MHC class I" refers to one of two main categories of major histocompatibility complex molecules. MHC class I (also referred to as "MHC I") molecules are present in every nucleated cell of the body. MHC class I functions to display epitopes to cytotoxic Cells (CTLs). In humans, MHC class I molecules consist of two polypeptide chains, alpha-and beta 2-microglobulin (b 2 m). Only the alpha chain is polymorphic, encoded by the HLA gene, while the b2m subunit is not polymorphic, encoded by the beta-2 microglobulin gene. In the context of the present invention, the term "MHC class II" refers to another major class of major histocompatibility complex molecules. MHC class II (also referred to as "MHC II") molecules are found only in a few specific cell types, including macrophages, dendritic cells and B cells, all of which are specialized Antigen Presenting Cells (APCs).

Preferably, the sequence variants of the fragments of the ZEBRA minimum domain as described above, in particular over their full length, have at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% amino acid sequence identity to the fragment of the ZEBRA minimum domain (residues 170 to 220 of SEQ ID NO: 3) without losing the cell penetrating ability of the cell penetrating peptide. In particular, a "fragment" of the minimum domain of ZEBRA as defined above is preferably understood as a truncated sequence thereof, i.e. an amino acid sequence truncated N-terminal, C-terminal and/or within the sequence compared to the amino acid sequence of the native sequence. Furthermore, such "fragments" of the ZEBRA minimum domain preferably have a length of from 5 to 50 amino acids total, preferably from 10 to 45 amino acids total, more preferably from 15 to 45 amino acids total.

More preferably, in the context of MHC class I and/or MHC class II molecules, fragments of the above cell-penetrating peptides or variants thereof also retain the ability of the peptides to present cargo molecules such as antigens or epitopes on the surface of cells such as antigen presenting cells. In the context of MHC class I and/or MHC class II molecules, the ability of a cell-penetrating peptide or a complex comprising said cell-penetrating peptide to present cargo molecules such as antigens or epitopes on the cell surface can be detected by standard methods known to those skilled in the art, including stimulation of MHC-restricted CD4 specific for these epitopes ⁺ Or CD8 ⁺ The ability of T cells to proliferate and/or function.

Preferred cell-penetrating peptides are those which,

ii) having an amino acid sequence comprising a fragment of the minimum domain of ZEBRA from the amino acid sequence according to SEQ ID NO:3 to residue 220, wherein, optionally, 1, 2, 3, 4 or 5 amino acids have been replaced, deleted and/or added without losing the cell penetrating ability of the peptide,

Preferably an amino acid sequence comprising at least one conservatively substituted amino acid compared to the reference sequence, meaning that a given amino acid residue is replaced by a residue having similar physicochemical characteristics.

Particularly preferred are preferred cell penetrating peptides

included in the sequence set forth relative to SEQ ID NO:3 with Ser at the equivalent position 189 of the ZEBRA amino acid sequence.

Thus, it is preferred that such preferred cell penetrating peptides have an amino acid sequence comprising a sequence according to the following general formula (a):

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

wherein 0, 1, 2, 3, 4 or 5 amino acids are substituted, deleted and/or added without losing the cell penetrating ability of the peptide, wherein

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

X ₂ r, K or H, preferably X ₂ R or K;

X ₃ y, W or F, preferably X ₃ Y, w or F;

X ₄ k, R or H, preferably X ₄ Is K or R;

X ₅ is N or Q;

X ₆ r, K or H, preferably X ₆ R or K;

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

X ₈ a, V, L, I or G, preferably X ₈ Is A or G;

X ₉ is S or T;

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

X ₁₁ k, R or H, preferably X ₁₁ Is K or R;

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

X ₁₄ a, V, L, I or G, preferably X ₁₄ Is A or G;

X ₁₅ 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 F, Y or W; and

X ₁₇ k, R or H, preferably X ₁₇ Is K or R.

Preferably, such a peptide, polypeptide or protein is either (entirely) composed of L-amino acids or (entirely) composed of D-amino acids, thereby forming a "reverse-reverse peptide sequence". The term "reverse-inverted (peptide) sequence" 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, e.g., jameson et al, nature,368, 744-746 (1994); brady et al, nature,368, 692-693 (1994)).

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₁ Is K.

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₂ Is R.

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₃ Is Y.

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₄ Is K.

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₅ Is N.

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₆ Is R.

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₇ Is V.

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₈ Is A.

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₉ Is S.

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₁₀ Is R.

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₁₁ Is K.

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₁₃ Is R.

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₁₄ Is A.

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₁₅ Is K.

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₁₆ Is F.

In particular embodiments, the cell penetrating peptide is generally defined by formula (A) above, wherein X ₁₇ Is K.

In a specific embodiment, the cell penetrating peptide is as defined generally in formula (a) above, wherein the amino acid at the position corresponding to position 12 relative to formula (a) is Ser (S).

Also particularly preferred are preferred cell penetrating peptides which

Comprising or consisting of a sequence selected from the group consisting of: 4 to 13 or a sequence variant thereof which does not lose the cell penetrating ability of the peptide, preferably the sequence variant has 0, 1, 2, 3, 4 or 5 amino acids substituted, deleted and/or added without losing the cell penetrating ability of the peptide.

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Thus, cell penetrating peptides are particularly preferred, which have a sequence comprising or consisting of a sequence according to SEQ ID NO:6 (CPP 3/Z13), SEQ ID NO:7 (CPP 4/Z14), SEQ ID NO:8 (CPP 5/Z15), or SEQ ID NO:11 (CPP 8/Z18) or a sequence variant thereof without losing the cell penetrating ability of the peptide, preferably a sequence variant having 0, 1, 2, 3, 4 or 5 amino acids replaced, deleted and/or added without losing the cell penetrating ability of the peptide. Furthermore, cell penetrating peptides are more preferred, which have a sequence comprising or consisting of a sequence according to SEQ ID NO:6 (CPP 3/Z13) or SEQ ID NO:7 (CPP 4/Z14) or a sequence variant thereof without losing the cell penetrating ability of the peptide, preferably a sequence variant having 0, 1, 2, 3, 4 or 5 amino acids replaced, deleted and/or added without losing the cell penetrating ability of the peptide. Furthermore, cell penetrating peptides are most preferred, which have a sequence comprising or consisting of a sequence according to SEQ ID NO:6 (CPP 3/Z13) or a sequence variant thereof without losing the cell penetrating ability of the peptide, preferably a sequence variant having 0, 1, 2, 3, 4 or 5 amino acids replaced, deleted and/or added without losing the cell penetrating ability of the peptide.

In a preferred embodiment, the cell penetrating peptide according to the invention has a sequence comprising or consisting of SEQ ID NO:6 (CPP 3/Z13).

In another preferred embodiment, the cell penetrating peptide according to the invention has a sequence comprising or consisting of SEQ ID NO:7 (CPP 4/Z14).

In another preferred embodiment, the cell penetrating peptide according to the invention has a sequence comprising or consisting of SEQ ID NO:8 (CPP 5/Z15).

In another preferred embodiment, the cell penetrating peptide according to the invention has a sequence comprising or consisting of SEQ ID NO:11 (CPP 8/Z18) amino acid sequence.

It will be appreciated by those skilled in the art that the primary amino acid sequence of the cell-penetrating peptide may be further post-translationally modified, e.g., by glycosylation or phosphorylation, without departing from the invention.

In certain embodiments, the cell penetrating peptide optionally comprises, in addition to its amino acid sequence as described above, any one or any combination of the following:

(i) Nuclear Localization Signal (NLS). Such signals are well known to the skilled person and are described in Nair et al (2003,Nucleic Acids Res.31 (1): 397-399)

(ii) Targeting peptides, including tumor homing peptides, such as those described in Kapore et al (2012, PLoS ONE 7 (4): e 35187) and http: is// crdd. Osdd. Net/raghava/tumorhape/general. Php? Those listed in

Preferably, the cell penetrating peptide is linked to an antigen or epitope and promotes cellular internalization of the antigen or epitope.

The complex comprised in the combination according to the invention may comprise one single cell penetrating peptide or more than one cell penetrating peptide. Preferably, the complex comprised by the combination according to the invention comprises no more than five cell penetrating peptides, more preferably the complex comprised by the combination according to the invention comprises no more than four cell penetrating peptides, even more preferably the complex comprised by the combination according to the invention comprises no more than three cell penetrating peptides, particularly preferably the complex comprised by the combination according to the invention comprises no more than two cell penetrating peptides, most preferably the complex comprised by the combination according to the invention comprises a single cell penetrating peptide.

Component b) -antigen/epitope

The complex comprised by the combination according to the invention comprises as component b) at least one antigen or epitope.

In general, the at least one antigen or epitope may be of any nature, for example it may be selected from: (i) a peptide, polypeptide or protein, (ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein or lipopeptide, (v) a glycolipid, (vi) a nucleic acid, and (vii) a small molecule drug or toxin. Thus, the at least one antigen or 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, an antisense oligonucleotide, decoy DNA, a plasmid), or a small molecule drug (e.g., cyclosporin a, paclitaxel, doxorubicin, methotrexate, 5-aminolevulinic acid), or any combination thereof (particularly if the complex comprised in the combination according to the invention comprises more than one antigen or epitope). Preferably, the at least one antigen or epitope comprised by the complex is a (poly) peptide.

As used herein, an "antigen" is any structural substance that serves as a target for a receptor for an adaptive immune response, in particular as a target for antibodies, T cell receptors and/or B cell receptors. An "epitope," also known as an "antigenic determinant," is a portion (or fragment) of an antigen that is recognized by the immune system, particularly by antibodies, T cell receptors, and/or B cell receptors. Thus, an antigen has at least one epitope, i.e., a single antigen has one or more than one epitope. In the context of the present invention, the term "epitope" is mainly used to denote T cell epitopes, which are presented on the surface of antigen presenting cells, where they bind to the Major Histocompatibility Complex (MHC). T cell epitopes presented by MHC class I molecules are typically, but not limited to, peptides from 8 to 11 amino acids in length, while MHC class II molecules present longer peptides, typically, but not limited to, from 12 to 25 amino acids in length.

Preferably, the complex comprises at least one fragment of an antigen, said fragment comprising at least one epitope of said antigen. As used herein, a "fragment" of an antigen comprises at least 10 contiguous amino acids of the antigen, preferably at least 15 contiguous amino acids of the antigen, more preferably at least 20 contiguous amino acids of the antigen, even more preferably at least 25 contiguous amino acids of the antigen, most preferably at least 30 contiguous amino acids of the antigen. "sequence variants" of an antigen or an epitope (or fragment) of an antigen are as defined above, i.e. sequence variants have an (amino acid) sequence which is at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% identical to the reference sequence. In the context of an antigen/antigen fragment/epitope, a "functional" sequence variant refers to an epitope, e.g. comprised by an antigen (fragment), that is, it is immunogenic, preferably has the same immunogenicity as the epitope comprised in a full-length antigen, without loss or elimination of function. In some embodiments, the amino acid sequence of an epitope, e.g., the amino acid sequence of an epitope comprised by a cancer/tumor antigen (fragment) as described herein, is not mutated and thus is identical to a (naturally occurring) reference epitope sequence.

Preferably, the complex comprised by the combination according to the invention comprises more than one antigen or epitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 antigens or epitopes, more preferably the complex comprised by the combination according to the invention comprises (at least) two or three antigens or epitopes, even more preferably the complex comprised by the combination according to the invention comprises (at least) four or five antigens or epitopes. If the complex comprised by the combination according to the invention comprises more than one antigen or epitope, it will be appreciated that said antigen or epitope is in particular also covalently linked in the complex comprised by the combination according to the invention, e.g. to another antigen or epitope and/or to component a), i.e. the cell penetrating peptide, and/or to component c), i.e. the TLR peptide agonist.

The various antigens or epitopes comprised by the complex may be the same or different. Preferably, the various antigens or epitopes comprised by the complex are different from each other, thereby providing a multi-antigen and/or multi-epitope complex.

Furthermore, more than one antigen or epitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 antigens or epitopes, are preferred, consecutively located in the complex comprised by the combination of the invention. This means in particular that all antigens and/or antigenic epitopes comprised by the complex are located in a sequence which is not interrupted by component a), i.e. the cell penetrating peptide, nor by component c), i.e. the TLR peptide agonist. More precisely, component a) and component c) are located in the complex, for example before or after this segment of all antigens and/or antigenic epitopes. Thus, a "multi-antigen domain" may be formed. As used herein, the term "multi-antigen domain" refers to a domain, such as a (poly) peptide, comprising epitopes of at least two (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or more than 9) different antigens or at least two (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or more than 9) different antigens. Preferably, a "multi-antigen domain" comprises fragments of at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more than 9) different antigens, wherein each fragment comprises at least one epitope. More preferably, a "multi-antigen domain" comprises fragments of two to five different antigens, wherein each fragment comprises at least one epitope. Even more preferably, a "multi-antigen domain" comprises fragments of (exactly) three or four different antigens, wherein each fragment comprises at least one epitope of an antigen.

Antigens and/or epitopes that are consecutively positioned in this manner may optionally be linked to each other, e.g. by spacer elements or linkers (e.g. as described below), which are neither component a), i.e. cell penetrating peptide, nor component c), i.e. TLR peptide agonist.

Alternatively, however, the various antigens and/or epitopes may also be located in any other way in the complex comprised by the combination according to the invention, e.g. component a) and/or component c) are located between two or more antigens and/or epitopes, i.e. one or more antigens and/or epitopes are located between component a) and component c) (and vice versa), and optionally one or more antigens and/or epitopes are located at the other end of each of component a) and/or component c).

It will be appreciated that many different antigens or epitopes associated with the same disease, in particular the same tumour, may advantageously be contained in a single complex. Alternatively, a number of different antigens or epitopes associated with the same disease, in particular the same tumor, may be distributed to sub-populations of different antigens or epitopes, in particular sub-populations that complement each other in the context of a certain disease, such as a tumor, whereby such different complexes comprising different sub-populations may advantageously be administered simultaneously to a subject in need thereof, e.g. in a single vaccine.

Preferably, at least one antigen or epitope will be presented on the cell surface in an MHC class I and/or MHC class II context and/or CD1 context, whereby presentation on the cell surface in an MHC class I and/or MHC class II context is preferred. The phrase "epitope presentation in the context of MHC class I" refers in particular to CD8 located in the groove of a cell surface MHC class I molecule ⁺ An epitope. The phrase "epitope presentation in the context of MHC class II" refers in particular to CD4 located in the groove of a cell surface MHC class II molecule ⁺ An epitope. The phrase "epitope presentation in the context of CD 1" particularly refers to a lipid epitope located in the molecular groove of cluster 1 on the cell surface.

Advantageously, the complex comprised by the combination according to the invention comprises a cell penetrating peptide and at least one antigen or antigenic epitope and allows the transport and presentation of said epitope on the cell surface of antigen presenting cells in MHC class I and MHC class II context and thus can be used for vaccination and immunotherapy.

Preferably, the complex comprised by the combination according to the invention comprises at least one antigen or epitope, which is at least one CD4 ⁺ Epitope and/or at least one CD8 ⁺ An epitope.

As used herein, the term "CD4 ⁺ Epitope "or" CD4 ⁺ Restriction epitope "means a epitope derived from CD4 ⁺ T-cell recognized epitopes, which consist in particular of antigen fragments located in the groove of MHC class II molecules. The combinations according to the invention comprise individual CD 4's in the complexes comprised therein ⁺ The epitope preferably consists of about 12 to 25 amino acids. It may also consist of, for example, about 8 to 25 amino acids or about 6 to 100 amino acids.

As used herein, the term "CD8 ⁺ Epitope "or" CD8 ⁺ Restriction epitope "refers to the epitope defined by CD8 ⁺ T-cell recognized epitopes, which consist in particular of antigen fragments located in the groove of MHC class I molecules. The combinations according to the invention comprise individual CD 8's in the complexes comprised therein ⁺ The epitope preferably consists of about 8 to 11 amino acids. It may also consist of, for example, about 8 to 15 amino acids or about 6 to 100 amino acids.

Preferably, the at least one antigen may comprise or the at least one epitope may consist of CD4 corresponding to an epitope of a cancer/tumor-associated antigen, a cancer/tumor-specific antigen or an antigenic protein from a pathogen ⁺ Epitope and/or CD8 ⁺ An epitope. More preferably, the at least one antigen may comprise or the at least one epitope may consist of CD4 corresponding to an epitope of a cancer/tumor-associated antigen or a cancer/tumor-specific antigen ⁺ Epitope and/or CD8 ⁺ An epitope. Even more preferably, the at least one antigen may comprise or the at least one epitope may consist of CD4 corresponding to an epitope of a tumor-associated antigen or a tumor-specific antigen ⁺ Epitope and/or CD8 ⁺ An epitope.

It is also preferred that the complex comprised by the combination according to the invention comprises at least two antigens or epitopes, whereinAt least one antigen or epitope comprises or consists of CD4 ⁺ An epitope, and at least one antigen or epitope of an antigen comprises or consists of CD8 ⁺ An epitope. It has now been determined that T _h Cells (CD 4) ⁺ ) Play a central role in anti-tumor immune responses, including both DC-permissive and tumor site CTL (CD 8) ⁺ ) Is to be used for the recruitment and maintenance of (a). Thus, the complex comprised by the combination according to the invention comprises at least two antigens or epitopes, wherein at least one antigen or epitope comprises or consists of CD4 ⁺ An epitope, and at least one antigen or epitope of an antigen comprises or consists of CD8 ⁺ Epitope providing for allowing simultaneous priming of CTL and T _h The overall immune response of the cell is therefore superior to that for only one CD8 ⁺ Epitope or against only one CD4 ⁺ Immunization of epitopes.

Preferably, the complex comprised by the combination according to the invention comprises at least two antigens or epitopes, wherein said at least two antigens or epitopes comprise or consist of at least two, e.g. 2, 3, 4, 5, 6, 7, 8, 9 or more than 9 CD4 s ⁺ Epitopes and/or at least two, e.g. 2, 3, 4, 5, 6, 7, 8, 9 or more than 9 CD8 ⁺ An epitope. Thus, the at least two antigens or epitopes are preferably different antigens or epitopes, more preferably the at least two antigens or epitopes are different from each other but relate to the same tumor. A multi-antigen vaccine will (i) avoid the generation of antigen-loss variants, (ii) target different tumor cells in a heterogeneous tumor mass, and (iii) avoid inter-patient tumor variability. Thus, the complex comprised by the combination according to the invention particularly preferably comprises at least four antigens or epitopes, in particular at least two CD8 ⁺ Epitope and at least two CD4 ⁺ An epitope. Such complexes comprised by the combination according to the invention induce multi-epitope CD8 CTL and CD 4T _h The cells act synergistically to combat tumor cells and promote effective anti-tumor immunity. T (T) _h The cells are also involved in maintaining long-term cellular immunity monitored after vaccination. Such complexes comprised by the combination according to the invention induce multipleCloning, polyepitopic immune response and multifunctional CD8 ⁺ And CD4 ⁺ T cells and thus induce potent anti-tumor activity.

Preferably, the complex comprised by the combination according to the invention comprises at least two antigens or epitopes, more preferably the complex comprised by the combination according to the invention comprises at least three antigens or epitopes, even more preferably the complex comprised by the combination according to the invention comprises at least four antigens or epitopes, particularly preferably the complex comprised by the combination according to the invention comprises at least five antigens or epitopes, most preferably the complex comprised by the combination according to the invention comprises at least six antigens or epitopes. The antigens or epitopes comprised by the complexes may be the same or different, preferably the antigens or epitopes comprised by the complexes are different from each other. Preferably, the complex comprises at least one CD4 ⁺ Epitope and at least one CD8 ⁺ An epitope.

Preferably, the complex comprised by the combination according to the invention comprises more than one CD4 ⁺ Epitopes, e.g. two or more CD4 from the same antigen or different antigens ⁺ Epitope, and preferably, CD 8-free ⁺ An epitope. It is also preferred that the complex comprised by the combination according to the invention comprises more than one CD8 ⁺ Epitopes, e.g. two or more CD8 from the same antigen or different antigens ⁺ Epitope, and preferably, CD 4-free ⁺ An epitope. Most preferably, however, the complex comprised by the combination according to the invention comprises (i) at least one CD4 ⁺ Epitopes, e.g. two or more CD4 from the same antigen or different antigens ⁺ An epitope, and (ii) at least one CD8 ⁺ Epitopes, e.g. two or more CD8 from the same antigen or different antigens ⁺ An epitope.

Although the at least one antigen or epitope may comprise any kind of antigen or epitope, e.g. one or more epitopes from cancer/tumor-associated antigens, cancer/tumor-specific antigens and/or antigen proteins from pathogens including viral, bacterial, fungal, protozoan and multicellular parasitic antigen proteins, cancer or tumor epitopes are preferred.

It will be appreciated that the skilled person will generally select the antigen or epitope depending on the disease to be treated. Thus, an antigen or epitope is typically associated with the disease to be treated. In the context of specific diseases, a large number of antigens are known in the art. For example, to treat a tumor/cancer, the skilled artisan selects tumor/cancer antigens (or epitopes), particularly tumor/cancer antigens (or epitopes) that are useful for a particular type of tumor/cancer. In some embodiments, patient-specific antigens may be tested/screened (e.g., by using an isolated sample to identify whether a cancer/tumor expresses a specific antigen) to determine whether the specific antigen in question is useful for treatment (or to identify useful antigens or epitopes for treatment).

Preferably, at least one antigen or epitope comprises or consists of at least one cancer or tumor epitope. More preferably, the at least one antigen or epitope of an antigen preferably comprises or consists of at least one epitope of a cancer/tumor-associated antigen or a cancer/tumor-specific antigen.

As used herein, a "cancer/tumor antigen/epitope" is an antigen/epitope produced by a cancer/tumor cell. Such epitopes are typically specific for (or associated with) a certain cancer/tumor. For example, cancer/tumor epitopes include glioma epitopes. In particular, cancer/tumor-associated (also referred to as cancer/tumor-associated) antigens (TAAs) are antigens expressed by both cancer/tumor cells and normal cells. For example, a TAA may be one or more surface proteins or polypeptides, nucleoproteins or glycoproteins, or fragments thereof, expressed by a tumor cell. For example, human tumor-associated antigens include differentiation antigens (e.g., melanocyte differentiation antigens), mutant antigens (e.g., p 53), overexpressing cell antigens (e.g., HER 2), viral antigens (e.g., human papilloma virus proteins), and cancer/testis (CT) antigens expressed in germ cells of the testes and ovaries but silenced in normal somatic cells (e.g., MAGE and NY-ESO-1). Many TAAs are not cancer or tumor specific and may also be found in normal tissues. Thus, these antigens may be present after birth (even before birth). Thus, the immune system may develop self-tolerance to these antigens.

In contrast, cancer/Tumor Specific Antigens (TSAs) are antigens that are specifically expressed by cancer/tumor cells, rather than by normal cells. TSA can be specifically recognized by neoantigen-specific T Cell Receptors (TCRs) in the context of Major Histocompatibility Complex (MHC) molecules. Thus, TSA includes in particular neoantigen. In general, a neoantigen is an antigen that was not previously present and is therefore "new" to the immune system. Neoantigens are typically caused by somatic mutations. In the context of cancer/tumor, cancer/tumor specific neoantigens are typically not present prior to cancer/tumor progression, and cancer/tumor specific neoantigens are typically encoded by somatic gene mutations in cancer cells/tumor cells. From an immunological perspective, tumor neoantigens are truly foreign proteins that are completely absent from normal human organs/tissues. For most human tumors without virus etiology studies, tumor neoantigens may be derived, for example, from various non-synonymous genetic alterations including Single Nucleotide Variations (SNV), insertions and deletions (indels), gene fusions, frameshift mutations, and Structural Variations (SV). For example, computer predictive tools known in the art can be used to identify tumor neoantigens, as disclosed in Trends in Molecular Medicine, november 2019, pages 980-992. Since the new antigens are novel to the immune system, the self-tolerance of these antigens is much lower than for cancer/tumor related antigens. However, the collection of tumor-specific mutations for each cancer appears to be unique. Thus, cancer/tumor specific antigens, in particular neoantigens, can be identified in subjects diagnosed with cancer by methods known to the skilled person, such as cancer genomic sequencing. Potential neoantigens can be predicted by methods known to the skilled artisan, such as cancer genome sequencing or deep sequencing techniques to identify mutations within the protein-encoding portion of the (cancer) genome. After identification, the corresponding cancer/tumor specific neoantigen and/or cancer/tumor specific neoepitope can be used in the complexes comprised by the combination according to the invention.

In some embodiments, the complex comprised by the combination according to the invention comprises one or more than one cancer/tumor-associated epitope and/or one or more than one cancer/tumor-associated antigen (but preferably no cancer/tumor-specific epitope). In other embodiments, the complex comprised by the combination according to the invention comprises one or more than one cancer/tumor specific epitope and/or one or more than one cancer/tumor specific antigen (but preferably no cancer/tumor associated epitope). The complex comprised by the combination according to the invention may also comprise both, (i) one or more than one cancer/tumor-associated epitope and/or one or more than one cancer/tumor-associated antigen, and (ii) one or more than one cancer/tumor-specific epitope and/or one or more than one cancer/tumor-specific antigen.

Suitable cancer/tumor epitopes can be retrieved, for example, from a database of cancer/tumor epitopes, for example from Van der BruggenP, stroobant V, vigneron N, van den Eynde b.peptide database: t cell-defined tumor anti-cancer immune 2013; URL: http: the human tumor antigens recognized by CD4+ or CD8+ T cells are divided into four groups or "Tantigen" from the database (TANTIGEN version 1.0, month 12, 1, 2009; developed by the bioinformatics core laboratory of the cancer vaccine center of the Dana-Farber cancer institute; URL: http:// cvc.dfci.harvard.edu/tadb /).

Specific examples of cancer/tumor antigens useful in the complexes comprised in the combination according to the invention include, but are not limited to, the following antigens: the prostate: prostate Specific Antigen (PSA), prostate Specific Membrane Antigen (PSMA), PAP, PSCA (PNAS 95 (4) 1735-17401998), prostamucin antigen (PMA) (Beckett and Wright,1995,Int.J.Cancer 62:703-710), prostase, her-2neu, SPAS-1; melanoma: TRP-2, tyrosinase, melan A/Mart-1, gplOO, BAGE, GAGE, GM gangliosides; breast: her2-neu, kinesin 2, TATA element regulator 1, tumor protein D52, MAGE D, ING2, HIP-55, TGF-1 anti-apoptotic factor, HOM-Mel-40/SSX2, epithelial antigen (LEA 135), DF31MUC1 antigen (Apostonopoulos et al, 1996immunol. Cell. Biol.74:457-464; pandey et al, 1995,Cancer Res.55:4000-4003); testis: MAGE-1, HOM-Mel-40/SSX2, NY-ESO-1; colorectal: EGFR, CEA; lung: MAGE D, EGFR ovarian Her-2neu; bladder: transitional Cell Carcinoma (TCC) (Jones et al, 1997,Anticancer Res.17:685-687); several cancers: epha2, epha4, PCDGF, HAAH, mesothelin; EPCAM; NY-ESO-1, glycoprotein MUC1 and NIUC10 mucin p5 (particularly mutant), EGFR; tumor of the miscellaneous type: cancer-associated serum antigen (CASA) and cancer antigen 125 (CA 125) (Kierkegaard et al, 1995, gynecol. Oncol. 59:251-254), epithelial glycoprotein 40 (EGP 40) (Kievit et al, 1997,Int.J.Cancer 71:237-245), squamous cell carcinoma antigen (SCC) (Lozza et al, 1997 Anticancer Res.17:525-529), cathepsin E (Mota et al, 1997,Am.J Pathol.150:1223-1229), tyrosinase in melanoma (Fishman et al, 1997 Cancer 79:1461-1464), nuclear antigen (PCNA) of cerebral spongiform hemangiomas (Notelet et al, 1997 Surg.Neurol.47:364-370), 35kD tumor-associated autoantigens in thyroid papillary carcinoma (Lucas et al, 1996 Anticancer Res.16:2493-2496), CDC27 (including mutant forms of proteins), triose phosphate isomerase antigen, 707-AP, A60 mycobacterial antigen (Macs et al, 1996,J.Cancer Res.Clin.Oncol.122:296-300), annexin II, AFP, ART-4, BAGE, beta-catenin/m, BCL-2, bcr-abl p190, bcr-abl p210, BRCA-1, BRCA-2, CA 19-9 (Tolliver and O' Brien,1997,South Med.J.90:89-90; tsuruta at al.,1997 Urol.Int.58:20-24), CAMEL, CAP-1, CASP-8, CDC27/m, CDK-4/m, CEA (Huang et al, super Rev. Vaccines (2002) 1:49-63), CT9, CT10, cyp-B, dek-cain, DAM-6 (MAGE-B2), DAM-10 (MAGE-B1) EphA2 (Zantek et al, cell Growth Diffen (1999) 10:629-38; carles-Kinch et al Cancer Res. (2002) 62:2840-7), ephA4 (Cheng et al, 2002,Cytokine Growth Factor Rev.13:75-85), tumor-associated TF (Thomsen-Friedenreich) antigen (Dahlenborg et al, 1997,Iht.J Cancer 70:63-71), ELF2M, ETV-AML 1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, gnT-V, gpl00 (Zajac et al, 1997,Iht.J Cancer 71:491-496), HAGE, HER2/neu, HLA-A 0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT, hTRT, iCE, apoptosis inhibitors (e.g., survivin), KH-1 adenocarcinoma antigen (Deshpande and Danishfefsky, 1997,Nature 387:164-166), 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/Melan-A (Kawakami and Rosenberg,1997, int. Rev. Immunol. 14:173-192), MC1R, MDM-2, myoglobin/m, MUC1, MUC2, MUM-1, MUM-2, MUM-3, neo-polyA polymerase, NA88-A, and MART-1 NY-ESO-1, NY-ESO-1a (CAG-3), PAGE-4, PAP, protease 3 (Molldrem et al, blood (1996) 88:2450-7; molldrem et al, blood (1997) 90:2529-34), P15, P190, pm1/RARα, PRAME, PSA, PSM, PSMA, RAGE, RAS, RCAS1, RU2, SAGE, SART-1, SART-2, SART-3, SP17, SPAS-1, TEL/AML1, TPI/m, tyrosinase, TARP, TRP-1 (gp 75), TRP-2/INT2, WT-1, or NY-ESO-ORF2 and CAMEL proteins derived from translation of the NY-ESO-1 and LAGE-1 genes. Many other cancer antigens are well known in the art.

