Vaccine Compositions Comprising Encoded Adjuvants

Abstract

The present invention relates to a vaccine composition comprising (1) a first set of one or more vectors comprising a nucleic acid encoding one or more adjuvants, the first set of one or more vectors being adenoviral vectors, and (2) an antigen or combination of antigens, or a nucleic acid encoding said antigen or combination of antigens, or a second set of one or more vectors comprising said nucleic acid. The present invention further relates to said vaccine composition for use in treating or preventing a disease. Furthermore, the present invention relates to a vaccine composition or vaccine kit for inducing an immune response comprising (1) a first nucleic acid encoding one or more adjuvants, or a first set of one or more vectors comprising said first nucleic acid, and (2) a second nucleic acid encoding an antigen or combination of antigens, or a second antigen or combination of antigens, or a second set of one or more vectors comprising said second nucleic acid, where (1) is administered to a patient at a first site, and (2) is administered to a patient at a second site, the first site being the same or within 20 cm of the second site, and the lymphatic systems of the first and second sites drain into the same lymph node. The present invention also relates to a vaccination regimen comprising a first administration step comprising administration of an antigen and an encoded adjuvant, and a second administration step comprising administration of an antigen and / or an encoded adjuvant.

Description

[Technical field]

[0001] The present invention relates to a vaccine composition comprising an antigen or a combination of an antigen and one or more encoded adjuvants.The present invention further relates to such a vaccine composition for use in cancer therapy. [Background technology]

[0002] The field of vaccines is rapidly advancing with the aim of inducing strong immune responses against various infectious and neoplastic diseases. In this context, genetic cancer vaccines may become an important tool in cancer treatment in the coming years.

[0003] Cancer vaccines must face the complexity of inducing T cell responses against tumor antigens that are either i) tumor-associated antigens (TAA) derived from self-proteins overexpressed in tumors, or ii) neo-antigens derived from mutated self-proteins. The most common genetic mutations in tumors are single nucleotide variants that cause a single amino acid change adjacent to an amino acid residue in the wild-type protein. Thus, most neo-antigens contain significant "self" components and are considered weak immunogens. To obtain a strong immune response, immune tolerance to "self" must be overcome. Summary of the Invention [Problem to be solved by the invention]

[0004] Adjuvants are components used in vaccines that help induce a strong immune response in vaccinated individuals. However, some strong adjuvants are associated with severe side effects. Therefore, optimizing the efficacy of cancer vaccines while minimizing toxicity is crucial. [Means for solving the problem]

[0005] The present invention is based on the discovery that the immune response to an antigen can be significantly increased when the antigen is co-administered with the encoded adjuvant.Unexpectedly, the present inventors have found that when a vaccine composition comprises one or more sets of adenovirus vectors, preferably a set of human adenovirus vectors that encode one or more adjuvants, the immune response to an antigen or a combination of antigens is amplified. Thus, the vaccine composition according to the present invention provides, inter alia: (i) enhancing the immune response to an antigen or combination of antigens, (ii) converting a suboptimal weak immune response into a stronger immune response, (iii) enabling an immune response to an antigen that would not otherwise elicit an immune response, (iv) converting an antigen from non-immunogenic to immunogenic, (v) enabling an immune response to TAAs, (vi) enabling an immune response to a single antigen or a combination of a small number of antigens, in particular TAAs or cancer neo-antigens, (vii) enabling a locally and temporally defined breakdown of immune tolerance to self-antigens, (viii) enabling limited systemic exposure of adjuvants, (ix) enabling limited non-specific activity of adjuvants, (x) enabling limited toxicity, (xi) enabling easy co-formulation of antigens and adjuvants, (xii) enabling simultaneous co-localized action of antigens and adjuvants.

[0006] In a first aspect, the present invention relates to a vaccine composition comprising: (1) a first set of one or more vectors comprising nucleic acids encoding one or more adjuvants, wherein the first set of one or more vectors are adenoviral vectors; and (2) a second set of one or more vectors comprising an antigen or combination of antigens, or a nucleic acid encoding said antigen or combination of antigens, or comprising said nucleic acid.

[0007] In a second aspect, the present invention relates to a vaccine composition according to the first aspect of the invention for use in the treatment or prevention of a disease.

[0008] In a third aspect, the present invention relates to a vaccine composition or vaccine kit for inducing an immune response to an antigen or combination of antigens comprising: (1) a first composition comprising nucleic acids encoding one or more adjuvants, or a first set of one or more vectors comprising said nucleic acids; and (2) a second composition comprising an antigen or combination of antigens, or a nucleic acid encoding said antigens or combination of antigens, or a second set of one or more vectors comprising said nucleic acids; wherein (1) is administered to a patient at a first site and (2) is administered to a patient at a second site, wherein the first site is within 20 cm of the second site, and wherein the lymphatic system of the first site drains into the same lymph nodes as the lymphatic system of the second site, or the first site and the second site are the same.

[0009] In a fourth aspect, the present invention relates to a vaccination regimen comprising a first and a second administration step, wherein (a) the first administration step comprises administration of a vaccine composition according to the first, second or third aspect of the invention, and (b) the second administration step comprises administration of (1) a first composition comprising nucleic acids encoding one or more adjuvants, or a first set of one or more vectors comprising said nucleic acids, and / or (2) a second composition comprising an antigen or a combination of antigens, or a second set of one or more vectors comprising nucleic acids encoding said antigens or combination of antigens. [Brief description of the drawings]

[0010] [Figure 1]A) Serum concentrations of anti-mCTLA4 (clone 9d9) in mice receiving Ad6, Ad5, GAd20 and ChAd68 vectors encoding 9D9 anti-mCTLA4 (10^8 viral particles, vp) measured 7 days after injection. B) Effect of encoded anti-CTLA4 on vaccine-induced T cell responses. C57Bl6 mice were vaccinated intramuscularly with GAd vaccines encoding seven CD8 T cell neo-antigens selected from the MC38 tumor model (vaccine, dose 2x10^7 vp) either alone or in combination (mix) with Ad encoding anti-mCTLA4 (Ad6, Ad5, GAd20 and ChAd68 vectors encoding 9D9 anti-mCTLA4, 10^8 vp each). The total response (number of IFNγ-producing T cells per million splenocytes) against the vaccine-encoded CD8 epitopes as measured in the five experimental groups by IFN-γ ELISpot assay is shown. [Diagram 2] Figure 2 shows the effect of encoded anti-mCTLA4 antibody (Ad-9d9) on vaccine-induced T cell responses when co-administered with the vaccine in one anatomical site as a mixture (mixed), administered closely (5 min time difference) in the same anatomical site as the mixture (separately), or administered in two separate sites (contralateral). C57Bl6 mice were vaccinated intramuscularly with GAd vaccine (vaccine, dose 2x10^7 vp) encoding seven CD8 T cell neo-antigens selected from the MC38 tumor model, and Ad6-9d9 (10^8 vp) ​​mixed with the vaccine was injected into the quadriceps of the mice, either administered separately in the same anatomical site or in two different anatomical sites (GAd vaccine in the left site and Ad-9d9 vaccine in the right contralateral site). The same vaccine dose was used for all three regimens. As a control, a group of mice received only the vaccine in the absence of Ad-9d9. Immune responses (number of IFNγ-producing T cells per million splenocytes) measured by IFN-γ ELISpot assay are shown. [Diagram 3]Figure 3 shows the effect of encoded anti-mCTLA4 antibody (Ad-9d9) on vaccine-induced T cell responses. A) BalBC mice were vaccinated intramuscularly (im) with GAd vaccine encoding 31 CT26 neoantigens (vaccine) alone or in combination with Ad6 encoding anti-mCTLA4 (Ad-9d9 at a dose of 10^8 vp) ​​(mixed) or in combination with anti-mCTLA4 antibody protein (9d9 Ab, 100ug) delivered intraperitoneally (ip). Responses against vaccine-encoded CD8 epitopes (light grey) and CD4 (dark grey) (number of IFN-γ producing T cells per million splenocytes) are shown in the three experimental groups (vaccine; vaccine+9d9 Ab; vaccine+Ad-9d9) by IFN-γ ELISpot assay. [Figure 4] Figure 4 shows the effect of encoded anti-mCTLA4 antibody (Ad6-9d9) on vaccine-induced T cell responses when co-administered with vaccine, as well as the impact of encoded anti-mCTLA4 on the anti-tumor efficacy of the vaccine. A) BalBC mice were intramuscularly (im) vaccinated with GAd vaccine encoding 62 CT26 neoantigens (vaccine) administered alone or in combination (mix) with Ad6 encoding anti-mCTLA4 (Ad-9d9 at a dose of 10^8 vp). The immune response (number of IFNγ producing T cells per million splenocytes) is shown. B) Anti-tumor efficacy of GAd-CT26-62 combined with anti-mPD1 and co-administered with Ad6-9d9 in combination with anti-mPD1. Treatment was started on day 0 for mice randomized according to tumor volume. Tumor growth over time is shown for individual mice belonging to the two different treatment groups. Antitumor response is assessed as the sum of complete and partial responses (≧40% tumor shrinkage). [Diagram 5]Figure 5 shows the concentration of anti-mCTLA4 antibodies in the serum of injected mice 7 days after injection of an Ad6 vector encoding anti-mCTLA4 (Ad-9d9, black) or a single dose of anti-mCTLA4 antibody protein (9d9 Ab, 100ug) injected subcutaneously (9d9 Ab sc, white) or intraperitoneally (9d9 Ab ip, dark grey). [Figure 6] Figure 6 shows the effect of encoded anti-CTLA4 to enhance the immunogenicity of a TAA-based GAd vaccine. BalBC mice were vaccinated intramuscularly with a GAd vaccine (vaccine, dose 5x10^8 vp) ​​encoding four TAAs selected from CT26 tumors, either alone or in combination (mix) with Ad6 encoding anti-mCTLA4 (vaccine+Ad-9d9). Responses (number of IFNγ-producing T cells per million splenocytes) against the encoded antigens (1-4) measured using a set of peptides covering the vaccine sequence are shown. [Figure 7] Figure 7 shows the effect of encoded anti-mCTLA4 in enhancing antibody responses to the TAA. hHer2 transgenic (Tg) mice were vaccinated intramuscularly with hHer2-encoding GAd vaccine (Ad-hHer2, dose 5x10^8 vp) ​​either alone or in combination (mix) with anti-mCTLA4-encoding Ad6 (Ad-hHer2 +Ad-9d9). Two weeks after immunization, serum was prepared from immunized mice and analyzed for the presence of Abs recognizing the TAA hHER2 / neu. Serum from wt mice was used as a positive control and was expected to show a positive response to hHer2. [Figure 8]Figure 8 shows the effect of encoded OX40L on vaccine-induced T cell responses. C57Bl6 mice were vaccinated intramuscularly with a GAd vaccine encoding seven selected CD8 T cell neo-antigens from the MC38 tumor model (vaccine, dose 2x10^7 vp) either alone or in combination (mix) with an Ad encoding OX40L (Ad-OX40L, 10^8 vp). As a positive control, one group of mice was co-administered with Ad-9d9. The total response (number of IFN-γ producing T cells per million splenocytes) to the vaccine-encoded CD8 epitopes as measured by IFN-γ ELISpot assay is shown. [Figure 9] Figure 9 shows the effect of encoding 9d9 and OX40L to break T cell tolerance to human Her2 in hHer2 Tg mice. Mice were vaccinated intramuscularly with GAd vaccine encoding h-Her2 alone or in combination with adenovirus encoding 9D9 (Ad-9D9) or OX40L (Ad-OX40L), or in combination with an equal mixture of Ad-9D9 and Ad-OX40L. T cell responses to hHer2 measured by IFN-γ ELISpot assay (number of T cells producing IFNγ per million splenocytes) are shown. [Figure 10] Figure 10 shows the effect of encoded ICOSL on vaccine-induced T cell responses. C57Bl6 mice were vaccinated intramuscularly with a GAd vaccine encoding seven selected CD8 T cell neo-antigens from the MC38 tumor model (vaccine, dose 2x10^7 vp) administered alone or in combination (mix) with Ad-encoded ICOSL (Ad-ICOSL, 10^8 vp). Total responses (number of IFN-γ producing T cells per million splenocytes) to vaccine-encoded CD8 epitopes as measured by IFN-γ ELISpot assay are shown. [Figure 11]Figure 11 shows the effect of encoded Ad6 anti-mCTLA4 on vaccine antitumor efficacy in adjuvant mono- and dual-dose regimens. Mice were inoculated subcutaneously with CT26 cells. One week later, animals were randomized according to tumor burden and treated with the combination of non-adjuvanted vaccine GAd-CT26-62 and anti-PD1 on day 0 (vaccine+anti-PD1), adjuvanted vaccine and encoded anti-CTLA4 mono-administration regimen (vaccine+Ad6-9d9+anti-PD1), or dual Ad6-9d9 co-administered with vaccine on day 0 and Ad6-9d9 on day 1 with the first dose of Ad6-9d9 (vaccine+Ad6-9d9 2x+anti-PD1). Tumor growth over time is shown. Antitumor response is evaluated as the sum of complete and partial responses (≥40% tumor shrinkage). [Figure 12] FIG. 12 shows serum concentrations of anti-hCTLA4 (ipilimumab) measured over time by ELISA assay in mice administered Ad6-Ipi (10^8 viral particles, vp). [Figure 13] Figure 13 shows the effect of a membrane-bound form of Ad6-encoded anti-mCTLA4 (Ad6-9d9TM) on the antitumor efficacy of the vaccine. Total responses (number of IFNγ-producing T cells per million splenocytes) to the vaccine-encoded CD8 epitope are shown for vaccine alone (Vaccine), Ad6-9d9 co-administered with vaccine, or Ad6-9d9TM co-administered with vaccine, as measured by FN-γ ELISpot assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, which may vary. It should also be understood that the terms used herein are only intended to describe specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the scope of the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0012] Preferably, the terms used herein are as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Koelbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland) and in "Pharmaceutical Substances: Syntheses, Patents, Applications" by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing,1999; the "Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals", edited by Susan Budavari et al., CRC Press, 1996, and theUnited States Pharmacopeia-25 / National Formulary-20, published by the UnitedStates Pharmcopeial Convention, Inc., Rockville Md., The term is defined as set forth in the "2001" International Conference on Comprehensive and Interdisciplinary Research (ICIR) Guidelines for the Prevention of Crucial Changes in Health and Safety of Children (ICC).

