Co-expression of constructs and immunostimulatory compounds

Abstract

The present invention relates to vectors, such as DNA plasmids, that contain multiple nucleic acid sequences engineered to co-express as separate molecules, including a first polypeptide that includes a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit that includes one or more antigens or portions thereof, and one or more immunostimulatory compounds.

Description

[Technical field]

[0001] The present invention relates to vectors, such as DNA plasmids, that contain multiple nucleic acid sequences of interest engineered to co-express as separate molecules, pharmaceutical compositions comprising such vectors, and the use of such vectors and such pharmaceutical compositions in the treatment or prevention of disease. [Background technology]

[0002] B cell (humoral / antibody-mediated) and T cell responses are important components of the defensive response against infection by pathogens. Specific antibodies against pathogen antigens mediate a wide range of effector functions, including: a) direct neutralization of toxins and pathogens, b) neutralization of pathogen virulence factors, c) binding to and capture of pathogens by mucin, d) activation of complement to mediate phagocytic clearance, degradation, or lysis of anti-pathogens, e) activation of opsonophagocytosis in neutrophils, f) induction of opsonophagocytosis in macrophages, g) activation of degranulation of natural killer (NK) cells to kill infected cells, h) enhancement of antigen updating, processing, and presentation by dendritic cells to T and B cells, and i) induction of degranulation of mast cells, basophils, and eosinophils in parasitic infections (L. Lu et al, Nat Rev Immunol 18(1), 2018, 46).

[0003] Complementing these activities, T cell responses are important for limiting viral replication and infection by killing infected cells, inducing apoptosis, releasing antiviral substances, and / or increasing intracellular lysis of already infected cells, thereby helping to prevent, reduce the severity, or cure disease. Moreover, effective and long-lasting responses in both immune systems usually require additional support from T-helper (Th1 and Th2) lymphocytes.

[0004] Cytotoxic T lymphocytes (CTLs) also play an important role in intracellular pathogens (F. Sheperd et al., Int J Mol Sci 21, 2020, 6144), where MHC class I-restricted CD8+ T cells are known to be important for clearing bacterial infections and providing protective immunity against various bacterial species. MHC class II-restricted CD4+ T cells support memory CD8+ T cell responses and are important for protective immunity against bacterial infections. Naive CD4+ T cells differentiate into subsets of cells with effector capabilities, such as T helper 1 (Th1) and Th2 cells. After binding to specific T cell epitopes on the surface of antigen-presenting cells (APCs), Th1 and Th2 cells provide specific soluble cytokine signals that regulate the balance between antibody and CTL immunity. Thus, effective immunization involves multiple antigen recognition events of specific pathogen immunogenic determinants (epitopes) by T helper cells, followed by molecular recognition by B cells, CTLs, or both.

[0005] Different types of lymphocytes (B cells, CTL, and Th cells) specifically recognize different types of epitopes of pathogens. B cell epitopes can be classified into linear epitopes and conformational epitopes, where linear epitopes are often part of conformational B cell epitopes in native proteins. Conformational epitopes are structural features exposed on the surface of pathogens, such as viral envelopes, bacterial outer membranes, and secreted bacterial toxins. T cell epitopes are short peptides derived from any protein of a pathogen that fit the host's antigen processing and MHC binding mechanisms, especially class I or class II MHC haplotype restriction mechanisms. It is estimated that suitable T cell epitopes occur at a frequency of 1 in 200-500 amino acid sequences, depending on the host population and pathogen.

[0006] A vaccine against a pathogen involves modifying the pathogen or parts of it so that it does not cause harm or disease, but so that when the host is confronted with the infectious agent, the immune system is able to neutralize it sufficiently before it can cause disease. For over 100 years, vaccines have been made by one of two approaches: by introducing a specific antigen to which the immune system responds directly, or by introducing a live attenuated infectious agent that replicates in the host without causing disease and then synthesizes antigens that stimulate the immune system.

[0007] Recently, a fundamentally new vaccination approach has been developed in which polynucleotide sequences (DNA or RNA) encoding antigens capable of stimulating an immune response are directly introduced into appropriate tissues, resulting in in situ production of the target antigen. This approach offers many potential advantages over traditional methods, including stimulation of both B and T cell responses, improved vaccine stability, absence of infectious agents, and relative ease of large-scale production.

[0008] Cancer treatment has improved over the past few decades, particularly with early detection and diagnosis, and the survival rate of cancer patients has increased significantly, but only about 60% of cancer patients are alive five years after diagnosis. Most cancer treatments currently used are surgery, radiation therapy, and cytotoxic chemotherapy, all of which have serious side effects. Recently, treatments using antibodies targeting known cancer-associated antigens have also been used.