In some embodiments, the cancer/tumor antigen or cancer/tumor epitope may be a recombinant cancer/tumor antigen or a recombinant cancer/tumor epitope. Such recombinant cancer/tumor antigens or recombinant cancer/tumor epitopes can be designed by introducing mutations that alter (add, delete or replace) specific amino acids in the total amino acid sequence of the native cancer/tumor antigen or native cancer/tumor epitope. The introduction of mutations does not alter the cancer/tumor antigen or cancer/tumor epitope too much to be universally applicable to mammalian subjects, preferably human or dog subjects, but is sufficient to alter it such that the resulting amino acid sequence breaks tolerance or is considered to be an exogenous antigen in order to generate an immune response. Another way may be to generate a consensus recombinant cancer/tumor antigen or cancer/tumor epitope having at least 85% and up to 99% amino acid sequence identity to its corresponding native cancer/tumor antigen or native cancer/tumor epitope; preferably at least 90% and up to 98% sequence identity; more preferably at least 93% and up to 98% sequence identity; or even more preferably at least 95% and up to 98% sequence identity. In certain instances, the recombinant cancer/tumor antigen or recombinant cancer/tumor epitope has 95%, 96%, 97%, 98% or 99% amino acid sequence identity to its corresponding native cancer/tumor antigen or cancer/tumor epitope. A natural cancer/tumor antigen is an antigen that is typically associated with a particular cancer or cancer tumor. Depending on the cancer/tumor antigen, the consensus sequence of the cancer/tumor antigen may be across mammalian species or within a subtype of species or across viral strains or serotypes. Some cancer/tumor antigens do not differ much from the wild-type amino acid sequence of the cancer/tumor antigen. The above methods may be combined such that the final recombinant cancer/tumor antigen or cancer/tumor epitope has a percent similarity to the natural cancer antigen amino acid sequence described above. In other embodiments, the amino acid sequence of the cancer/tumor antigen epitope described herein is not mutated and thus is identical to the reference epitope sequence.

Preferably, the at least one cancer/tumor antigen or epitope (e.g., comprised in a multi-antigen domain) is selected from tumors or cancers, including endocrine tumors, gastrointestinal tumors, genitourinary and gynecological tumors, head and neck tumors, hematopoietic tumors, skin tumors, breast and respiratory tumors. Preferably, at least one tumor epitope, or at least one TAA, or at least one TSA of the multi-antigen domain of the invention is selected from tumors and/or cancers, including breast cancer, including triple negative breast cancer, biliary tract cancer; bladder cancer; brain cancers, including glioblastoma and medulloblastoma; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; stomach cancer; gastrointestinal stromal tumor (GIST), appendiceal cancer, cholangiocarcinoma, carcinoid, gastrointestinal colon cancer, extrahepatic cholangiocarcinoma, gallbladder carcinoma, gastric (stomach) carcinoma, gastrointestinal carcinoid, colorectal or metastatic colorectal cancer, hematological tumors including acute lymphoblastic and myeloid leukemia; t cell acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic myelogenous leukemia, multiple myeloma; AIDS-related leukemia and adult T-cell leukemia lymphoma; intraepithelial tumors, including bowden and paget's disease; liver cancer; lung cancer, including non-small cell lung cancer, lymphomas, including hodgkin's disease and lymphocytic lymphomas; neuroblastoma; glioblastoma, oral cancer, including squamous cell carcinoma; ovarian cancer, including ovarian cancer derived from epithelial cells, stromal cells, germ cells, and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas include leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancers, including melanoma, meckel cell carcinoma, kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma; testicular cancer, including germ tumors, such as seminomas, non-seminomas (teratomas, choriocarcinomas), interstitial tumors, and germ cell tumors; thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma; and renal cancers, including adenocarcinoma and wilms' cell neoplasm.

In particular, the at least one cancer/tumor antigen or epitope (e.g., comprised in a multi-antigen domain) is preferably selected from tumors or cancers, including colorectal cancer, metastatic colorectal cancer, pancreatic cancer, or breast cancer, including Triple Negative Breast Cancer (TNBC). The term "triple negative breast cancer" as used herein refers to breast cancers that lack Estrogen Receptor (ER), progesterone receptor (PgR) and HER2 expression, all of which are molecular targets for therapeutic agents. TNBC accounts for 10% to 20% of invasive breast cancer cases and comprises more than one molecular subtype. In general, patients with TNBC have a relatively poor prognosis compared to patients with other breast cancer subtypes, due to the inherent invasive clinical behavior and lack of accepted molecular therapeutic targets. Triple negative breast cancer is a phenotype whose major components in molecular assays are basal-like tumors, normal breast-like tumors, and recently recognized unusual but interesting low molecular subtypes of claudin, including also BRCA 1-deficient subtypes. TAAs expressed by TNBC include, for example, MAGE-A3, MUC-1, PRAME, ASCL2 and NY-ESO-1.

The term "pancreatic cancer" or "pancreatic cancer" as used herein relates to cancers derived from pancreatic cells. Preferably, pancreatic cancer as used herein refers to pancreatic adenocarcinoma, including pancreatic ductal adenocarcinoma and morphological variants thereof, such as adenosquamous cell carcinoma, mucinous/mucinous carcinoma, undifferentiated/poorly differentiated carcinoma, printed-circuit cell carcinoma, myeloid carcinoma, and hepatolike carcinoma. Pancreatic cancer is a fatal disease with poor prognosis and an increasing incidence. Pancreatic cancer is a typical disease of the elderly. The case where patients were diagnosed before age 30 is extremely rare, with 90% of newly diagnosed patients being over age 55, most being 70 and 80 years old, with male morbidity being higher than female. Pancreatic cancer is characterized by the expression of tumor-associated antigens, including mesothelin, survivin, and NY-ESO-1.

As used herein, colorectal cancer (CRC, also referred to as "bowel cancer") is a cancer that includes colon cancer and rectal cancer (CC). These two cancers share many common characteristics, but the origin of the cancers is different. According to Siegel, r., c.desantis and a.jemal, colorectal cancer statistics,2014.CA Cancer J Clin,2014.64 (2): p.104-17, in the united states, 2006 through 2010, the incidence of tumor sites was slightly more important in the proximal colon (first and middle part of the colon). About 19 cases were found in 100000 people, accounting for 42% of cases. Next, the incidence of rectal cancer was 28% and the incidence of distal colon (bottom of colon) was 10 out of 100000. Anatomically, the term "colorectal cancer" includes (i) colon cancers, such as blind bowel cancer (including ileocecal carcinoma), appendiceal carcinoma, ascending colon cancer, hepatic carcinoma, transverse colon carcinoma, splenic carcinoma, descending colon carcinoma, sigmoid colon carcinoma (including sigmoid colon (curved) carcinoma), and colon overlap carcinoma; (ii) Cancers of the colorectal-sigmoid junction, such as colorectal cancer and colorectal sigmoid cancer; and (iii) rectal cancer, such as rectal ampulla cancer.

Preferably, the colorectal cancer is colon cancer, such as blind bowel cancer (including ileocecal carcinoma), appendiceal carcinoma, ascending colon cancer, hepatic carcinoma, transverse colon cancer, splenic carcinoma, descending colon cancer, sigmoid colon cancer (including sigmoid colon (curved) carcinoma), or a combination thereof.

It is also preferred that the colorectal cancer is a cancer of the colorectal sigmoid junction, such as (i) colorectal cancer or (ii) colorectal sigmoid cancer. Furthermore, colorectal cancer is also preferred to be rectal cancer, such as rectal ampulla cancer.

Colorectal cancer includes different cell types, such as cell types, colorectal cancer including colorectal adenocarcinoma, colorectal stromal tumor, primary colorectal lymphoma, colorectal leiomyosarcoma, colorectal melanoma, colorectal squamous cell carcinoma, and colorectal carcinoid, such as cecum, appendix, ascending colon, transverse colon, descending colon, sigmoid colon, and/or colorectal carcinoid. Thus, preferred types of colorectal cancers include colorectal adenocarcinoma, colorectal stromal tumor, primary colorectal lymphoma, colorectal leiomyosarcoma, colorectal melanoma, colorectal squamous cell carcinoma, and colorectal carcinoid, such as carcinoid of cecum, appendix, ascending colon, transverse colon, descending colon, sigmoid colon, and/or rectum. More preferably, the colorectal cancer is colorectal adenocarcinoma or colorectal carcinoid. Even more preferably, the colorectal cancer is colorectal adenocarcinoma. Thus, the at least one tumor or cancer epitope of the complex may be selected from any colorectal cancer cell type disclosed above.

Since colorectal cancer expresses different TAAs or TSAs according to the tumor stage of the TMN stage system, the at least one tumor or cancer epitope (of the multiple antigen domains) of the complex preferably comprises, for example, TAAs or TSAs of the following primary tumor stage ("T" stage): TX-primary tumor was not assessed, T0-evidence of no primary tumor, ta-non-invasive papillary carcinoma, tis-carcinoma in situ: intraepithelial or lamina propria invasion, T1-tumor invasion into submucosa, T2-tumor invasion into muscularis propria, T3-tumor invasion into pericolorectal tissue through muscularis propria, T4 a-tumor penetration to visceral peritoneal surface, T4 b-tumor direct invasion or attachment to other organs or structures; the following lymph node stage ("N" stage): NX-regional lymph nodes cannot be assessed, N0-no regional lymph node metastasis, N1-1 to 3 regional lymph node metastases, where N1 a-1 regional lymph node metastases, N1 b-2 to 3 regional lymph node metastases, N1 c-no regional lymph node metastases with tumor deposition in subserosal, mesenteric or non-peritoneal pericolonic or perirectal tissue, N2-4 or more than 4 lymph node metastases, where N2 a-4 to 6 regional lymph node metastases, N2 b-7 or more than 7 regional lymph node metastases; and the following distant metastasis phases ("M" phase): m0-distantly related metastasis and M1-distantly related metastasis, wherein M1 a-distantly related metastasis is localized to one organ or site (e.g., liver, lung, ovary, non-regional nodule) and M1 b-distantly related metastasis to more than 1 organ/site or peritoneum. The staging may be integrated into the following digital staging of colorectal cancer: phase 0: tis, N0, M0; stage I: t1, N0, M0 or T2, N0, M0; stage IIA: t3, N0, M0; IIB phase: t4a, N0, M0; IIC phase: 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, M a and IVB phases: any T, any N, M b. Briefly, in stage 0, the cancer does not grow beyond the inner layers of the colon or rectum; in stage I, cancer has spread from the mucosa to the muscle layer; in stage II, the cancer has spread through the muscle layer to the serosa of nearby organs; in stage III, the cancer has spread to nearby lymph nodes or cancer cells have spread to tissue near lymph nodes; in stage IV, cancer has spread to other parts of the body through the blood and lymph nodes.

Various tumor-associated antigens have been reported for the types and stages of colorectal cancer cells described above, including, for example, CEA, MAGE, MUC1, survivin, WT1, RNF43, TOMM34, VEGFR-1, VEGFR-2, KOC1, ART4, KRas, epCAM, HER-2, COA-1SAP, TGF-beta RII, p53, ASCL2, and SART 1-3 (see, e.g., world J Gastroenterol 2018, 12, 28; 24 (48): 5418-5432). Thus, at least one cancer/tumor epitope/antigen of the complex is preferably an antigen (an epitope) selected from EpCAM, HER-2, MUC-1, TOMM34, RNF43, KOC1, VEGFR, βhcg, survivin, CEA, ASCL2, tgfβr2, p53, KRas, OGT, mesothelin, CASP5, COA-1, MAGE, SART, IL rα2, ASCL2, NY-ESO-1, MAGE-A3, PRAME, WT 1.

Melanoma associated antigen (MAGE)

Mammalian members of the MAGE (melanoma-associated antigen) gene family were originally described as fully silenced in normal adult tissues, except for male germ cells and some of their placenta. In contrast, these genes are expressed in various tumors. Thus, the complex preferably comprises an antigen of the MAGE family ("MAGE" antigen) or an epitope thereof. Of the MAGE families, MAGE-A3 and MAGE-D4 are particularly preferred, with MAGE-A3 being particularly preferred. The normal function of MAGE-A3 in healthy cells is unknown. MAGE-A3, which may also be referred to as cancer/testis antigen 1.3, for example, is a tumor specific protein and has been identified on many tumors. The amino acid sequence of MAGE-A3 is as follows:

Thus, the complex preferably comprises a sequence according to SEQ ID NO:14 or a fragment or variant thereof.

Interval skin protein

The proteins that the mesothelin was originally identified as reacting with antibodies called "mAb K1" in ovarian cancer are tumor antigens that are highly expressed in many human cancers, including malignant mesothelioma and pancreatic, ovarian adenocarcinoma, and lung adenocarcinoma. The amino acid sequence of mesothelin according to UniProtKB Q13421 is shown below:

thus, the complex preferably comprises a sequence according to SEQ ID NO:15 or a fragment or variant thereof.

Survivin protein

Survivin, also known as baculovirus-containing apoptosis inhibitor repeat 5 or BIRC5 (UniProtKB O15392), is a member of the family of apoptosis Inhibitors (IAPs). Survivin functions to inhibit caspase activation, resulting in down-regulation of apoptosis or programmed cell death. The amino acid sequence of survivin is shown below:

thus, the complex preferably comprises a sequence according to SEQ ID NO:16 or a fragment or variant thereof.

Several epitopes of survivin are known to the skilled person. Preferred survivin epitopes preferably comprised in the complex include the following epitopes (the epitope sequences shown below are fragments of the above survivin sequences; the following epitope sequences may refer to one epitope or more than one (overlapping) epitope):

Thus, the complex preferably comprises a sequence according to SEQ ID NO: 17.

Thus, it is preferred that the complex comprises an epitope of survivin. More preferably, the complex comprises a polypeptide having a sequence according to SEQ ID NO:16, or a fragment thereof of at least 10 amino acids in length (preferably at least 15 amino acids, more preferably at least 20 amino acids, even more preferably at least 25 amino acids, most preferably at least 30 amino acids), or a peptide thereof having a functional sequence variant with at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity. Even more preferably, the complex comprises a polypeptide having a sequence according to SEQ ID NO:17, and a peptide of the amino acid sequence of seq id no.

The complex may further comprise a fragment of survivin comprising at least one epitope, e.g. SEQ ID NO:18:

particularly preferably, the complex comprises a polypeptide having a sequence according to SEQ ID NO:18 or a peptide thereof having a functional sequence variant with at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity.

NY-ESO-1

NY-ESO-1 (also known as "cancer/testicular antigen 1", or "esophageal squamous cell carcinoma of new york 1", uniProtKB P78358) is a well-known cancer-testicular antigen (CTA) that is re-expressed in many cancer types. NY-ESO-1 elicits spontaneous humoral and cellular immune responses and is characterized by a restricted expression pattern, making it a good candidate target for cancer immunotherapy. NY-ESO-1 specific immune responses have been observed in various cancer types. The amino acid sequence of NY-ESO-1 is shown below:

preferably, at least one tumor epitope of the complex is an epitope of an antigen selected from the group consisting of mesothelin, survivin and NY-ESO-1. For example, the at least one tumor epitope of the complex is an epitope selected from the group consisting of mesothelin, survivin, or mesothelin and NY-ESO-1 or survivin and NY-ESO-1. In some embodiments, at least one tumor antigen/epitope of the complex comprises an epitope of an antigen mesothelin or NY-ESO-1 or survivin or fragment thereof or sequence variant thereof.

For example, complexes comprising multiple antigen domains comprising at least one, e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than ten epitopes selected from at least one, two, or all of the foregoing antigens, e.g., mesothelin, survivin, and NY-ESO-1, may be particularly useful in the case of pancreatic cancer.

PRAME

PRAME (melanoma antigen preferentially expressed in tumors, uniProtKB P78395) is also known as cancer testis antigen 130 (CT 130), MAPE (melanoma antigen preferentially expressed in tumors) and OIP4 (OPA interacting protein 4) are members of the Cancer Testis Antigen (CTA) family. PRAME expression in normal somatic tissues is epigenetically limited to adult germ cells and is underexpressed in testes, epididymis, endometrium, ovary and adrenal glands. Like the CTA member NY-ESO-1, PRAME was identified as an immunogenic tumor-associated antigen in melanoma and since its discovery, its expression has been demonstrated in a variety of solid and hematological malignancies, including triple negative breast cancers. The amino acid sequence of PRAME is shown below:

thus, the complex preferably comprises a sequence according to SEQ ID NO:20 or a fragment or variant thereof.

ASCL2 (bristle-less squama homolog 2)

ASCL2 is a basic helix-loop-helix transcription factor, critical for the proliferation of trophoblast cells during placental development. ASCL2 was found to be a putative proliferation modulator, over-expressed in intestinal neoplasia. The amino acid sequence of ASCL2 is as follows:

Thus, the complex preferably comprises a sequence according to SEQ ID NO:21 or a fragment or variant thereof.

Several epitopes of ASCL2 are known to the skilled person. Preferred ASCL2 epitopes preferably comprised in the complex include the following (the epitope sequences shown below are fragments of the above ASCL2 sequences and are thus shown underlined in the above ASCL2 sequences; each of the following epitope sequences may refer to one epitope or more than one (overlapping) epitope):

thus, the complex preferably comprises a sequence according to SEQ ID NO:22 and/or the amino acid sequence according to SEQ ID NO:23, and a sequence of amino acids thereof.

Thus, it is preferred that the complex comprises an epitope of ASCL 2. More preferably, the complex comprises a polypeptide having a sequence according to SEQ ID NO:21, or a fragment thereof of at least 10 amino acids in length (preferably at least 15 amino acids, more preferably at least 20 amino acids, even more preferably at least 25 amino acids, most preferably at least 30 amino acids), or a peptide thereof having an amino acid sequence of a functional sequence variant having at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity. Even more preferably, the complex comprises a polypeptide having a sequence according to SEQ ID NO:22 and/or a peptide according to the amino acid sequence of SEQ ID NO:23, and a peptide of the amino acid sequence of 23.

The complex may further comprise a fragment of ASCL2 comprising at least one epitope, as set forth in SEQ ID NO:24:

particularly preferably, the complex comprises a polypeptide having a sequence according to SEQ ID NO:24 or a peptide thereof having a functional sequence variant with at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity.

Mucin-1 (MUC-1)

MUC-1 (UniProtKB P15941) is a human epithelial mucin that acts on cell adhesion. The amino acid sequence of MUC-1 is shown below:

thus, the complex preferably comprises a sequence according to SEQ ID NO:25 or a fragment or variant thereof.

Several epitopes of MUC-1 are known to the skilled artisan. Preferred MUC-1 epitopes preferably included in the complex include the following (the epitope sequences shown below are fragments of the above MUC-1 sequences and are therefore shown underlined in the above MUC-1 sequences; each of the following epitope sequences may refer to one epitope or more than one (overlapping) epitope):

thus, the complex preferably comprises a sequence according to SEQ ID NO:26 and/or the amino acid sequence according to SEQ ID NO: 27.

Transforming growth factor beta receptor 2 (TGF beta R2)

Tgfβ receptors are single transmembrane serine/threonine kinase receptors. They exist as several different isomers. TGF-beta R2 (UniProtKB P37137) is a transmembrane protein with a protein kinase domain that forms a heterodimeric complex with another receptor protein and binds TGF-beta. This receptor/ligand complex phosphorylates proteins and then enters the nucleus and regulates transcription of a set of genes associated with cell proliferation.

Thus, the complex preferably comprises a sequence according to SEQ ID NO:28 or a fragment or variant thereof.

Carcinoembryonic antigen (CEA)

CEA is an intracellular adhesion glycoprotein. CEA is usually produced in gastrointestinal tissue during fetal development, but ceases to be produced 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 shown below:

thus, the complex preferably comprises a sequence according to SEQ ID NO:29 or a fragment or variant thereof.

Several epitopes of CEA are known to the skilled artisan. Preferred CEA epitopes preferably included in the complex include the following (the epitope sequences shown below are fragments of the above CEA sequences and are thus underlined in the above CEA sequences; each of the following epitope sequences may refer to one epitope or more than one (overlapping) epitope):

Thus, the complex preferably comprises a sequence according to SEQ ID NO:30 and/or the amino acid sequence according to SEQ ID NO: 31.

Thus, it is preferred that the complex comprises an epitope of CEA. More preferably, the complex comprises a polypeptide having a sequence according to SEQ ID NO:29, or a fragment thereof of at least 10 amino acids in length (preferably at least 15 amino acids, more preferably at least 20 amino acids, even more preferably at least 25 amino acids, most preferably at least 30 amino acids), or a peptide thereof having an amino acid sequence of a functional sequence variant having at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity. Even more preferably, the complex comprises a polypeptide having a sequence according to SEQ ID NO:30 and/or a peptide according to the amino acid sequence of SEQ ID NO:31, and a peptide of the amino acid sequence of seq id no.

The complex may further comprise a fragment of CEA comprising at least one epitope as set forth in SEQ ID NO:32:

particularly preferably, the complex comprises a polypeptide having a sequence according to SEQ ID NO:32 or a peptide thereof having a functional sequence variant with at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity.

P53

P53 (UniProtKB P04637) is a tumor suppressor protein and has the effect of preventing genomic mutation. P53 has a variety of anti-cancer functional mechanisms and plays a role in apoptosis, genomic stability and inhibition of angiogenesis. In its anticancer effect, p53 acts through several mechanisms: when DNA is continuously damaged, it can activate DNA repair proteins; it can prevent growth by maintaining the cell cycle at the G1/S regulatory point recognized by DNA damage; and it can trigger apoptosis.

Thus, the complex preferably comprises a sequence according to SEQ ID NO:33 or a fragment or variant thereof.

Ke Ersi ton Ras (KRAS)

GTPase KRAS, also known as V-Ki-ras2 Ke Ersi, is a rat sarcoma viral oncogene homolog and KRAS, performs a fundamental function in normal tissue signaling, and mutation of the KRAS gene is a fundamental step in many cancer developments. Like other members of the ras subfamily, KRAS proteins are gtpases and early participants in many signal transduction pathways. KRAS is typically attached to the cell membrane because of the presence of an isoprene group at its C-terminus. The amino acid sequence of KRas is shown below:

thus, the complex preferably comprises a sequence according to SEQ ID NO:34 or a fragment or variant thereof.

Several epitopes of KRas are known to the skilled person. Preferred Ke Ersi ton Pas epitopes preferably included in the complex include the following (the epitope sequences shown below are fragments of the Ke Ersi ton Pas sequence described above, and thus underlined in the Ke Ersi ton Pas sequence described above; the following epitope sequences may refer to one epitope or more than one (overlapping) epitope):

thus, it is preferred that the complex comprises a sequence according to SEQ ID NO:35, and a sequence of amino acids.

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

OGT (O-linked N-acetylglucosamine (GlcNAc) transferase, O-GlcNAc transferase, OGT enzyme, O-linked N-acetylglucosamine transferase, uridine diphosphate-N-acetylglucosamine: polypeptide beta-N-acetylglucosamine transferase, protein O-linked beta-N-acetylglucosamine transferase, uniProtKB O15294) is a compound of the formula UDP-N-acetyl-D-glucosamine: protein-O- β -N-acetyl-D-glucosamine transferase) is a enzyme with the system name "UDP-N-acetyl-D-glucosamine: an enzyme of the protein-O-beta-N-acetyl-D-glucosamine transferase ". OGT catalyzes the addition of a single N-acetylglucosamine to serine or threonine residues of intracellular proteins at the O-glycosidic bond. OGT is part of many biological functions in the human body. OGT increases the rate of IRS1 phosphorylation (at serine 307 and serine 632/635), decreases insulin signaling, and the glycosylated component of insulin signaling by inhibiting threonine 308 phosphorylation of AKT1, involved in insulin resistance in muscle cells and adipocytes. Furthermore, OGT catalyzes the intracellular glycosylation of serine and threonine residues following the addition of N-acetylglucosamine. Studies have shown that OGT alleles are critical for embryogenesis and that OGT is essential for intracellular glycosylation and embryonic stem cell viability. OGT also catalyzes post-translational modification of transcription factors and RNA polymerase II, however the specific function of such modifications is largely unknown. The sequence of OGT is shown below:

Thus, the complex preferably comprises a sequence according to SEQ ID NO:36 or a fragment or variant thereof.

Caspase 5 (CASP 5)

Caspase 5 (UniProtKB P51878) is an enzyme that proteolytically cleaves other proteins at aspartic acid residues, belonging to the family of cysteine proteases known as caspases. It is an inflammatory caspase that acts in the immune system together with caspase 1, caspase 4 and murine caspase 4 homologous caspase 11. The amino acid sequence of CASP5 is shown below:

thus, the complex preferably comprises a sequence according to SEQ ID NO:37 or a fragment or variant thereof.

Colorectal tumor associated antigen-1 (COA-1)

COA-1 was strongly expressed by colorectal and melanoma cells (no data is available) as identified in 2003 by Maccallli et al (Maccallli, C., et al, identification of a colorectal tumor-associated antigen (COA-1) recognetized by CD4 (+) T-lyhocytes. Cancer Res,2003.63 (20): p.6735-43). Its mutations may interfere with differential recognition of tumor and normal cells. The amino acid sequence of COA-1 (UniProtKB Q5T 124) is shown below:

Thus, the complex preferably comprises a sequence according to SEQ ID NO:38 or a fragment or variant thereof.

Squamous cell carcinoma antigen (SART) recognized by T cells

Among the SART family, SART-3 is most preferred. Thus, the complex preferably comprises an antigen of the SART family ("SART" antigen) or an epitope thereof; more preferably, the complex comprises SART-3 or an epitope thereof. The squamous cell carcinoma antigen recognized by T cell 3 has tumor epitopes capable of inducing HLA-A 24-restricted and tumor-specific cytotoxic T lymphocytes in cancer patients. SART-3 is thought to be involved in the regulation of mRNA splicing.