[0013] Unless the context otherwise requires, throughout this specification and the claims that follow, the word "comprise" and variations such as "comprises" and "comprising" are to be understood as implying the inclusion of the stated features, integers, or steps, or groups of features, integers, or steps, but not the exclusion of other features, integers, steps, or groups of integers or steps. In the following passages, the various aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect, unless expressly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature indicated as being preferred or advantageous.

[0014] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether above or below, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0015] definition Below are definitions of some of the terms frequently used herein, which will have their respective defined and preferred meanings throughout the remainder of the specification whenever they are used.

[0016] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to be understood as polymeric or oligomeric macromolecules made from nucleotide monomers. A nucleotide monomer is composed of a nucleobase, a pentose sugar (such as, but not limited to, ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, nucleic acids are formed through phosphodiester bonds between individual nucleotide monomers. In the context of the present invention, preferred nucleic acid molecules include, but are not limited to, ribonucleic acid (RNA), modified RNA, deoxyribonucleic acid (DNA), and mixtures thereof, such as RNA-DNA hybrids. Nucleic acids can be chemically synthesized, for example, by the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584).

[0017] As used herein, the terms "protein," "peptide," "polypeptide," "peptide" and "polypeptide" are used interchangeably throughout. These terms are used in the context of the present invention to refer to both naturally occurring peptides, such as naturally occurring proteins, and synthetic peptides, which may contain natural or unnatural amino acids.

[0018] In the context of the present invention, the term "immune response" includes cellular and humoral immune responses.

[0019] In the context of the present invention, the term "antigen" is used to refer to any structure recognized by molecules of the immune response, such as antibodies, T cell receptors (TCRs), etc. Preferred antigens are cellular proteins or fragments thereof associated with a particular disease. Antigens are recognized by highly variable antigen receptors (B cell receptors or T cell receptors) of the adaptive immune system and may induce humoral or cellular immune responses. Antigens that induce such responses are also called "immunogens." Some proteins within cells, whether foreign or cellular, are processed into smaller peptides and presented by the major histocompatibility complex (MHC).

[0020] The term "vector" as used herein refers to a polynucleotide or mixture of polynucleotides and proteins that can introduce foreign genetic material (especially DNA or RNA) into a cell (preferably a mammalian cell) and can be replicated and / or expressed in the cell. Examples of vectors include, but are not limited to, plasmids, cosmids, phages, viruses or artificial chromosomes. An expression vector may contain a "replicon" polynucleotide sequence that facilitates autonomous replication of the expression vector in a host cell. Once inside a host cell, the expression vector can replicate independently of or simultaneously with the host chromosomal DNA, resulting in the generation of several copies of the vector and its inserted DNA. When a replication-incompetent expression vector is used (often for safety reasons), the vector may not replicate but may simply direct the expression of a nucleic acid. Depending on the type of expression vector, the expression vector may be lost from the cell, e.g., expressing only transiently an antigen or adjuvant encoded by the nucleic acid, or may be stable in the cell. An expression vector usually contains an expression cassette, e.g., the necessary elements that allow transcription of a nucleic acid into an mRNA molecule.

[0021] The terms "adenoviral vector" and "adenovecter" are used interchangeably throughout this application.

[0022] The term "adeno-associated virus" (AAV) refers to viruses belonging to the Parvoviridae family, which may include several genera that can be subdivided into the Parvoviridae family, which includes Parvovirus, Erythrovirus, Dependovirus, Amdovirus, and Bocavirus, and the Densoviridae family, which includes Densovirus, Iteravirus, Brevidensovirus, Pephdensovirus, and Contravirus. The unique life cycle of AAV and its ability to infect both non-dividing and dividing cells with persistent expression make it an attractive vector. An additional attractive feature of wild-type AAV virus is the lack of apparent pathogenicity.

[0023] The terms "adeno-associated viral vector" or "AAV vector" are used interchangeably throughout this application.

[0024] The vaccine composition described in the present invention comprises an antigen or a combination of antigens, or a nucleic acid encoding said antigen or combination of antigens, or one or more vectors comprising said nucleic acid, and may further comprise one or more encoded adjuvants, and may further comprise stabilizers, further adjuvants, antibiotics, and preservatives.

[0025] With respect to the vaccine composition according to the invention, the term "antigen" refers to one or more proteins or fragments thereof that are delivered to a subject to induce an immune response. The antigen may be delivered in the form of a protein or may be encoded, and the nucleic acid encoding the antigen may or may not be included in a vector.

[0026] The term "adjuvant" is used in the context of the present invention to refer to an agent that enhances, stimulates, activates, strengthens, or modulates the immune response to an antigen contained in the vaccine composition. Examples of such adjuvants include, but are not limited to, cytokines, cytokine analogs, cytokine receptors, regulators of checkpoint molecules, synthetic polynucleotide adjuvants (e.g., polyarginine or polylysine), interferon (IFN) gene activators, antagonists of indoleamide 2,3-dioxygenase (IDO), adenosine deaminase (ADA) or growth factor-activated receptor gamma coactivator 1-alpha (PGC-1), and the like. Preferred adjuvants are selected from the group consisting of agonists of OX40, preferably OX40L, agonists of ICOS, preferably ICOSL, agonists of CD40, preferably CD40L, and antagonistic CTLA-4 specific antibodies or antibody-like proteins. In the context of the present invention, the vaccine composition comprises one or more encoded adjuvants. Therefore, in the context of the vaccine composition according to the present invention, the term adjuvant refers to an encoded adjuvant.In the vaccine composition of the first aspect of the present invention, one or more adjuvants are encoded by the nucleic acid contained in an adenoviral vector, preferably a human adenoviral vector.In the vaccine composition or vaccine kit for use in the third aspect of the present invention and in the vaccination regimen of the fourth aspect of the present invention, the delivery of one or more encoded adjuvants is not limited to a viral vector.

[0027] Those skilled in the art are familiar with various suitable methods for delivering coded antigen and / or adjuvant.Delivery can be achieved, for example, by DNA, particularly plasmid DNA; RNA, particularly in vitro transcribed (IVT) RNA, non-replicating messenger RNA, and / or self-amplifying RNA (SAM); virus vector; alphavirus vector, Venezuelan equine encephalitis (VEE) virus vector, Sindbis (SIN) virus vector, Semliki Forest virus (SFV) virus vector, and preferably by adenovirus vector, poxvirus vector, vaccinia virus vector or modified vaccinia Ankara (MVA) vector, simian or human cytomegalovirus (CMV) vector, lymphocytic choriomeningitis virus (LCMV) vector, retrovirus or lentivirus vector, replication-competent or replication-incompetent.

[0028] When the antigen or adjuvant is encoded by RNA, administration is accomplished as naked nucleic acid or as a complex with a carrier.RNA can also be administered in combination with a stabilizing agent, such as RNase inhibitor.Useful carriers according to the present invention include lipid-containing carriers, such as cationic lipids, liposomes, micelles, lipid nanoparticles, and lipid-polymer hybrid nanoparticles.Preferred carriers for administration of RNA are lipid nanoparticles or lipid-polymer hybrid nanoparticles. Typical lipid nanoparticle formulations consist of pH-responsive lipids or cationic lipids with tertiary or quaternary amines to encapsulate polyanionic mRNA; neutral helper lipids such as zwitterionic lipids [i.e., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)] and / or sterol lipids (i.e., cholesterol) to stabilize the lipid bilayer of lipid nanoparticles and enhance mRNA delivery efficiency; and polyethylene glycol (PEG) lipids to reduce nonspecific absorption of plasma proteins and improve colloidal stability in biological environments by forming a hydration layer on the nanoparticles. Lipid-polymer hybrid nanoparticles consist of a biodegradable mRNA-loaded polymer core coated with a lipid layer. Typically, lipid envelopes are organized into lipid bilayers or lipid monolayers that contain a mixture of cationic or ionic lipids, helper lipids, and pegylated lipids (Guevara et al., 2020, Advances in Lipid Nanoparticles form RNA-Based Cancer Immunotherapy. Front. Chem. 8:589-959).

[0029] The term "immunomodulator" refers to a compound selected from the group consisting of regulators of checkpoint molecules and cytokines or cytokine analogs. In the context of the present invention, the immunomodulator may be administered in combination with, before or after, or simultaneously with the vaccine composition of the present invention. Thus, when an immunomodulator is present, in addition to the adjuvant, the immunomodulator is a further component of the vaccine composition. It is preferred that the adjuvant is coded and the immunomodulator is administered as a protein. A preferred immunomodulator is selected from the group consisting of an antagonistic CTLA-4 specific antibody or antibody-like protein, an antagonistic PD-1 specific antibody or antibody-like protein, and IL-2 or an analog thereof.

[0030] The term "antibody" is used in the context of the present invention to refer to a glycoprotein belonging to the immunoglobulin superfamily. Antibodies refer to protein molecules that may be produced by plasma cells and are used by the immune system to identify and neutralize foreign bodies such as bacteria and viruses. Antibodies recognize antigens, which are unique parts of foreign targets. The term "antibody" refers to a molecule having the overall structure of an antibody, for example an IgG antibody. In general, references to IgG include IgG1, IgG2, IgG3, and IgG4, unless otherwise defined. An IgG antibody molecule is a Y-shaped molecule that contains four polypeptide chains, two heavy chains and two light chains. Each light chain is composed of two domains, the N-terminal domain known as the variable or VL domain (or region) and the C-terminal domain known as the constant (or CL) domain (constant kappa (Cκ) or constant lambda (Cλ) domain). Each heavy chain is composed of four domains. The N-terminal domain of the heavy chain is known as the variable (or VH) domain (or region), followed by the first constant domain (CH1), the hinge region, and the second and third constant domains (CH2 and CH3). In an assembled antibody, the VL and VH domains combine to form the antigen-binding site. The CL and CH1 domains also combine to maintain the association of one heavy chain with one light chain. The heterodimer of two heavy and light chains associates through the interaction of the CH2 and CH3 domains and between the hinge regions of the two heavy chains. The term "antibody" as used herein also includes molecules that may have chimeric domain replacements (i.e., at least one domain is replaced by a domain from a different antibody), such as an IgG1 antibody that contains an IgG3 domain (e.g., the CH3 domain of IgG3). In addition, the term generally refers to multispecific, e.g., bispecific or trispecific, antibodies. The term antibody also includes molecules that have one or more mutations in the heavy chain constant domain.

[0031] The term "antibody-like molecule" as used within the context of this specification includes antibody derivatives and antibody mimetics.