[0009] Cancer immunotherapy, or cancer vaccines, which target cancer cells with the help of a patient's own immune system, has attracted attention in recent years, and may reduce or even eliminate the side effects associated with conventional cancer treatments.

[0010] Therefore, there is a need for efficient drugs and medicines that can be used both for the treatment or prevention of infectious diseases and for the treatment or prevention of cancer.

[0011] A vaccibody construct is a dimeric fusion protein consisting of two polypeptides, each of which comprises a targeting unit targeting antigen-presenting cells, a dimerization unit, and an antigen unit comprising one or more antigens or portions thereof, and which, after administration to a subject (e.g., an animal or a human), efficiently generates an immune response against the antigen or a portion thereof, e.g., an epitope, contained in the antigen unit. In another embodiment, a vaccibody construct is a multimeric fusion protein consisting of multiple polypeptides, each of which comprises a targeting unit targeting antigen-presenting cells, a multimerization unit, and an antigen unit comprising one or more antigens or portions thereof, and which, after administration to a subject, has been shown to efficiently generate an immune response against the antigen or a portion thereof, e.g., an epitope, contained in the antigen unit.

[0012] The vaccibody construct may be administered to a subject in the form of a polynucleotide encoding the polypeptide, e.g., a polynucleotide contained in a vector, such as a DNA plasmid. After administration to a host cell, e.g., a muscle cell of a subject, a polypeptide is expressed that forms a multimeric fusion protein, e.g., a dimer, due to the multimerization unit, e.g., dimerization unit. Summary of the Invention

[0013] The inventors have made the surprising observation that it is possible to enhance the overall immune response of a vaccibody by co-expressing one or more immunostimulatory compounds from the same vector in which the vaccibody is expressed.

[0014] In a first aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more antigens or portions thereof; and (b) one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds. Regarding a vector comprising The vector allows for the co-expression of the first polypeptide and one or more immunostimulatory compounds as separate molecules.

[0015] In one embodiment, the vector of the invention comprises a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more disease-associated antigens or portions thereof. Such vectors may be used, for example, in the form of a pharmaceutical composition comprising such vector and a pharma- ceutically acceptable carrier or diluent, for the prophylactic or therapeutic treatment of a disease, for example, for the therapeutic treatment of cancer, or for the prophylactic or therapeutic treatment of an infectious disease, by administering the composition to a subject in need of such prophylactic or therapeutic treatment. [Brief description of the drawings]