IL13Rα2

IL13Rα2 binds interleukin 13 (IL-13) with very high affinity (thus it can be sequestered), but is not allowed to bind IL-4. It acts as a negative regulator of IL-13 and IL-4, however its mechanism is still uncertain. The amino acid sequence of IL13 ra 2 is as follows:

thus, the complex preferably comprises a sequence according to SEQ ID NO:39 or a fragment or variant thereof.

Several epitopes of IL13 ra 2 are known to the skilled person. Preferred IL13Rα2 epitopes that are preferably comprised in the complex include the following (the epitope sequences shown below are fragments of the above-described IL13Rα2 sequence and are thus underlined in the IL13Rα2 sequence; the following epitope sequences may refer to one epitope or more than one (overlapping) epitope):

Thus, the complex preferably comprises a sequence according to SEQ ID NO: 40.

KOC1

KOC1 (UniProtKB O00425), also known as insulin-like growth factor 2mRNA binding protein 3 (IGF 2BP 3), IMP3, KOC1, VICKZ3, is an mRNA binding protein. However, there is no expression data available, the sequence of which is shown below:

thus, the complex preferably comprises a sequence according to SEQ ID NO:41 or a fragment or variant thereof.

TOMM34

TOMM34 (UniProtKB Q15785) is involved in the delivery of precursor proteins into mitochondria. The skilled person knows a number of epitopes thereof, which may be selected from the amino acid sequences shown below:

thus, the complex preferably comprises a sequence according to SEQ ID NO:42 or a fragment or variant thereof.

RNF43

RNF43 (UniProtKB Q68DV 7) is a RING-type E3 ubiquitin ligase, expected to contain transmembrane domains, protease-related domains, extracellular domains and cytoplasmic RING domains. RNF43 is thought to down regulate Wnt signaling, with expression of RNF43 resulting in increased ubiquitination of frizzled receptors, altering their subcellular distribution, resulting in decreased surface levels of these receptors. The skilled artisan knows many of its epitopes, the amino acid sequence of RNF43 is shown below:

Thus, the complex preferably comprises a sequence according to SEQ ID NO:43 or a fragment or variant thereof.

Vascular Endothelial Growth Factor (VEGF)/Vascular Endothelial Growth Factor Receptor (VEGFR)

Vascular endothelial growth factor (VEGF, uniProtKB P15692), originally referred to as Vascular Permeability Factor (VPF), is a cellular signaling protein that stimulates angiogenesis and vasculogenesis. When blood circulation is inadequate, it is part of the restoration tissue oxygenation system. The normal function of VEGF is to produce new blood vessels during embryonic development, to produce new blood vessels after injury, to produce muscle after exercise, and to produce new blood vessels (side branch circulation) to bypass occluded blood vessels. There are three major subtypes of VEGF receptor (VEGFR), namely VEGFR1 (UniProtKB P17948), VEGFR2 (UniProtKB P35968) and VEGFR3 (UniProtKB P35916). The sequences of VEGF, VEGFR1, VEGFR2 and VEGFR3 are incorporated herein by reference. Thus, the complex preferably comprises the amino acid sequences of VEGF, VEGFR1, VEGFR2 and VEGFR3 described herein, or fragments or variants thereof.

Human chorionic gonadotrophin beta subunit (beta hCG)

Human chorionic gonadotropin (hCG) is a hormone produced by the embryo after implantation. Some cancerous tumors produce this hormone; thus, an elevated level measured when the patient is not pregnant may be diagnosed as cancer. hCG is a heterodimer with the same alpha subunit as Luteinizing Hormone (LH), follicle Stimulating Hormone (FSH), thyroid Stimulating Hormone (TSH) and a β subunit unique to hCG. The hCG gonadotrophin β subunit (β -hCG) contains 145 amino acids and is encoded by six highly homologous genes. Thus, the complex preferably comprises the amino acid sequence of β -hCG described herein or a fragment or variant thereof.

EpCAM

EpCAM (UniProtKB P16422) is a glycoprotein that mediates cell adhesion. The amino acid sequence of EpCAM is as follows:

thus, the complex preferably comprises a sequence according to SEQ ID NO:44 or a fragment or variant thereof.

Several epitopes of EpCAM are known to the skilled person. Preferred EpCAM epitopes preferably included in the complex include the following (the epitope sequences shown below are fragments of the EpCAM sequences described above, and are thus underlined in EpCAM sequences; the following epitope sequences may refer to one epitope or more than one (overlapping) epitope):

thus, the complex preferably comprises a sequence according to SEQ ID NO:45 or a fragment or variant thereof.

HER-2/neu

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

thus, the complex preferably comprises a sequence according to SEQ ID NO:46 or a fragment or variant thereof.

As described above, suitable cancer/tumor epitopes of Her-2 are known from the literature or can be identified by using a database of cancer/tumor epitopes, for example according to Van der Bruggen P, stroobant V, vigneron N, van den Eynde B. Peptide database: t cell-defined tumor anti-cancer immune 2013; URL: www.cancerimmunity.org/peptide/, wherein human tumor antigens recognized by CD4+ or CD8+ T cells are grouped into four major groups according to their expression pattern, or "Tantigen" according to the database (TANTIGEN version 1.0, month 1, 2009; developed by Bioinformatics Core at Cancer Vaccine Center, dana-Farber Cancer Institute; URL: cvc.dfci.harvard.edu/tadb /).

WT1

WT1 (nephroblastoma protein, uniProtKB P19544) transcription factor plays an important role in cell development and cell survival. The gene encoding WT1 is characterized by a complex structure, located on chromosome 11. It is involved in cell growth and differentiation and has a strong influence on successive stages of body function. The WT1 gene may, for example, undergo a number of different mutations, or may be overexpressed without mutations. The molecular basis of diseases such as wilms's cell neoplasm is congenital WT1 mutation, whereas somatic mutation of this gene occurs in acute and chronic myeloid leukemia, myelodysplastic syndrome, and some other hematological neoplasms, such as acute lymphoblastic leukemia. Increased expression of this gene was observed in leukemia and solid tumors, but without mutations. The amino acid sequence of WT1 is shown below:

thus, the complex preferably comprises a sequence according to SEQ ID NO:47 or a fragment or variant thereof.

Preferably, the complex comprises 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 IL13rα2. More preferably, the complex comprises 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, TGF βR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART and IL13Rα2. These antigens are particularly useful in colorectal cancer. It is also preferred that the complex comprises at least one tumor antigen, or a fragment thereof, or a sequence variant of a tumor antigen, or a sequence variant of a fragment thereof, selected from EpCAM, HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhcg, survivin, CEA, tgfβr2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART and IL13rα2. It is also preferred that the complex comprises at least one tumor antigen selected from ASCL2, epCAM, HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, CEA, TGF βR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART and IL13Rα2, or a fragment thereof, or a sequence variant of a tumor antigen, or a sequence variant of a fragment thereof.

Preferably, the complex comprises at least one tumor epitope which is an epitope of an antigen selected from EpCAM, MUC-1, survivin, CEA, KRas, MAGE-A3, IL13 ra 2 and ASCL2, for example an epitope according to any of SEQ ID NOs 45, 26, 27, 17, 30, 31, 35, 40, 22 and 23; more preferably, the at least one tumour epitope is an epitope selected from EpCAM, MUC-1, survivin, CEA, KRas, MAGE-A3 and ASCL2, for example an epitope according to any one of SEQ ID NOs 45, 26, 27, 17, 30, 31, 35, 22 and 23; even more preferably, the at least one tumor epitope is an epitope of an antigen selected from EpCAM, MUC-1, survivin, CEA, and ASCL2, for example an epitope according to any one of SEQ ID NOs 45, 26, 27, 17, 30, 31, 22, and 23; most preferably, the at least one tumor epitope is an epitope of an antigen selected from EpCAM, survivin, CEA and ASCL2, for example an epitope according to any one of SEQ ID NOs 45, 17, 30, 31, 22 and 23.

In some embodiments, the at least one tumor epitope of the complex is an epitope selected from the group consisting of MAGE-A3, MUC-1, PRAME, ASCL2, and NY-ESO-1, preferably the at least one tumor epitope of the complex is an epitope selected from the group consisting of MAGE-A3, MUC-1, PRAME, ASCL2, preferably the at least one tumor epitope of the complex is an epitope selected from the group consisting of MAGE-A3, MUC-1, PRAME, preferably the at least one tumor epitope of the complex is an epitope selected from the group consisting of MAGE-A3, MUC-1, ASCL2, preferably the at least one tumor epitope of the complex is an epitope selected from the group consisting of MAGE-A3, ASCL2, PRAME, preferably the at least one tumor epitope of the complex is an epitope selected from the group consisting of MAGE-A3, MUC-1, NY-ESO-1. In one embodiment, more preferably the multi-antigen domain of the complex comprises at least one epitope of the antigen MAGE-A3, or ASCL2, or MUC1, or PRAME, or NY-ESO-1.

Also preferably, the complex comprises

i) EpCAM or a functional sequence variant thereof (e.g., according to SEQ ID NO: 45).

ii) one or more epitopes of MUC-1 or a functional sequence variant thereof (e.g. according to SEQ ID NO:26 and/or an epitope according to SEQ ID NO: 27) an epitope of 27).

iii) One or more epitopes of survivin or a functional sequence variant thereof (e.g. according to SEQ ID NO: 17) an epitope of 17);

iv) one or more epitopes of CEA or a functional sequence variant thereof (e.g., according to SEQ ID NO:30 and/or an epitope according to SEQ ID NO: 31).

v) one or more than one epitope of KRas or a functional sequence variant thereof (e.g. according to SEQ ID NO: 31). And/or

vi) one or more than one epitope of MAGE-A3 or a functional sequence variant thereof.

Also preferably, the complex comprises

v) one or more than one epitope of KRas or a functional sequence variant thereof (e.g. according to SEQ ID NO: 35) an epitope of 35).

vi) one or more than one epitope of MAGE-A3 or a functional sequence variant thereof; and/or

vii) one or more than one epitope of ASCL2 or a functional sequence variant thereof (e.g. according to SEQ ID NO:22 and/or an epitope according to SEQ ID NO: 23).

Also preferably, the complex comprises

-EpCAM fragments or functional sequence variants thereof comprising one or more than one epitope;

-a MUC-1 fragment comprising one or more than one epitope or a functional sequence variant thereof;

-survivin fragments comprising one or more than one epitope or functional sequence variants thereof;

-a CEA fragment comprising one or more than one epitope or a functional sequence variant thereof;

-a KRas fragment comprising one or more than one epitope or a functional sequence variant thereof; and/or

-a MAGE-A3 fragment comprising one or more than one epitope or a functional sequence variant thereof.

Also preferably, the complex comprises

-a KRas fragment comprising one or more than one epitope or a functional sequence variant thereof;

-a MAGE-A3 fragment comprising one or more than one epitope or a functional sequence variant thereof; and/or

An ASCL2 fragment comprising one or more than one epitope or a functional sequence variant thereof.

As used herein, a "fragment" of an antigen comprises at least 10 contiguous amino acids of the antigen, preferably at least 15 contiguous amino acids of the antigen, more preferably at least 20 contiguous amino acids of the antigen, even more preferably at least 25 contiguous amino acids of the antigen, most preferably at least 30 contiguous amino acids of the antigen. Thus, a fragment of EpCAM comprises at least 10 contiguous amino acids of EpCAM (SEQ ID NO: 44), preferably at least 15 contiguous amino acids of EpCAM (SEQ ID NO: 44), more preferably at least 20 contiguous amino acids of EpCAM (SEQ ID NO: 44), even more preferably at least 25 contiguous amino acids of EpCAM (SEQ ID NO: 44), most preferably at least 30 contiguous amino acids of EpCAM (SEQ ID NO: 44); the fragment of MUC-1 comprises at least 10 contiguous amino acids of MUC-1 (SEQ ID NO: 25), preferably at least 15 contiguous amino acids of MUC-1 (SEQ ID NO: 25), more preferably at least 20 contiguous amino acids of MUC-1 (SEQ ID NO: 25), even more preferably at least 25 contiguous amino acids of MUC-1 (SEQ ID NO: 25), most preferably at least 30 contiguous amino acids of MUC-1 (SEQ ID NO: 25); fragments of survivin comprise at least 10 contiguous amino acids of survivin (SEQ ID NO: 16), preferably at least 15 contiguous amino acids of survivin (SEQ ID NO: 16), more preferably at least 20 contiguous amino acids of survivin (SEQ ID NO: 16), even more preferably at least 25 contiguous amino acids of survivin (SEQ ID NO: 16), most preferably at least 30 contiguous amino acids of survivin (SEQ ID NO: 16); the CEA fragment comprises at least 10 contiguous amino acids of CEA (SEQ ID NO: 29), preferably at least 15 contiguous amino acids of CEA (SEQ ID NO: 29), more preferably at least 20 contiguous amino acids of CEA (SEQ ID NO: 29), even more preferably at least 25 contiguous amino acids of CEA (SEQ ID NO: 29), most preferably at least 30 contiguous amino acids of CEA (SEQ ID NO: 29); fragments of KRAS comprise at least 10 contiguous amino acids of KRAS (SEQ ID NO: 34), preferably at least 15 contiguous amino acids of KRAS (SEQ ID NO: 34), more preferably at least 20 contiguous amino acids of KRAS (SEQ ID NO: 34), even more preferably at least 25 contiguous amino acids of KRAS (SEQ ID NO: 34), most preferably at least 30 contiguous amino acids of KRAS (SEQ ID NO: 34); and fragments of MAGE-A3 comprise at least 10 contiguous amino acids of MAGE-A3 (SEQ ID NO: 14), preferably at least 15 contiguous amino acids of MAGE-A3 (SEQ ID NO: 14), more preferably at least 20 contiguous amino acids of MAGE-A3 (SEQ ID NO: 14), even more preferably at least 25 contiguous amino acids of MAGE-A3 (SEQ ID NO: 14), most preferably at least 30 contiguous amino acids of MAGE-A3 (SEQ ID NO: 14). Furthermore, the fragment of ASCL2 comprises at least 10 consecutive amino acids of ASCL2 (SEQ ID NO: 21), preferably at least 15 consecutive amino acids of ASCL2 (SEQ ID NO: 21), more preferably at least 20 consecutive amino acids of ASCL2 (SEQ ID NO: 21), even more preferably at least 25 consecutive amino acids of ASCL2 (SEQ ID NO: 21), most preferably at least 30 consecutive amino acids of ASCL2 (SEQ ID NO: 21).

The functional sequence variant of such a fragment has at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% of the same (amino acid) sequence as the reference sequence and the epitope function of at least one, preferably all, of the epitopes comprised by the fragment is maintained.

In preferred embodiments, the complex does not comprise any epitope of HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGF βR2, p53, KRas, OGT, CASP5, COA-1, SART, or IL13Rα2. In a more preferred embodiment, the complex does not comprise any epitope of ASCL2, HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGF βR2, p53, KRas, OGT, CASP5, COA-1, SART or IL13Rα2.

In a preferred embodiment, the complex comprises

One or more than one epitope of EpCAM or a functional sequence variant thereof (e.g. an epitope according to SEQ ID No. 45);

one or more than one epitope of MUC-1 or a functional sequence variant thereof (e.g. an epitope according to SEQ ID NO:26 and/or an epitope according to SEQ ID NO: 27);

One or more than one epitope of CEA or a functional sequence variant thereof (e.g.an epitope according to SEQ ID NO:30 and/or an epitope according to SEQ ID NO: 31); and

-one or more than one epitope of MAGE-A3 or a functional sequence variant thereof.

In this preferred embodiment, the complex preferably does not comprise any epitope of HER-2, TOMM34, RNF43, KOC1, VEGFR, βhCG, survivin, TGF βR2, p53, KRas, OGT, CASP5, COA-1, SART or IL13Rα2. More preferably, in this preferred embodiment, the complex does not comprise any epitopes of ASCL2, HER-2, TOMM34, RNF43, KOC1, VEGFR, βhCG, survivin, TGF βR2, p53, KRas, OGT, CASP5, COA-1, SART or IL13Rα2.

In another preferred embodiment, the complex comprises

One or more than one epitope of EpCAM or a functional sequence variant thereof (e.g. an epitope according to SEQ ID NO: 45);

One or more than one epitope of KRAS or a functional sequence variant thereof (e.g.an epitope according to SEQ ID NO: 35).

In this preferred embodiment, the complex preferably does not comprise any epitopes of HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGF βR2, p53, OGT, CASP5, COA-1, MAGE, SART or IL13Rα2. More preferably, in this preferred embodiment, the complex does not comprise any epitope of ASCL2, HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGF βR2, p53, OGT, CASP5, COA-1, MAGE, SART or IL13Rα2.

In various preferred embodiments, the complex comprises

one or more than one epitope of survivin or a functional sequence variant thereof (e.g. an epitope according to SEQ ID NO: 17);

-one or more than one epitope of MAGE-a35 or a functional sequence variant thereof.

In this preferred embodiment, the complex preferably does not comprise any epitopes of HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGF βR2, p53, KRas, OGT, CASP5, COA-1, SART or IL13Rα2. More preferably, in this preferred embodiment, the complex does not comprise any epitopes of ASCL2, HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, SART or IL13Rα2.

In a preferred embodiment, the complex comprises

one or more than one epitope of survivin or a functional sequence variant thereof (e.g. an epitope according to SEQ ID NO: 17); and

Such a complex preferably does not comprise any epitope of EpCAM, HER-2, TOMM34, RNF 43, KOC1, VEGFR, IDCG, CEA, TGF βR2, p53, KRas, OGT, CASP5, COA-1, SART or IL13Rα2. More preferably, such a complex does not comprise any epitope of ASCL2, epCAM, HER-2, TOMM34, RNF 43, KOC1, VEGFR, beta hCG, CEA, TGF beta R2, p53, KRas, OGT, CASP5, COA-1, SART or IL13Rα2.

More preferably, the complex comprises

one or more than one epitope of survivin or a functional sequence variant thereof (e.g. an epitope according to SEQ ID NO: 17); and/or

One or more than one epitope of CEA or a functional sequence variant thereof (e.g.an epitope according to SEQ ID NO:30 and/or an epitope according to SEQ ID NO: 31).

Such a complex preferably does not comprise any epitopes of HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGF βR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Rα2. More preferably, such a complex does not comprise any epitope of ASCL2, HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Rα2.

More preferably, the complex comprises

one or more than one epitope of ASCL2 or a functional sequence variant thereof (e.g. an epitope according to SEQ ID NO:22 and/or an epitope according to SEQ ID NO: 23); and/or

Such a complex preferably does not comprise any epitopes of HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGF βR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Rα2.

More preferably, the complex comprises

Such a complex preferably does not comprise any epitopes of HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Rα2.

Particularly preferably, such a complex comprises

It is also particularly preferred that the complex comprises

one or more than one epitope of MUC-1 or a functional sequence variant thereof (e.g. an epitope according to SEQ ID NO:26 and/or an epitope according to SEQ ID NO: 27); and

Such a complex preferably does not comprise any epitope of HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGF βR2, p53, KRas, OGT, CASP, COA-1, MAGE, SART or IL13Rα2. More preferably, such a complex does not comprise any epitope of ASCL2, HER-2, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGF βR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Rα2.

In a most preferred embodiment, the complex comprises

One or more than one epitope of CEA or a functional sequence variant thereof (e.g.an epitope according to SEQ ID NO:30 and/or an epitope according to SEQ ID NO: 31);

one or more than one epitope of EpCAM or a functional sequence variant thereof (e.g. an epitope according to SEQ ID NO: 41); and

one or more than one epitope of ASCL2 or a functional sequence variant thereof (e.g.an epitope according to SEQ ID NO:22 and/or an epitope according to SEQ ID NO: 23).

In another most preferred embodiment, the complex comprises in the N-to C-terminal direction:

Such a complex preferably does not comprise any epitopes of HER-2, epCAM, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART or IL13Rα2.

Even more preferably, the complex comprises in the N-to C-terminal direction:

i) Has the sequence according to SEQ ID NO:29, or a fragment thereof of at least 10 amino acids in length (preferably at least 15 amino acids, more preferably at least 20 amino acids, even more preferably at least 25 amino acids, most preferably at least 30 amino acids), or a peptide thereof having a functional sequence variant with at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity;

ii) has a sequence according to SEQ ID NO:16, or a fragment thereof of at least 10 amino acids in length (preferably at least 15 amino acids, more preferably at least 20 amino acids, even more preferably at least 25 amino acids, most preferably at least 30 amino acids), or a peptide thereof having a functional sequence variant with at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity; and

iii) Has the sequence according to SEQ ID NO:21, or a fragment thereof of at least 10 amino acids in length (preferably at least 15 amino acids, more preferably at least 20 amino acids, even more preferably at least 25 amino acids, most preferably at least 30 amino acids), or a peptide thereof having a functional sequence variant with at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity.

Such complexes preferably do not comprise any other antigens or other epitopes than CEA, survivin and ASCL2, more preferably such complexes do not comprise any other (tumour) epitopes.

Preferably, in such a complex, (i) has a sequence according to SEQ ID NO:29 or a fragment or variant thereof is directly linked to (ii) a peptide having an amino acid sequence according to SEQ ID NO:16 or a fragment or variant thereof; and (ii) has a sequence according to SEQ ID NO:16 or a fragment or variant thereof is directly linked to (iii) a peptide having an amino acid sequence according to SEQ ID NO:21 or a fragment or variant thereof.

More preferably, the complex comprises in the N-to C-terminal direction:

i) Has the sequence according to SEQ ID NO:32, or a peptide thereof having a functional sequence variant with at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity;

ii) has a sequence according to SEQ ID NO:18, or a peptide thereof having a functional sequence variant with at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity; and

iii) Has the sequence according to SEQ ID NO:24, or a peptide thereof having a functional sequence variant with at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity.

Preferably, in such a complex, (i) has a sequence according to SEQ ID NO:32 or a variant thereof is directly linked to (ii) a peptide having an amino acid sequence according to SEQ ID NO:18 or a variant thereof; and (ii) has a sequence according to SEQ ID NO:18 or a variant thereof is directly linked to (iii) a peptide having an amino acid sequence according to SEQ ID NO:24 or a variant thereof.

Most preferably, the multi-antigen domain of the complex comprises or consists of a polypeptide having a sequence according to SEQ ID NO:48, or a peptide having a (functional) sequence variant with at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity. In a most preferred embodiment, the complex does not comprise any other antigen or other epitope other than CEA, survivin, and ASCL2, more preferably does not comprise any other (tumor) epitope.

Component c) -TLR peptide agonists

In the complexes comprised by the combinations according to the invention, TLR peptide agonists result in improved targeting of the vaccine to dendritic cells as well as self-adjuvanticity. The physically linked TLR peptide agonist provides an enhanced immune response by simultaneously stimulating antigen presenting cells, particularly dendritic cells, that internalize, metabolize and display antigen, with at least one antigen or epitope in the complex comprised by the CPP and the combination according to the invention.

As used in the context of the present invention, a "TLR peptide agonist" is an agonist of a Toll-like receptor (TLR), i.e. it binds to a TLR and activates it, in particular to generate a biological response. Furthermore, TLR peptide agonists are peptides, polypeptides or proteins as defined above. Preferably, the TLR peptide agonist comprises from 10 to 150 amino acids, more preferably from 15 to 130 amino acids, even more preferably from 20 to 120 amino acids, particularly preferably from 25 to 110 amino acids, most preferably from 30 to 100 amino acids.

Toll-like receptors (TLRs) are transmembrane proteins characterized by an extracellular domain, a transmembrane domain, and a cytoplasmic domain. Horseshoe-shaped extracellular domains containing leucine-rich repeats (LRRs) are involved in recognizing common molecular patterns from different microorganisms. Toll-like receptors include TLR1 to 10. Compounds capable of activating TLR receptors and modifications and derivatives thereof are well documented in the art. TLR1 can be activated by bacterial lipoproteins and their acetylated forms, TLR2 can also be activated by gram positive bacterial glycolipids, LPS, LP a, LTA, pili, outer membrane proteins, heat shock proteins from bacteria or hosts, and mycobacterial lipoarabinomannans. TLR3 may be activated by dsRNA, in particular dsRNA of viral origin, or by the chemical compound poly (LC). TLR4 can be activated by gram-negative LPS, LTA, heat shock proteins from host or bacterial sources, viral coat or envelope proteins, paclitaxel or its derivatives, hyaluronic acid containing oligosaccharides and fibronectin. TLR5 may be activated by bacterial flagella or flagellin. TLR6 may be activated by mycobacterial lipoproteins and group B streptococcus thermolabile soluble factors (GBS-F) or staphylococcal modulators. TLR7 can be activated by imidazoquinoline. TLR9 may be activated by unmethylated CpG DNA or chromatin-IgG complexes.

Preferably, the TLR peptide agonist comprised by the complex comprised by the combination according to the invention is an agonist of TLR1, 2, 4, 5, 6 and/or 10. TLR expression is on the cell surface (TLR 1, 2, 4, 5, 6 and 10) or on the membrane of intracellular organelles such as endosomes (TLR 3, 4, 7, 8 and 9). The natural ligand for the endosomal receptor was originally a nucleic acid-based molecule (except TLR 4). Cell surface expressed TLRs 1, 2, 4, 5, 6 and 10 recognize the molecular patterns of extracellular microorganisms (Monie, t.p., bryant, c.e. et al 2009:Activating immunity:Lessons from the TLRs and NLRs.Trends Biochem.Sci.34 (11), 553-561). TLRs are expressed on several cell types, but virtually all TLRs are expressed on dendritic cell DCs, enabling these proprietary cells to target all possible pathogens and danger signals.

However, TLRs 2, 4 and 5 are constitutively expressed on dendritic cell DC surfaces. Thus, the TLR peptide agonist comprised by the complex comprised by the combination according to the invention is more preferably a peptide agonist of TLR2, TLR4 and/or TLR 5. Even more preferably, the TLR peptide agonist is a TLR2 peptide agonist and/or a TLR4 peptide agonist. Particularly preferably, the TLR peptide agonist is a TLR4 peptide agonist. Most preferably, the TLR peptide agonist is a TLR peptide agonist, which is both a TLR2 and a TLR4 agonist. TLR2 can detect various ligands for bacteria, viruses, parasites and fungi. Ligand specificity is generally determined by the interaction of TLR2 with other TLRs, such as TLR1, 6 or 10, or non-TLR molecules such as the C-lectin receptor (lectin-1), CD14 or CD 36. The formation of heterodimers with TLR1 enables TLR2 to identify triacylglycerols or lipopeptides derived from (fungal) bacterial sources, such as Pam3CSK4 and peptidoglycans (PGA; gay, n.j. And ganloff, m. (2007): structure and function of Toll receptors and their ligands, annu. Rev. Biochem.76, 141-165; spohn, r., buttett-Beckmann, u. Et al (2004): synthetic lipopeptide adjuvants and Toll-like receptor 2-Structure-activity references. Vaccine 22 (19), 2494-2499). Heterodimerization of TLR2 and 6 enables detection of diacyl lipopeptides and zymosan. Lipopolysaccharide (LPS) and its derivatives are ligands for TLR4, and flagellin or entolimod (CBLB 502) is a ligand for TLR5 (Bryant, C.E., sprmg, D.R. et al (2010) The molecular basis ofthe host response to lipopolysaccharide. Nat. Rev. Microbiol.8 (1), 8-14).

TLR2 interacts with a wide and structurally diverse array of ligands, including molecules expressed by microorganisms and fungi. Various TLR2 agonists have been identified, including natural and synthetic lipopeptides (e.g., mycoplasma fermentum macrophage activated lipopeptides (MALP-2)), peptidoglycans (PGs, e.g., those from staphylococcus aureus), lipopolysaccharides from various bacterial strains (LPS), polysaccharides (e.g., zymosan), glycosyl phosphatidyl-inositol-anchoring structures from gram positive bacteria (e.g., lipoteichoic acid (LTA) and lipoarabinomannan from mycobacteria, and lipomannans from mycobacterium tuberculosis). Some viral determinants may also be triggered by 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.2009:10 (11): 1200-7). Bacterial lipopeptides are structural components of the cell wall. They consist of an acylated s-glyceryl cysteine moiety to which the peptide can be bound via a cysteine residue. Examples of bacterial lipopeptide TLR2 agonists include MALP-2 and its synthetic analog dipalmitoyl-S-glyceryl cysteine (Pam ₂ Cys) or tripalmitoyl-S-glyceryl cysteine (Pam) ₃ Cys)。

A variety of ligands interact with TLR4, including monophosphoryl lipid a (MPLA), lipopolysaccharide (LPS), mannan (candida albicans), glycoinositol phospholipids (trypanosoma), viral envelope proteins (RSV and MMTV) and endogenous antigens from minnesota/salmonella R595, including fibrinogen and heat shock proteins. Such TLR4 agonists are described, for example, in Akira S, uematsu S, takeuchi o.pathen recognition and innate immunity cell.2006, 24: 124 (4): 783-801 or Kumar H, kawai T, akira s.toll-like receptors and innate immunity, biochem Biophys Res com.2009, 10 months 30 days 388 (4): 621-5. LPS found in the outer membrane of gram-negative bacteria is the most widely studied TLR4 ligand. Suitable LPS-derived TLR4 agonist peptides are described, for example, in WO 2013/120073 (A1).