[0032] The term "antibody mimic" refers to a compound that can specifically bind to an antigen in a manner similar to an antibody, but is not structurally related to an antibody. Typically, an antibody mimic is an artificial peptide or protein with a molecular weight of about 3-20 kDa that contains one, two or more exposed domains that specifically bind to an antigen. Typically, such an antibody mimic contains at least one variable peptide loop attached to both ends of a protein scaffold. This dual structural constraint greatly increases the binding affinity of the antibody-like protein to a level comparable to that of an antibody. The length of the variable peptide loop usually consists of 10-20 amino acids. The scaffold protein can be any protein with good solubility properties. Preferably, the scaffold protein is a small globular protein. Examples include, inter alia, LACI-D1 (lipoprotein-associated coagulation inhibitor); affilins, such as human gamma B crystallins or human ubiquitin; cystatins; Sac7D from Sulfolobus Acidocaldarius; lipocalins and anticalins derived from lipocalins; DARPins (designed ankyrin repeat domains); SH3 domains of Fyn; Kunitz domains of protease inhibitors; monobodies, such as the 10th type III domain of fibronectin; adnectins: knottins (cysteine ​​knot mini-proteins); atrimers; evibodies, such as CTLA4-based binders, affibodies, such as the three-helix bundle of the Z domain of protein A from Staphylococcus aureus; Trans-bodies, such as human transferrin; These include tetranectins, e.g., monomeric or trimeric human C-type lectin domains; microbodies, e.g., trypsin inhibitor II; affilins; and armadillo repeat proteins. Nucleic acids and small molecules are also sometimes considered antibody mimics (aptamers), but are not considered artificial antibodies, antibody fragments, or fusion proteins composed of these. General advantages over antibodies include better solubility, tissue penetration, heat and enzymatic stability, and relatively low production costs.

[0033] The term "binding" according to the present invention preferably relates to specific binding. The term "binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., a target or antigen). Unless otherwise stated, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for a partner Y can generally be expressed as a dissociation constant (Kd). "Specific binding" means that a binding moiety (e.g., an antibody) binds stronger to a target, such as an epitope, for which it is specific, compared to its binding to another target. A binding moiety binds a first target stronger than a second target if it binds to the first target with a lower dissociation constant (Kd) than the dissociation constant of the second target. The dissociation constant (Kd) of a target to which a binding moiety specifically binds is more than 10-fold, preferably more than 20-fold, more preferably more than 50-fold, even more preferably more than 100-fold, more than 200-fold, more than 500-fold or more than 1000-fold lower than the dissociation constant (Kd) of a target to which a binding moiety does not specifically bind.

[0034] That is, the term "Kd" (measured in "mol / L" (sometimes abbreviated as "M")) is intended to refer to the dissociation equilibrium constant of a particular interaction between a binding moiety (e.g., an antibody or fragment thereof) and a target molecule (e.g., an antigen or epitope thereof). Affinity can be measured by common methods known in the art, including, but not limited to, surface plasmon resonance-based assays (such as BIAcore assays); quartz crystal microbalance assays (such as Attana assays); enzyme-linked immunosorbent assays (ELISAs); and competitive assays (such as RIAs). Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high affinity antibodies generally bind antigens faster and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

[0035] Typically, the antibody or antibody mimetic will have sufficient binding affinity, e.g., between 500 nM and 1 pM, i.e., about 500 nM, about 450 nM, about 400 nM, about 350 nM, about 300 nM, about 250 nM, about 200 nM, about 150 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, or about 1 pM, e.g., 500 nM, 450 nM, 400 nM, 350 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 50 nM, 10 The antibodies bind to the target with a Kd value of 0.1 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM or 1 pM.

[0036] The term "immunoglobulin (Ig)" as used herein refers to immunity-conferring glycoproteins of the immunoglobulin superfamily. "Surface immunoglobulins" are membrane-bound by a transmembrane region, for example, on effector or endothelial cells, and include, but are not limited to, molecules such as the neonatal Fc receptor, B cell receptor, T cell receptor, class I and II major histocompatibility complex (MHC) proteins, beta 2 microglobulin (β2M), CD3, CD4, and CD8.

[0037] The term "antibody derivative" as used herein refers to a molecule that contains at least the domains it is defined to contain, but does not have the overall structure of an antibody such as IgA, IgD, IgE, IgG, IgM, IgY or IgW, but is still capable of binding to a target molecule. Said derivative may be, but is not limited to, a functional (i.e. target-binding, in particular specific target-binding) antibody fragment or a combination thereof. The present invention also relates to antibodies to which additional antibody domains, such as additional variable domains, have been added. Thus, the term antibody derivative also includes multispecific (bispecific, trispecific, tetraspecific, pentaspecific, hexavalent, etc.) and multivalent (bivalent, trivalent, tetravalent, etc.) antibodies.

[0038] Bispecific antibodies exist in multiple formats (Brinkmann and Kontermann, Mabs2017, Vol. 9, No. 2, 182-212). An example of a bispecific antibody consisting of only an antigen-binding domain is the bivalent Fab (bi-Fab). Another example is a format that contains only a variable domain (Fv) and no constant domain. Formats that contain only variable domains have the advantage of a very low molecular weight, resulting in good tumor penetration, which is important for oncological applications. Due to the lack of a constant domain that mediates binding to FcRn, such formats have a short plasma half-life.

[0039] The term "epitope", also known as antigenic determinant, is used in the context of the present invention to refer to a segment of an antigen, preferably a peptide bound by a molecule of the immune system, such as a B cell receptor, a T cell receptor, or an antibody. An epitope to which an antibody or a B cell binds is called a "B cell epitope", and an epitope to which a T cell binds is called a "T cell epitope". In this context, the term "binding" preferably refers to an epitope with a binding constant between an antibody or a T cell receptor (TCR) and the respective epitope of 1×10 5 M -1 More than 1×10 6 M -1 , 1×10 7 M -1 , 1×10 8 M -1The above relates to specific binding, which is defined as binding where the binding constant is 0.5-0.5. The skilled artisan is familiar with methods for determining binding constants (see, for example, Caoili, SE (2012) Advances in Bioinformatics Vol. 2012). Preferably, the specific binding of an antibody to an epitope is mediated by the Fab (fragment, antigen binding) region of the antibody, the specific binding of a B cell is mediated by the Fab region of the antibody that is specifically bound to the B cell receptor, and the specific binding of a T cell is mediated by the variable (V) region of the T cell receptor. T cell epitopes are presented on the surface of antigen-presenting cells, where they bind to major histocompatibility (MHC) molecules. There are at least two different classes of MHC molecules, referred to as MHC class I and II. Epitopes presented through the MHC-I pathway induce responses by cytotoxic T lymphocytes (CD8+ cells), whereas epitopes presented through the MHC-II pathway induce responses by helper T cells (CD4+ cells). T cell epitopes presented by MHC class I molecules are usually peptides of 8-12 amino acids in length, whereas T cell epitopes presented by MHC class II molecules are usually peptides of 13-17 amino acids in length. MHC class III molecules also present non-peptide epitopes as glycolipids. Thus, the term "T cell epitope" refers preferably to peptides of 8-11 or 13-17 amino acids in length that can be presented by either MHC class I or MHC class II molecules. Epitopes are usually composed of chemically active surface groups of amino acids, which may or may not have sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0040] In the context of the present invention, the terms "CTLA4-specific antibody" and "anti-CTLA4 antibody" are used interchangeably.

[0041] In the context of the present invention, the term "antagonist antibody" refers to an antibody that can inhibit the biological activity of the molecule to which it binds. When an antagonist antibody binds to a specific receptor, it can block or weaken the signal transduction pathway downstream of the receptor, or compete with the receptor ligand. Those skilled in the art are well aware that the determination of antagonist activity depends on multiple parameters, such as the assay or cell type used. In the context of the present invention, an antagonist antibody specific for CTLA-4 is characterized by the following activity: abolishing the negative signal transduction of T cell response mediated by CTLA4, i.e., abolishing the inhibitory effect of CTLA4 signal transduction on T cell activation, resulting in the enhancement of immune response.

[0042] In the context of the present invention, the term "agonist antibody" refers to an antibody that binds to a receptor and activates the downstream signaling pathway of the receptor in a manner equivalent to the receptor ligand. An example of an agonist antibody is CP-870,893, which binds to and activates the receptor CD40. Those skilled in the art are well aware that the determination of agonist activity depends on multiple parameters, such as the assay or cell type used.

[0043] In the context of the present invention, the term "agonist ligand" refers to a soluble ligand that binds to a receptor and activates the downstream signal transduction pathway of the receptor. An example of an agonist ligand is OX40L, which binds to and activates the receptor OX40.

[0044] The term "tumor associated antigen (TAA)" is used in the context of the present invention to refer to an antigen derived from a self-protein that is overexpressed in tumors, i.e., a protein that is not expressed at all or is expressed only at low levels in healthy tissue and has increased expression levels in tumor tissue. TAA can be a full-length protein or a fragment thereof.

[0045] The term cancer-testis (CT) antigens refers to a group of proteins grouped together by their importance in development and cancer immunotherapy. Generally, the expression of these proteins is restricted to male germ cells in adult animals. However, in cancer, these developmental antigens are often re-expressed. Thus, they represent a category of tumor-associated antigens. CT antigens have been reported in several tumors, including melanoma, liver cancer, lung cancer, bladder cancer, and pediatric tumors such as neuroblastoma. A regularly updated list of CT antigens can be found at http: / / www.cta.lncc.br / index.php. Important CT antigens in cancer therapy include MAGE-A1, MAGE-A3, MAGE-A4, NY-ESO-1, PRAME, CT83, and SSX2.

[0046] The term "neoantigen" is used in the context of the present invention to refer to an antigen that is not present in normal / germline cells, but is present in transformed cells, particularly cancerous cells. A neoantigen may comprise one or more, e.g., 2, 3, 4, 5 or more, neoepitopes. The length of each neoantigen contained in the antigen of the present invention is preferably selected in such a way as to ensure that it is unlikely to contain an epitope that occurs in normal / germline cells. Typically, this can be ensured in that the neoantigen comprises no more than 12 amino acids C- and / or N-terminal to the amino acid change that generated the neoepitope.

[0047] The mutated cancer proteins that constitute neoantigens are generated by mutations occurring at the DNA level and may include: a) one or more single aa changes caused by one or more point mutations representing nonsynonymous single nucleotide variations (SNVs); and / or b) a non-wild-type amino acid sequence caused by a frameshift peptide or an insertion / deletion resulting in an in-frame insertion of one or more non-wild-type amino acids or a deletion of one or more wild-type amino acids; and / or c) a non-wild-type amino acid sequence caused by a mutation that results in an alteration of an exon boundary or intron retention; and / or d) Mutant cancer proteins produced by gene fusion events.

[0048] Neoantigens that are the result of one or more single amino acid changes caused by non-synonymous SNV point mutations in the genome are referred to in the context of the present invention as single amino acid mutant peptides.

[0049] The term "frameshift peptide" is used in the context of the present invention to refer to the complete, non-wild-type translation product of a protein-coding segment of a nucleic acid, including an insertion or deletion mutation that causes a shift in the open reading frame (ORF).

[0050] The term "open reading frame" or "ORF" is used in the context of the present invention to refer to a sequence of nucleotides that can be translated into a string of consecutive amino acids. Typically, an ORF contains a start codon and the subsequent region is usually a multiple of three nucleotides in length, but does not contain a stop codon (TAG, TAA, TGA, UAG, UAA, or UGA) within a given reading frame. An ORF encodes a protein in which the translated amino acids form a peptide-linked chain.

[0051] Neoantigens that are the result of non-wild-type amino acid sequences caused by mutations resulting in altered exon boundaries or intron retention are referred to in the context of the present invention as splice site variant peptides.

[0052] Neo-antigens that are the result of mutant oncoproteins generated by gene fusion events are referred to in the context of the present invention as read-through mutant peptides.

[0053] The term "cytokine analogue" is used in the context of the present invention to refer to a cytokine that has been modified to exhibit improved physicochemical properties, such as being more robust, having favorable pharmacokinetic properties, having an extended half-life, being more suitable for certain delivery systems and formulations, or having enhanced or more selective biological activity. Cytokine analogues may contain amino acid changes compared to the unmodified cytokine or may contain post-translational modifications, such as, for example, pegylation.

[0054] The term "expression cassette" is used in the context of the present invention to refer to a nucleic acid molecule comprising at least one nucleic acid sequence to be expressed, e.g., a nucleic acid encoding an antigen or a part thereof of the present invention, operably linked to transcriptional and translational control sequences. Preferably, the expression cassette comprises cis-regulatory elements for efficient expression of a given gene, e.g., a promoter, an initiation site, and / or a polyadenylation site. Preferably, the expression cassette comprises all additional elements required for expression of a nucleic acid in the patient's cells. Thus, a typical expression cassette comprises a promoter operably linked to the nucleic acid sequence to be expressed, as well as efficient polyadenylation of the transcript, a ribosome binding site, and signals required for translation termination. Additional elements of the cassette may include, for example, enhancer or intron elements. The expression cassette also preferably comprises a transcription termination region downstream of the encoded antigen to provide efficient termination. The termination region may be obtained from the same gene as the promoter sequence or from a different gene.