[0016] [Figure 1] Co-expression elements for use in vectors of the present invention. Shown is an IRES co-expression element for use in vectors of the present invention, which is inserted between two coding regions. Once mRNA is produced, two ribosomes (T) can begin translation at two separate sites on the mRNA, resulting in the formation of two proteins (A and B). A and B can be, for example, a first polypeptide and an immunostimulatory compound. [Diagram 2] Co-expression elements for use in vectors of the invention. Shown is a 2A self-cleaving peptide co-expression element for use in vectors of the invention, which is inserted between two genes. After transcription, one ribosome translates the mRNA and two proteins (A and B) are formed. The top of the figure shows how a fusion protein is formed when the 2A peptide sequence is not part of the coding sequence. A and B are, for example, a first polypeptide and an immunostimulatory compound. [Diagram 3]Co-expression elements for use in vectors of the invention Figure 3a shows a bidirectional promoter (P) co-expression element for use in vectors of the invention, which is located between two coding regions. One mRNA is produced, two ribosomes (T) can initiate translation in different directions, and two proteins (A and B) are formed. A and B can be, for example, a first polypeptide and an immunostimulatory compound. Figure 3a shows two promoters (P) located in front of two coding regions, i.e., co-expression elements for use in vectors of the invention. Two mRNAs are produced, two ribosomes (T) can initiate translation on the two different mRNAs, and two proteins (A and B) are formed. A and B can be, for example, a first polypeptide and an immunostimulatory compound of the invention. [Figure 4] Embodiments of the First Polypeptide Embodiments of the first polypeptide encoded by the first nucleic acid sequence contained in the vector of the present invention are shown. [Diagram 5] Expression and secretion levels of proteins encoded by DNA plasmids Figure 1 shows the protein expression and secretion levels of the first polypeptide encoded by DNA plasmids VB4194, VB4168, VB4169 and VB4170 detected in the supernatants of HEK293 cells transfected with said DNA plasmids by enzyme-linked immunosorbent assay (ELISA) using mouse a-human IgG CH3 domain capture Ab (MCA878G) and a-human MIP-1α biotinylated capture Ab (BAF270). Lipo neg ctrl (=only cells treated with the transfection agent Lipofectamine serving as a negative control). [Figure 6]Expression and secretion levels of proteins encoded by DNA plasmids. Protein expression and secretion levels of the immunostimulatory compound FLTL3 encoded by DNA plasmids VB4168, VB4169 and VB4170 detected in the supernatants of HEK293 cells transfected with the DNA plasmids by ELISA using mouse a-human FLT3L capture Ab (MAB608) and mouse a-human FLT3L biotinylated detection Ab (BAF308). [Figure 7] Expression and secretion levels of proteins encoded by DNA plasmids. Figure 1 shows the protein expression and secretion levels of the immunostimulatory compound GM-CSF encoded by DNA plasmids VB4169 and VB4170 detected in the supernatants of HEK293 cells transfected with the DNA plasmids by ELISA using rat a-mouse GM-CSF capture Ab (MAB415) and goat a-mouse GM-CSF biotinylated detection Ab (BAM215). [Figure 8] Expression and secretion levels of proteins encoded by DNA plasmids. Figure 1 shows the protein expression and secretion levels of the immunostimulatory compound CCL5 encoded by DNA plasmid VB4170 detected in the supernatants of HEK293 cells transfected with the DNA plasmids by ELISA using rat a-mouse CCL5 capture Ab (MAB4781) and goat a-mouse CCL5 biotinylated detection Ab (BAF478). [Figure 9] Immunogenicity of DNA Plasmids The immunogenicity of DNA plasmids VB4194, VB4168 and VB4169 in mice administered these plasmids is shown in comparison with the negative control VB1026, as measured by IFN-γ secretion from T cells (total T cell response). [Figure 10] Immunogenicity of DNA Plasmids The immunogenicity of DNA plasmids VB4194, VB4168 and VB4169 in mice administered these plasmids is shown in comparison to the negative control VB1026 by measuring TNF-α secretion from T cells (total T cell response). [Figure 11] Immunogenicity of DNA Plasmids The immunogenicity of DNA plasmids VB4194, VB4168 and VB4169 in mice administered these plasmids is shown in comparison to the negative control VB1026 by measuring IFN-γ+TNF-α co-secretion from T cells (total T cell response). [Figure 12] Immunogenicity of DNA Plasmids The immunogenicity of DNA plasmids VB4194, VB4168 and VB4169 in mice administered these plasmids is shown in comparison to the negative control VB1026 by measuring IFN-γ secretion from CD8+ T cells (CD4+ T cell depleted samples). [Figure 13] Immunogenicity of DNA Plasmids The immunogenicity of DNA plasmids VB4194, VB4168 and VB4169 in mice administered these plasmids is shown in comparison to the negative control VB1026 by measuring TNF-α secretion from CD8+ T cells (CD4+ T cell depleted samples). [Figure 14] Immunogenicity of DNA Plasmids The immunogenicity of DNA plasmids VB4194, VB4168 and VB4169 in mice administered these plasmids is shown in comparison to the negative control VB1026 by measuring IFN-γ + TNF-α secretion from CD8+ T cells (CD4+ T cell depleted samples). [Figure 15] Expression and secretion levels of proteins encoded by DNA plasmids Figure 1 shows the protein expression and secretion levels of the first polypeptide encoded by DNA plasmid VB4202 detected in the supernatants of HEK293 cells transfected with said DNA plasmids by ELISA using mouse a-human IgG CH3 domain capture Ab (MCA878G) and a-human MIP-1α biotinylated capture Ab (BAF270). Lipo (=only cells treated with the transfection agent Lipofectamine serving as a negative control). [Figure 16]Expression and secretion levels of proteins encoded by DNA