TLR5 is initiated by (i) a region of a flagellin molecule expressed by almost all motile bacteria; or (ii) initiated by entolimod (CBLB 502). Thus, (i) a flagellin, or a peptide or protein derived from a flagellin and/or a flagellin variant or fragment; or (ii) entolimod (CBLB 502) are also suitable as TLR peptide agonists for inclusion in the complex.

Thus, examples of TLR peptide agonists include the TLR2 lipopeptide agonist MALP-2, pam ₂ Cys and Pam ₃ Cys or modifications thereof, different forms of TLR4 agonist LPS, such as neisseria meningitidis wild-type L3-LPS and mutant pentaacylated LpxL1-LPS, and TLR5 agonist flagellin.

However, it is preferred that the complex comprises a TLR peptide agonist that is neither a lipopeptide nor a lipoprotein, nor a glycopeptide nor a glycoprotein, more preferably that the complex comprises a TLR peptide agonist that is a typical peptide, polypeptide or protein as defined herein.

In some embodiments, the TLR peptide agonist is a (naturally-occurring) protein, or a fragment thereof having a variant of at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity. Such fragments may have a minimum length of at least 20 or 25, preferably at least 30 or 35, more preferably at least 40 or 50, even more preferably 60 or 70, yet more preferably at least 80 or 90, for example at least 100 amino acids. In particular, the fragments exhibit TLR agonist function. A fragment of a protein may advantageously be selected such that it provides a "TLR agonist domain" of the protein, but preferably does not include any other domain of the protein (other than a TLR agonist domain). Thus, in some embodiments, the TLR agonist does not comprise another immunologically active domain (other than the TLR agonist domain), more preferably the TLR agonist does not comprise another biologically active domain (other than the TLR agonist domain). For example, in some embodiments, the TLR agonist is not a flagellin (which also includes domains other than TLR agonist functionality). However, in some embodiments, the TLR agonist may be a fragment of a flagellin, including a TLR agonist domain of a flagellin (but without other domains of a flagellin).

Preferred TLR2 peptide agonists are annexin II or immunomodulatory fragments thereof (with TLR agonist function), as described in detail in WO2012/048190A1 and U.S. patent application 13/0331546, particularly preferred SEQ ID NOs: 7 or a fragment or variant thereof.

Thus, comprising or consisting of a sequence according to SEQ ID NO:49 or an amino acid sequence that hybridizes to SEQ ID NO:49, preferably as component c) comprised by the complex, i.e. as TLR peptide agonist, at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% of the same sequence variant.

SEQ ID NO:49 (TLR 2 peptide agonist Anaxa)

According to SEQ ID NO: a particularly preferred functional sequence variant of the TLR peptide agonist of 49 is according to SEQ ID NO:50, TLR peptide agonist:

thus, a sequence according to SEQ ID NO:50 or a sequence variant thereof, particularly preferably as component c) comprised by the complex, i.e. as at least one TLR peptide agonist.

With respect to TLR4, TLR peptide agonists are particularly preferred, which correspond particularly to motifs that bind to TLR4, in particular (i) peptides that mimic the natural LPS ligand (RS 01: gln-Glu-Ile-Asn-Ser-Tyr and RS09: ala-Pro-His-Ala-Leu-Ser) and (ii) fibronectin-derived peptides. The cellular glycoprotein Fibronectin (FN) has multiple isoforms produced by alternative splicing of three exons from a single gene. One of these isoforms is the additional domain a (EDA), which interacts with TLR 4.

Other suitable TLR peptide agonists comprise the fibronectin EDA domain or fragment or variant thereof. Such suitable fibronectin EDA domains or fragments or variants thereof are disclosed in EP 1 913 954 B1, EP 2 476 A1, US 2009/0220532 A1, and WO 2011/101332 A1. Thus, a sequence according to SEQ ID NO:40 or a sequence variant thereof, is preferably comprised as component c), i.e. as at least one TLR peptide agonist, in a complex comprised in a combination according to the invention.

SEQ ID NO:52 (TLR 4 peptide agonist EDA)

Another suitable TLR peptide agonist comprises or consists of Hp91 or a fragment or variant thereof, as described herein. Hp91 is a TLR4 agonist, as described for example in US 9539321 B2, and has the following amino acid sequence:

furthermore, high mobility group box 1 (HMGB 1) and peptide fragments thereof are assumed to be TLR2 agonists, in particular as enhancers of TLR2 mediated inflammatory activity. Such HMGB1 derived peptides are for example disclosed in US 2011/0236406 A1. Thus, a sequence according to SEQ ID NO:51 or a sequence variant thereof, as component c), i.e. as at least one TLR peptide agonist, is comprised in a complex comprised in a combination according to the invention:

HMGB1 and peptide fragments thereof are useful as TLR agonists alone or in combination with (other) TLR2/TLR4 peptide agonists as enhancers of TLR2 mediated inflammatory activity. Thus, in some embodiments, the complex may, for example, comprise as part of TLR agonist Δ30hmgb1 or any immunomodulatory fragment thereof, such as those disclosed in WO2006/083301 A1, and a TLR2/TLR4 peptide agonist such as ANAXA (SEQ ID NO: 49) or a sequence variant thereof as set forth in SEQ ID NO:50 combinations. Thus, in addition to the TLR peptide agonist disclosed in Δ30HMGB1 (SEQ ID NO: 51) above, the complex may comprise any immunomodulatory fragment thereof, or any peptide Hp-1-Hp-166 disclosed in WO2006/083301 A1, preferably Hp-31, hp-46, hp-106. For example, the complex may comprise at least the TLR peptide agonists EDA (SEQ ID NO: 52) and Δ30HMGB1 (SEQ ID NO: 51), or EDA (SEQ ID NO: 52) and Hp-31, or Hp-46, or Hp-106, preferably the complex comprises at least the TLR peptide agonists ANAXA (SEQ ID NO:49 or 50) and Δ30HMGB1 (SEQ ID NO: 51), or ANAXA (SEQ ID NO:49 or 50) and Hp-31, or Hp-46, or Hp-106. The use of any such combination may be advantageous if a stronger self-adjuvanticity of the complex is desired.

Preferably, the complex comprised by the combination according to the invention comprises a single TLR agonist. In other embodiments, a complex comprised of a combination according to the invention may comprise more than one TLR peptide agonist, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 TLR peptide agonists, more preferably a complex comprised of a combination according to the invention comprises (at least) two or three TLR peptide agonists, even more preferably a complex comprised of a combination according to the invention comprises (at least) four or five TLR peptide agonists. If a complex comprises more than one TLR peptide agonist, it is understood that said TLR peptide agonist is also, in particular, covalently linked in the complex comprised in the combination according to the invention, e.g. to another TLR peptide agonist and/or component a), i.e. a cell penetrating peptide, and/or component b), i.e. an antigen or an epitope of an antigen.

In particularly preferred embodiments, the complex comprised by the combination according to the invention comprises one single TLR peptide agonist. In particular, in this particularly preferred embodiment, the complex comprised by the combination according to the invention comprises one single TLR peptide agonist and there is no other component having TLR agonist properties than said one single TLR peptide agonist.

The various TLR peptide agonists comprised by the complex may be the same or different. Preferably, the complexes comprise various TLR peptide agonists that are different from each other.

Furthermore, more than one antigen or epitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 antigens or epitopes, or more TLR peptide agonists, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 TLR agonists, are preferred, consecutively located in the complex comprised by the combination of the invention. This means in particular that all TLR peptide agonists comprised by the complex are located in a sequence which is not interrupted by component a), i.e. the cell penetrating peptide, nor by component b), i.e. the at least one antigen or epitope. Instead, component a) and component b) are located in a complex, for example before or after a stretch of such all TLR peptide agonists. However, TLR peptide agonists that are consecutively positioned in this way may be linked to each other, e.g. by a spacer element or linker that is neither component a), i.e. the cell penetrating peptide, nor component b), i.e. at least one antigen or epitope.

Alternatively, however, the various TLR peptide agonists may also be located in any other way in the complex comprised by the combination according to the invention, e.g. component a) and/or component b) are located between two or more TLR peptide agonists, i.e. one or more TLR peptide agonists are located between component a) and component b) (and vice versa), and optionally one or more TLR peptide agonists are located at the other end of each of component a) and/or component b).

It will be appreciated that many different TLR peptide agonists that activate the same or different TLR receptors may advantageously be contained in a single complex. Alternatively, a number of different TLR peptide agonists that activate the same or different TLR receptors may be partitioned into subgroups of different TLR peptide agonists that activate the same or different TLR receptors, which subgroups are comprised in different complexes, whereby different complexes comprising different subgroups may advantageously be administered simultaneously, e.g. in a single vaccine, to a subject in need thereof.

Connection and arrangement of components a), b) and c) in a complex

In the complexes comprised in the combination according to the invention, components a), b) and c) are covalently linked, i.e. the bonds between two of the three components a), b) and c) of the complex are covalent bonds. Preferably, two of the three components a), b) and c) of the complex are covalently linked to each other (i.e. "first" and "second" components), and the third of the three components a), b) and c) is covalently linked to the first of the three components a), b) and c) or the second of the three components a), b) and c). Therefore, linear molecules are preferably formed. However, it is also conceivable that each of the three components a), b) and c) is covalently linked to two other components of the three components a), b) and c).

"covalent attachment" (also referred to as covalent bond) as used in the context of the present invention refers to chemical bonds involving sharing of electron pairs between atoms. "covalent attachment" (also known as covalent bonding) refers to a stable balance of attractive and repulsive forces between atoms, particularly when they share electrons. For many molecules, the sharing of electrons allows each atom to acquire a complete shell, corresponding to a stable electronic structure. Covalent bonds include a variety of interactions such as sigma-bonds, pi-bonds, metal-to-metal bonds, covalent interactions, and three-centered double electron bonds. Thus, the complex comprised by the combination according to the invention may also be referred to as "compound", in particular it may be referred to as "molecule".

Preferably, in the complex comprised in the combination according to the invention, components a), b) and c) are covalently linked by chemical coupling in any suitable way known in the art, such as a cross-linking method. It should be noted, however, that many known chemical crosslinking methods are non-specific, i.e. they do not direct the coupling point to any specific site on components a), b) and c). Thus, the use of non-specific cross-linking agents may attack the functional site or spatially block the active site, rendering the fusion component of the complex biologically inactive. Blocking potentially reactive groups by using suitable protecting groups is considered to be known to the skilled artisan. Alternatively, powerful and versatile oxime and hydrazone ligation techniques can be used, which are chemoselective entities useful for crosslinking of components a), b) and c). Such connection techniques are described, for example, in Rose et al (1994), JACS 116, 30.

The connection between two of the three components a), b) and c) of the complex comprised by the combination according to the invention may be direct or indirect, i.e. the two components are directly adjacent, or they may be connected by additional components of the complex, such as spacer elements or linkers.

The complex comprised by the combination according to the invention may optionally comprise a spacer element or linker which is typically a non-immune moiety, preferably cleavable, and which links components a) and b) and/or components a) and C), and/or components b) and C), and/or links a continuous antigen or epitope of an antigen, and/or links a continuous TLR peptide agonist, and/or links a continuous cell penetrating peptide, and/or it may be located at the C-terminal part of components b) and/or C). In addition to the attachment of the components, the linker or spacer element may preferably provide other functions and is preferably cleavable, more preferably cleavable naturally within the target cell, e.g. by enzymatic cleavage. However, such other functions do not include, inter alia, any immune functions. Examples of other functions, particularly with regard to linkers in fusion proteins, can be found in Chen x. Et al, 2013: fusion Protein Linkers: property, design and functionality. Adv Drug Deliv Rev.65 (10): 1357-1369, wherein e.g. in vivo cleavable linkers are also disclosed. Furthermore, chen x. Et al, 2013: fusion Protein Linkers: property, design and functionality. Adv Drug Deliv Rev.65 (10): 1357-1369 also disclose various joints, such as flexible joints and rigid joints, as well as joint design tools and databases, which may be used in the composites comprised by the combinations of the invention or for designing joints for use in the composites.

The spacer element may be a peptide or a non-peptide, preferably the spacer element is a 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 amino acid 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 an unnatural amino acid sequence, e.g., an amino acid sequence resulting from conservative amino acid substitutions of the natural flanking regions or a sequence with a known protease cleavage site. In some embodiments, the peptide spacer does not comprise any Cys (C) residues. In some embodiments, the linker sequence comprises at least 20%, more preferably at least 40%, even more preferably at least 50% glycine or β -alanine residues. Suitable linker sequences can be readily selected and prepared by those skilled in the art. They may consist of D and/or L amino acids.

In some embodiments, the complex comprised by the combination according to the invention may comprise spacer elements or linkers, in particular peptide spacer elements, between components a) and b) and/or between components a) and c) and/or between components b) and c). The skilled artisan can select such a peptide spacer element such that after the complex comprising the cell penetrating peptide and cargo molecule has been internalized, it can be cleaved by the cell machinery.

Techniques for linking two of the three components a), b) and c) are well documented in the literature and may depend on the nature of at least one antigen or epitope. For example, the linkage between two of the three components a), b) and c) may be achieved by cleavable disulfide bonds, by full step solid phase synthesis or solution phase or solid phase fragment coupling, stable amide, thiazolidine, oxime and hydrazine linkages, disulfide bonds, stable thiomaleimide linkages, peptide bonds (including peptide bonds between amino acids of fusion proteins), or electrostatic or hydrophobic interactions.

Preferably, the at least one antigen or epitope comprised by the complex, and any optional spacer element or linker comprised by the complex, are of peptide nature. More preferably, all components of the complex comprised by the combination according to the invention, such as cell penetrating peptides, at least one antigen or epitope that is a peptide, polypeptide or protein, at least one TLR peptide agonist and any optional peptide linker or spacer, are linked by peptide bonds in the complex comprised by the combination according to the invention. Most preferably, the complex comprised by the combination according to the invention is thus a peptide, polypeptide or protein, e.g. a fusion protein, e.g. a recombinant fusion protein.

Components a), b) and c) may be arranged in any manner in the complex comprised by the combination according to the invention.

In particular, if the complex comprises more than one cell penetrating peptide and/or more than one antigen or epitope and/or more than one TLR peptide agonist, then more than one cell penetrating peptide may be positioned in a discontinuous manner, i.e. at least one antigen or epitope (component b)) and/or at least one TLR peptide agonist (component c)) may interrupt the sequence of a continuously positioned cell penetrating peptide, and/or the cell penetrating peptide may be positioned in an alternating manner with component b) and/or with component c). Similarly, more than one antigen or epitope may be localized in a discontinuous manner, i.e. at least one cell penetrating peptide (component a)) and/or at least one TLR peptide agonist (component c)) may interrupt a sequence of consecutively localized antigens or epitopes, and/or antigens or epitopes may be localized alternately with component a) and/or with component c). Similarly, more than one TLR peptide agonist may be localized in a discontinuous manner, i.e. at least one cell penetrating peptide (component a)) and/or at least one antigen or epitope (component b)) may interrupt a sequence of a TLR peptide agonist that is localized consecutively, and/or a TLR peptide agonist may be localized in an alternating manner with component a) and/or with component b).

However, it is preferred that more than one cell penetrating peptide is located in a continuous manner in the complex comprised by the combination of the invention, and/or that more than one antigen or epitope is located in a continuous manner in the complex comprised by the combination of the invention, and/or that more than one TLR peptide agonist is located in a continuous manner in the complex comprised by the combination of the invention. This means in particular that all individual units of a certain component comprised by the complex, i.e. all cell penetrating peptides, all antigens or epitopes or all TLR peptide agonists, are located in a sequence which is not interrupted by either of the other two components. In contrast, the other two components are located in the complex, for example before or after the sequence of all individual units of a segment of the particular component. However, the individual units of the particular component which are positioned consecutively in this way may be connected to one another, for example by spacer elements or joints which are not of the other two components described herein.

It is particularly preferred that each of components a), b) and c) is positioned in a continuous manner.

Preferably, all three components a), b) and c) are linked by a backbone/backbone linkage, thus specifically yielding a complex backbone comprising a backbone of one or more than one cell penetrating peptide, a backbone of one or more than one antigen or epitope and a backbone of one or more than one TLR peptide agonist. In other words, the backbone of one or more cell penetrating peptides, the backbone of one or more antigens or epitopes, and the backbone of one or more TLR peptide agonists constitute the backbone of the complex, optionally together with other components, such as linkers, spacer elements, etc. Thus, the following arrangement of components a), b) and C) is preferred, in particular if at least one antigen or epitope is a peptide, polypeptide or protein, whereby the preferred arrangement is shown below in the N-terminal to C-terminal direction of the complex backbone, and wherein all three components a), b) and C) are linked by a backbone/backbone bond, and may optionally be linked by a linker, spacer or another additional component:

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

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

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

(epsilon) component b) (at least one antigen or epitope of an antigen) -component a) (cell penetrating peptide) -component c) (at least one TLR peptide agonist); or alternatively

(ζ) component b) (at least one antigen or epitope of an antigen) -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 by a backbone/backbone linkage, preferably at least one antigen or epitope is located at the C-terminus of the cell penetrating peptide, whereby the cell penetrating peptide and at least one antigen or epitope are optionally linked by other components, such as a linker, spacer or by at least one TLR peptide agonist. Therefore, this corresponds to the arrangements (α), (β) and (γ) in the above-described arrangement, that is, the arrangements (α), (β) and (γ) are more preferable according to the above-described arrangement.

Even more preferably, the at least one antigen or epitope is located at the C-terminus of the cell penetrating peptide, whereby the cell penetrating peptide and the at least one antigen or epitope are optionally linked by other components, such as a linker, spacer element, but not by at least one TLR peptide agonist. Therefore, this corresponds to the arrangements (α) and (β) in the above-described arrangement, that is, the arrangements (α) and (β) in the above-described arrangement are even more preferable. Particularly preferably, the complex comprised by the combination according to the invention is a recombinant polypeptide or recombinant protein, and components a) to C) are located in the N-terminal to C-terminal direction (N-terminal to C-terminal direction) of the complex backbone in the following order:

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

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

wherein the components may be linked by other components, in particular by linkers or spacer elements.

In some embodiments, the at least one antigen or epitope (or multi-antigen domain) of a complex is located C-terminal to a cell-penetrating peptide of the complex, wherein the cell-penetrating peptide and the at least one antigen or epitope (or multi-antigen domain) are optionally linked by other components such as a linker, spacer element, or by a TLR peptide agonist of the complex.

Preferred exemplary complexes of the combinations of the invention are polypeptides or proteins, wherein

a) The cell penetrating peptide has a sequence comprising or consisting of a sequence according to SEQ ID NO:6 (CPP 3/Z13), SEQ ID NO:7 (CPP 4/Z14), SEQ ID NO:8 (CPP 5/Z15) or SEQ ID NO:11 An amino acid sequence of (CPP 8/Z18) or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity (without losing the cell penetrating ability of the peptide);

b) The at least one antigen or epitope is a peptide, polypeptide or protein, and preferably comprises or consists of at least one cancer/tumor epitope; and

c) The TLR peptide agonist is a TLR2 peptide agonist and/or a TLR4 peptide agonist.

For the combination according to the invention, it is particularly preferred that the complex comprises or consists of a polypeptide having a sequence according to SEQ ID NO:54, or a (functional) sequence variant thereof having at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity.

In some embodiments, the complex may comprise a polypeptide having a sequence according to SEQ ID NO:55 or a functional sequence variant thereof having at least 70% or at least 75%, preferably at least 80% or at least 85%, more preferably at least 90% or at least 95%, even more preferably at least 97% or at least 98%, particularly preferably at least 99% sequence identity.

STING agonists

An Interferon (IFN) gene stimulatory factor (STING) is a 379 amino acid protein belonging to the family of nucleic acid sensors that is an adapter to cytoplasmic DNA signals. STING is expressed in a variety of endothelial and epithelial cell types, as well as in hematopoietic cells, such as T cells, macrophages and Dendritic Cells (DCs). STING is part of the innate immune response to cytoplasmic nucleic acids and acts as a DNA sensor and signaling molecule. STING is essential for controlling transcription of many host defense genes after recognition of Cyclic Dinucleotides (CDNs) in the cytosol of cells, including type I IFN and other pro-inflammatory cytokines. In its basal state STING exists as a dimer with its N-terminal domain anchored in the Endoplasmic Reticulum (ER) and its C-terminal domain located in the cytosol. The Cyclic Dinucleotide (CDN) produced by the protein cyclic GMP-AMP synthase (cGAS) is the natural ligand for STING (Ablasser et al, nature 498, 380-384, 2013). Binding of CDN to STING induces conformational changes, which allow for binding and activation of TANK-binding kinase (TBK 1) and interferon regulatory factor 3 (IRF 3) and relocation from ER to perinuclear endosomes (Liu et al Science 347, issue 6227, 2630-1-2630-14, 2015). Phosphorylation of the transcription factors IRF3 and NF-kB by TBK1 results in the expression of a variety of cytokines, including type I IFN. The production of type I IFN results in activation of dendritic cells, efficient cross priming of cd8+ T cells against tumor antigens, and migration of tumor-specific cd8+ T cells into the tumor. Thus, therapeutic strategies that activate STING innate immune response pathways to restore type I IFN signaling may increase tumor immunogenicity—making immune cold tumors "hot-started" that do not respond to replacement therapies.

As used herein, the term "STING agonist" refers to a compound that induces, activates, stimulates, enhances or prolongs STING activity. For example, an overview is provided in Ding et al 2020 (C.Dmg, Z.Song, A.Shen, T.Chen, A.Zhang.Small molecules targeting the innate immune cGAS-STING-TBK1signaling pathway. Acta pharm. Sin. B (2020), 10.1016/j. Apsb.2020.03.001, published on 13 th month line of 2020). In particular, specific examples of STING agonists described in Ding et al 2020 (incorporated herein by reference) may be used as STING agonists in the context of the present invention.

In some embodiments, the term "STING agonist" includes compounds that act indirectly (i.e., do not interact directly with STING), such as cyclic GMP-AMP synthase (cGAS) agonists. Activated cGAS then synthesizes 2',3' -cGAMP, which then acts as an agonist of STING. Non-limiting examples of cGAS agonists include

Spherical Nucleic Acids (SNAs) exhibiting dsDNA at high surface densities, particularly SNA nanostructures carrying 45bp IFN-mimetic dsDNA oligonucleotides, such as those described in Alexander Stegh, DDIS-03.DEVELOPMENT OF A NOVEL CLASS OF cGAS AGONISTS TO TRIGGER STING PATHWAY-DEPENDENT INNATE IMMUNE RESPONSES AGAINST GLIOBLASTOMA, neuro-Oncology, volume 21, journal of journal 62019, month 11, page vi65, incorporated herein by reference; and

G3-YSD, which is a 26-mer DNA sequence from the HIV-1 RNA genome (Herzner AM. et al 2015.Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA.Nat Immunol.16 (10): 1025-33).

In other embodiments, the term "STING agonist" refers only to compounds that interact directly with STING.

Various (direct) STING agonists are known in the art. In general, (direct) STING agonists can be divided into Cyclic Dinucleotide (CDN) and non-CDN STING agonists. CDN STING agonists are inspired by the natural ligand 2'3' -cGAMP for STING. Examples of non-CDN STING agonists include small molecule STING agonists, where "Compound 7" is described in L.Conales et al (L.Corrales, L.H.Glickman, S.M.McWhirter, D.B.Kanne, K.E.Sivick, G.E.Katibah et al Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and immunity, cell Rep,11 (2015), pp.1018-1030), and is considered a prototype structural model for the development of non-CDN small molecule STING agonists. Preferably, however, the (direct) STING agonist is a Cyclic Dinucleotide (CDN) -based STING agonist.

Non-limiting examples of STING agonists useful in the context of the present invention include:

STING agonists described in WO 2014/093936 A1, WO 2014/189805 and WO 2014/189806, in particular ADU-S100 (Aduro),

STING agonists, in particular MK-1454 (Merck), as described in WO 2017/027646 A1 and WO 2018/118664 A1,

STING agonists, in particular E-7766 (Eisai), as described in WO 2018/152450 A1, WO 2018/152453 A1 and WO 2020/036199 A1,

-MK-2118(Merck)

-BMS-986301(Bristol-Myers Squibb)，

an analogue of IMSA-101 (ImmuneSensor Therapeutics inc.) cGAMP,

SB-11285 (Spring Bank Pharmaceuticals), small molecule-nucleic acid hybrid STING agonists,

SYNB-1891 (Synlogic), a nonpathogenic strain of E.coli (E.coli) bacteria, which has been designed to express STING,

GSK-3745417 (GlaxoSmithKline), which is considered a synthetic non-CDN STING agonist with a dimeric ABZI scaffold,

non-CDN STING agonist TAK-676 (Takeda), and

non-CDN small molecule STING agonist TTI-10001 (Trillium Therapeutics inc.).

Thus, the STING agonist may be selected from ADU-S100, MK-1454, E-7766, MK-2118, BMS-986301, IMSA-101, SB-11285, SYNB-1891, GSK-3745417, TAK-676 and TTI-10001. Of these agonists, ADU-S100 is preferred.

Preferably, STING agonists are compounds described in WO 2018/060323 A1, which is incorporated herein by reference, or compounds described in WO 2018/172206, which is incorporated herein by reference.

More preferably, the STING agonist is a compound of formula I

/>

Wherein the method comprises the steps of

R ¹ Selected from H, F, -O-C _1-3 Alkyl groupOH, and

R ² is H, or

R ² is-CH ₂ -，R ¹ is-O-together with-CH ₂ -O-bridge, and

R ³ is a purine nucleobase selected from the group consisting of purine, adenine, guanine, xanthine, hypoxanthine, through N thereof ⁹ A nitrogen linkage is provided, wherein the nitrogen linkage,

or a solvate or hydrate thereof, or a salt thereof.

Even more preferably, the STING agonist is a compound of formula Ia

Wherein R is ¹ And R is ² As defined in relation to formula I or a solvate or hydrate thereof or a salt thereof.

Even more preferably, the STING agonist is a compound of formula Ib

Or a solvate or hydrate thereof, or a salt thereof.

More preferably, the STING agonist is a compound of formula ia.1

Or a solvate or hydrate thereof.

Still more preferably, the STING agonist is a compound of formula ia.2 (STING 2)

Or a solvate or hydrate thereof.

Furthermore, it is still more preferred that the STING agonist is a compound of formula ia.3

Or a solvate or hydrate thereof.

Furthermore, it is still more preferred that the STING agonist is a compound of formula ib.1

Or a solvate or hydrate thereof.

Also preferred, STING agonists are compounds of formula (II) (as described in WO 2018/172206):

wherein the method comprises the steps of

Base group ¹ And a base ² Independently selected from the group consisting of purine, adenine, guanine, xanthine and hypoxanthine, by N thereof ⁹ The nitrogen atom is connected with the nitrogen atom,

or a salt thereof.