[0055] The term "operably linked" is used in the context of the present invention to refer to an arrangement of elements in which the elements described as operably linked are configured to perform their normal function. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter is operably linked to one or more transgenes if the promoter affects the transcription of one or more transgenes. In addition, regulatory elements operably linked to a coding sequence can affect the expression of the coding sequence. Regulatory elements need not be contiguous with the coding sequence as long as they function to direct its expression. Thus, for example, there may be intervening sequences between the promoter sequence and the coding sequence that are not translated but are transcribed, and such promoter sequences can still be considered to be "operably linked" to the coding sequence.

[0056] The term "pharmaceutical formulation" or "pharmaceutical composition" as used in the context of the present invention is intended to encompass a vaccine composition according to the invention, i.e. an antigen or a combination of antigens (protein or coded), one or more adjuvants (protein or coded), optionally an immunomodulatory agent, and a pharma- ceutical acceptable carrier and / or excipient.

[0057] As used in the context of the present invention, "pharmaceutical acceptable" means approved by a regulatory agency of the Federal or state government or listed in the United States Pharmacopeia or approved by other generally recognized pharmacopoeias for use in animals, and more specifically, in humans.

[0058] The term "pharmaceutical acceptable carrier" as used herein refers to a pharmacologically inactive substance, such as, but not limited to, a diluent, excipient, surfactant, stabilizer, physiological buffer or vehicle, with which a therapeutically active ingredient is administered. Such pharmaceutical carriers may be liquid or solid. Liquid carriers include, but are not limited to, sterile liquids, such as saline solutions in water and oils, including, but not limited to, those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions, aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Saline is a preferred carrier when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0059] Suitable pharmaceutical "excipients" include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0060] "Surfactants" include, but are not limited to, anionic, cationic, and nonionic surfactants such as sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, and polysorbates such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80.

[0061] "Stabilizers" include, but are not limited to, mannitol, sucrose, trehalose, albumin, and protease and / or nuclease antagonists.

[0062] "Physiological buffers" that may be used in the context of the present invention include, but are not limited to, sodium chloride solution, demineralized water, and suitable organic or inorganic buffers, such as, for example, but not limited to, phosphate buffer, citrate buffer, Tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4(2hydroxyethyl)piperazino]ethanesulfonic acid), or MOPS buffer (3morpholino-1propanesulfonic acid). In general, the choice of the respective buffer depends on the desired molar concentration of the buffer. Phosphate buffers are suitable for injections, infusions, etc.

[0063] An "effective amount" or "therapeutically effective amount" is an amount of a therapeutic agent sufficient to achieve its intended purpose. The effective amount of a given therapeutic agent will vary depending on factors such as the nature of the agent, the route of administration, the size and species of the animal receiving the therapeutic agent, and the purpose of administration. The effective amount in a particular case can be empirically determined by those skilled in the art according to methods established in the art.

[0064] As used herein, "treating," "treat," "treatment," or "therapy" of a disease or disorder means achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing the onset of symptoms characteristic of the disease being treated; (c) inhibiting the worsening of symptoms characteristic of the disease being treated; (d) limiting or preventing the recurrence of the disorder in individuals who previously suffered from the disorder; and (e) limiting or preventing the recurrence of symptoms in individuals who previously had symptoms of the disorder.

[0065] ASPECTS OF THE PRESENT EMBODIMENTS AND PREFERRED EMBODIMENTS In a first aspect, the present invention relates to a vaccine composition comprising: (1) a first set of one or more vectors comprising nucleic acids encoding one or more adjuvants, wherein the first set of one or more vectors are adenoviral vectors; and (2) a second set of one or more vectors comprising an antigen or combination of antigens, or a nucleic acid encoding said antigen or combination of antigens, or comprising said nucleic acid.

[0066] vector The first set of vectors is preferably a human adenovirus vector, more preferably a replication-incompetent human adenovirus vector. The first set of vectors is preferably a group C human adenovirus vector. Group C (also called species C) of human adenovirus includes hAd1, hAd2, hAd5, hAd6 and hAd57. In a preferred embodiment, the first set of vectors is selected from the group consisting of hAd6, hAd57 and hAd5. In some embodiments, the first set of vectors is selected from hAd6 and hAd5. Preferably, the first set of vectors is selected from hAd6 and hAd57, more preferably hAd6.

[0067] When an antigen or combination of antigens is encoded, the antigen or combination of antigens is encoded by a nucleic acid not included in the first set of one or more vectors.

[0068] The vaccine composition preferably comprises a second set of one or more vectors comprising nucleic acids encoding the antigen or combination of antigens. The second set of vectors can be envisaged to be adenovirus vectors or adeno-associated virus (AAV) vectors.

[0069] The one or more adjuvants are encoded by nucleic acids contained in a first set of one or more vectors, and the antigen or combination of antigens (if encoded by nucleic acids contained in a vector) is contained in a second set of one or more vectors, in other words, the antigen is not encoded by nucleic acids contained in the first set of one or more vectors.

[0070] Preferably, the second set of vectors are replication-competent or replication-incompetent adenovirus vectors, preferably replication-incompetent adenovirus vectors. Preferably, the adenovirus vectors are derived from great apes, preferably non-human great apes. Preferred non-human great apes from which the adenoviruses are derived are chimpanzees (Pan), preferably bonobos (Pan paniscus) and common chimpanzees (Pan troglodytes), gorillas (Gorilla) and orangutans (Pongo). In a preferred embodiment, the second set of vectors are adenovirus vectors derived from chimpanzees, bonobos or gorillas, most preferably adenovirus vectors derived from gorillas. Typically, naturally occurring non-human great ape adenoviruses are isolated from fecal samples of the respective great apes.

[0071] The most preferred vector is a non-replicating adenoviral vector based on the gorilla adenovirus vector.

[0072] Other suitable vectors are hAd4, hAd5, hAd6, hAd7, hAd11, hAd26, hAd35, hAd49, hAd57, ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd55, ChAd63, ChAd73, ChAd82, ChAd83, ChAd146, ChAd147, PanAd1, PanAd2, and PanAd3 vectors, or non-replicating adenoviral vectors based on the replication-competent Ad4 and Ad7 vectors. Human adenoviruses hAd4, hAd5, hAd6, hAd7, hAd11, hAd26, hAd35, hAd49 and hAd57 are well known in the art.Vectors based on the naturally occurring ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63 and ChAd82 are described in detail in WO 2005 / 071093. Vectors based on the naturally occurring PanAd1, PanAd2, PanAd3, ChAd55, ChAd73, ChAd83, ChAd146, and ChAd147 are described in detail in WO 2010 / 086189.

[0073] Preferred AAV vectors are based on an AAV serotype selected from the group consisting of AAV-1, AAV-2, AAV-2-AAV-3 hybrid, AAV-3a, AAV-3b, AAV-4, AAV-5, AAV-6, AAV-6.2, AAV-7, AAV-8, AAV-9, AAV-10, AAVrh.10, AAV-11, AAV-12, AAV-13 and AAVrh32.33.

[0074] antigen The antigen or combination of antigens may be delivered in the form of protein or may be encoded by nucleic acid. The nucleic acid may or may not be included in a vector. In some embodiments, the antigen or combination of antigens is encoded by RNA and delivered by lipid nanoparticles or lipid-polymer hybrid nanoparticles. The antigen or combination of antigens is preferably encoded by the nucleic acid included in the second set of vectors.

[0075] In preferred embodiments of all aspects of the invention, the antigen or combination of antigens is selected from a cancer antigen, a viral antigen, a bacterial antigen and a fungal antigen, or the combination of antigens comprises one or more antigens selected from the group consisting of a cancer antigen, a viral antigen, a bacterial antigen and a fungal antigen.

[0076] In a preferred embodiment of all aspects of the present invention, the antigen or antigen combination elicits no immune response or only a suboptimal immune response in the subject in the absence of one or more adjuvants encoded by the human adenoviral vector of the first set of vectors. In other words, in a preferred embodiment, the antigen or antigen combination is a weak antigen, i.e. an antigen with low immunogenicity. Factors that affect the immunogenicity of an antigen are foreignness (the antigen must be recognizable as non-self), molecular size, chemical composition and heterogeneity, and the ability to form a complex with MHC molecules on the cell surface and be presented. As mentioned above, tumor-associated antigens and tumor neo-antigens are often weak antigens. A suboptimal immune response is also called a "weak immune response". Those skilled in the art are familiar with how to quantify an immune response and determine whether to classify an immune response as "suboptimal" or "no" immune response. In particular, the immune response is quantified by analyzing T cell responses to the antigen or antigen combination. Activation of T cells in response to an antigen or combination of antigens can be analyzed by measuring cytokine secretion, in particular the secretion of IFNγ, IL-2, TNF-α, IL-4, IL-5, and / or IL-13. In a preferred embodiment, the immune response is measured by measuring the activation of T cells in response to an antigen or combination of antigens. 6 It is quantified by measuring the number of IFNγ-producing T cells per splenocyte.An exemplary assay that can be used to determine immune response is the IFN-γ ELISpot assay described in Example 11.Humoral immune response can be analyzed by measuring serum antibody levels against antigens.

[0077] A "suboptimal" immune response is preferably achieved by inducing a splenic 6 This is defined as less than 600, less than 500, less than 400, less than 300, less than 200, and most preferably less than 150 IFNγ producing T cells per cell.

[0078] An immune response of "none" is preferably determined by measuring 10 splenocytes. 6This is defined as less than 100, less than 60, less than 40, more preferably less than 30 IFNγ producing T cells per individual.

[0079] The inventors have discovered that when administration of an antigen or combination of antigens (alone or in combination with a systemically administered non-encoded adjuvant, i.e., a protein adjuvant) results in a "suboptimal" immune response, co-administration of one or more adenoviral vector-encoded adjuvants with the same antigen or the same combination of antigens significantly increases the immune response, particularly in increasing responses that are no longer classified as "suboptimal" (Figures 3, 8, 9).

[0080] Furthermore, the inventors have discovered that where administration of an antigen or combination of antigens (alone or in combination with a systemically administered non-encoded adjuvant) essentially does not generate an immune response (i.e., "no" immune response), co-administration of one or more adenoviral vector-encoded adjuvants with the same antigen or the same combination of antigens does generate an immune response (Figures 4, 6, 7).

[0081] The inventors further discovered that when administration of an antigen or combination of antigens (either alone or in combination with a systemically administered non-encoded adjuvant) generates an adequate immune response (i.e., an immune response stronger than an immune response classified as "suboptimal"), co-administration of one or more adenoviral vector-encoded adjuvants with the same antigen or the same combination of antigens generates an even stronger immune response.

[0082] Surprisingly, the inventors discovered that the described effects depended on the type of adenovirus used to encode the adjuvant. Human adenovirus vectors, especially human group C adenovirus vectors, resulted in higher levels of adjuvant (Figure 1A) and increased immune responses (Figure 1B). The adenovirus vectors hAd5, hAd6, and hAd57 (which have very high sequence similarity to hAd6) proved to be particularly advantageous.

[0083] The inventors have also shown that providing the encoded adjuvant, preferably in a human adenoviral vector, leads to reduced systemic exposure compared to the same adjuvant administered as a protein (Figure 5). This demonstrates the improved safety of the encoded adjuvant, particularly the adjuvant encoded in the adenoviral vector. Without wishing to be bound by theory, the inventors propose that adenoviral vectors, particularly human adenoviral vectors, more particularly human group C adenoviral vectors, more particularly hAd5, hAd6 and hAd57, even more particularly hAd6 and hAd57, most particularly hAd6, generate sufficiently high local levels of adjuvant so that immune response is increased without the concomitant high systemic levels of adjuvant.

[0084] In a preferred embodiment, the antigen or combination of antigens comprises or consists of one or more cancer antigens selected from tumor associated antigens (TAA) and / or cancer neo-antigens.

[0085] In preferred embodiments, the TAA is specific for a particular tumor type, in particular bladder cancer, head and neck cancer, non-small cell lung cancer (NSCLC), melanoma, thymoma, colon cancer; breast cancer, ovarian cancer, liver cancer; or kidney cancer. In some embodiments, the TAA is characterized by a protein that is not expressed at all or is expressed only at low levels in healthy tissues and has increased expression levels in tumor tissues. A common class of TAAs is, for example, cancer-testis (CT) antigens. Generally, the expression of these proteins is restricted to male germ cells in adult animals. However, in cancer, these developmental antigens are often re-expressed.

[0086] In preferred embodiments, the cancer neo-antigen is selected from the group consisting of single amino acid mutant peptides, frameshift peptides, intron read-through mutant peptides, and splice site mutant peptides. In some embodiments, the cancer neo-antigen is a fragment of a mutant protein expressed in cancer tissue, which fragment contains a central non-wild type amino acid caused by a mutation (one or more non-synonymous single nucleotide mutations) flanked on either side by the respective wild type amino acid sequence (preferably 12 amino acids on each side). In some embodiments, the cancer neo-antigen may contain multiple non-wild type amino acids.

[0087] Similarly, the nucleic acids encoding the combination of antigens can be present in a single vector or distributed among multiple vectors in a second set of vectors. The single antigens can be linked head to tail with or without a linker. If present, the linker between the antigens or antigen groups can be derived from naturally occurring multidomain proteins or generated by design. Linkers include flexible linkers and / or in vivo cleavable linkers that can be processed by cellular proteases. Suitable linker sequences are well known in the art and preferably comprise or consist of 1-10 amino acids. The linker preferably consists of or comprises small amino acids such as Ser and Gly.

[0088] In preferred embodiments of all aspects of the invention, the second set of vectors comprises nucleic acids encoding at least 1, at least 3, at least 5, at least 8, at least 10, at least 20, at least 30, at least 40, at least 50 TAAs.

[0089] In preferred embodiments of all aspects of the invention, the second set of vectors comprises nucleic acids encoding at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100 cancer neoantigens.

[0090] Generally, preventive or therapeutic vaccination against viral, bacterial or fungal infections does not require as many different antigens to be effective as vaccination in the treatment of proliferative diseases. Nevertheless, there are some viruses, e.g. HIV, that have a large epitope diversity, especially in their coat proteins. Multiple antigens can be included to induce a broad immune response. In preferred embodiments of all aspects of the invention, the second set of vectors comprises nucleic acids encoding at least 1, at least 3, at least 5, at least 8, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100 viral, bacterial or fungal antigens.

[0091] Typically, a vaccine composition containing more antigens will induce a stronger immune response than a vaccine composition containing only a few antigens, where "a few antigens" refers to 10 or fewer antigens, particularly 5 or fewer antigens.

[0092] Adjuvants The vaccine composition according to the first aspect of the present invention may contain one encoded adjuvant or several encoded adjuvants. The nucleic acids encoding one or more adjuvants may be present in a single vector or may be distributed among multiple vectors of the first set of vectors. For example, if the adjuvant is an antibody, the heavy chain may be encoded in one vector and the light chain in another vector, or the heavy and light chains may be encoded in the same vector. If there are multiple encoded adjuvants, they may be included in a single vector or a series of vectors.

[0093] In all aspects of the invention, the one or more encoded adjuvants can be membrane-bound or soluble. Those skilled in the art know that membrane-bound adjuvants are encoded by nucleic acids that contain a transmembrane domain and an ER sorting signal. In a preferred embodiment of all aspects of the invention, the one or more adjuvants are selected from regulators of checkpoint molecules, cytokines, preferably IL-2, IL-1β, IL-7, IL-15, IL-18, GM-CFS, and INF-γ, or cytokine analogs, cytokine receptors, preferably CD25 (IL-2α receptor), synthetic polynucleotide adjuvants, polyamino acid adjuvants, preferably polyarginine or polylysine, activators of interferon genes, preferably STING (stimulator of interferon genes, also known as MITA and MPYS), adenosine deaminase (ADA), or growth factor-activated receptor gamma coactivator 1-α (PGC-1α). In a preferred embodiment, the regulator of a checkpoint molecule is selected from the group consisting of an agonist of a tumor necrosis factor (TNF) receptor superfamily member or an agonist of a B7-CD28 superfamily member, where preferably the agonist is a (soluble) ligand or agonist antibody or antibody-like protein (e.g. CP-870,893 for CD40); and an antagonist of PD-1, PD-L1, A2AR, B7-H3 (e.g. MGA271), B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, TIGIT or VISTA, where preferably the antagonist is an antagonist antibody or antibody-like protein. In a preferred embodiment, the agonist of a TNF receptor superfamily member is CD27, CD40 (e.g. CP-870,893), OX40, GITR or CD137. In a preferred embodiment, the B7-CD28 superfamily member agonist is CD28 or ICOS.

[0094] In a preferred embodiment of all aspects of the present invention, one or more adjuvants are selected from the group consisting of an agonist of OX40, preferably OX40L, an agonist of ICOS, preferably ICOSL, an agonist of CD40, preferably CD40L, and an antagonistic CTLA-4 specific antibody or antibody-like protein, where the antagonistic CTLA-4 specific antibody or antibody-like protein may be soluble or may include a transmembrane domain and an ER sorting signal, i.e., a membrane-bound antibody.In some embodiments, the transmembrane domain is the mouse transmembrane domain shown in SEQ ID NO:4.In a preferred embodiment, the transmembrane domain is the human transmembrane domain shown in SEQ ID NO:5.

[0095] In preferred embodiments of all aspects of the invention, the one or more adjuvants are: (1) Antagonistic CTLA-4-specific antibodies or antibody-like proteins; (2) an agonist of OX40, preferably OX40L; (3) an agonist of ICOS, preferably ICOSL; (4) an agonist of CD40, preferably CD40L; (5) an antagonistic CTLA-4 specific antibody or antibody-like protein and an agonist of OX40, preferably OX40L; (6) antagonistic CTLA-4 specific antibodies or antibody-like proteins and agonists of ICOS, preferably ICOSL; (7) antagonistic CTLA-4 specific antibodies or antibody-like proteins and agonists of CD40, preferably CD40L; (8) an agonist of OX40, preferably OX40L, and an agonist of ICOS, preferably ICOSL; (9) an agonist of OX40, preferably OX40L, and an agonist of CD40, preferably CD40L; or (10) An agonist of ICOS, preferably ICOSL, and an agonist of CD40, preferably CD40L.

[0096] In preferred embodiments of all aspects of the invention, the antagonistic CTLA-4 specific antibody or antibody-like protein is ipilimumab.

[0097] In a preferred embodiment of all aspects of the invention, one or more of the adjuvants comprises a transmembrane domain and an ER sorting signal, in other words, when the encoded adjuvant is expressed it becomes a membrane-bound protein.

[0098] The expression and function of the CTLA-4 receptor molecule are intrinsically linked to T cell activation. CTLA4 is upregulated soon after T cell receptor (TCR) engagement (signal 1), and its expression peaks 2–3 days after activation. CTLA4 weakens TCR signaling by competing with the costimulatory molecule CD28 for binding to the B7 ligands B7-1 (CD80) and B7-2 (CD86), for which CTLA4 has higher avidity and affinity. Since both B7-1 and B7-2 provide positive costimulatory signals to T cells engaging the TCR (signal 1) via CD28 (signal 2), it is necessary to inhibit the interaction of CTLA4 with both molecules. Thus, anti-CTLA-4 antibodies that block the inhibitory activity of CTLA-4 enhance T cell activation. Anti-CTLA4 antibodies (ipilimumab; BMS) have been successfully developed as a cancer immunotherapy based on the induction of long-lasting protection for some melanoma patients. However, the therapeutic potential of systemic delivery of anti-CTLA-4 antibodies is limited by the significant side effects associated with immunotherapy.

[0099] OX40L in the context of the present invention refers to OX40 ligand (human OX40: NP_003317, mouse OX40L: NP_033478). OX40L is a ligand for OX40 (also known as CD134 or TNFRSF4) and is stably expressed on many antigen-presenting cells, such as DC2 (a subtype of dendritic cells), macrophages, and activated B lymphocytes. Binding of OX40L to OX40 provides a survival signal for T cells and allows the development of memory T cells.

[0100] ICOSL in the context of the present invention refers to ICOS ligand (human ICOSL: NP_056074, mouse ICOSL: NP_056605).

[0101] CD40L in the context of the present invention refers to CD40 ligand (human CD40L: NP_000065, mouse CD40L: NP_035746).

[0102] In the examples, mouse versions of ICOSL, CD40L, and OX40L were used.

[0103] In some embodiments, the adjuvant is an antibody that is encoded as one contiguous amino acid sequence including a 2A sequence, which allows for the production of separate heavy and light chains. In some embodiments, the adjuvant is an antibody that is encoded as one contiguous amino acid sequence including a first signal peptide, a heavy chain, a furin site, a 2A sequence, a second signal peptide, and a light chain. Such constructs were used for ipilimumab and 9D9 in the Examples section.

[0104] We demonstrated that the adjuvant activity of the encoded adjuvant is significantly superior to that of a protein adjuvant delivered systemically by intraperitoneal injection (Figure 3). Without being bound by any theory, these results indicate that co-administration of an adjuvant encoded in an adenoviral vector ensures that the antigen and adjuvant are co-localized in time for effective immunostimulatory activity. Furthermore, we demonstrated that the encoded adjuvant significantly reduces the concentration of adjuvant in serum compared to protein adjuvant injected subcutaneously or intraperitoneally (Figure 5). Thus, the adjuvant effect of the encoded adjuvant is achieved with very limited systemic exposure.

[0105] All terms used in relation to the following aspects of the invention have the meanings defined in relation to the first aspect of the invention unless specifically defined otherwise. Furthermore, all embodiments specified in relation to the first aspect that are applicable to other aspects are also envisaged for those aspects unless specifically defined otherwise.

[0106] In a second aspect, the present invention relates to a vaccine composition according to the first aspect of the invention for use in the treatment or prevention of a disease.

[0107] In a preferred embodiment, the vaccine composition is for use in the treatment of a proliferative disease in a subject. Preferably, the proliferative disease is a cancer and / or a tumor.

[0108] In general, the tumor is preferably at least stage Tis or T1 (excluding Tx and T0), and preferably at least stage T2, T3 or T4. It may be at the same time of all stages N (e.g., Nx or N0) and M (e.g., M0), and in a preferred embodiment, at least stage N1, N2 or N3 and / or M1). This refers to the TNM classification, which defines the tumor stage as follows: T: Size of the primary tumor or direct spread Tx: Tumor not assessable Tis: Carcinoma in situ T0: No evidence of tumor T1, T2, T3, T4: Evidence of primary tumor, increasing in size and / or extension with increasing stage N: The extent of metastasis to regional lymph nodes Nx: Lymph nodes not assessable N0: No regional lymph node metastasis N1: Regional lymph node metastasis present; in some locations, the tumor has spread to the nearest lymph node or a small number of regional lymph nodes N2: The tumor has spread to a stage between N1 and N3 (N2 is not used for all locations) N3: Tumor has spread to more distant lymph nodes or to many regional lymph nodes (N3 is not used for all sites) M: Presence of distant metastasis M0: No distant metastasis M1: Metastasis to distant organs (beyond regional lymph nodes)

[0109] Exemplary stages that are particularly envisioned to benefit from the present invention are Ti and any of N (preferably N1, N2, or N3), and any of M (preferably M1), T1 and any of N (preferably N1, N2, or N3), and any of M (preferably M1), T2 and any of N (preferably N1, N2, or N3), and any of M (preferably M1), T3 and any of N (preferably N1, N2, or N3), and any of M (preferably M1), and T4 and any of N (preferably N1, N2, or N3), and any of M (preferably M1). The presence and spread of tumors in patients can be detected using imaging methods, such as computed tomography (CT) scans, magnetic resonance imaging (MRI), isotopic diagnostics using radioactive tracers detected by scintigraphy in positron emission tomography (PET), or a combination thereof. Imaging methods can also be combined with other methods, such as ultrasound, endoscopy, mammography, detection of biomarkers in blood, fine needle biopsy, or a combination thereof. The size of tumors detectable by imaging methods varies depending on the method used, with isotope imaging methods measuring approximately 1.5 cm in diameter, CT and MRI measuring approximately 3 mm in diameter, and PET-based methods measuring approximately 7 mm in diameter (Erdi. (2012) Molecular Imaging and Radionuclide Therapy 21(1): 23).

[0110] Preferably, the presence of a tumor ("evidence") has been determined by a method selected from the group consisting of detection of circulating tumor cell free DNA, computed tomography (CT) scan, magnetic resonance imaging (MRI), isotopic diagnosis using radioactive tracers detected by positron emission tomography (PET) scintigraphy, and combinations of the above. In one embodiment, one or more of the aforementioned methods, or a combination thereof, is combined with a method from the group consisting of ultrasound, endoscopy, mammography, detection of biomarkers in the blood, fine needle biopsy, and any combination thereof.