More preferably, in the compound of formula (II), the base ¹ And a base ² Is adenine such that the STING agonist is represented by the structure of formula (II-1):

still more preferably, in the compound of formula (II), the base is ¹ Is adenine, base ² Is guanine, such that the STING agonist is represented by the structure of formula (II-2):

still more preferably, inIn the compound of formula (ID), the base ¹ Is guanine, base ² Is adenine, such that the STING agonist is represented by the structure of formula (II-3):

still more preferably, in the compound of formula (II), the base is ¹ Is adenine, base ² Is hypoxanthine such that the STING agonist is represented by the structure of formula (II-4):

the STING agonist may also be a substantially pure (Sp, sp), (Rp, rp), (Sp, rp) or (Rp, sp) stereoisomer of a compound represented by any of the above structural formulas I, ia, ib, ia.1, ia.2, ia3, ib.1, II-1, II-2, II-3, and II-4, or a salt thereof. Preferably, the STING agonist is a substantially pure (Rp, rp) stereoisomer of a compound represented by any one of formulas I, ia, ib, ia.1, ia.2, ia.3, ib.1, II-1, II-2, II-3, and II-4 above, or a salt thereof. The term "substantially pure" as used herein refers to one (Rp, rp), (Rp, sp), (Sp, rp) or (Sp, sp) diastereomer, with a purity of at least 75% in terms of phosphorus atoms relative to the other possible diastereomers. In preferred embodiments, a substantially pure compound is a compound that is at least 85% pure, at least 90% pure, at least 95% pure, at least 97% pure, and at least 99% pure.

In some embodiments, the STING agonist is a pharmaceutically acceptable salt of a compound of any one of formulas I, ia, ib, ia.1, ia.2, ia.3, ib.1, II-1, II-2, II-3, and II-4 above. The expression "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by salt formation with a base. Pharmaceutically acceptable salts can be synthesized from the parent compound containing an acidic moiety by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid forms of these compounds with a sufficient amount of the appropriate base in water or in an organic diluent such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile or mixtures thereof. Alternatively, salts may be prepared by ion exchange, for example by treating an aqueous solution of a compound of the invention (in free acid or salt form) with a cation exchanger. In some embodiments, the STING agonist is a sodium salt of a compound represented by any one of formulas I, ia, ib, ia.1, ia.2, ia.3, ib.1, II-1, II-2, II-3, and II-4 above.

In the combination according to the invention, preferably the complex comprises or consists of a sequence according to SEQ ID NO:55 or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity, and the STING agonist is ADU-S100.

It is also preferred in a combination according to the invention that the complex comprises or consists of a polypeptide according to SEQ ID NO:55 or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity, and the STING agonist is a compound selected from the group consisting of compounds represented by formulae ia.1, ia.2, ia.3, ib.1, II-1, II-2, II-3 and II-4, or a solvate or hydrate thereof (as described above).

More preferably, the complex comprises or consists of a sequence according to SEQ ID NO:55 or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity, and the STING agonist is ADU-S100.

Even more preferably, the complex comprises or consists of a sequence according to SEQ ID NO:55 or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity, and the STING agonist is a compound selected from the group consisting of compounds represented by formulae ia.1, ia.2, ia.3, ib.1, II-1, II-2, II-3 and II-4, or a solvate or hydrate thereof (as described above).

In general, with respect to sequence variants, the higher the percent identity, the more preferred the sequence variants. Thus, 100% identity is most preferred, e.g. according to SEQ ID NO:54 or 55. Similarly, compounds of formulas Ia.1, ia.2, ia.3, ib.1, II-1, II-2, II-3 and II-4 are generally preferred over their solvates or hydrates.

Thus, the present invention also provides STING agonists as described above for use in combination with a complex, particularly for use in medicine. Preferably, STING agonists are as described above. Preferred medical uses are as follows, such as the prevention and/or treatment of cancer.

Furthermore, the present invention provides STING agonists for use in combination with a complex as described above, in particular for use in medicine. Preferably, STING agonists are as described above. Preferred medical uses are as follows, for example in the prevention and/or treatment of cancer.

Medical application

As mentioned above, the combination of (i) STING agonist and (ii) complex (and any optional other components) according to the invention may be used in medicine, in particular as a medicament.

As described herein and shown in the accompanying examples, the combination of (i) STING agonists and (ii) complexes comprising cell penetrating peptides, at least one antigen or epitope and TLR peptide agonists improves CD4 and CD 8T cell responses, enhances antigen specific CD 8T cells, increases intratumoral immunogenicity, and results in a significant increase in survival and reduced tumor growth. This demonstrates the synergistic effect of STING agonists and complexes acting together, which significantly increases the anti-tumor effect of each of their components administered as independent therapies.

The combination according to the invention can be used for a variety of diseases. Preferably, the combinations described herein are used (for the preparation of a medicament) to prevent, treat or stabilize a disease or disorder, such as a disease or disorder that can be treated by immunotherapy, including cancer, infectious diseases, autoimmune disorders, hematological disorders, and transplant rejection. Thus, combinations described herein for preventing, treating or stabilizing a disease or disorder, such as a disease or disorder that can be treated by immunotherapy, including cancer, infectious diseases, autoimmune disorders, hematological disorders, and transplant rejection, are preferred.

In the context of the present invention, it is particularly preferred that the combination according to the invention as described herein is used for the prevention and/or treatment of cancer or tumors.

Accordingly, the present invention also provides a method for treating cancer or for initiating, enhancing or prolonging an anti-tumor response in a subject in need thereof, comprising administering to the subject an effective amount of a combination of the invention as described above (i.e., an effective amount of (i) a STING agonist, and an effective amount of (ii) a complex comprising a cell penetrating peptide, at least one antigen or epitope of an antigen, and a TLR peptide agonist, and optionally (iii) an effective amount of any optional other active agent). In addition, the invention provides a method of increasing tumor antigen specific T cell infiltration of a tumor in a patient, the method comprising administering to a patient having a tumor or cancer a combination of the invention as described above (i.e., (i) a STING agonist, and (ii) a complex comprising a cell penetrating peptide, at least one antigen or epitope thereof, and a TLR peptide agonist, and optionally (iii) any optional other active agent. Furthermore, the present invention provides a combination therapy for the prevention and/or treatment of cancer, wherein the combination therapy comprises administration of a combination of the invention as described above (i.e. (i) a STING agonist, and (ii) a complex comprising a cell penetrating peptide, at least one antigen or epitope of an antigen, and a TLR peptide agonist, and optionally (iii) any optional other active agent.

The term "disease" as used in the context of the present invention is intended to be generally synonymous with the terms "disorder" and "symptom" (as in a medical condition), and is used interchangeably, as all of these reflect abnormal (non-physiological or pathological) conditions of the human or animal body or a part thereof that impair normal functioning, and are generally manifested by distinguishing disorders and symptoms, and result in a shortened duration or reduced quality of life for the human or animal.

Preferred diseases for treatment and/or prophylaxis by use of a complex comprising a cell penetrating peptide, at least one antigen or epitope and at least one TLR peptide agonist as described herein include cancer, hematological disorders, infectious diseases, autoimmune diseases and transplant rejection. Thus, it is preferred to treat and/or prevent cancer and infectious diseases, more preferred to treat and/or prevent cancer. For cancers, preferably endocrine, gastrointestinal, genitourinary or gynecological, breast, head and neck, hematopoietic, skin, breast or respiratory tumors, colorectal cancers, for example metastatic colorectal cancers, are preferred.

Preferably, a combination of a STING agonist as described herein and a complex comprising a cell penetrating peptide, at least one antigen or epitope and at least one TLR peptide agonist as described herein is useful (e.g., for the preparation of a medicament) for the prevention, treatment and/or amelioration of cancer or neoplastic disease, including disease caused by defective apoptosis. The cancer may be a solid tumor, a blood cancer or a lymphatic cancer. Cancers may be benign or metastatic. Further preferred examples of cancers to be treated include brain cancer, prostate cancer, breast cancer, ovarian cancer, esophageal cancer, lung cancer, liver cancer, kidney cancer, melanoma, intestinal cancer, lung cancer, head and neck squamous cell cancer, chronic myelogenous leukemia, colorectal cancer, gastric cancer, endometrial cancer, myelogenous leukemia, lung squamous cell cancer, acute lymphoblastic leukemia, acute myelogenous leukemia, bladder tumor, promyelocytic leukemia, non-small cell lung cancer and sarcoma. Particularly preferred is a complex comprising a cell penetrating peptide as described herein, at least one antigen or epitope and at least one TLR peptide agonist for use in the treatment of colorectal cancer.

In some embodiments, the cancer/tumor may be selected from breast cancer, including triple negative breast cancer, biliary tract cancer; bladder cancer; brain cancers, including glioblastoma and medulloblastoma; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; stomach cancer; gastrointestinal stromal tumor (GIST), appendiceal cancer, cholangiocarcinoma, carcinoid, gastrointestinal colon cancer, extrahepatic cholangiocarcinoma, gallbladder carcinoma, gastric (stomach) carcinoma, gastrointestinal carcinoid, colorectal or metastatic colorectal cancer, hematological tumors including acute lymphoblastic and myeloid leukemia; t cell acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic myelogenous leukemia, multiple myeloma; AIDS-related leukemia and adult T-cell leukemia lymphoma; intraepithelial tumors, including bowden and paget's disease; liver cancer; lung cancer, including non-small cell lung cancer, lymphomas, including hodgkin's disease and lymphocytic lymphomas; neuroblastoma; glioblastoma, oral cancer, including squamous cell carcinoma; ovarian cancer, including ovarian cancer derived from epithelial cells, stromal cells, germ cells, and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas include leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancers, including melanoma, meckel cell carcinoma, kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma; testicular cancer, including germ tumors, such as seminomas, non-seminomas (teratomas, choriocarcinomas), interstitial tumors, and germ cell tumors; thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma; and renal cancers, including adenocarcinoma and wilms' cell neoplasm.

In some embodiments, the cancer/tumor may be selected from the group consisting of an auditory schwannoma, anal cancer, astrocytoma, basal cell tumor, behcet's syndrome, bladder cancer, blastoma, bone cancer, brain metastasis tumor, brain cancer (glioblastoma), breast cancer (breast cancer), burkitt's lymphoma, carcinoid, cervical cancer, colon cancer, colorectal cancer, uterine malignancy, craniopharyngeal pipe tumor, CUP syndrome, endometrial cancer, gall bladder cancer, genital tumor including genitourinary tract cancer, glioblastoma, glioma, head/neck tumor, liver cancer, histiocytic lymphoma, hodgkin's syndrome or lymphoma and non-hodgkin's lymphoma, pituitary tumor, intestinal cancer (including small intestine tumor) and gastrointestinal tract tumor, kaposi's sarcoma, kidney cancer, renal epithelial cancer, breast cancer or laryngeal carcinoma, leukemia (including acute myelogenous leukemia (lung cancer), erythroleukemia, acute Lymphocytic Leukemia (ALL), chronic Myelogenous Leukemia (CML) and chronic leukemia), eyelid (l), eyelid cancer (=l), liver cancer, small-cell carcinoma (=small-cell tumor), small-cell carcinoma of the esophagus, small-sized lung cancer, small-cell carcinoma of the esophagus, small-sized lung cancer Pancreatic epithelial cancer (=pancreatic cancer), penile cancer, stem cancer, laryngeal cancer, pituitary tumor, plasmacytoma, prostate cancer (=prostate tumor), rectal cancer, rectal tumor, kidney cancer, renal epithelial cancer, retinoblastoma, sarcoma, schneeberger disease, skin cancer, such as melanoma or non-melanoma skin cancer, including basal cell carcinoma and squamous cell carcinoma, as well as psoriasis, pemphigus vulgaris, soft tissue tumor, spinal tumor, gastric cancer, testicular cancer, laryngeal cancer, thymoma, thyroid cancer, tongue cancer, urinary tract cancer, uterine cancer, vaginal cancer, various virus-induced tumors, such as papillomavirus-induced cancers (e.g., cervical cancer = cervical cancer), adenocarcinomas, herpes virus-induced tumors (e.g., burkitt lymphoma, EBV-induced B cell lymphoma, cervical cancer), hepatitis B-induced tumors (hepatocellular carcinoma), HTLV-1 and ht2-induced lymphomas and vulvar cancers.

Preferably, the patient treated with the combination of the invention suffers from colorectal cancer (CRC), in particular advanced colorectal cancer (CRC) or advanced metastatic colorectal cancer (mCRC), wherein the term "advanced" CRC, mCRC comprises stage IIIC: t4a, N2a, M0 or T3-T4a, N2b, M0 or T4b, N1-N2, M0; stage IVA: any T, any N, M a and IVB phases: any T, any N, M b (according to TNM staging), wherein the CRC or mCRC tumor may be e.g. "microsatellite stable" (MSS), or microsatellite unstable "(MSI), preferably the CRC or mCRC tumor is MSS.

In the context of the present invention, the terms "therapy" and "therapeutic" preferably refer to having at least some minimal physiological effect when administered to a living body. For example, the physiological effect of administering a "therapeutic" anti-tumor compound may be to inhibit tumor growth, or to reduce tumor size, or to prevent tumor recurrence. Preferably, in the treatment of cancer or a neoplastic disease, compounds that inhibit tumor growth or reduce tumor size or prevent tumor recurrence are considered therapeutically effective. Thus, the term "anti-neoplastic agent" preferably refers to any therapeutic agent that has a therapeutic effect on a tumor, neoplastic disease or cancer.

The components of the combination of the invention described herein, i.e., (i) STING agonists and (ii) complexes comprising a cell penetrating peptide, at least one antigen or epitope, and a TLR peptide agonist (and any optional other components), are typically administered as a combination therapy. This means that even if one component (STING agonist or complex) is not, e.g. administered on the same day as the other component (STING agonist or other of complex), their treatment regimens are often closely related. This means that "combination" in the context of the present invention does not particularly include starting treatment with one component (STING agonist or complex) after the end of treatment with the other component (STING agonist or complex). Thus, treatment "ending" means in particular that the active ingredient no longer exerts its effect-i.e. the "treatment" may in particular end a few minutes, hours or days after the last administration of the active ingredient, depending on the time at which the active ingredient exerts its effect. More generally, "associative" treatment regimens of STING agonists and complexes-thus, combinations of STING agonists and complexes-refer to

(i) Not every administration of STING agonist (and complete STING agonist therapy) has been completed for more than one week (preferably more than 3 days, more preferably more than 2 days, even more preferably more than one day) before the first administration of the complex (complete therapy with the complex) is initiated; or alternatively

(ii) Not every administration of the complex (complete therapy with the complex) has been completed for more than one week (preferably more than 3 days, more preferably more than 2 days, even more preferably more than one day) before the first administration of the STING agonist (and complete STING agonist therapy) is initiated.

For example, in a combination of a STING agonist described herein and a complex described herein comprising a cell penetrating peptide, at least one antigen or epitope, and at least one TLR peptide agonist, one component (STING agonist or complex) may be administered once per week and the other component (the other of STING agonist or complex) may be administered once per month. To achieve a "combination" in the sense of the present invention in this example, the components administered monthly will be administered at least once within the same week, with the other components administered weekly also being administered.

As mentioned above, administration of STING agonists and/or complexes comprised in a combination according to the invention may require multiple sequential administrations, e.g. multiple injections. Thus, administration may be repeated at least twice, for example once as a primary immune injection, the latter as a booster injection.

In particular, STING agonists and/or complexes comprised in a combination according to the invention may be administered repeatedly (or consecutively). STING agonists and/or complexes comprised in a combination according to the invention may be administered repeatedly or consecutively for at least 1 week, 2 weeks, 3 weeks or 4 weeks; 2, 3, 4, 5, 6, 8, 10 or 12 months; or a period of 2 years, 3 years, 4 years, or 5 years. For example, STING agonists included in a combination according to the invention may be administered twice daily, once every two days, once every three days, once weekly, once every two weeks, once every three weeks, once monthly or once every two months. For example, the complex comprised in the combination according to the invention may be administered twice daily, once every two days, once every three days, once weekly, once every two weeks, once every three weeks, once monthly or once every two months. Preferably, the complex and/or STING agonist comprised in the combination according to the invention may be administered repeatedly, for example once per week or once every two weeks (once).

Preferably, STING agonists and/or complexes comprised in a combination according to the invention may be administered on the same day. For example, a STING agonist and/or complex included in a combination according to the invention may be repeatedly administered (as described above; e.g., weekly), and on those days of administration of the complex, the STING agonist is also administered. Furthermore, on the day of such combined administration, an optional third component may also be administered.

According to the invention, in a combination of a STING agonist as described herein and a complex comprising a cell penetrating peptide, at least one antigen or epitope of an antigen, and at least one TLR peptide agonist as described herein, the STING agonist and complex are preferably administered at about the same time.

As used herein, "about simultaneously" refers to, in particular, simultaneous administration, or administration of the complex immediately after administration of the STING agonist, or administration of the STING agonist immediately after administration of the complex. The skilled artisan will understand that "immediately following" includes the time required to prepare the second application, particularly the time required to expose and disinfect the second application site and to properly prepare the "applicator" (e.g., syringe, pump, etc.). Simultaneous administration also includes if the administration periods of the STING agonist and the complex overlap, or if, for example, one component (STING agonist or complex) is administered over a longer period of time, such as 30 minutes, 1 hour, 2 hours, or even more than 2 hours, for example by infusion, while the other component (STING agonist or complex) is administered for such a long period of time. If different dosage forms, different routes of administration and/or different sites of administration are used, it is particularly preferred to administer the STING agonist and the complex at about the same time.

Preferably, the STING agonist comprised by the combination according to the invention and the complex (and optionally other active compounds) comprised by the combination according to the invention are administered in therapeutically effective amounts. As used herein, a "therapeutically effective amount" is an amount sufficient to alleviate symptoms of the disease or disorder being treated and/or to prevent symptoms of the disease or disorder being prevented. In other words, a "therapeutically effective amount" refers to an amount of the complex and/or STING agonist that is sufficient to significantly induce positive improvement of a disease or disorder, i.e., an amount of the complex and/or STING agonist, that elicits a biological or pharmaceutical response in a tissue, system, animal or human being sought. The term also includes amounts of the complex and/or STING agonist sufficient to slow down the disease process, in particular slow down or inhibit tumor growth or infection, thereby eliciting the sought response, in particular such a response may be an immune response against the antigen or epitope comprised in the complex (i.e. "an inhibitory effective amount"). At the same time, however, the "therapeutically effective amount" is preferably small enough to avoid serious side effects, that is to say to allow a reasonable relationship to be established between advantage and risk. Determination of these limitations is generally within the scope of sound medical judgment. The "therapeutically effective amount" of the complex and/or STING agonist will also vary depending on the particular condition being treated, as well as the age and physical condition of the patient being treated, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the activity of the particular component (STING agonist and complex), the severity of the condition, the duration of the treatment, the nature of the concomitant therapy, the particular pharmaceutically acceptable carrier used, and like factors, all of which are within the knowledge and experience of the practitioner.

Thus, the dose administered to an individual in a single dose or multiple doses will vary depending on a variety of factors including pharmacokinetic properties, condition and characteristics of the subject (sex, age, weight, health, body type), the extent of the symptoms, concurrent therapy, frequency of treatment, and desired effect.

The complex comprised by the combination according to the invention and the STING agonist (and optionally other active compounds) comprised by the combination according to the invention may be administered by various routes of administration, for example systemic or local (e.g. intratumoral). Systemic routes of administration generally include, for example, transdermal, oral, and parenteral routes, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal, and intraperitoneal routes, and/or intranasal routes of administration. Routes of topical administration include, for example, administration at the affected site, such as intratumoral administration.

Preferably, the complex comprised by the combination of the invention and the agonist comprised by the combination of the invention are administered systemically. In some embodiments, the STING agonist is administered intratumorally and the complex is administered systemically.

For systemic administration, parenteral routes of administration are preferred. More preferably, the complex comprised by the combination according to the invention and the STING agonist comprised by the combination according to the invention are administered by intravenous, intradermal, subcutaneous, intramuscular, intranasal or intranodal routes. Even more preferably, the complex comprised by the combination of the invention and the STING agonist comprised by the combination of the invention are administered intravenously or subcutaneously. In some embodiments, the complex comprised by the combination of the invention and the STING agonist comprised by the combination of the invention are administered intramuscularly.

Preferably, the complex comprised by the combination of the invention and the STING agonist comprised by the combination of the invention are administered by the same route of administration, preferably by the same systemic route of administration, more preferably by the same parenteral route of administration, even more preferably intravenously or subcutaneously.

In some embodiments, the complex comprised by the combination of the invention and the STING agonist comprised by the combination of the invention are administered by different routes. For example, STING agonists are administered intratumorally, and the complex is administered systemically, e.g., intramuscularly or subcutaneously. In some embodiments, the complex comprised by the combination of the invention and the STING agonist comprised by the combination of the invention are administered by different systemic routes. For example, STING agonists may be administered intravenously, and the complex administered systemically, e.g., intramuscularly or subcutaneously.

Composition and method for producing the same

As described above, (i) STING agonists and (ii) complexes may be provided in different compositions. In some embodiments, (i) the STING agonist and (iii) the optional third component (other than the complex and STING agonist) may be provided in different compositions. In some embodiments, (ii) the complex and (iii) the optional third component (other than the complex and STING agonist) may be provided in different compositions. For example, (i) STING agonists; (ii) The complex and (iii) optionally a third component (other than the complex and STING agonist) may be provided in different compositions.

In some embodiments, (i) STING agonist and (ii) complex may be included in the same composition. In some embodiments, (i) the STING agonist and (iii) the optional third component (other than the complex and STING agonist) may be included in the same composition. In some embodiments, (ii) the complex and (iii) optionally a third component (other than the complex and STING agonist) may be included in the same composition. For example, (i) STING agonists; (ii) The complex and (iii) optionally a third component (other than the complex and STING agonist) may be included in the same composition.

Thus, the invention also provides a combination of compositions wherein the first composition comprises a STING agonist as described above (but preferably does not comprise a complex as described above); the second composition comprises a complex as described above (but preferably not a STING agonist as described above). Each of these compositions may optionally comprise an optional third component (other than the complex and STING agonist). However, in some embodiments, neither the composition comprising the STING agonist nor the composition comprising the complex further comprises an optional third component (other than the complex and STING agonist). In this case, the optional third component (other than the complex and STING agonist) may be included in a different composition.

In addition, the present invention provides a composition comprising a STING agonist as described above and a complex as described above. Such compositions may optionally further comprise an optional third component (in addition to the complex and STING agonist). However, in some embodiments, the optional third component (other than the complex and STING agonist) may be included in a different composition.

Thus, in another aspect, the invention also provides a composition comprising

(i) STING agonists

(ii) A complex comprising:

a) Cell penetrating peptide;

b) At least one antigen or epitope; and

c) A TLR peptide agonist, which is a compound of formula (i),

wherein the components a) to c) comprised by the complex are covalently linked.

In particular, such compositions according to the invention comprise (i) a STING agonist as described above and (ii) a complex as described above. In other words, preferred embodiments of the STING agonists described above (in the context of the combination according to the invention) are also preferred in the composition according to the invention. Thus, preferred embodiments of the above-described complexes (in the context of the combination according to the invention) are also preferred in the composition according to the invention.

Typically, the composition may be a pharmaceutical composition and/or a vaccine composition. In particular, such a composition is preferably a (pharmaceutical) composition, optionally comprising a pharmaceutically acceptable carrier and/or carrier, excipient, buffer, stabilizer or other materials well known to the person skilled in the art. The terms "pharmaceutical formulation" and "pharmaceutical composition" as used in the context of the present invention particularly refer to formulations in a form which allows for a clear and effective biological activity of the active ingredient and which are free of other ingredients which are toxic to the subject to which the formulation is to be administered. In some embodiments, the (pharmaceutical) composition does not comprise additional active components (e.g., "active" with respect to cancer treatment) other than the STING agonist and/or complex (and/or optional third component(s) (other than complex and STING agonist)).

As further components, the (pharmaceutical) composition may in particular comprise a pharmaceutically acceptable carrier and/or vehicle. In the context of the present invention, a pharmaceutically acceptable carrier generally comprises a liquid or non-liquid matrix of the (pharmaceutical) composition. If the (pharmaceutical) composition is provided in liquid form, the carrier is typically pyrogen-free water; isotonic saline or buffer (aqueous solutions), such as phosphate, citrate, and the like, buffer solutions. In particular for injection of (pharmaceutical) compositions, water or preferably a buffer, more preferably an aqueous buffer, comprising a sodium salt, preferably at least 30mM sodium salt, a calcium salt, preferably at least 0.05mM calcium salt, and optionally a potassium salt, preferably at least 1mM potassium salt may be used. According to a preferred embodiment, the sodium, calcium and optionally potassium salts may be present in the form of their halides, for example chloride, iodide or bromide, in the form of their hydroxides, carbonates, bicarbonates or sulphates etc. Examples of sodium salts include, but are not limited to, for example NaCl, naI, naBr, na ₂ CO ₃ 、NaHCO ₃ 、Na ₂ SO ₄ Examples of optional potassium salts include, for example KCl, KI, KBr, K ₂ CO ₃ 、KHCO ₃ 、K ₂ SO ₄ Examples of calcium salts include, for example, caCl ₂ 、CaI ₂ 、CaBr ₂ 、CaCO ₃ 、CaSO ₄ 、Ca(OH) ₂ . In addition, the buffer may contain an organic anion of the above cation. According to a more preferred embodiment, the buffer suitable for injection purposes as defined above may comprise a buffer selected from sodium chloride (NaCl), calcium chloride (CaCl) ₂ ) And optionally a salt of potassium chloride (KCl), wherein other anions may be present in addition to chloride. CaCl (CaCl) ₂ It is also possible to replace it with another salt, for example KCl. Typically, the concentration of salt in the injection buffer is at least 30mM sodium chloride (NaCl), at least 1mM potassium chloride (KCl), and at least 0.05mM calcium chloride (CaCl) ₂ ). The injection buffer may be hypertonic, isotonic or hypotonic with respect to the specific reference medium, i.e. the buffer may have a higher, the same or a lower salt content with respect to the specific reference medium, wherein preferably such a concentration of the above mentioned salts may be used which does not cause cell damage due to osmosis or other concentration effects. The reference medium is, for example, a liquid produced in an "in vivo" method, such as blood, lymph, cytosolic liquid, or other body fluids, or, for example, a liquid, which may be used as a reference medium in an "in vitro" method, such as a commonly used buffer or liquid. Such usual buffers or liquids are known to the skilled person. Saline (0.9% NaCl) and ringer-lactate solution are particularly preferred as liquid matrices. In some embodiments, the (pharmaceutical) composition further comprises arginine, such as L-arginine.

However, one or more than one compatible solid or liquid filler or diluent or encapsulating compound may also be used in (pharmaceutical) compositions, which are suitable for administration to a subject to be treated. The term "compatible" as used herein means that these components of the (pharmaceutical) composition are capable of being mixed with the complex as defined above in such a way that no interaction occurs which would significantly reduce the pharmaceutical effect of the (pharmaceutical) composition under typical use conditions. Of course, pharmaceutically acceptable carriers, fillers and diluents must be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to a subject to be treated. Some examples of compounds that may be used as pharmaceutically acceptable carriers, fillers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch or potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate; powdery tragacanth; malt; gelatin; a lipid; 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 cocoa butter; polyols, such as polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.

In some embodiments, the (pharmaceutical) composition may be administered by injection or infusion techniques. The sterile injectable form of the (pharmaceutical) composition may be an aqueous or oleaginous suspension. These suspensions may be formulated according to techniques 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 suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Among the acceptable carriers and solvents that can be used are water, ringer's solution and physiological saline. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents, which are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as tweens, span and other emulsifying agents or bioavailability enhancers, are commonly used in the preparation of pharmaceutically acceptable solid, liquid or other dosage forms, and may also be used in the formulation of (pharmaceutical) compositions.

For parenteral injection, the active ingredient is preferably in the form of a parenterally acceptable aqueous solution which is pyrogen free and has suitable pH, isotonicity and stability. Those skilled in the art are able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, ringer's injection, lactated ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, if desired.

The (pharmaceutical) compositions described herein may also be administered orally in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions or solutions. For oral tablets, common carriers include lactose and corn starch. A lubricant, such as magnesium stearate, is also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is mixed with emulsifying and suspending agents. If desired, certain sweeteners, flavoring agents or coloring agents may also be added.

The (pharmaceutical) composition may also be administered topically, especially when the therapeutic target comprises an area or organ that is easily accessible by topical administration, e.g. a disease comprising skin or any other accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these regions or organs. For topical application, (pharmaceutical) compositions may be formulated as suitable ointments, wherein the active ingredient is suspended or dissolved in one or more carriers. Carriers for topical application include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water. Alternatively, the (pharmaceutical) composition may be formulated as a suitable lotion or cream. In the context of the present invention, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetostearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

In this case, the prescription of the treatment, e.g. the decision of the dosage etc. When using the above (pharmaceutical) compositions, it is often the responsibility of the average physician and other doctors, and it is often considered that the disease to be treated, the condition of the individual patient, the site of administration, the method of administration and other factors known to the physician.