[0111] In preferred embodiments of the second aspect, the cancer is selected from the group consisting of malignant neoplasms of the lips, oral cavity, pharynx, digestive tract, respiratory tract, intrathoracic organs, bone, articular cartilage, skin, mesothelial tissue, soft tissue, breast, female reproductive organs, male reproductive organs, urinary tract, brain and other parts of the central nervous system, thyroid, endocrine glands, lymphatic tissue, and hematopoietic tissue. Generally, it is preferred that the subject has a tumor at a TNM stage as described above.

[0112] In one embodiment, tumors are characterized by lesions at least about 3 mm in diameter, preferably at least 7 mm in diameter, more preferably at least 1.5 cm in diameter.

[0113] In a preferred embodiment, the vaccine composition is administered in combination with one or more immunomodulatory agents, more particularly anti-PD1. The one or more immunomodulatory agents, particularly anti-PD1, are preferably administered as a protein.

[0114] It is envisioned that administration of one or more immunomodulatory agents may be initiated prior to initiation of administration of the vaccine composition, or after initiation of administration of the vaccine composition, or simultaneously with initiation of administration of the vaccine composition.

[0115] In another embodiment of the second aspect of the invention, a vaccine composition is provided for the treatment of an infectious disease, such as a viral, bacterial or fungal infection.

[0116] In a third aspect, the present invention relates to a vaccine composition or vaccine kit for inducing an immune response to an antigen or combination of antigens comprising: (1) a first composition comprising a nucleic acid encoding one or more adjuvants, or a first set of one or more vectors comprising said nucleic acids; and (2) a second composition comprising an antigen or combination of antigens, or a nucleic acid encoding an antigen or combination of antigens, or a second set of one or more vectors comprising said nucleic acids; wherein (1) is administered to a patient at a first site and (2) is administered to a patient at a second site, wherein the first site is within 20 cm of the second site, and wherein the lymphatic system of the first site drains into the same lymph nodes as the lymphatic system of the second site, or the first site and the second site are the same.

[0117] In the context of the present invention, the expression "inducing an immune response" refers to a cellular immune response and / or a humoral immune response as described herein. In some embodiments, a vaccine composition or vaccine kit is provided for use in the treatment or prevention of a disease, preferably a proliferative disease or an infectious disease, more preferably a cancer.

[0118] The antigen or combination of antigens may be delivered in the form of a protein or may be delivered encoded, and the nucleic acid encoding the antigen or combination of antigens may or may not be included in a vector. One or more adjuvants may be encoded, and the nucleic acid encoding the one or more adjuvants (i.e., the first nucleic acid) may or may not be included in a vector. The nucleic acid encoding the one or more adjuvants (i.e., the first nucleic acid) may be one molecule or one or more molecules, such as two or more nucleic acid molecules. It is known to those skilled in the art that terms such as "one nucleic acid molecule," "two nucleic acid molecules," and the like do not indicate an absolute number of nucleic acid molecules, but rather an amount of different nucleic acid molecules (i.e., nucleic acid molecules having different sequences). When the adjuvant is an antibody, the heavy chain may be encoded by one nucleic acid molecule and the light chain may be encoded by another nucleic acid molecule, or the heavy and light chains may be encoded by one nucleic acid molecule. When there are multiple encoded adjuvants, they may be encoded by one nucleic acid molecule or several nucleic acid molecules. Similarly, the nucleic acid encoding one or more adjuvants (i.e., the first nucleic acid) may be present in a single vector or distributed among two or more vectors of the first set of vectors. For example, if the adjuvant is an antibody, the heavy chain may be encoded in one vector and the light chain in another vector, or the heavy and light chains may be encoded in the same vector. When there are multiple encoded adjuvants, they may be included in a single vector or a series of vectors. In some embodiments of the third aspect of the invention, the one or more adjuvants and / or antigens or combinations of antigens are encoded by RNA and delivered by lipid nanoparticles or lipid-polymer hybrid nanoparticles. In a preferred embodiment of the third aspect of the invention, the one or more adjuvants and / or antigens or combinations of antigens are encoded by nucleic acids included in the first set of vectors and the second set of vectors, respectively. Most preferably, the vectors are as described for the first aspect of the invention.

[0119] Surprisingly, the inventors have found that the efficacy of the encoded adjuvant is lost when the antigen and adjuvant are administered at distant locations where the lymphatic systems of both locations do not drain into the same lymph node, such as the contralateral limb (Figure 2). Without wishing to be bound by theory, the inventors hypothesize that for effective immunostimulation, it is important that the antigen and adjuvant act simultaneously and in close proximity, particularly within one lymph node. This can be achieved by using an encoded adjuvant, preferably an adjuvant encoded in an adenoviral vector, more preferably an adjuvant encoded in a human adenoviral vector. Furthermore, if the antigen, preferably the encoded antigen and the encoded adjuvant, are administered as a mixture or in close proximity (draining into the same lymph node), the simultaneous action in close proximity is enhanced. When both the antigen and the adjuvant are encoded, the nucleic acid sequences encoding the antigen and the adjuvant are not contained within the same molecule, e.g., not contained within the same vector or on the same RNA molecule. The vaccine composition or vaccine kit of the third aspect of the present invention may comprise the first and second compositions as a mixture or as two separate components. In other words, the vaccine composition or vaccine kit may be formulated for simultaneous or separate administration of the first and second compositions. The components of the first and second compositions may be contained within one composition, but are separate molecules. The first nucleic acid and the second nucleic acid are not contained in the same nucleic acid molecule. The first set of one or more vectors and the second set of one or more vectors are different sets of vectors. Thus, the antigen and adjuvant are delivered as separate molecules, but these separate molecules are delivered in close temporal and spatial proximity.

[0120] In preferred embodiments, the first site is within 17.5 cm, 15 cm, 12.5 cm, 10 cm, 7.5 cm, 5 cm, 2.5 cm, 1 cm, 0.5 cm, 0.25 cm, or 0.1 cm of the second site. In the most preferred embodiment, the first site and the second site are the same. One skilled in the art will recognize when (1) and (2) are administered as a mixture, the first site and the second site are the same, and there is no time interval between administration of (1) and (2). It is envisioned that (1) and (2) are administered by intramuscular, subcutaneous, intradermal, intraperitoneal or intrathoracic injection. In a preferred embodiment, (1) and (2) are administered by the same route, for example, both are administered by intramuscular injection. However, administration does not necessarily have to be by the same route, as long as (1) is administered to a first and second tissue, and the lymphatic systems of the first and second tissues drain into the same lymph node.

[0121] In a preferred embodiment of the third aspect of the invention, (1) and (2), i.e., the encoded adjuvant and the (protein or encoded) antigen, are administered within a time interval of 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 3 minutes or less, or 1 minute or less. In a most preferred embodiment of the third aspect of the invention, (1) and (2) are administered to the patient as a mixture, i.e., (1) and (2) are administered simultaneously to the same location.

[0122] Furthermore, the simultaneous action in close proximity can be enhanced by using an encoded adjuvant that contains a transmembrane domain and an ER sorting signal. Such adjuvants bind to the membrane when expressed. Unlike soluble adjuvants, they cannot diffuse, but are bound to the cells in which they are expressed, thus promoting close action. Furthermore, membrane-bound adjuvants only exert a local effect, limiting the undesirable effects of systemic exposure of soluble adjuvants. Thus, in a preferred embodiment, the adjuvant contains a transmembrane domain and an ER sorting signal. Examples of membrane-bound adjuvants include membrane-bound forms of OX40L, CD40L, ICOSL, or anti-CTLA4.

[0123] The vaccine composition or vaccine kit for inducing an immune response against an antigen or a combination of antigens is preferably for use in treating a disease in a subject.The disease may be an infectious disease or a proliferative disease, preferably a proliferative disease.Preferably, the proliferative disease is cancer and / or tumor.

[0124] In preferred embodiments of the second and third aspects, viral vectors, particularly adenoviral vectors comprising nucleic acids encoding one or more adjuvants, are administered to a subject, particularly a human subject, preferably by intramuscular administration, at a viral particle load (vp) of 10^10vp or more, 2x10^10vp or more, 4x10^10vp or more, and 10^11vp or less, 8x10^10vp or less, 6x10^10vp or less, and adenoviral vectors encoding an antigen or combination of antigens are administered to a subject, particularly a human subject, preferably by intramuscular administration, at a viral particle load (vp) of 5x10^10vp or more, 6x10^10vp or more, 7x10^10vp or more, 8x10^10vp or more, and 2x10^11vp or less, 10^11vp or less, 9x10^10vp or less.

[0125] In a fourth aspect, the present invention relates to a vaccination regimen comprising a first and a second administration step, wherein (a) the first administration step comprises administration of a vaccine composition according to the first, second or third aspect of the invention, and (b) the second administration step comprises administration of (1) a first composition comprising nucleic acids encoding one or more adjuvants, or a first set of one or more vectors comprising said nucleic acids, and / or (2) a second composition comprising an antigen or a combination of antigens, or a nucleic acid encoding said antigen or combination of antigens, or a second set of one or more vectors comprising said nucleic acids.

[0126] The vaccine composition administered in the first administration step may be a vaccine composition provided according to the first aspect of the invention.The vaccine composition administered in the first administration step may be a vaccine composition provided for use according to the second or third aspect of the invention.

[0127] The one or more coded adjuvants administered in the first and second administration steps can be the same or different, preferably the same. They can be selected from the adjuvants described in relation to the first aspect of the present invention. In a preferred embodiment, the one or more adjuvants included in the first and second vaccine compositions are selected from the group consisting of OX40 agonist, preferably OX40L, ICOS agonist, preferably ICOSL, CD40 agonist, preferably CD40L, and antagonistic CTLA-4 specific antibody or antibody-like protein, where the antagonistic CTLA-4 specific antibody or antibody-like protein can be soluble or can include a transmembrane domain and an ER sorting signal, i.e., a membrane-bound antibody.

[0128] If an antigen or combination of antigens is administered in the second administration step (protein or encoded), the antigen or combination of antigens is the same as in the first administration step.

[0129] When the second administration step involves administration of an antigen (protein or encoded), that administration can be described as a prime boost regimen.

[0130] In some embodiments, the vaccination regimen is a heterologous prime boost regimen using two different viral vectors. In such embodiments, the first and second administrations are preferably separated by at least one week, preferably six weeks.

[0131] It is preferred that both the first and second administration steps include administration of a first set of one or more vectors comprising nucleic acids encoding one or more adjuvants. In other words, both the first and second administration steps include administration of one or more encoded adjuvants, the encoded adjuvants being included in a vector. It is further preferred that both the first and second administration steps include administration of a second set of one or more vectors comprising nucleic acids encoding an antigen or combination of antigens. In other words, both the first and second administration steps include administration of an encoded antigen or combination of antigens, the encoded antigen or combination of antigens being included in a vector.

[0132] The first and second sets of vectors of the second administration step may be viral vectors selected from the group consisting of an alphavirus vector, a Venezuelan Equine Encephalitis (VEE) virus vector, a Sindbis (SIN) virus vector, a Semliki Forest virus (SFV) virus vector, a simian or human cytomegalovirus (CMV) vector, a lymphocytic choriomeningitis virus (LCMV) vector, a retroviral or lentiviral vector, an adenovirus vector, an AAV vector, a poxvirus vector, a vaccinia virus vector, or a modified vaccinia Ankara (MVA) vector.

[0133] It is preferred that the first set of vectors ("adjuvant vectors") of the first and second administration steps are adenoviral vectors. More preferably, they are human adenoviral vectors, preferably selected from those described for the first set of vectors of the first aspect of the invention. In a preferred embodiment, the first set of vectors of the first administration step is a different adenoviral vector, preferably a different human adenoviral vector, from the first set of vectors of the second administration step.

[0134] It is further preferred that the second set of vectors ("antigen vectors") of the first and second administration steps are selected from those described for the second set of vectors of the first aspect of the invention. However, the second set of vectors of the first administration step is different from the second set of vectors of the first administration step. In other words, the second set of vectors ("antigen vectors") of the first and second administration steps are different vectors but contain the same antigen or combination of antigens.

[0135] In a preferred embodiment of the first administration step, the first set of vectors (the "adjuvant vectors") are human adenoviral vectors and the second set of vectors (the "antigen vectors") are adenoviral vectors.

[0136] In a preferred embodiment of the second administration step, the first set of vectors ("adjuvant vectors") are adenoviral, AAV or MVA vectors, preferably adenoviral vectors, and the second set of vectors ("antigen vectors") are MVA vectors.