Thus, (pharmaceutical) compositions generally comprise a therapeutically effective amount of the components of the (pharmaceutical) composition, in particular the complex and/or STING agonist. The (pharmaceutical) composition may be used in humans as well as in veterinary medicine, preferably in human medicine, generally as a pharmaceutical composition or as a vaccine.

The (pharmaceutical) composition, in particular the vaccine composition or formulation, may be administered as a pharmaceutical formulation, which may contain the complex described herein and/or any form of STING agonist described herein. For example, (pharmaceutical) compositions, particularly vaccine compositions, or formulations may also be administered as pharmaceutical formulations, which may comprise antigen presenting cells (e.g. dendritic cells) loaded with complexes in any of the forms described herein.

The vaccine and/or composition may also be formulated as (pharmaceutical) compositions and unit dosages thereof, particularly with adjuvants, immunomodulatory substances, carriers, diluents or excipients conventionally used, as described above and below, and in this form may be used as solids, such as tablets or filled capsules, or as liquids, such as solutions, suspensions, emulsions, elixirs or filled capsules, all for oral use, or in the form of sterile injectable solutions for parenteral (including subcutaneous and intradermal) use by injection or continuous infusion.

In the context of the present invention, in particular in the context of (pharmaceutical) compositions and vaccines, the injectable composition may be based on injectable sterile saline or phosphate buffered saline or other injectable carriers known in the art. Such (pharmaceutical) compositions and unit dosage forms thereof may comprise the components in conventional proportions, with or without other active compounds or components, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be used.

Examples of suitable adjuvants and/or immunomodulatory substances in the context of the present inventionScraper(Corixa), aluminum-based minerals (commonly known as alum) including aluminum compounds, ASO1-4, MF59, calcium phosphate, liposomes, iscom, polyinosinic acid: polycytidylic acid (poly IC), including its stable form of poly ICLC (Hiltonol), cpG oligodeoxynucleotide, granulocyte-macrophage colony stimulating factor (GM-CSF), lipopolysaccharide (LPS), montanide, polylactide co-glycolide (PLG), flagellin, sapogenin (QS 21), aminoalkyl glucamide compounds (e.g., RC 529), bi-component antimicrobial peptides with synthetic oligodeoxynucleotides (e.g., IC 31), imiquimod, resiquimod, immune Stimulating Sequences (ISS), monophosphoryl lipid a (MPLA), and fibroblast stimulating lipopeptides (FSL 1).

Compositions, particularly pharmaceutical compositions and vaccines, can be liquid formulations including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups and elixirs. The composition may also be formulated as a dry product for reconstitution with water or other suitable carrier prior to use. Such liquid formulations may contain additives including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles, and preservatives. Suspending agents include, but are not limited to, sorbitol syrup, methyl cellulose, dextrose/syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Preservatives include, but are not limited to, methyl or propyl parahydroxybenzoate and sorbic acid. Dispersing or wetting agents include, but are not limited to, poly (ethylene glycol), glycerol, bovine serum albumin,

Compositions, particularly pharmaceutical compositions and vaccines, may also be formulated as depot formulations, which may be administered by implantation or intramuscular injection.

The compositions, particularly pharmaceutical compositions and vaccines, may also be solid compositions, which may be in the form of tablets or lozenges formulated 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. Binders include, but are not limited to, syrup, gum arabic, gelatin, sorbitol, tragacanth, starch mucilage, and polyvinylpyrrolidone. 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 dodecyl sulfate. The tablets may be coated according to methods well known in the art.

The compositions, particularly pharmaceutical compositions and vaccines, may also be administered in a sustained release form or from a sustained release drug delivery system.

Furthermore, the compositions, particularly pharmaceutical compositions and vaccines, may be adapted for delivery by repeated administration.

Other materials for use in compositions, particularly pharmaceutical compositions and vaccines, or for their preparation, and formulation processing techniques, etc. are known to the skilled person.

Preferably, the composition is a vaccine. In the context of the present invention, the term "vaccine" refers to a biological agent that provides innate and/or adaptive immunity, typically against a specific disease, preferably cancer. Thus, the vaccine specifically supports the innate and/or adaptive immune response of the immune system of the example to be treated. For example, an antigen or epitope of a complex described herein generally results in or supports an adaptive immune response in a patient to be treated, and a TLR peptide agonist of a complex described herein can result in or support an innate immune response.

The vaccine may further comprise a pharmaceutically acceptable carrier, adjuvant and/or excipient as defined above for the (pharmaceutical) composition. In the specific case of a vaccine, the choice of pharmaceutically acceptable carrier will in principle depend on the mode of administration of the vaccine. As mentioned above, the vaccine may be administered, for example, systemically or locally. More preferably, the vaccine may be administered by intravenous, intratumoral, intradermal, subcutaneous or intramuscular route. Thus, the vaccine is preferably formulated in liquid (or sometimes solid) form. The appropriate amount of vaccine to be administered can be determined by routine experimentation in animal models. Such models include, but are not limited to, rabbit, sheep, mouse, rat, dog, and non-human primate models. Preferred unit dosage forms for injection include sterile solutions of water, physiological saline, or mixtures thereof. The pH of this solution should be adjusted to about 7.4. Suitable injection carriers include hydrogels, controlled or sustained release devices, polylactic acid and collagen matrices. Suitable pharmaceutically acceptable carriers for topical administration include those suitable for use in lotions, creams, gels and the like. If the vaccine is administered orally, tablets, capsules, etc. are the preferred unit dosage forms. Pharmaceutically acceptable carriers for preparing unit dosage forms useful for oral administration are well known in the art. The choice will depend on secondary considerations such as taste, cost and storability, which are not critical for the purposes of the present invention and can be easily selected by a person skilled in the art.

The vaccine may additionally comprise one or more auxiliary substances to further enhance immunogenicity. Thereby preferably a synergistic effect of STING agonists and/or complexes as defined above with auxiliary substances, which may optionally be comprised in a vaccine as described above is achieved. In this regard, various mechanisms may be considered depending on various types of auxiliary substances. For example, compounds that allow Dendritic Cell (DC) maturation, such as lipopolysaccharide or TNF- α, form a first class of suitable auxiliary substances. In general, any agent that affects the immune system in the form of a "danger signal" (LPS, GP96, etc.) or a cytokine, such as GM-CSF, that can specifically enhance and/or affect the immune response generated by STING agonists or complexes can be used as an adjunct. Particularly preferred cofactors are cytokines, such as monokines, lymphokines, interleukins or chemokines, which further promote an 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, INF-alpha, IFN-beta, INF-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors such as hGH.

In general, the (pharmaceutical) compositions described herein, in particular vaccines, can be used in medicaments for the above-mentioned medical uses. In particular, it is useful for the prevention and/or treatment of diseases or disorders including, for example, cancer, hematological disorders, infectious diseases, autoimmune diseases and transplant rejection, with cancer being preferred, as described above.

Kit for detecting a substance in a sample

In a further aspect, the invention also provides a kit, in particular a kit of parts, comprising

(i) STING agonists

(ii) A complex comprising:

a) Cell penetrating peptide;

b) At least one antigen or epitope; and

c) A TLR peptide agonist, which is a compound of formula (i),

wherein the components a) to c) comprised by the complex are covalently linked.

In particular, such a kit according to the invention comprises (i) a STING agonist as described above (in the context of a combination according to the invention) and (ii) a complex as described above (in the context of a combination according to the invention). In other words, preferred embodiments of the STING agonists described above (in the context of a combination according to the invention) are also preferred in a kit according to the invention. Thus, preferred embodiments of the above-described complexes (in the context of a combination according to the invention) are also preferred in a kit according to the invention.

The various components of the kit may be packaged in one or more containers. The above components may be provided in lyophilized or dried form or dissolved in a suitable buffer. For example, the kit may comprise a (pharmaceutical) composition comprising a STING agonist as described above and a (pharmaceutical) composition comprising a complex as described above, e.g. each composition in a separate container.

As described above, (i) STING agonist and (ii) complex may be contained in the same container (e.g., syringe). In some embodiments, (i) the STING agonist and (iii) the optional third component (other than the complex and STING agonist) may be contained in the same container (e.g., syringe). In some embodiments, (ii) the complex and (iii) the optional third component (other than the complex and STING agonist) may be contained in the same container (e.g., syringe). For example, (i) STING agonists; (ii) The complex and (iii) optionally a third component (other than the complex and STING agonist) may be contained in the same container (e.g., syringe).

In some embodiments, (i) STING agonist and (ii) complex may be provided in different containers (e.g., different syringes). In some embodiments, (i) the STING agonist and (iii) the optional third component (other than the complex and STING agonist) may be provided in different containers (e.g., different syringes). In some embodiments, (ii) the complex and (iii) the optional third component (other than the complex and STING agonist) may be provided in different containers (e.g., different syringes). For example, (i) STING agonists; (ii) The complex and (iii) optionally the third component (other than the complex and STING agonist) may be provided in different containers (e.g., different syringes).

The kit may also comprise other reagents including, for example, preservatives, growth media and/or buffers, washes, etc. for storing and/or reconstituting the above components.

Furthermore, the kit according to the invention may optionally comprise instructions for use. Preferably, the kit further comprises a package insert or label with instructions for treating a disease described herein, such as cancer.

Such a kit may preferably be used in the medicaments described herein, in particular in the prevention and/or treatment of cancer as described herein.

Brief description of the drawings

Hereinafter, a brief description of the drawings will be given. These drawings are intended to illustrate the invention in more detail. However, they are not intended to limit the subject matter of the present invention in any way.

Throughout the legend, the letter "K" represents a complex comprising a cell penetrating peptide, at least one antigen or epitope, and a TLR peptide agonist, such as Z13Mad25Anaxa (SEQ ID NO: 55) or ATP128 (SEQ ID NO: 54), as shown in the various examples section.

Figure 1 shows that the combined administration of complex (K) comprising cell penetrating peptide, at least one antigen or epitope and TLR peptide agonist of example 1 with STING agonist (STING) modulates CD4 and CD 8T extracellular Zhou Yingda in tumor-free mice. C57BL/6 mice received twice at two week intervals a complex comprising a cell penetrating peptide, at least one antigen or epitope, a TLR peptide agonist (K), STING agonist (STING), or a combination of both. (a) vaccination schedule. (B) Serum IFN-a levels measured 4 hours and 24 hours after the first vaccination. (C) One week after the second vaccination, circulating HPV-E7-specific CD 8T cells were detected by multimeric staining. One week after the third vaccination, mice were sacrificed and CD8 (D-E) or CD4 (F-K) T cell responses were analyzed by flow cytometry. (D) frequency of CD 8T cells in spleen cells. (E) Percentage of PMA restimulated CD 8T cells producing cytokines. (F) frequency of CD 4T cells in spleen cells. Frequency of Treg (G), th17 (H), th1 (I) and Th2 (J) in spleen CD 4T cells. (K) ratio of Th1/Th2 splenic CD 4T cells. (L) in vivo cytotoxicity of RAHYNIVTF-specific CD 8T cells was measured by transfer method using spleen cells loaded with RAHYNIVTF peptide. (M) RAHYNIVTF specific CD 8T cell TCR viability was measured by ex vivo ELIspot.

Figure 2 shows that example 2 administration of a complex (K) comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist in combination with STING agonist (STING) is well tolerated in tumor bearing mice. (A-B) Back transplantation 10 in C57BL/6 mice ⁵ TC-1 cells. When the tumor is visible, the mice are treated with two administrations: a complex (K) comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist, STING agonist (STING) or a combination thereof, spaced one week apart. Body temperature (a) and body weight (B) of the mice were measured at the indicated time points.

FIG. 3 shows the process of example 3Phenotype of circulating HPV-specific CD 8T cells in TC-1 tumor bearing mice. Back transplantation 10 in C57BL/6 mice ⁵ TC-1 cells. When the tumor is visible, the mice are treated with two administrations: a complex (K) comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist, STING agonist (STING), or a combination of both, spaced one week apart. One week after the last treatment, HPV-specific CD 8T cell responses were analyzed in the mouse blood. Frequency (a) and number (B) of circulating HPV-specific CD 8T cells as measured by flow cytometry.

Figure 4 shows the effect of example 3 administration of a complex (K) comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist in combination with STING agonist (STING) on CD 8T cells. Back transplantation 10 in C57BL/6 mice ⁵ TC-1 cells. When the tumor is visible, the mice are treated with two administrations: a complex (K) comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist, STING agonist (STING), or a combination of both, spaced one week apart. One week after the last treatment, mice were sacrificed and tumors were harvested and analyzed for the presence and phenotype of CD 8T cells by FACS staining. The frequency and total number of total (A-B) and HPV-specific (C-D) CD 8T cells in tumor-infiltrating leukocytes are shown.

FIG. 5 shows the monitoring of tumor-infiltrating HPV-specific CD 8T cells function after ex vivo stimulation of bone marrow-derived dendritic cells (BMDCs) loaded with HPV peptides by measuring the expression of IFNγ, TNFα and degranulation marker CD107 α in example 3. Tumor-infiltrating cd45+ cells were co-cultured ex vivo with HPV peptide-loaded BMDCs for 6 hours. Measuring the production of antigen-specific cytokines by intracellular staining; representative FACS plots and frequencies of cytokine production in CD 8T cells are shown.

FIG. 6 shows intracellular production of granzyme B (GzB) after a short ex vivo TIL culture in the presence of a Golgi stop inhibitor of example 3. Cd45+ tumor infiltrating cells were cultured ex vivo with Golgi inhibitor for 4 hours. Monitoring granzyme B production by intracellular staining; the frequency and total number of total (A-B) and HPV-specific (C-D) CD 8T cells that produce granzyme B are described.

FIG. 7 shows the phenotype of HPV-specific CD 8T cells circulating in TC-1 tumor-bearing mice of example 3, i.e., splenic HPV-specific CD 8T cells producing cytokines or GzB were observed at very low frequencies in all different treatments. For this purpose, spleen cells were re-stimulated ex vivo with HPV-derived peptides. The frequency of HPV-specific CD 8T cells producing cytokine (a) and secreting granzyme B (B) is shown.

FIG. 8 shows the measurement of the expression of activation and depletion markers of total (A) or HPV-specific (B) CD 8T cells by flow cytometry in example 3. Frequency of total (C-E-G) or HPV-specific (D-F-H) CD 8T cell CD38 (C-D), NKG2Da (E-F) or TCF-1 (G-H) expression. Co-expression of PD-1, granzyme B and TCF-1 on HPV-specific CD 8T cells (I).

FIG. 9 shows the phenotype of circulating HPV-specific CD 8T cells in TC-1 tumor-bearing mice of example 3, i.e., activation of circulating HPV-specific CD 8T cells as measured by flow cytometry).

Figure 10 shows that example 4 administration of a complex (K) comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist in combination with STING agonist (STING) modulates intratumoral CD 4T cells in a TC-1 model. Back transplantation 10 in C57BL/6 mice ⁵ TC-1 cells. When the tumor is visible, the mice are treated with two administrations: a complex (K) comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist, STING agonist (STING), or a combination of both, spaced one week apart. One week after the last treatment, mice were sacrificed, blood and tumors were taken, and the presence and phenotype of CD 4T cells were analyzed by FACS staining. Frequency and total number of total (a-B) and Treg (C-D) CD 4T cells in tumor infiltrating leukocytes. Ratio between tumor-infiltrating CD 8T cells and total (E) or Treg (F) CD 4T cells. (G) Frequency of tregs and non-tregs in tumor-infiltrating CD 4T cells. Frequencies of Th1 (H), th2 (I) and Th17 (K) in tumor-infiltrating CD 4T cells. (J) ratio between Th1 and Th2 tumor infiltrating CD 4T cells. (L) tumor-infiltrating CD45+ cells were co-cultured ex vivo with BMDC loaded with HPV peptide for 6 hours. Measuring the production of antigen-specific cytokines by intracellular staining; the frequency of cytokines in CD 4T cells is shown.

Fig. 11 shows that example 5 administration of a complex (K) comprising a cell penetrating peptide, at least one antigen or epitope of an antigen and a TLR peptide agonist in combination with STING agonist (STING) modulates the Tumor Microenvironment (TME). Back transplantation 10 in C57BL/6 mice ⁵ TC-1 cells. When the tumor is visible, the mice are treated with 2 administrations: a complex (K) comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist, STING agonist (STING), or a combination of both, spaced one week apart. One week after the last treatment, mice were sacrificed and tumors were taken and analyzed for tumor microenvironment by FACS staining. (A) The proportion of different cell populations in cd45+ tumor infiltrating cells, each circle represents 1% of the cd45+ cell population. (B) ratio of different dendritic cell populations. Proportion of type 1 (C) or type 2 (D) tumor-associated macrophages (TAMs) in CD45+ tumor infiltrating cells. (E) the ratio between M1 and TAM 2. Proportion of monocyte myeloid derived suppressor cells MDSC (F), granulocyte MDSC (G) and neutrophil (H) in cd45+ tumor infiltrating cells.

Figure 12 shows that example 6 administration of a complex (K) comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist in combination with a STING agonist (STING ga) modulates intratumoral expression of PD-L1 and MHC-I. Back transplantation 10 in C57BL/6 mice ⁵ TC-1 cells. When the tumor is visible, the mice are treated with 2 administrations: a complex (K) comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist, STING agonist (STING), or a combination of both, spaced one week apart. One week after the last treatment, mice were sacrificed and tumors were taken and analyzed for tumor microenvironment by FACS staining. Expression level (mean MFI) of PD-L1 on CD45- (A) and CD45+ (B). Percentages of PD-L1 in immersed TAM1 (C) and TAM2 (D). Expression levels of H2-Kb (E) and H2-Db (F) on CD 45-tumor infiltrating cells. MHC-II in CD11b+ cells ^hi Expression level (G) and frequency (H). Two independent experimental pools (n=7) are shown, mann-whitney test p < 0.05, p < 0.01, p < 0.001.

FIG. 13 shows example 7 administration of a group comprising cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist (K) and STING agonist (STING Ga)And (3) a combined anti-tumor effect. Back transplantation 10 in C57BL/6 mice ⁵ TC-1 cells (A-B). When the tumor is visible, mice are treated twice, one week apart, and monitored for tumor growth (a) and mouse survival (B).

Figure 14 shows the anti-tumor effect of example 8 administration of a complex (K) comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist in combination with a STING agonist (STING ga 2). Back transplantation 10 in C57BL/6 mice ⁵ TC-1 cells. When the tumor is visible, the mice are treated twice with a complex comprising a cell penetrating peptide, at least one antigen or epitope and TLR peptide agonist (K), one week apart, and/or are treated with systemic administration of STING agonist (STING 2) on days 6, 10, 13 and 17. Tumor growth (a) and mouse survival (B) were monitored.

Figure 15 shows that example 8 administration of a complex (K) comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist in combination with a STING agonist (STING ga 2) modulates CD 8T extracellular Zhou Yingda. Circulating HPV-specific CD 8T cells measured by multimeric staining one week after the second vaccination were shown as percentage of cd8+ T cells (a) and number of HPV-specific CD 8T cells per ml of blood (B).

FIG. 16 shows that ATP128 immunogenicity as detected in the mouse model in example 9, and that the combination of STINGA and ATP128 (K) induced CEA-specific CD 8T cell responses. Subcutaneous implantation in the back of female C57BL/6J mice 5X 10 ⁵ And (3) MC38-CEA tumor cells. Mice were vaccinated with 10 nanomolar ATP128, 25 μg STING agonist (ADU-S100), or a combination of both, on days 6 and 13 post tumor implantation. Both ATP128 and STING agonists were subcutaneously injected at the bottom of the tail. One week after the second vaccination, mouse blood was collected from the tail vein and the frequency and total number of CEA-specific CD 8T cells was analyzed by flow cytometry using custom designed multimers, wherein specific epitopes of CEA in C57BL/6 mice were predicted and designed. ATP128 vaccination elicits CEA-specific CD 8T cells, which can be monitored using custom dextran staining (a). Custom CEA-dextran staining was performed 1 week after the 2 nd vaccination. The combination of sting ga and ATP128 induces CEA tA heterologous CD 8T cell response (B, C).

Figure 17 shows the function of example 10 to enhance CD 8T and CD 4T cell peripheral responses in tumor-free mice by administering a combination of complex (K) comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist, and a STING agonist (STING). (A) C57BL/6 mice received twice at two week intervals the administration of the Z13Mad39Anaxa vaccine, STING agonist or a combination of both. One week after the second vaccination, circulating (left) and spleen (right) SITNFEKL-specific CD 8T cells were measured by multimeric staining. (B) SIINFEKL-specific CD 8T cell TCR viability was measured by ex vivo ELIspot (upper panel). Antigen-specific cytokines produced by CD 8T cells were measured by intracellular staining after ex vivo stimulation with SIINFEKL peptide (bottom panel). (C) One week after the second vaccination Treg in spleen CD 4T cells was measured by flow cytometry (FoxP 3 ⁺ )、Th1(T-bet ⁺ )、Th2(GATA-3 ⁺ ) And a Th1/Th2 ratio. Antigen-specific cytokines produced by CD 4T cells were measured by intracellular staining after ex vivo stimulation with ISQAVHAAHAEINEAGR (OVA-CD 4) peptide. A representative experiment (n=5) is shown with p < 0.05, p < 0.01, p < 0.001 for the mann-whitney test or bi-directional analysis of variance.

Figure 18 shows that example 11 administration of a complex (K) comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist in combination with STING agonist (STING) inhibited B16-OVA tumor growth. Will 10 ⁵ The B16-OVA cells were intravenously injected into C57BL/6 mice. On days 3 and 10 after tumor injection, mice are treated with two administrations of a complex comprising a cell penetrating peptide, at least one antigen or epitope, and a TLR peptide agonist (K), STING agonist (STING), or a combination of both. On day 20, the lungs were perfused to clear blood, the number of tumor metastases was counted and the lung infiltrating lymphocytes were analyzed. (a) vaccination schedule. (B) number of metastasis nodules per lung and representative picture. (C) The frequency of SIINFEKL (OVA) -specific CD 8T cells and the expression of granzyme B in tumor-infiltrating leukocytes were measured by flow cytometry. After ex vivo stimulation with SIINFEKL peptide (SEQ ID NO: 57) in the presence of Golgi inhibitors Antigen-specific cytokine production by CD 8T cells was measured by intracellular staining. Measuring the production of antigen-specific cytokines by intracellular staining; the frequency of cytokines in CD 8T cells is shown. (D) Treg (FoxP 3) was measured by flow cytometry ⁺ ) And a Th1/Th2 ratio. Antigen-specific cytokine production of CD 4T cells was measured by intracellular staining after ex vivo stimulation with ISQAVHAAHAEINEAGR (OVA-CD 4) peptide (SEQ ID NO: 59) in the presence of a Golgi inhibitor. Measuring the production of antigen-specific cytokines by intracellular staining; the frequency of cytokines in CD 4T cells is shown. Two independent experiments (B) or a collection of representative experiments (C-D) are shown (n.gtoreq.7), with Mannich test p < 0.05, p < 0.01, p < 0.001.

FIG. 19 shows the phenotype and function of B16-OVA tumor-bearing mice in example 11 in T cells specific for Zhou Kangyuan. One week after the last treatment, antigen-specific CD 8T cell responses were analyzed in the blood and spleen of mice. (A) The frequency and number of SIINFEKL (OVA) -specific CD 8T cells in circulation were measured by flow cytometry. (B) Spleen cells were stimulated ex vivo with (or without granzyme B) SIINFEKL (OVA) peptide (SEQ ID NO: 57) and cytokines and granzyme B produced by CD 8T cells were measured by intracellular staining. (C) Spleen cells were stimulated ex vivo with ISQAVHAAHAEINEAGR (OVA-CD 4) peptide (SEQ ID NO: 59) and antigen-specific cytokine production was measured by intracellular staining. A representative experiment (n=7) p < 0.05, p < 0.01, p < 0.001 is shown.

Examples

In the following, specific examples are presented that illustrate various embodiments and aspects of the invention. However, the scope of the invention should not be limited by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and practice the present invention. However, the scope of the invention is not limited by the exemplary embodiments, which are intended as illustrations of individual aspects of the invention, and functionally equivalent methods are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description, the accompanying drawings and the examples below. All such modifications fall within the scope of the appended claims.

Method

A mouse

Female C57BL/6J mice were purchased from Charles River Laboratories (L' arbresles, france). All animals used were 6 to 10 weeks of age at the time of the experiment. These studies have been reviewed and approved by institutions and state veterinary authorities in accordance with the swiss federal animal protection agency.

Vaccine

Vaccine constructs were designed and produced by Genscript in E.coli (E.Coli) by itself. Vaccines were prepared by dilution in vaccine buffer and administered by subcutaneous (s.c.) injection of 10 nanomoles in a volume of 100 μl. Z13Mad25Anaxa (SEQ ID NO: 55) contains the CD4 and CD8 epitopes from HPV-16 and was used in a TC-1 tumor model. ATP128 (SEQ ID NO: 54) contains CEA, survivin and ASCL2 epitopes and is used in MC-38 CEA tumor models.

STING agonists

The following STING agonists were used: ADU-S100 (Aduro; also referred to as "STINGa") was used in examples 1 to 7 and 9; and a different STING agonist (called "STING 2") was used in example 8.

Sting (ADU-S100) has the following structural formula (III):

sting 2 has the following structural formula ia.2:

ADU-S100 (Aduro) was resuspended in DMSO and diluted with 1 XPhosphate buffered saline (PBS, gibco) prior to injection. Sting ga 2 was resuspended in 1 x phosphate buffered saline (PBS, gibco) prior to injection.

Cell lines

TC-1 cells derived from a cell line of lung epithelial cells transfected with HPV16E6/E7 and c-H-ras oncogene maintained at RPMI 1640Glutamax supplemented with 10% heat-inactivated Fetal Calf Serum (FCS), 100U/ml penicillin/streptomycin (P/S), 1mM sodium pyruvate, MEM NEAA and 0.4mg/ml geneticin G418 ^tm Is a kind of medium.

MC-38C 57BL/6 mouse colon adenocarcinoma cell lines have been transduced with retroviral constructs containing cDNA encoding the human carcinoembryonic antigen (CEA) gene. CEA expressed by MC-38-CEA-1 clone has a molecular weight of 180kDa, similar to that of natural CEA. MC-38-CEA-1 clones as used herein express high levels of CEA on their cell surfaces (Hand et al 1993,Cancer Immunol Immunother.36:65-75). MC-38-CEA-1 cells (MC-38-CEA-1 culture-190115 for tumor transplants) were cultured in DMEM medium 1640glutamax (Life technologies) with 10% high inactivated fetal bovine serum (Life technologies) using standard laboratory techniques.

The B16-OVA cell line is provided by Bertrand Huard at the university of Klebur-Alps, french. The cell line was derived from murine melanoma cells transfected with OVA maintained at RPMI 1640 Glutamax supplemented with 10% heat-inactivated Fetal Calf Serum (FCS), 100U/ml penicillin/streptomycin (P/S), 1mM sodium pyruvate, MEM NEAA and 1mg/ml geneticin G418 ^tm Is a kind of medium.

In vivo tumor experiments

Subcutaneous implantation of the back of C57BL/6 mice 1X 10 ⁵ TC-1 tumor cells were collected and mice were graded according to tumor size on day 6 of tumor implantation. Alternatively, C57BL/6J mice were given 1X 10 intravenous injections ⁵ B16-OVA cells. Mice were vaccinated twice (on days 6 and 13 after tumor implantation) by subcutaneous injection of 10 nanomolar vaccine at the bottom of the tail. While vaccinating, mice received 25 μg of STING agonist, administered by 2×50 μl subcutaneous injection on each side of the lower back. Alternatively, while vaccinating, mice received 10 μg of STING agonist 2, administered by 2×100 μl subcutaneous injection on each side of the lower back.

Alternatively, the female C57BL/6J mice were subcutaneously transferred on their backsPlant 5×10 ⁵ MC38-CEA tumor cells and vaccinated twice at the bottom of the tail (on days 6 and 13 after tumor implantation) by subcutaneous injection of 10 nanomolar vaccine, 25 μg STINGa (ADU-S100) or a combination of both.