[0137] Surprisingly, the inventors have discovered that re-administration of adjuvant alone, preferably adjuvant encoded in an adenoviral vector, enhances the antitumor effect of neo-antigen vaccines (Figure 10). Thus, in some embodiments, the second administration step comprises administration of an adjuvant, preferably an adjuvant encoded in an adenoviral vector, more preferably an adjuvant encoded in a human adenoviral vector, rather than an antigen. In such embodiments, the first set of vectors is preferably the same in the first and second administration steps (i.e., the same adjuvant in the same vector). Furthermore, in such embodiments, the first and second administration steps are preferably performed with an interval of about one day. Preferably, the first and second administrations are performed via the same route.

[0138] In a preferred embodiment of the vaccination regimen, the first and / or second administration step further comprises the administration of at least one immunomodulatory agent.

[0139] In another aspect, the present invention relates to a pharmaceutical preparation or composition comprising the vaccine composition according to the first aspect and a pharma- ceutical acceptable carrier and / or excipient. The pharmaceutical preparation or composition may further comprise at least one immunomodulator. The present invention also relates to said pharmaceutical preparation or composition for use in the prevention or treatment, in particular the treatment, of a proliferative disease in a subject.

[0140] To prepare the pharmaceutical composition of the present invention, the pharma- ceutically acceptable carrier can be either solid or liquid. The solid form of the composition includes powder, tablet, pill, capsule, lozenge, cachet, suppository, and dispersible granule. The solid excipient can be one or more substances that can also function as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials. In the case of powder, the excipient is preferably a finely divided solid that is mixed with the finely divided inhibitor of the present invention. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in an appropriate ratio and compressed into the desired shape and size. Suitable excipients are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, and the like. To prepare suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active ingredient is dispersed homogeneously therein, as by stirring. The molten homogeneous mixture is then poured into suitable sized molds and allowed to cool, thereby solidifying. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0141] Liquid form compositions include solutions, suspensions, and emulsions, for example, water, saline, aqueous glucose solution, glycerol solution, or water / propylene glycol solution.For parenteral injection (for example, intravenous, intraarterial, intraosseous injection, intramuscular, subcutaneous, intraperitoneal, intradermal, and intrathecal injection), liquid preparations can be formulated as, for example, a solution in aqueous polyethylene glycol solution.Saline is a preferred carrier when administering pharmaceutical compositions intravenously.

[0142] Preferably, the pharmaceutical composition is in unit dosage form.In this form, the composition can be divided into unit doses containing an appropriate amount of active ingredient.The unit dosage form can be a packaged composition, and the package contains individual amounts of the composition, such as packaged tablets, capsules, and powders in vials or ampoules.The unit dosage form can also be a capsule, injection vial, tablet, cachet, or lozenge itself, or any of the appropriate number of these in packaged form. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0143] In addition, such pharmaceutical compositions may also contain other pharmacologically active substances.

[0144] In another aspect, the present invention relates to a vaccine kit comprising, in separate packages: (i) a vaccine composition according to the first aspect; and (ii) at least one immunomodulatory agent.

[0145] In the present context, the adjuvant is a coded adjuvant and the immunomodulatory agent is preferably a protein.

[0146] In yet another aspect, the present invention relates to a method for treating or preventing a proliferative disease or infectious disease, preferably a proliferative disease, comprising administering to a patient in need thereof an effective amount of a vaccine composition according to the first aspect of the invention or a vaccine composition as described in relation to the third aspect of the invention.

[0147] In preferred embodiments of any of the above aspects, the one or more immunomodulatory agents is a cytokine selected from IL-2, IL-1β, IL-7, IL-12, IL-15, IL-18, GM-CFS, and INF-γ; or a cytokine analog selected from analogs of IL-2, IL-1β, IL-7, IL-12, IL-15, IL-18, GM-CFS, and INF-γ; a modulator of a checkpoint molecule selected from the group consisting of an agonist of a tumor necrosis factor (TNF) receptor superfamily member, an agonist of a B7-CD28 superfamily member; an antagonist of PD-1, PD-L1, A2AR, B7-H3 (e.g., MGA271), B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, or VISTA.

[0148] In preferred embodiments of any of the above aspects, the at least one immunomodulatory agent is selected from an antagonistic CTLA-4-specific antibody or antibody-like protein, an antagonistic PD-1-specific antibody or antibody-like protein, and / or IL-2 or an analog thereof. The one or more immunomodulatory agents are preferably administered as a protein. EXAMPLES

[0149] Example 1: Intramuscular administration of Ad-encoded α-mCTLA4 together with an adenoviral vaccine encoding a tumor neoantigen enhances vaccine-induced T cell responses, but with significantly different efficiency (Ad6, Ad5 >> GAd20 and ChAd68) (Figure 1).

[0150] For example, mice were vaccinated with GAd vaccines encoding seven CD8 T cell neo-antigens selected from the MC38 tumor model (Yadav et al., Nature. 2014 Nov 27;515(7528):572-6; D'Alise et al, Nat. Commun. 2019 Jun19;10(1):2688) either alone or mixed with different adenoviral vectors (clone 9d9, SEQ ID NO:1) encoding anti-mCTLA4 (Ad6-9d9; Ad5-9d9; GAd20-9d9; ChAd68-9d9). The adenoviral vectors encoding anti-mCTLA4 were administered in a dose of 10^8 vp (10 8 The vaccine was administered at a dose of 1000 ng / mL (viral particles) per 1000 ng / mL, which corresponds to the dose administered to human patients in clinical practice. Measurement of the levels of circulating encoded anti-mCTLA4 after Ad injection (day 7) in the different groups showed higher levels of anti-mCTLA4 when encoded by Ad6 and Ad5 compared to GAd20 and ChAd68 (Figure 1A). Pools of peptides corresponding to the sequences of each neo-antigen present in the vaccine vector were used as antigens, and immune responses were measured by ex vivo IFN-γ ELISpot assay 2 weeks after vaccination. Vaccine immunogenicity was enhanced in the presence of encoded anti-mCTL4 expressed by Ad6 and Ad5, but not by GAd20 and ChAd68 (Figure 1B).

[0151] Example 2: The effect of Ad-encoded α-mCTLA4 in enhancing vaccine-induced T cell responses requires co-administration with the vaccine (FIG. 2). To understand whether the effect of Ad-encoded α-mCTLA4 requires co-administration in a mixture with a vaccine, C57Bl6 mice were vaccinated with a GAd vaccine encoding seven selected CD8 neo-antigens from the MC38 tumor model administered with Ad6-α-mCTLA4 in three different regimen modalities: i) co-administration as a mixture in one anatomical site (quadriceps muscle), ii) administration as two separate doses adjacent within 5 min in the same anatomical site as i) and iii) administration as separate doses in two contralateral distant sites. Immune responses were measured by ex vivo IFN-γ ELISpot assays 2 weeks after vaccination, showing that the adjuvant effect was lost when the vaccine and Ad6-α-mCTLA4 were administered as separate components (Figure 2). Adenoviral vectors encoding the adjuvant α-mCTLA4 were administered at a dose of 10^8 vp. The best effect on enhancing immune responses was achieved when the vaccine and adjuvant Ad6-9d9 were co-administered as a mixture.

[0152] Example 3: Intramuscular coadministration of an adenoviral vector encoding anti-mCTLA4 with an adenoviral vector encoding a murine tumor neoantigen enhances vaccine-induced T cell responses (CD8 and CD4) and outperforms the same antibody delivered systemically as a protein (Figure 3).

[0153] In this example, mice were vaccinated with a polyneoantigen GAd vaccine encoding 31 neoantigens selected from the CT26 murine tumor (D'Alise et al, Nat. Commun. 2019 Jun 19;10(1):2688). The vaccine was administered intramuscularly alone (10^8 vp) ​​or co-administered with Ad6 anti-mCTLA4 encoding anti-mouse CTLA4 (clone 9d9) at a dose of 10^8 vp. Parallel groups of mice were treated with the same vaccine in combination with anti-mCTLA4 (clone 9d9) protein (BioXcell) administered intraperitoneally. Immune responses were measured 2 weeks later by ex vivo IFN-γ ELISpot assays by using as antigens a set of peptides corresponding to the sequence of each neoantigen present in the vaccine vector. Ad-encoded anti-mCTLA4 antibodies co-administered with the GAd neoantigen vaccine increased both vaccine-induced CD8+ and CD4+ T cell responses against tumor neoantigens (Figure 3). This effect was more potent than that observed in the presence of anti-m-CTLA4 delivered as a protein.

[0154] Example 4: Anti-CTLA4-encoded adenoviral vector enhances the immune response of a genetic vaccine encoding 62 neoantigens in two separate expression cassettes and exhibits stronger anti-tumor activity (Figure 4).

[0155] The performance of the encoded adjuvants was also tested with more complex constructs encoding more neo-antigens (Figure 4A). For this purpose, a GAd vaccine vector (named GAd-CT26-62) was used that encodes 62 neo-antigens identified in the murine colon cancer cell line CT26 as disclosed in WO2020 / 099614 A1. Mice were vaccinated intramuscularly with a low dose (2x10^7 vp) of GAd-CT26-62, administered alone or co-administered with Ad6-anti-CTLA4 encoding anti-mouse anti-CTLA4 (clone 9D9) at a dose of 10^8 vp. The immune response was evaluated 2 weeks later by ex-vivo IFN-γ ELISpot assay by using as antigen a set of peptides corresponding to the sequence of each neo-antigen present in the vector. Co-administration of adenovector-encoded anti-mCTLA4 antibodies with GAd-CT26-62 increased vaccine-induced T cell responses against tumor neo-antigens (Figure 4A). The same combination was also tested in a CT26 cancer mouse model to evaluate the effect on the antitumor activity of an adenoviral vector encoding anti-CTLA4 co-administered with the GAd vaccine compared to the effect of the vaccine alone (no adjuvant) in the presence of anti-mPD1 treatment (clone RMP1-14 BioXcell). The results showed that the addition of vaccine-encoded Ad6-9d9 as an adjuvant enhanced the antitumor activity of the vaccine and anti-mPD1 (Figure 4B).

[0156] Example 5: Compared to systemic and local delivery of antibody drugs, systemic exposure to anti-CTLA4 is limited when delivered by adenoviral vectors (Figure 5).

[0157] In this example, mice were injected with an Ad6 vector encoding anti-mCTLA4 (Ad-9d9) at a dose of 10^8 vp or with a single intraperitoneal or subcutaneous injection of the same anti-mCTLA4 antibody (9d9 Ab, 100 μg). Measurement of circulating anti-mCTLA4 serum levels after Ad6 administration demonstrated very limited systemic exposure compared to injection of anti-mCTLA4 as a protein (clone 9D9 BioXcell), supporting the improved biosafety of the encoded antibody (Figure 5).

[0158] Example 6: Intramuscular co-administration of an adenoviral vector encoding anti-CTLA4 with an adenoviral vector encoding a murine surrogate tumor-associated antigen (TAA) reverses immune tolerance (Figure 6).

[0159] To investigate the efficacy of adenoviral vectors encoding anti-mCTLA4 in circumventing immune tolerance of tumor-associated antigens (TAA), we selected surrogate TAA genes belonging to an antigen family expressed in mouse CT26 tumors but not in healthy tissues. Vectors encoding four mouse TAAs (Slc9b1, Psg17, Gm773, Tcp11x2) preceding the human tissue plasminogen activator (TPA) signal peptide were generated and injected in vivo alone or mixed with a 10^8 vp dose of Ad6-encoded anti-mCTLA4. Immune responses were measured by ex-vivo IFN-γ ELISpot assays 2 weeks after vaccination using a set of peptides corresponding to the sequences of the vaccine vector-encoded TAA as antigens. The results showed that the immune response was significantly enhanced when Ad-9D9 was co-injected with the vaccine TAA (Figure 6).

[0160] Example 7: Adenoviral vector encoding a tumor-associated antigen Adenoviral vector encoding anti-CTLA4 co-administered intramuscularly with a vaccine also increases the antibody response to self-antigens (Figure 7). In this example, the effect of adenoviral vectors encoding anti-mCTLA4 in increasing vaccine-induced antibody responses was also investigated. hHer2 transgenic (Tg) mice, a well-known mouse model that is resistant to hHer2 and widely used for testing Her2 vaccines, were immunized by injection with hHer2-encoding GAd vaccine alone or mixed with Ad6-9d9 at a dose of 10^8 vp. Sera prepared from immunized mice were analyzed by ELISA against hHer2 protein to measure antibody levels after treatment. The results showed that while the vaccine alone induced low levels of antibodies against hHer2, a relevant increase in antibody responses was observed in the presence of the encoded anti-mCTL4 expressed on Ad6.