Measuring tumor size with diameter measuring instrument when tumor volume reaches 1000mm ³ At this time, mice were euthanized. Tumor volumes were calculated using the following formula:

v=length×length×wide bottom×pi/6

Mice bearing B16-OVA tumors were sacrificed on day 20, lungs were perfused with saline solution and the number of lung metastases counted.

Cell preparation

Bone marrow derived DCs (BMDCs) were prepared from C57BL/6 mice by extracting bone marrow from tibia and femur and culturing the DCs in BMDC medium (DMEM glutamine supplemented with 10% FCS, 100U/ml P/S, 50. Mu.M beta. -mercaptoethanol, 10mM HEPES, 0.116mg/ml L-arginine, MEM NEAA, and 10ng/ml GM-CSF). At 37℃with 5% CO ₂ After the next 3 days, half the volume of fresh medium was added. On day 6, floating cells were recovered, resuspended in BMDC medium and cultured alone. BMDCs were taken on day 9 and used for T cell stimulation in vitro.

TC-1 tumors were excised at day 20 post-implantation and tumor-infiltrating leukocytes (TILs) were purified using a Miltenyi tumor dissociation kit according to the manufacturer's instructions. Briefly, tumor tissues were cut into small pieces and resuspended in DMEM medium containing tumor dissociating enzymes (Miltenyi). The solid tumor procedure was used to digest the tumor on gentle MACS (Miltenyi) with a heating system. Enzyme digestion was stopped by adding cold PBS 0.5% bsa solution and keeping the cells on ice. The digested tumor was passed through 70 μm to remove remaining undigested tissue. Cd45+ cells were purified using CD45TIL microbeads (Miltenyi) according to the manufacturer's protocol. Purified cd45+ cells were used for flow cytometry staining or ex vivo T cell stimulation.

Prior to collection, B16-OVA tumor bearing mice were perfused with saline solution to remove blood from the lungs. Lung Infiltrating Leukocytes (LILs) were purified using Miltenyi's mouse tumor dissociation kit according to the manufacturer's instructions.

Peripheral blood and spleen mononuclear cell suspensions from mice were isolated using Ficoll-Paque gradient (GE Healthcare) prior to flow cytometry analysis, in vitro stimulation, or TCR viability assays.

Ex vivo T cell restimulation

TIL, LIL or spleen cells were counted and seeded 1X 10 under each condition, respectively ⁵ Or 2X 10 ⁶ Individual cells. Cells were incubated with HPV-CD4, HPV-CD8, OVA-CD8 or OVA-CD4 epitope peptide for 6 hours in the presence of Golgi stop (BD biosciences) and anti-CD 107a, with PMA/ionomycin as positive control or without any stimulators as negative control. After washing, cells were stained with cell surface antigen and fixable reactive dye, and then, after fixation and permeabilization according to the manufacturer's instructions (BD biosciences), cells were stained with intracellular cytokines.

In vivo cytotoxicity assay

Untreated spleen cells were taken and incubated for 1.5 hours at 37℃in DMEM complete medium with or without HPV-E7 CD8 epitope peptide (SEQ ID NO: 56). The loaded and unloaded spleen cells were then stained with the Cell Tracer Violet (CTV) or CFSE (both from ThermoFisher Scientific), respectively, as per the manufacturer's instructions. The spleen cells were then mixed in a 1:1 ratio and a total of 5X 10 by intravenous injection ⁶ Individual cells were transferred to pre-vaccinated mice. After 20 hours of cell transfer, spleen cells were taken and survival of CTV or CFSE stained cells was assessed by flow cytometry. The percentage of antigen-specific killing was calculated using the following formula: % antigen-specific killing = (1- (vaccinated peptide) ⁺ Peptide ^- Ratio of (2) to untreated peptide ⁺ Peptide ^- Ratio of (x) 100).

In vitro TCR viability assay

One week after the second vaccination, spleens were excised and spleen cells were isolated (see above). Will be 1X 10 ⁶ Individual cells/wells were seeded in IFN-. Gamma.ELIspot plates and O/N was stimulated with reduced concentrations of RAHYNIVTF (SEQ ID NO: 56) or SIINFEKL (SEQ ID NO: 57) peptides. The ELIspot plate was then displayed according to the manufacturer's instructions and calculatedPercent maximum response relative to the highest concentration of stimulating peptide.

Antibody and flow cytometry

The following antibodies were used: CD45 (30-F11), CD11B (M1/70), KLRG1 (2F 1), CD103 (M290), NKG2a (20 d 5), ly6C (AL-21), ly6G (1A 8), PD-L1 (MIH 5), I-A/I-E (M5/114), CD11C (HL 3), PDCA1 (927), CD64 (X54-5/7.1), B220 (RA 3-6B 2), CD24 (M1/69), CD4 (GK 1.5), CD25 (3C 7), CD3 (500A 2), NKp46 (29A1.4), TNF-alpha (MP 6-XT 22), IFN-gamma (XMG 1.2), H2-KB (AF 6-88.5) and H2-Db (28-14-8) are from BD biosciences; tim3 (RMT 3-23), PD-1 (29 F.1A12), CD38 (90), gr-1 (RB 6-8C 5), CD206 (C068C 2), CD68 (FA-11) from BioLegend; ki67 (solA 15), foxP3 (FJK-16 s), T-bet (4B 10), GATA-3 (TWAJ) and ROR γt (AFKJS-9) are from ThermoFisher Scientific; granzyme B (REA 226) is from Miltenyi; CD8 (KT 15) is from MBL. DEAD cells were stained with LIVE/DEAD yellow or aqueous fluorescent reactive dye (Life Technologies) and excluded from analysis. Murine MHC peptide multimers were from Immulex (Coben Harroot, denmark). Cells were analyzed using an Attune NxT flow cytometer (ThermoFisher Scientific) Kaluza (Beckman Coulter) software.

Quantitative detection of serum interferon-alpha

Blood was collected from the tail vein of the mice and serum was centrifuged using a Starstedt tube. The concentration of IFN- α cytokines was measured using a commercial ELISA kit according to the manufacturer's recommendations (PBL Assay Science).

Statistical analysis

Statistical analysis was performed using Prism software (GraphPad), and if p < 0.05, statistical significance was considered.

Example 1: combination of STING agonist and vaccine complex modulates T cell response

In preclinical tumor models and ongoing clinical trials, STING agonists are typically administered by intratumoral (i.t.) injection to stimulate the Tumor Microenvironment (TME). In contrast, in this experiment, systemic administration of STING agonists in combination with a complex comprising a cell penetrating peptide, at least one antigen or epitope and at least one TLR peptide agonist was studied.

To assess the immunogenicity of this combination, tumor-free C57BL/6 mice were vaccinated (on days 0 and 14) twice at 2 week intervals by subcutaneous injection of 10 nanomolar Z13Mad25Anaxa (an exemplary complex comprising a cell penetrating peptide, at least one antigen or epitope, and a TLR peptide agonist comprising Human Papillomavirus (HPV) -derived CD4 and CD8 epitopes). At about the same time as vaccination, mice received 25 μg of STING agonist ADU-S100, administered by subcutaneous injection at 2×50 μl on each side of the lower back. Serum was collected 4 hours and 24 hours after the first vaccination and IFN- α concentration was determined by ELISA. Whole blood was collected on day 21 and stained by multimeric flow cytometry for antigen-specific CD 8T cell measurement. Meanwhile, spleen was taken and analyzed by flow cytometry for ex vivo stimulation and intracellular cytokine production. Alternatively, spleen cells are used for TCR viability assays.

The schedule and results are shown in figure 1. Fig. 1A shows the schedule of the experiment. Unlike vaccination with a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist, this complex induces only local inflammation, systemic treatment with STINGa induces a potent but transient systemic type I interferon response, characterized by high IFN- α serum levels that peak at 4 hours after injection and have declined after 24 hours (fig. 1B). The systemic response is not affected by concomitant injection of a complex comprising a cell penetrating peptide, at least one antigen or epitope, and a TLR peptide agonist. Although vaccination with Z13Mad25Anaxa was able to elicit circulating HPV-E7-specific CD 8T cells, the data showed that the combination with sting treatment further increased the frequency of antigen-specific CD 8T cells (fig. 1C). Furthermore, sting treatment resulted in a higher proportion of spleen CD 8T cells after re-stimulation with isolated PMA/Luo Numei, increased cytokine secretion, indicating better cell function (fig. 1D-E). In addition, the combination of a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist with a STING agonist also modulated the CD 4T cell response, slightly increasing the proportion of spleens and deeply altering their polarization. Indeed, significantly higher proportions of helper T cells 1 (Th 1) and Thl7 and lower proportions of Treg and Th2 CD 4T cells were found in the combination treated mice, resulting in positive Th1/Th2 and Th17/Th2 ratios (fig. 1F-K). In summary, a combination of a complex comprising a cell penetrating peptide, at least one antigen or epitope of an antigen and a TLR peptide agonist with a STING agonist enhances CD4 and CD 8T cell responses, enhancing antigen specific CD 8T cells. In addition to their frequency, combination therapy of complexes comprising cell penetrating peptides, at least one antigen or epitope and TLR peptide agonists with STINGa also highly enhances the effector function of antigen-specific CD 8T cells. In vivo killing experiments performed one week after inoculation showed a significant 2.5-fold increase in antigen-specific cytotoxicity in mice treated with a combination comprising cell penetrating peptide, at least one antigen or epitope of antigen and TLR peptide agonist, and STINGa (fig. 1L). Furthermore, ex vivo stimulation with reduced concentrations of HPV-CD8 peptide showed significantly higher TCR viability for complex-STINGa sensitized T cells (fig. 1M).

Example 2: safety and tolerability of combined administration of STING agonists and vaccine complexes

Systemic injection of STING agonists resulted in an effective systemic type I interferon response. Since this may lead to adverse side effects, the safety and tolerability of combined administration of STING agonists and vaccine complexes was investigated.

For this, 10 was transplanted on the back of C57BL/6 mice ⁵ And TC-1 tumor cells. When the tumor is visible, the mice are treated with two administrations: (i) A complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist (Z13 Mad25 Anaxa); (ii) STING agonists; or (iii) a combination of both, spaced one week apart, substantially as described in example 1. Body temperature and body weight of the mice were measured at the time points shown in fig. 2.

The results are shown in FIG. 2. Shortly after administration to TC-1 tumor-bearing mice, neither single nor combination treatments caused significant changes in body temperature (fig. 2A) or body weight (fig. 2B), confirming the safety and tolerability of combined administration of STING agonists and complexes comprising cell penetrating peptides, at least one antigen or epitope of antigen, and TLR peptide agonists.

Example 3: combination of STING agonist and vaccine complex improves antigen-specific CD8 in TC-1 tumor bearing mice T cell response

TC-1 is a well-known cold tumor model characterized by very low infiltration rates of CD4 and CD 8T cells. To investigate the effect of administering a STING agonist and a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist (Z13 Mad25 Anaxa), antigen specific CD 8T cell responses were assessed in a TC-1 cold tumor model.

For this, 10 was transplanted on the back of C57BL/6 mice ⁵ And TC-1 tumor cells. When the tumor is visible, the mice are treated with two administrations: (i) A complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist (Z13 Mad25 Anaxa); (ii) STING agonists; or (iii) combinations thereof, spaced one week apart, substantially as described in example 1. One week after the last treatment, mice were sacrificed, tumors were excised, and analyzed for the presence and phenotype of CD 8T cells by FACS staining.

TC-1 tumor cells were low in immunogenicity, and very low ratios and numbers of circulating HPV-specific CD 8T cells were found in vehicle-treated mice (FIG. 3). Similar to observations in tumor-free mice, vaccination with a complex comprising a cell penetrating peptide, at least one antigen or epitope of an antigen, and a TLR peptide agonist significantly increases the peripheral HPV-specific response, while sting monotherapy is not effective, the combination therapy increases the number of antigen-specific CD 8T cells.

Next, the ability of HPV-specific CD 8T cells to infiltrate TC-1 tumors was investigated. TC-1 is a well known cold tumor model, finding very few total or HPV-specific CD 8T cells in control tumors, whether in proportion-their proportion to tumor infiltrating leukocytes is below 1% -or total (FIGS. 4A-D). Vaccination with a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist induces a significant increase in CD 8T cell tumor infiltration, of which more than 50% are HPV-specific. Notably, HPV-specific CD 8T cells were present in large numbers in tumors, although the percentage in blood was quite low, indicating that the measurement of peripheral responses was only partially predictive of intratumoral results (fig. 3A-B). Sting monotherapy did not modulate CD 8T cell tumor infiltration nor HPV-specific ratios, and thus was different from the results observed in the spleen of tumor-free mice (fig. 1). Interestingly, the combination of a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist with a STING agonist showed a synergistic effect, increasing CD 8T cell infiltration and HPV-specific ratio. This demonstrates that systemic administration of STING agonists can modulate the intratumoral effects of a complex comprising a cell penetrating peptide, at least one antigen or epitope of an antigen, and a TLR peptide agonist.

Furthermore, the functionality of tumor-infiltrating HPV-specific CD 8T cells was monitored by measuring expression of ifnγ, TNFo and degranulation marker CD107 α following in vitro stimulation with bone marrow-derived dendritic cells (BMDCs) loaded with HPV peptides, and significant increases in HPV-specific cytokine production and degranulation of CD 8T cells were found in mice treated with a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist compared to the control group or the STINGa monotherapy group (fig. 5). Interestingly, vaccination with a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist increases in particular the proportion of multifunctional CD 8T cells which are capable of producing ifnγ, tnfα and/or CD107 α simultaneously. The combination with sting further increases the function of CD 8T cells and more importantly the frequency of multifunctional cells.

To further characterize the function of tumor-infiltrating T cells, intracellular production of granzyme B (GzB) was also measured after a short in vitro TIL culture in the presence of Golgi inhibitors, as GzB is one of the primary weapons of CD 8T cells to eliminate cancer cells. The frequency and number of total or HPV-specific CD 8T cells producing GzB was found to be significantly higher in mice vaccinated with complexes comprising cell penetrating peptide, at least one antigen or epitope and TLR peptide agonist compared to vehicle or STINGa treatment (fig. 6). Although the frequency of GzB positives in HPV-specific CD 8T cells was not altered, combination with STINGa further increased their total number. Importantly, in contrast to the intratumoral compartments, very low frequencies of splenic HPV-specific CD 8T cells producing cytokines or GzB were observed in all different treatments (fig. 7), demonstrating that CD 8T cells are not systemically activated.

The efficacy of cancer-specific T cells is often limited by tumor-induced failure. Thus, the expression of activation and depletion markers on intratumoral and peripheral CD 8T cells was subsequently analyzed. T cell failure is a gradual process, ultimately leading to loss of cell function, which can be monitored by the gradual expression of failure markers. While most of the tumor-infiltrating CD 8T cells expressed only PD-1 or none of the depletion markers at all in control and STINGa single treated mice, most of the CD 8T cells, particularly HPV-specific cells, expressed the depletion markers PD-1 and Tim-3 in mice vaccinated with a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist or in combination treated mice (fig. 8A-B). Interestingly, in the combination group, a lower proportion of CD 8T cells co-expressed PD-1 and Tim-3, indicating a less depleted phenotype, which correlates with a higher proportion of cytokine-secreting cells. In addition, a higher proportion of mice vaccinated with complexes comprising cell penetrating peptide, at least one antigen or epitope, and TLR peptide agonist showed CD 8T cells that also expressed CD38 (fig. 8C-D), NKg2a (fig. 8E-F), and TCF-1 (fig. 8G-H), which are other markers associated with reduced T cell function. FIG. 8I shows the co-expression of PD1, granzyme B and TCF-1 on HPV-specific CD 8T cells.

Similar to the functional analysis, peripheral CD 8T cells showed a less depleted phenotype, most cells expressed only PD-1, and some cells still expressed the early activation marker KLRG1 (fig. 9), indicating that depletion was obtained within TME.

In summary, vaccination with a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist highly increases HPV-specific CD 8T cell tumor infiltration and functionality, while sting ga monotherapy is not effective, combination therapy further enhances the effect of a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist. However, intratumoral CD 8T cells have a partially depleted phenotype that is less pronounced in combination treated mice than mice vaccinated with a complex comprising a cell penetrating peptide, at least one antigen or epitope, and a TLR peptide agonist alone.

Example 4: combination of STING agonist and vaccine complex modulates intratumoral CD 4T cell response

Because of regulatory T cells (tregs), CD 4T cells are generally considered to be immunosuppressive cells only, and immunotherapy studies have focused extensively on CD 8T cells, ignoring CD 4T cells. However, recent studies underscore the importance of CD 4T cells, particularly Th1 and Th17 polarization, for developing appropriate anti-Tumor CD 8T cell responses (Melssen, M. And C.L.Slingluff, jr. (2017), "Vaccines targeting helper T ceHs for cancer immunotherapeutic.": curr Opin Immunol 47:85-92; muranski, P. Et al (2008), "Tumor-specific Th17-polarized ceHs eradicate large established melanoma." Blood 112 (2): 362-373).

In view of this, CD4T cells within the tumor were next monitored. For this purpose, the TC-1 tumor model described in example 3 was used.

The results are shown in FIG. 10. The data show that tumor infiltration of CD4T cells was significantly increased in mice treated with a combination comprising cell penetrating peptide, at least one antigen or epitope, and a TLR peptide agonist (Z13 Mad25 Anaxa) and STING agonist (STING) compared to all other groups (fig. 10A-B). Unlike what was observed in the spleen, STINGa monotherapy had no effect on intratumoral CD4T cell recruitment. Interestingly, this increased CD4T cell infiltration was not caused by tregs, as their percentage was greatly reduced in the combination treated mice, but rather by effector CD4T cells (fig. 10C, D and G). The ratio between intratumoral CD8 and total or regulatory CD4T cells is typically used as a predictor of TME immune status. Vaccination with a complex comprising a cell penetrating peptide, at least one antigen or epitope of an antigen, and a TLR peptide agonist induced a higher CD8/CD 4T cell ratio compared to the vehicle or sting treatment group (fig. 10E). Although combination therapy produced a CD8/CD 4T cell ratio similar to vaccination with a complex comprising a cell penetrating peptide, at least one antigen or epitope, and a TLR peptide agonist, it induced a significantly higher CD8/Treg ratio (fig. 10F), highlighting the lower immunosuppressive TME. Further analysis showed that in the combination treated mice, most of the intratumoral CD4T cells were T-bet+Th1, while only the smallest fraction was GATA-3+Th2 cells, resulting in a positive Th1/Th2 ratio (FIG. 10H-J). In contrast to the spleen, a slight decrease in intratumoral Th17 CD4T cells was observed in the combination treated mice (fig. 10K). Because Th1 CD4T cells are generally characterized by IFN-gamma and TNF-alpha production, cytokine production was measured by flow cytometry after ex vivo restimulation with HPV peptide-loaded BMDC. However, in contrast to CD 8T cells, no production of IFN- γ or TNF- α by intratumoral CD4T cells was detected (fig. 10L).

Example 5: the combination of STING agonist and vaccine complex modulates Tumor Microenvironment (TME)

Although T cells are the primary target of immunotherapy due to their ability to directly kill cancer cells, TMEs are a very complex network of different immune cell types capable of promoting or inhibiting cancer growth. In view of this, the composition of TMEs was deeply profiled to obtain a complete overview of their immune status. For this, a TC-1 tumor model was used as described above.

The results are shown in FIG. 11. As previously described, TC-1 is a cold tumor model characterized by very low CD4 and CD 8T cell infiltration, the sum of which accounts for less than 2% of tumor infiltrating cd45+ cells in vehicle-treated mice (fig. 11A). The most prominent cell type is Tumor Associated Macrophages (TAMs), accounting for up to 75% of infiltrates, particularly immunosuppressive TAM2, which is associated with promotion of tumor growth in different cancer types. Myeloid Derived Suppressor Cells (MDSCs), whose role is less clear and which are associated with tumor promotion or control depending on the type of cancer, account for another 15%, monocyte type (MDSCs) predominate. Other cell types found less frequently were dendritic cells (DC, 7%), B cells (2%), NK and NKT cells (1.5%) and neutrophils (1%). Vaccination with a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist induces a dramatic change in TME, characterized by a strong increase in CD 8T cells and DC frequency and the appearance of non-Treg CD 4T cells. Interestingly, the increase in DC infiltration of dendritic cells was also characterized by an increase in the proportion of monocytic dendritic cells (moDC) (fig. 11B), a special DC phenotype, which has been described as differentiating only under inflammatory conditions and has been shown to activate an anti-tumor T cell response (Kuhn, s. Et al (2015), "Monocyte-Derived Dendritic Cells Are Essential for CD (+) T Cell Activation and Antitumor Responses After Local immunology.," Front Immunol 6:584). Although the TAMl compartment remained largely unchanged, TAM2 frequency was significantly reduced, resulting in a higher TAM1/TAM2 ratio (fig. 11C-E). In contrast, by seeding with a complex comprising a cell penetrating peptide, at least one antigen or epitope, and a TLR peptide agonist, the frequency of MDSCs was increased, while granulocyte MDSCs remained essentially unchanged (fig. 11F-G). The opposite effects on TAM2 and mdsc indicate a possible cell repolarization, since both populations of monocyte origin are known for their plasticity and ability to change differentiation state depending on the circumstances.

Similar to observations of CD 8T cell infiltration and phenotype, systemic administration of STINGa alone did not affect TME composition, which was essentially the same as vehicle-treated mice. However, in combination therapy STING agonists show a synergistic effect with vaccination with complexes comprising cell penetrating peptide, at least one antigen or epitope and TLR peptide agonist, further expanding CD8 and non Treg CD 4T cell infiltration by a factor of 2.5 while reducing TAM2 frequency, resulting in a more inflammatory environment.

Example 6: combination of STING agonist and vaccine complex modulates intratumoral expression of PD-L1 and MHC

The PD-1/PD-L1 axis is the primary pathway leading to T cell failure, thereby inhibiting the anti-tumor effect of antigen-specific CD 8T cells, and PD-L1 expression on tumor cells has been shown to be up-regulated upon intratumoral treatment with STING agonists. Furthermore, down-regulation of MHC-I expression on tumor cells is one of the main mechanisms of immune evasion. Thus, as described above, intratumoral expression of PD-L1 and MHC-I and MHC-II was also monitored in the TC-1 tumor model.

The results are shown in FIG. 12. Increased PD-L1 expression was found when treated with a complex comprising a cell penetrating peptide, at least one antigen or epitope thereof, and a TLR peptide agonist (Z13 Mad25 Anaxa) alone or in combination with STINGa compared to vehicle or STINGa subcutaneous monotherapy (fig. 12A-B). The increased expression of PD-L1 in both CD 45-and CD45+ cell compartments highlights that both tumor cells and immune cells can promote T cell failure. The primary immune cell population expressing PD-L1 was identified as two types of TAMs (fig. 12C-D). Furthermore, regarding MHC-I expression on tumor cells, both H2-Kb and H2-Db allele expression are upregulated by tumor cells when treated with a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist alone or in combination with STINGa, as compared to vehicle and STINGa monotherapy (fig. 12E-F), excluding this immune evasion mechanism and indicating that vaccination with a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist may even promote epitope presentation by tumor cells. Also, vaccination with complexes comprising cell penetrating peptide, at least one antigen or epitope of antigen and TLR peptide agonist alone or in combination with STINGa increases MHC-II expression on cd11b+ cells compared to vehicle and STINGa monotherapy (fig. 12G-H), thereby promoting presentation of the epitope to CD 4T cells.

Taken together, these results underscores the deep modulation of TME induced by vaccination with a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist, which is able to convert a cold tumor to heat, and the synergistic effect of STING agonist treatment in combination with the vaccine complex, which further increases intratumoral immunogenicity, although no effect was observed with STING agonist monotherapy.

Example 7: antitumor effects of STING agonist and vaccine complex combinations

Next, the anti-tumor therapeutic effect of a combination comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist, and a STING agonist is evaluated in a TC-1 tumor model.

Back transplantation 10 in C57BL/6 mice ⁵ TC-1 cells. When the tumor is visible, in the therapeutic setting of the TC-1 tumor model, at weekly intervals using (i) a complex comprising a cell penetrating peptide, at least one antigen or epitope, and a TLR peptide agonist (Z13 Mad25 Anaxa); (ii) STING agonists; or (iii) a combination of both, the mice are treated twice as described above.

The results are shown in FIG. 13. In the TC-1 model, two vaccinations with complexes comprising cell penetrating peptide, at least one antigen or epitope and TLR peptide agonist resulted in a significant delay in tumor progression and an increase in median survival (fig. 13A-B). Although sting monotherapy has only a small effect on tumor growth, its combination with a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist significantly increases this effect, thereby significantly delaying tumor progression and increasing median survival.

Example 8: antitumor effects of vaccine complexes in combination with different STING agonists

Next, the anti-tumor therapeutic effect of a combination comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist (Z13 Mad25 Anaxa) with different STING agonists was evaluated in a TC-1 tumor model. STING agonist STING 2 was used instead of STING agonist ADU-S100 (Aduro) used in the above experiments.

Similar to that described above, will be 10 ⁵ The TC-1 cells were transplanted into the back of C57BL/6 mice and divided into different groups (control (untreated), complexes comprising cell penetrating peptide, at least one antigen or epitope and TLR peptide agonist (Z13 Mad25 Anaxa), STING agonist STING Ga 2, and combinations thereof. For treatment with a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist (Z13 Mad25 Anaxa), mice were injected subcutaneously 10 nanomolar on day 6 (when tumor is visible) and on day 13 after tumor implantation with a cell penetrating peptide, at least one antigen or epitope, and TLR peptideThe agonist complex (Z13 Mad25 Anaxa) was vaccinated. For treatment with STING agonist STING ga 2, mice were systemically (subcutaneously) administered 10 μg of STING agonist STING ga 2 on days 6, 10, 13 and 17. Whole blood was collected on day 20 and stained by multimeric flow cytometry for antigen-specific CD 8T cell measurement.

As shown in fig. 14, monotherapy with a complex comprising a cell penetrating peptide, at least one antigen or epitope, and a TLR peptide agonist increased survival and reduced tumor growth, while STING agonist monotherapy provided only a slight improvement. However, the combination of the two showed a synergistic effect of significantly increasing survival and decreasing tumor growth. This demonstrates the results of systemic administration of different STING agonists as described in example 7 above.

As shown in fig. 15, HPV-specific CD 8T cells capable of eliciting circulation are seeded with a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist. However, the combination with STING agonist treatment further increased the frequency of antigen specific CD 8T cells, confirming the results of the different STING agonists described in example 1.

Example 9: immunogenicity of ATP128 in mice; the combination of sting ga and ATP128 induces CEA-specific CD8 T cell response

Subcutaneous implantation of 5X 10 in the dorsal part of female C57BL/6J mice ⁵ And (3) MC38-CEA tumor cells. Mice were vaccinated with 10 nanomolar ATP128, 25 μg sting agonist (ADU-S100), or a combination of both, on days 6 and 13 post tumor implantation. Both the ATP128 vaccine and STING agonist were subcutaneously injected at the bottom of the tail. One week after the second vaccination, mouse blood was collected from the tail vein and the frequency and total number of CEA-specific CD 8T cells was analyzed by flow cytometry using custom designed multimers.

The results are shown in FIG. 16. IFN-g Elispot assays were performed one week after the third vaccination with ATP 128. ATP128 vaccination elicited CEA-specific CD 8T cells, which could be monitored by multimeric staining (fig. 16A). One week after the second vaccination, multimeric staining was performed. The data show that addition of STING agonist to ATP128 enhances CEA-specific CD 8T cell responses (fig. 16b, c).

Example 10: combination of STING agonist and vaccine complex enhances CD 8T and CD 4T refinement in tumor-free mice Function of extracellular peripheral response

The immunogenicity of the combination was evaluated in tumor-free C57BL/6 mice similarly to example 1, but using a different complex (Z13 Mad39Anaxa; SEQ ID NO: 58). Briefly, tumor-free C57BL/6 mice were vaccinated twice (on days 0 and 7) at weekly intervals with subcutaneous injections of 10 nanomoles of Z13Mad39Anaxa (SEQ ID NO:58, an exemplary complex comprising a cell penetrating peptide, at least one antigen or epitope, and a TLR peptide agonist, serum was collected 4 hours and 24 hours after the first vaccination, and IFN- α concentrations were determined by ELISA, whole blood was collected on day 14, and splenocytes for ex vivo stimulation and intracellular cytokine production were analyzed by flow cytometry for spleen cell or spleen cell viability determination by flow cytometry.