[0161] Example 8: Adenoviral vector encoding mOX40L co-administered with an Ad-based neoantigen vaccine enhances immunogenicity (Figure 8).

[0162] In this example, mice were vaccinated with GAd vaccines encoding seven CD8 T cell neo-antigens selected from the MC38 tumor model, either alone or in combination with adenovirus Ad6 encoding anti-mCTLA4 (Ad-9d9) and adenovirus Ad6 encoding mOX40L (Ad-OX40L). Adenovirus vectors encoding anti-mCTLA4 and mOX40L were administered at a dose of 10^8 vp. Immune responses were measured 2 weeks after vaccination in each experimental group by ex-vivo IFN-γ ELISpot assay using a pool of peptides corresponding to the sequences of each neo-antigen present in the vaccine vector as antigens. As a result, Ad6 encoding OX40L showed a strong effect of enhancing vaccine immunogenicity at the same level as Ad-9d9.

[0163] Example 9: Use of two encoded adjuvants anti-mCTLA4 and OX40L enhances vaccine efficacy against TAA in a stringent T cell tolerance mouse model (Figure 9).

[0164] In this example, the effect of anti-mCTLA4 and Ad-OX40L-encoded adenoviral vectors was investigated in a stringent T cell tolerance mouse model to human Her2. hHer2-resistant hHer2 transgenic (Tg) mice were immunized with hHer2-encoded GAd vaccine alone, with hHer2-encoded GAd vaccine mixed with Ad6-9d9 or Ad6 OX40L at a dose of 10^8vp, or with hHer2-encoded GAd vaccine together with a mixture of the two adjuvants. In each experimental group, immune responses were measured by ex vivo IFNγ ELISpot assay two weeks after vaccination, showing the effect of breaking T cell tolerance to human Her2 when the two encoded adjuvants were co-administered with the vaccine.

[0165] Example 10: Adenoviral vector encoding ICOSL co-administered with an Ad-based neoantigen vaccine enhances immunogenicity (Figure 10).

[0166] In this example, mice were vaccinated with GAd vaccines encoding seven CD8 T cell neo-antigens selected from the MC38 tumor model, either alone or mixed with the adenovirus Ad6 encoding mouse ICOS-L (Ad-ICOSL), at a dose of 10^8 vp. The immune response was measured 2 weeks after vaccination by ex vivo IFN-γ ELISpot assay for each experimental group using a pool of peptides corresponding to the sequences of each neo-antigen present in the vaccine vector as antigen. The results showed an enhancement of vaccine-induced T cell responses by the encoded Ad-ICOSL.

[0167] Example 11: Readministration of adenoviral vectors encoding anti-CTLA4 enhances the antitumor effect of a GAd neoantigen vaccine in combination with anti-PD1 (Figure 11).

[0168] In a mouse model of well-established CT26 tumors, the impact of the encoded adjuvant anti-CTLA4 on the antitumor activity of a GAd vaccine in combination with a checkpoint inhibitor (anti-PD1) was tested in a single-dose (vaccine+Ad6-9d9, day 0) and double-dose (vaccine+Ad6-9d9 on day 0, Ad6-9d9 on day 1) regimen. Tumor-bearing mice were treated on day 0 with a GAd vaccine vector encoding 62 CT26 neo-antigens (GAd-CT26-62) alone or co-administered with Ad6-anti-CTLA4 encoding anti-mCTLA4 (clone 9D9 10^8vp) in the presence of anti-mPD1 (clone RMP1-14 BioXCell). In parallel, a group of mice received a second dose of Ad6-anti-CTLA4 the next day. The results showed that vaccination with the encoded Ad6-9d9 as an adjuvant enhanced the antitumor activity of the vaccine and anti-PD1, and the highest antitumor response rate was observed in mice that received two doses of Ad6-9d9.

[0169] Example 12: Measurement of circulating anti-hCTLA4 in mice after injection with Ad6 encoding human anti-CTLA4 (Figure 12).

[0170] The sequence of anti-hCTLA4 ipilimumab (SEQ ID NO:2) was encoded into Ad6 and tested in vivo to evaluate its expression by Ad6. C57Bl6 mice were injected with Ad6-ipilimumab at a dose of 10^8 vp. Circulating anti-hCTLA4 levels were measured over time after Ad injection, showing detectable and good expression of the encoded ipilimumab, with a peak observed at day 7 after Ad injection.

[0171] Example 13: Ex vivo IFN-γ ELISpot assay IFN-γ ELISpot assays were performed on single cell suspensions of spleens. MSIP S4510 plates (Millipore, Billerica, MA) were coated with 10 μg / ml anti-mouse IFN-γ antibody (Cat. Number: CT317-C; U-CyTech) and incubated overnight at 4°C. After washing the plates and blocking with medium to avoid background, mouse splenocytes were seeded in duplicate at two different cell densities and stimulated overnight with a single 25-mer peptide or a peptide pool at a final concentration of 1 μg / ml. Peptide diluents dimethylsulfoxide (Sigma-Aldrich) and concanavalin A (Sigma-Aldrich) were used as negative and positive controls, respectively. Plates were subsequently developed by incubation with biotinylated anti-mouse IFN-γ antibody (1 / 100 dilution, Cat. Number: CT317-D; U-CyTech), conjugated streptavidin-alkaline phosphatase (1 / 2500 dilution, Cat. Number 554065; BD Biosciences), and finally 5-bromo-4-chloro-3-indoyl-phosphate / nitroblue tetrazolium one-step solution (Thermo Fisher Scientific). Plates were analyzed using an automated enzyme linked immunosorbent-spot assay video analysis system automated plate reader. ELISpot data were expressed as IFN-γ SFC per million splenocytes. An ELISpot reaction was considered positive if all of the following conditions occurred: (i) IFN-γ production was present in ConA-stimulated wells, (ii) the number of spots seen in positive wells was three times the number detected in mock control wells (dimethyl sulfoxide), and (iii) at least 30 specific spots per million splenocytes.

[0172] Example 14: Adenoviral vectors encoding membrane-bound anti-CTLA4 co-administered with Ad-based neoantigen vaccines enhance vaccine immunogenicity C57Bl6 mice were vaccinated with a GAd vaccine (vaccine, dose 2x10^7 vp) encoding seven CD8 T cell neo-antigens selected from the MC38 tumor model and administered Ad6 (SEQ ID NO:3) encoding a membrane-bound form of 9d9 anti-mCTLA4 (Ad-9d9TM) at a dose of 10^8 vp. Membrane binding was achieved by appending a transmembrane domain segment to the C-terminus of the 9d9 heavy chain of SEQ ID NO:2. As a positive control, a group of mice was co-administered with the vaccine and Ad6-9d9. Similar to the soluble form of 9d9, the membrane-bound form also enhanced vaccine immunogenicity as measured by IFN-γ ELISpot assay (Figure 13).

[0173] Drawing Terminology Blood levels of encoded αCTLA4 splenocytes splenocytes Vaccine mix mixture separate Contralateral Tumor volume anti-PD1 anti-PD1 response days sc subcutaneous ip intraperitoneal Response to TAA Pool Antibodies anti-hHER2 anti-hHER2 T cell response to hHER2 hHER2 Tg mice Blood level Ipi encoded

Claims

1. (1) A first set of one or more vectors comprising nucleic acids encoding one or more adjuvants, wherein the first set of one or more vectors is an adenovirus vector, and (2) an antigen or combination of antigens, or a nucleic acid encoding said antigen or combination of antigens, or a second set of one or more vectors comprising said nucleic acid, A vaccine composition comprising the same.

2. The vaccine composition according to claim 1, wherein the first set of one or more vectors is a human adenovirus vector.

3. The vaccine composition according to claim 2, wherein the human adenovirus vector is selected from the group consisting of hAd6, hAd5, and hAd57, or is selected from hAd6 and hAd57, or is hAd6.

4. The vaccine composition according to any one of claims 1 to 3, wherein the antigen or combination of antigens is encoded by a nucleic acid not contained in the first set of one or more vectors.

5. The vaccine composition according to any one of claims 1 to 3, wherein the second set of one or more vectors comprises a second set of one or more vectors comprising a nucleic acid encoding an antigen or combination of antigens, or the second set of one or more vectors is an adenoviral vector, or is derived from a non-human great ape, more preferably from a chimpanzee, bonobo, or gorilla, and most preferably from a gorilla.

6. The one or more adjuvants are a. regulators of immune checkpoint molecules, selected from the group consisting of: - an agonist of a tumor necrosis factor (TNF) receptor superfamily member, or an agonist of a B7-CD28 superfamily member, or an agonist of CD27, CD40, OX40, GITR, CD137, CD28 or ICOS, wherein the agonist is a ligand or an antagonist antibody or antibody-like protein (e.g., CP-870,893 for CD40), and - an antagonist of PD-1, PD-L1, A2AR, B7-H3 (e.g., MGA271), B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, TIGIT or VISTA, or the antagonist is an (antagonist) antibody or antibody-like protein. b. A cytokine, or IL-2, IL-1β, IL-7, IL-15, IL-18, GM-CFS, or INF-γ, and / or a cytokine analog c. A cytokine receptor, or CD25 (IL-2α receptor), d. An activator of the interferon (IFN) gene, or STING, e. Adenosine deaminase (ADA) or proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), f. A polynucleotide adjuvant, The vaccine composition according to claim 5, selected from the group consisting of

7. One or more adjuvants are an agonist of OX40, or OX40L, an agonist of ICOS, or ICOS-L, an agonist of CD40, or CD40L, and an antagonistic CTLA-4 specific antibody or antibody-like protein, wherein the antagonistic CTLA-4 specific antibody or antibody-like protein may be soluble or may contain a transmembrane domain and an ER sorting signal, The vaccine composition according to claim 6, selected from the group consisting of

8. One or more adjuvants contain a transmembrane domain and an ER sorting signal, The vaccine composition according to claim 7.

9. The antigen or combination of antigens does not induce any immune response in the subject or induces only a suboptimal immune response in the absence of an adjuvant, The vaccine composition according to claim 4.

10. The antigen or combination of antigens is a. A tumor-associated antigen (TAA), or a TAA specific for a particular tumor type, and / or b. A cancer neoantigen, or a cancer neoantigen selected from the group consisting of single amino acid variant peptides, frameshift peptides, read-through variant peptides, and splice site variant peptides, The vaccine composition according to claim 9, comprising one or more cancer antigens selected from or consisting of one or more cancer antigens

11. The vaccine composition according to claim 10, for use in the treatment or prevention of a disease, or for use in the treatment of a proliferative disease or cancer.

12. (1) A first nucleic acid encoding one or more adjuvants, or A first set of one or more vectors containing the first nucleic acid, A first composition comprising, and (2) An antigen or combination of antigens, or A second composition comprising a second nucleic acid encoding an antigen or combination of antigens, or a second set of one or more vectors comprising said second nucleic acid, A vaccine composition or vaccine kit for inducing an immune response against an antigen or combination of antigens, comprising: wherein a. (1) is administered to the patient at a first site and (2) is administered to the patient at a second site, where the first site is within 20 cm, 17.5 cm, 15 cm, 12.5 cm, 10 cm, 7.5 cm, 5 cm, 2.5 cm, 1 cm, 0.5 cm, 0.25 cm or 0.1 cm of the second site, and the lymphatic system of the first site drains into the same lymph nodes as the lymphatic system of the second site, or the first site and the second site are the same, and optionally b. The adjuvant comprises a transmembrane domain and an ER sorting signal. A vaccine composition or vaccine kit.

13. (1) and (2) are administered by intramuscular, subcutaneous, intradermal, intraperitoneal or intrathoracic injection, or (1) and (2) are administered by intramuscular, subcutaneous, intradermal, intraperitoneal or intrathoracic injection and are administered by the same route. The vaccine composition or vaccine kit according to claim 12.

14. (1) and (2) are administered within a time interval of 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 3 minutes or less, or 1 minute or less. The vaccine composition or vaccine kit according to claim 13.

15. a. The first administration step comprises administration of the vaccine composition according to any one of claims 1 or 14, and b. The second administration step comprises (1) a first composition comprising a first nucleic acid encoding one or more adjuvants, or a first set of one or more vectors comprising said first nucleic acid, and / or (2) an antigen or combination of antigens, or a second nucleic acid encoding an antigen or combination of antigens, or a second set of one or more vectors comprising said second nucleic acid, comprising administration of A vaccination regimen comprising first and second administration steps.

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