The results are shown in fig. 17. Circulating (left) and spleen (right) SIINFEKL-specific CD 8T cells were measured by multimeric staining one week after the second vaccination, as shown in figure 17A. Figure 17B shows SIINFEKL-specific CD 8T cell TCR viability measured by ex vivo ELIspot (upper panel) and antigen-specific cytokine production by CD 8T cells measured by intracellular staining after ex vivo stimulation with SIINFEKL peptide (lower panel). FIG. 17C shows Treg (FoxP 3) in spleen CD 4T cells ⁺ )、Th1(T-bet ⁺ ) And Th2 (GATA-3) ⁺ ) Is a ratio of Th1/Th2 (measured by flow cytometry one week after the second vaccination). In addition, antigen-specific cytokine production by CD 4T cells by use of ISQAVHA was shownAHAEINEAGR (OVA-CD 4) peptide (SEQ ID NO: 59) was measured by intracellular staining after in vitro stimulation.

In summary, similar modulation of CD8 and CD 4T cell responses was observed as in example 1 using different complexes (Z13 Mad39Anaxa containing CD4 and CD8 epitopes derived from Ovalbumin (OVA) in this example and Z13Mad25Anaxa containing HPV-E7 epitopes in example 1). This confirms that the modulation of T cell responses is independent of antigen cargo. Z13Mad39Anaxa vaccination elicited multifunctional CD8 and CD4 antigen-specific T cells that produced IFNγ and TNFα upon ex vivo stimulation with specific peptides (FIG. S2). In summary, the addition of STING agonists to complexes comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist profoundly affects the frequency and quality of CD 8T cell responses and polarization of CD 4T cells to Th 1.

Example 11: combination of STING agonist and vaccine complex inhibits B16-OVA tumor growth

The anti-tumor effect of a combination comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist (Z13 Mad39 Anaxa) and STING agonist (STING) was then evaluated in a B16-OVA lung metastasis model.

Briefly, 10 will be ⁵ The B16-OVA cells were intravenously injected into C57BL/6 mice. Starting three days after tumor cell intravenous injection, mice were vaccinated at weekly intervals with two exemplary complexes comprising a cell penetrating peptide, at least one antigen or epitope, and a TLR peptide agonist (Z13 Mad39Anaxa; SEQ ID NO: 58), a STING agonist (STING Ga), or a combination of both. On day 20 (10 days after the last vaccination), the lungs were perfused to clear blood, the number of lung metastases was counted, and Lung Infiltrating Lymphocytes (LILs) were analyzed.

The results are shown in FIGS. 18 and 19. Fig. 18A shows an experimental schedule. The number of metastasis nodules per lung shown in fig. 18B indicates that Z13Mad39Anaxa vaccination significantly reduced the number of metastases, while sting monotherapy was not effective, in combination with KISIMA significantly further reduced the number of metastases. In addition, the presence and function of Lung Infiltrating Lymphocytes (LILs) were analyzed by flow cytometry. Vaccination induced multifunctional OVA-specific CD 8T cell infiltration characterized by expression of granzyme B (GzB), ifnγ and tnfα (fig. 18C), which was significantly increased in the STINGa combination. Similar low-amplitude increases in T cell phenotype and functionality were observed in the periphery (blood and spleen), indicating the general recruitment of antigen-specific T cells to tumor sites, as shown in fig. 19A and B. As observed in tumor-free mice (example 10), a complex comprising a cell penetrating peptide, at least one antigen or epitope and a TLR peptide agonist (Z13 Mad39Anaxa; SEQ ID NO: 58) and STING agonist (STING) modulate the polarization of CD 4T cells within the tumor, reducing the presence of tregs while increasing the Th1/Th2 ratio (fig. 18D). Ex vivo stimulation with OVA peptide highlighted the presence of functional antigen-specific CD 4T cells in the spleen but not in the lung, suggesting that helper CD 8T cell responses occurred ubiquitously in secondary lymphoid organs (fig. 18D and 19C).

Taken together, these results demonstrate that combination therapy comprising a cell penetrating peptide, at least one antigen or epitope of an antigen, and a TLR peptide agonist, with a STING agonist promotes intratumoral infiltration of antigen specific effector CD 8T cells and function of peripheral CD 4T cells, thereby inhibiting growth of B16-OVA tumors.

Sequence and SEQ ID numbering table (sequence Listing):

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Claims

1. a combination of (i) a STING agonist and (ii) a complex,

the complex comprises:

a) Cell penetrating peptide;

b) At least one antigen or epitope; and

c) A TLR peptide agonist, which is a compound of formula (i),

wherein the components a) to c) comprised by the complex are covalently linked.

2. The combination according to claim 1, wherein the complex is a (recombinant) polypeptide or a (recombinant) protein.

3. The combination of any one of the preceding claims, wherein the cell penetrating peptide

(1) The length of the amino acid sequence of the peptide having a total of 5 to 50 amino acids, preferably a total of 10 to 45 amino acids, more preferably a total of 15 to 45 amino acids; and/or

(2) Having an amino acid sequence comprising a fragment of the minimum domain of ZEBRA, or a variant thereof, said minimum domain being selected from the group consisting of: 3 to residue 220, wherein, optionally, 1, 2, 3, 4 or 5 amino acids have been substituted, deleted and/or added without losing the cell penetrating ability of the peptide.

4. A combination according to claim 3, wherein the cell penetrating peptide has a sequence comprised in a sequence equivalent to the sequence according to SEQ ID NO:3, ser (S) at position 189 of the ZEBRA amino acid sequence of seq id no.

5. The combination of claim 3 or 4, wherein the amino acid sequence of the cell penetrating peptide comprises:

according to the following general formula (A):

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

X ₂ r, K or H, preferably X ₂ R or K;

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

X ₄ k, R or H, preferably X ₄ Is K or R;

X ₅ is N or Q;

X ₆ r, K or H, preferably X ₆ R or K;

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

X ₈ a, V, L, I or G, preferably X ₈ Is A or G;

X ₉ is S or T;

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

X ₁₁ k, R or H, preferably X ₁₁ Is K or R;

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

X ₁₄ a, V, L, I or G, preferably X ₁₄ Is A or G;

X ₁₅ 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 F, Y or W; and

X ₁₇ k, R or H, preferably X ₁₇ Is K or R.

6. The combination according to any one of claims 3 to 5, wherein the cell penetrating peptide has a sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 4 to 13, or a sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity without losing the cell penetrating ability of the peptide.

7. The combination of claim 6, wherein the cell penetrating peptide has a sequence comprising or consisting of a sequence according to SEQ ID NO:6 (CPP 3/Z13), SEQ ID NO:7 (CPP 4/Z14), SEQ ID NO:8 (CPP 5/Z15) or SEQ ID NO:11 (CPP 8/Z18), or a sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity without losing the cell penetrating ability of the peptide; preferably, the cell penetrating peptide has a sequence comprising or consisting of a sequence according to SEQ ID NO:6 (CPP 3/Z13), or a sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity without losing the cell penetrating ability of the peptide.

8. The combination according to any one of the preceding claims, wherein at least one antigen or epitope of an antigen is a peptide, polypeptide or protein.

9. The combination according to any one of the preceding claims, wherein the complex comprises more than one antigen or epitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 antigens or epitopes.

10. The combination according to claim 9, wherein more than one antigen or epitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 antigens or epitopes, are located consecutively in the multi-antigen domain of the complex.

11. The combination according to any one of the preceding claims, wherein at least one antigen or epitope is at least one CD4 ⁺ Epitope and/or at least one CD8 ⁺ An epitope.

12. The combination according to any one of the preceding claims, wherein at least one antigen or epitope comprises or consists of at least one tumor or cancer epitope.

13. The combination according to claim 12, wherein at least one tumor epitope is selected from the following tumors: endocrine tumors, gastrointestinal tumors, genitourinary tumors, gynecological tumors, breast cancer, head and neck tumors, hematopoietic tumors, skin tumors, breast tumors, and respiratory tumors.

14. The combination according to claim 12 or 13, wherein at least one tumor or cancer epitope is selected from the following tumors or cancers: gastrointestinal tumors, including anal cancer, appendiceal cancer, cholangiocarcinoma, carcinoid tumor, gastrointestinal colon cancer, extrahepatic cholangiocarcinoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), hepatocellular carcinoma, pancreatic cancer, rectal cancer, colorectal cancer, or metastatic colorectal cancer.

15. The combination according to any one of the preceding claims, wherein at least one antigen or epitope is selected from a tumor-associated antigen, a tumor-specific antigen or a tumor neoantigen, preferably wherein at least one antigen or epitope is selected from a tumor-associated antigen, a tumor-specific antigen or a tumor neoantigen of colorectal cancer or metastatic colorectal cancer.

16. The combination according to any one of claims 12 to 15, wherein at least one tumor or cancer epitope 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, TGF βR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART, IL Rα2, ASCL2, NY-ESO-1, MAGE-A3, PRAME and WT1.

17. The combination according to any one of claims 12 to 16, wherein at least one tumor or cancer epitope is an epitope of an antigen selected from the group consisting of: ASCL2, epCAM, MUC-1, survivin, CEA, KRas, MAGE-A3 and IL13 ra 2, preferably at least one tumor epitope is an epitope of an antigen selected from the group consisting of: ASCL2, epCAM, MUC-1, survivin, CEA, KRas, and MAGE-A3, more preferably, at least one tumor epitope is an epitope of an antigen selected from the group consisting of: ASCL2, epCAM, MUC-1, survivin and CEA, even more preferably, at least one tumor epitope is an epitope of an antigen selected from the group consisting of: ASCL2, epCAM, survivin, and CEA.

18. The combination of any one of the preceding claims, wherein the complex comprises a multi-antigen domain comprising epitopes of at least two different antigens.

19. The combination of claim 18, wherein the multi-antigen domain of the complex comprises at least one epitope of survivin.

20. The combination of claim 18 or 19, wherein the multi-antigen domain of the complex comprises a sequence consisting of a sequence according to SEQ ID NO:16, or a fragment thereof of at least 10 amino acids in length, or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

21. The combination according to any one of claims 18 to 20, wherein the multi-antigen domain of the complex comprises a polypeptide having a sequence according to SEQ ID NO:18, or a (functional) sequence variant thereof having at least 70%, 75%, 80% or 85% sequence identity.

22. The combination according to any one of claims 18 to 21, wherein the multi-antigen domain of the complex comprises a sequence consisting of a sequence according to SEQ ID NO:17, or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

23. The combination according to any one of claims 18 to 22, wherein the multi-antigen domain of the complex comprises at least one epitope of CEA.

24. The combination according to any one of claims 18 to 23, wherein the multi-antigen domain of the complex comprises a polypeptide having a sequence according to SEQ ID NO:29, or a fragment thereof of at least 10 amino acids in length, or a peptide thereof having a (functional) sequence variant of at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

25. The combination according to any one of claims 18 to 24, wherein the multi-antigen domain comprises a polypeptide having a sequence according to SEQ id no:32, or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

26. The combination according to any one of claims 18 to 25, wherein the multi-antigen domain of the complex comprises a polypeptide having a sequence according to SEQ ID NO:30 and/or a peptide having an amino acid sequence according to SEQ ID NO:31, and a peptide of the amino acid sequence of seq id no.

27. The combination according to any one of claims 18 to 26, wherein the multi-antigen domain of the complex comprises at least one epitope of ASCL 2.

28. The combination according to any one of claims 18 to 27, wherein the multi-antigen domain of the complex comprises a polypeptide having a sequence according to SEQ ID NO:21, or a fragment thereof of at least 10 amino acids in length, or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

29. The combination according to any one of claims 18 to 28, wherein the multi-antigen domain of the complex comprises a sequence consisting of a sequence according to SEQ ID NO:24, or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

30. The combination according to any one of claims 18 to 29, wherein the multi-antigen domain comprises a polypeptide consisting of a polypeptide according to SEQ ID NO:22 and/or having an amino acid sequence according to SEQ ID NO:23, and a peptide of the amino acid sequence of 23.

31. The combination according to any one of claims 18 to 30, wherein the multi-antigen domain comprises

a) One or more than one epitope of EpCAM or a functional sequence variant thereof;

b) One or more than one epitope of MUC-1 or a functional sequence variant thereof;

c) One or more than one epitope of survivin or a functional sequence variant thereof;

d) One or more than one epitope of CEA or a functional sequence variant thereof;

e) One or more than one epitope of KRas or a functional sequence variant thereof; and/or

f) One or more than one epitope of MAGE-A3 or a functional sequence variant thereof;

g) One or more than one epitope of ASCL2 or a functional sequence variant thereof.

32. The combination according to any one of claims 18 to 31, wherein the multi-antigen domain comprises

d) One or more than one epitope of CEA or a functional sequence variant thereof; and

f) One or more than one epitope of MAGE-A3 or a functional sequence variant thereof.

33. The combination according to any one of claims 18 to 32, wherein the multi-antigen domain comprises

e) One or more than one epitope of KRas or a functional sequence variant thereof.

34. The combination according to any one of claims 18 to 33, wherein the multi-antigen domain comprises

35. The combination according to any one of claims 18 to 34, wherein the multi-antigen domain comprises

36. The combination of any one of claims 18 to 35, wherein the multi-antigen domain comprises

c) One or more than one epitope of survivin or a functional sequence variant thereof; and/or

37. The combination according to any one of claims 18 to 36, wherein the multi-antigen domain comprises

d) One or more than one epitope of CEA or a functional sequence variant thereof.

38. The combination according to any one of claims 18 to 37, wherein the multi-antigen domain comprises

b) One or more than one epitope of MUC-1 or a functional sequence variant thereof; and/or

39. The combination according to any one of claims 18 to 38, wherein the multi-antigen domain comprises

a) One or more than one epitope of EpCAM or a functional sequence variant thereof; and/or

40. The combination according to any one of claims 18 to 39, wherein the multi-antigen domain comprises

d) One or more than one epitope of CEA or a functional sequence variant thereof; and/or

41. The combination according to any one of claims 18 to 30, wherein the multi-antigen domain comprises

-one or more than one epitope of survivin or a functional sequence variant thereof; and

-one or more epitopes of CEA or a functional sequence variant thereof.

42. The combination according to any one of claims 18 to 30, wherein the multi-antigen domain comprises

One or more than one epitope of ASCL2 or a functional sequence variant thereof.

43. The combination according to any one of claims 18 to 30, wherein the multi-antigen domain comprises

-one or more epitopes of CEA or a functional sequence variant thereof; and

one or more than one epitope of ASCL2 or a functional sequence variant thereof.

44. The combination according to any one of claims 18 to 30, wherein the multi-antigen domain comprises

-one or more than one epitope of survivin or a functional sequence variant thereof;

-one or more epitopes of CEA or a functional sequence variant thereof; and

one or more than one epitope of ASCL2 or a functional sequence variant thereof.

45. The combination according to any one of claims 18 to 30 and 44, wherein the multi-antigen domain comprises in the N-to C-terminal direction:

-one or more epitopes of CEA or a functional sequence variant thereof;

one or more than one epitope of ASCL2 or a functional sequence variant thereof.

46. The combination of any one of claims 18 to 30, 44 and 45, wherein the multi-antigen domain comprises in the N-to C-terminal direction:

-having a sequence according to SEQ ID NO:29, or a fragment thereof of at least 10 amino acids in length, or a peptide thereof having a (functional) sequence variant of at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity;

-having a sequence according to SEQ ID NO:16, or a fragment thereof of at least 10 amino acids in length, or a peptide thereof having a (functional) sequence variant of at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity; and

-having a sequence according to SEQ ID NO:21, or a fragment thereof of at least 10 amino acids in length, or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

47. The combination of any one of claims 18 to 30 and 44 to 46, wherein the amino acid sequence represented by SEQ ID NO:29 or a fragment or variant thereof is directly linked to a peptide consisting of the amino acid sequence according to SEQ ID NO:16 or a fragment or variant thereof; and consists of a sequence according to SEQ ID NO:16 or a fragment or variant thereof is directly linked to a peptide having the amino acid sequence according to SEQ ID NO:21 or a fragment or variant thereof.

48. The combination according to any one of claims 18 to 30 and 44 to 47, wherein the multi-antigen domain comprises a sequence consisting of a sequence according to SEQ ID NO:32 or a (functional) sequence variant having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity; consists of a sequence according to SEQ ID NO:18 or a (functional) sequence variant having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity; and consists of a sequence according to SEQ ID NO:24 or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

49. The combination according to any one of claims 18 to 30 and 44 to 48, wherein the multi-antigen domain of the complex comprises a sequence consisting of a sequence according to SEQ ID NO:48, or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

50. The combination of any one of the preceding claims, wherein the TLR peptide agonist is a TLR2, TLR4 and/or TLR5 peptide agonist.

51. The combination of any one of the preceding claims, wherein the TLR peptide agonist is a TLR2 peptide agonist and/or a TLR4 peptide agonist.

52. The combination of any one of the preceding claims, wherein the TLR peptide agonist is annexin II or an immunomodulatory fragment thereof.

53. The combination of any one of the preceding claims, wherein the TLR peptide agonist comprises or consists of the amino acid sequence according to SEQ ID NO:49 or 50 or a (functional) sequence variant having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

54. The combination of any one of the preceding claims, wherein the TLR agonist comprises or consists of the amino acid sequence of SEQ ID NO:7 or a fragment or variant thereof.

55. The combination of any one of the preceding claims, wherein the TLR agonist comprises or consists of the amino acid sequence according to SEQ ID NO:52 or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

56. The combination of any one of the preceding claims, wherein the TLR agonist comprises or consists of the amino acid sequence according to SEQ ID NO:53 or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

57. The combination of any one of the preceding claims, wherein the TLR agonist comprises or consists of the amino acid sequence according to SEQ ID NO:51 or an immunomodulatory fragment or (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

58. The combination according to any one of the preceding claims, wherein the complex comprises more than one TLR peptide agonist, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 TLR peptide agonists.

59. The combination according to any one of the preceding claims, wherein at least one antigen or epitope (or multi-antigen domain) of the complex is located C-terminal to the cell-penetrating peptide of the complex, wherein the cell-penetrating peptide and the at least one antigen or epitope (or multi-antigen domain) are optionally linked by other components, such as a linker, spacer element, or by a TLR peptide agonist of the complex.

60. The combination of any one of the preceding claims, wherein the complex is a polypeptide or protein, wherein

a) The cell penetrating peptide has a sequence comprising or consisting of a sequence according to SEQ ID NO:6 (CPP 3/Z13), SEQ ID NO:7 (CPP 4/Z14), SEQ ID NO:8 (CPP 5/Z15) or SEQ ID NO:11 An amino acid sequence of (CPP 8/Z18) or of a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity (without losing the cell penetrating ability of the peptide);

b) At least one antigen or epitope is a peptide, polypeptide or protein, and preferably comprises or consists of at least one cancer epitope; and

61. The combination according to any one of the preceding claims, wherein the complex is a recombinant polypeptide or recombinant protein and components a) to C) are located in the N-terminal→c-terminal direction of the complex backbone in the following order:

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

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

wherein the components may be connected by other components, in particular by means of linkers or spacer elements.

62. The combination of any one of the preceding claims, wherein the complex comprises or consists of a sequence according to SEQ ID NO:54 or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

63. A combination according to any one of the preceding claims, wherein the STING agonist is a Cyclic Dinucleotide (CDN) -based STING agonist.

64. A combination according to any one of the preceding claims, wherein the STING agonist is selected from ADU-S100, MK-1454, E-7766, MK-2118, BMS-986301, IMSA-101, SB-11285, SYNB-1891, GSK-3745417, TAK-676 and TTI-10001.

65. A combination according to any one of claims 1 to 63, wherein the STING agonist is a compound of formula I

Wherein the method comprises the steps of

R ¹ Selected from H, F, -O-C _1-3 Alkyl and OH, and

R ² is H, or

R ² is-CH ₂ -and R ¹ is-O-together with-CH ₂ -O-bridge, and

or a solvate or hydrate thereof, or a salt thereof.

66. The combination of claim 65, wherein the STING agonist is a compound of formula Ia

Or a solvate or hydrate thereof, or a salt thereof.

67. The combination of claim 65, wherein the STING agonist is a compound of formula Ib

Or a solvate or hydrate thereof, or a salt thereof.

68. A combination according to claim 66, wherein the STING agonist is a compound of formula ia.1

Or a solvate or hydrate thereof.

69. A combination according to claim 66, wherein the STING agonist is a compound of formula ia.2

Or a solvate or hydrate thereof.

70. A combination according to claim 66, wherein the STING agonist is a compound of formula ia.3

Or a solvate or hydrate thereof.

71. The combination according to claim 67, wherein the STING agonist is a compound of formula ib.1

Or a solvate or hydrate thereof.

72. A combination according to any one of claims 1 to 63, wherein the STING agonist is a compound of formula II:

wherein the method comprises the steps of

or a salt thereof.

73. The combination according to claim 72, wherein the STING agonist is a compound of formula II-1

Or a solvate or hydrate thereof.

74. A combination according to claim 72, wherein the STING agonist is a compound of formula II-2

Or a solvate or hydrate thereof.

75. The combination according to claim 72, wherein the STING agonist is a compound of formula II-3

Or a solvate or hydrate thereof.

76. The combination according to claim 72, wherein the STING agonist is a compound of formula II-4

Or a solvate or hydrate thereof.

77. A combination according to any one of claims 1 to 63, wherein the STING agonist is a substantially pure (Sp, sp), (Rp, rp), (Sp, rp) or (Rp, sp) stereoisomer of a compound as defined in any one of claims 65 to 76, or a salt thereof, which stereoisomer has a purity of at least 90% relative to the other potential diastereomers.

78. A combination according to any one of claims 1 to 63, wherein the STING agonist is a substantially pure (Rp, rp) stereoisomer of a compound as defined in any one of claims 65 to 76, or a salt thereof, which stereoisomer is at least 90% pure relative to the other potential diastereomers.

79. A combination according to any one of claims 1 to 63, wherein the STING agonist is a pharmaceutically acceptable salt of a compound as defined in any one of claims 65 to 78.

80. A combination according to any one of claims 1 to 63 wherein the STING agonist is the sodium salt of a compound as defined in any one of claims 68 to 71 or 73 to 76.

81. A combination according to any one of the preceding claims wherein the STING agonist and the complex are comprised in the same composition.

82. A combination according to any one of claims 1 to 80, wherein the STING agonist and the complex are provided in different compositions.

83. The combination of any one of claims 1 to 64, 80 and 81, wherein the complex comprises or consists of a sequence according to SEQ ID NO:55, or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity, and wherein the STING agonist is ADU-S100.

84. The combination of any one of claims 1 to 63, 68 to 71, 73 to 76, 80 and 81, wherein the complex comprises or consists of a polypeptide according to SEQ ID NO:55 or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity, and wherein the STING agonist is a compound selected from the group consisting of compounds represented by formulae ia.1, ia.2, ia.3, ib.1, II-1, II-2, II-3 and II-4, or solvates or hydrates thereof.

85. The combination of any one of claims 1 to 64, 80 and 81, wherein the complex comprises or consists of a sequence according to SEQ ID NO:54, or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity, and wherein the STING agonist is ADU-S100.

86. The combination of any one of claims 1 to 63, 68 to 71, 73 to 76, 80 and 81, wherein the complex comprises or consists of a polypeptide according to SEQ ID NO:54 or a (functional) sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity, and wherein the STING agonist is a compound selected from the group consisting of compounds represented by formulae ia.1, ia.2, ia.3, ib.1, II-1, II-2, II-3 and II-4, or solvates or hydrates thereof.

87. The combination according to any one of the preceding claims for use in medicine.

88. A combination according to any one of the preceding claims for use in the prevention and/or treatment of cancer.

89. The combination for use according to claim 87 or 88, wherein the STING agonist and the complex are administered at approximately the same time.

90. The combination for use according to any one of claims 87-89, wherein the STING agonist and the complex are administered via the same route of administration.

91. The combination for use according to any one of claims 95 to 99, wherein the STING agonist and the complex are administered via different routes of administration.

92. The combination for use according to any one of claims 87 to 91, wherein the STING agonist is administered systemically.

93. The combination for use according to any one of claims 87 to 92, wherein the complex is administered systemically.

94. The combination for use according to any one of claims 87 to 93, wherein the complex is administered repeatedly.

95. The combination for use according to any one of claims 87 to 94, wherein the STING agonist is administered repeatedly.

96. The combination for use according to any one of claims 87 to 95, wherein the STING agonist and the complex are administered on the same day.

97. A kit comprising

(i) STING agonists

(ii) A complex comprising:

a) Cell penetrating peptide;

b) At least one antigen or epitope; and

c) A TLR peptide agonist, which is a compound of formula (i),

wherein components a) to c) are covalently linked.

98. A kit according to claim 97, wherein the STING agonist is as defined in any one of claims 63 to 80 and 83 to 86.

99. The kit of claim 97 or 98, wherein the complex is as defined in any one of claims 2 to 62 and 83 to 86.

100. The kit of any one of claims 97 to 99, further comprising a package insert or label, for example with instructions for treating cancer, in particular by using a combination of STING agonist and complex.

101. The kit of any one of claims 97 to 100 for use in medicine.

102. The kit according to any one of claims 97 to 101 for use in the prevention and/or treatment of cancer.

103. A composition comprising:

(i) STING agonists

(ii) A complex comprising:

a) Cell penetrating peptide;

b) At least one antigen or epitope; and

c) A TLR peptide agonist, which is a compound of formula (i),

wherein components a) to c) are covalently linked.

104. A composition according to claim 103, wherein STING agonist is as defined in any one of claims 63 to 80 and 83 to 86.

105. The composition according to claim 103 or 104, wherein the complex is as defined in any one of claims 2 to 62 and 83 to 86.

106. The composition of any one of claims 103-105, wherein the composition comprises a pharmaceutically acceptable carrier.

107. The composition of any one of claims 103-106, wherein the composition is a vaccine.

108. The composition of any one of claims 103-107 for use in medicine.

109. The composition according to any one of claims 103-108 for use in the prevention and/or treatment of cancer.

110. A method for treating cancer or initiating, enhancing or prolonging an anti-tumor response in a subject in need thereof, comprising administering to the subject an effective amount of

(i) STING agonists

(ii) A complex comprising:

a) Cell penetrating peptide;

b) At least one antigen or epitope; and

c) A TLR peptide agonist, which is a compound of formula (i),

wherein components a) to c) are covalently linked.

111. A method for increasing tumor antigen-specific T cell infiltration of a tumor in a patient, the method comprising administering to a patient having a tumor or cancer

(i) STING agonists

(ii) A complex comprising:

a) Cell penetrating peptide;

b) At least one antigen or epitope; and

c) A TLR peptide agonist, which is a compound of formula (i),

wherein components a) to c) are covalently linked.

112. A combination therapy for the prevention and/or treatment of cancer, wherein the combination therapy comprises administration of

(i) STING agonists

(ii) A complex comprising:

a) Cell penetrating peptide;

b) At least one antigen or epitope; and

c) A TLR peptide agonist, which is a compound of formula (i),

wherein components a) to c) are covalently linked.

113. The method of claim 110 or 111, or the combination therapy of claim 112, wherein the subject has cancer or a tumor.

114. The method or combination therapy of claim 113, wherein the subject has an endocrine tumor, a gastrointestinal tumor, a genitourinary or gynecological tumor, a breast cancer, a head and neck tumor, a hematopoietic tumor, a skin tumor, a breast or respiratory tumor, preferably colorectal cancer, such as metastatic colorectal cancer.

115. A complex as defined in any one of claims 1 to 62 for use in combination with a STING agonist.

116. A complex for use according to claim 114, wherein the STING agonist is a STING agonist as defined in any one of claims 63 to 80.

117. A STING agonist for use in combination with a complex as defined in any one of claims 1 to 62.

118. A STING agonist for use according to claim 116, wherein the STING agonist is as defined in any one of claims 63 to 80.