plasmids. Figure 1 shows the protein expression and secretion levels of the immunostimulatory compound GM-CSF encoded by DNA plasmid VB4202 detected in the supernatants (1:1000 dilution) of HEK293 cells transfected with the DNA plasmids by ELISA using rat a-mouse GM-CSF capture Ab (MAB415) and goat a-mouse GM-CSF biotinylated detection Ab (BAM215). [Figure 17] Immunogenicity of DNA Plasmids The immunogenicity of DNA plasmids VB4194 and VB4202 in mice administered these plasmids is shown in comparison to the negative control VB1026 by measuring IFN-γ secretion from T cells (total T cell response). [Figure 18-1] Immunogenicity of DNA Plasmids Flow cytometry evaluation used to assess dendritic cell (DC) responses at the single cell level in mice administered DNA plasmids VB1026, VB4194 and VB4202. The figure shows the gating strategy used to define DCs. A. All events were examined using time parameters to exclude hydrodynamic misalignment. B. Exclusion of doublets was performed using side scatter (SSC) height and area parameters. [Figure 18-2] Immunogenicity of DNA Plasmids Flow cytometry evaluation used to assess dendritic cell (DC) responses at the single cell level in mice administered DNA plasmids VB1026, VB4194 and VB4202. The figure shows the gating strategy used to define DCs. C. Forward scatter (FSC) height and area parameters were used to exclude doublets. D. Dead cells, neutrophils and T cells were excluded and CD45+ immune cells were used for further analysis. [Figure 18-3]Immunogenicity of DNA Plasmids Flow cytometry evaluation was used to assess dendritic cell (DC) responses at the single cell level in mice administered DNA plasmids VB1026, VB4194 and VB4202. The figure shows the gating strategy used to define DCs. E. MHCII expressing cells were gated and used for further analysis. F. B cells and plasmacytoid (p)DCs were excluded from the analysis. [Figure 18-4] Immunogenicity of DNA Plasmids Flow cytometry evaluation was used to assess dendritic cell (DC) responses at the single cell level in mice treated with DNA plasmids VB1026, VB4194 and VB4202. The figure shows the gating strategy used to define DC. G. DC were defined as CD24+. H. Based on the expression of CD11b and CD64, total DC were divided into monocyte-derived (mo)DC and classical (c)DC. [Figure 18-5] Immunogenicity of DNA Plasmids Flow cytometry evaluation was used to assess dendritic cell (DC) responses at the single cell level in mice administered DNA plasmids VB1026, VB4194 and VB4202. The figure shows the gating strategy used to define DCs. I. Classical DCs were divided into cDC1 and cDC2 based on the expression of XCR1 and CD172a markers. [Figure 19] Immunogenicity of DNA plasmids The percentage of live CD45+ cells at the injection site 1, 2, and 4 days after intramuscular injection of DNA plasmid VB4202 compared with DNA plasmid VB4194 (comparison) and VB1026 (negative control). No EP No vacc indicates the results for the group of mice that did not receive plasmid or electroporation. [Figure 20]Immunogenicity of DNA plasmids The percentage of DCs among live CD45+ cells at the injection site 1, 2, and 4 days after intramuscular injection of DNA plasmid VB4202, compared with DNA plasmids VB4194 (comparison) and VB1026 (negative control). No EP No vacc indicates the results for the mouse group that was not administered plasmid or electroporated. [Figure 21] Immunogenicity of DNA plasmids The percentage of cDC1 cells among live CD45+ cells at the administration site 1, 2, and 4 days after intramuscular administration of DNA plasmid VB4202, compared with DNA plasmid VB4194 (comparison) and VB1026 (negative control). No EP No vacc indicates the results for the mouse group that was not administered plasmid or electroporated. [Figure 22] Immunogenicity of DNA plasmids The percentage of moDC cells among live CD45+ cells at the injection site 1, 2, and 4 days after intramuscular injection of DNA plasmid VB4202, compared with DNA plasmid VB4194 (comparison) and VB1026 (negative control). No EP No vacc indicates the results for the mouse group that was not administered plasmid or electroporated. [Figure 23] Expression and secretion levels of proteins encoded by DNA plasmids Figure 1 shows the protein expression and secretion levels of the first polypeptide encoded by DNA plasmids VB1020, VB4195 and VB4196 detected in the supernatants of HEK293 cells transfected with said DNA plasmids by ELISA using mouse a-human IgG CH3 domain capture Ab (MCA878G) and a-human MIP-1α biotinylated capture Ab (BAF270) and lipofectamine (=cells treated with only the transfection agent lipofectamine serving as a negative control). [Figure 24]Expression and secretion levels of proteins encoded by DNA plasmids. Protein expression and secretion levels of the immunostimulatory compound FLT3L encoded by DNA plasmids VB4195 and VB4196 detected in the supernatants (1:500 dilution) of HEK293 cells transfected with the DNA plasmids by ELISA using mouse a-human FLT3L capture Ab (MAB608) and mouse a-human FLT3L biotinylated detection Ab (BAF308). Lipofectamine (=cells treated only with the transfection agent Lipofectamine serving as a negative control), supernatants of cells treated only with Lipofectamine were not diluted for ELISA. [Diagram 25] Expression and secretion levels of DNA plasmid-encoded proteins. Protein expression and secretion levels of the immunostimulatory compound GM-CSF encoded by DNA plasmid VB4196 detected in the supernatants (1:500 dilution) of HEK293 cells transfected with said DNA plasmids by ELISA using rat a-mouse GM-CSF capture Ab (MAB415) and goat a-mouse GM-CSF biotinylated detection Ab (BAM215). Lipofectamine (=cells treated only with the transfection agent Lipofectamine serving as negative control), supernatants of cells treated only with Lipofectamine were not diluted for ELISA. [Figure 26-1] Expression and secretion of intact proteins encoded by DNA plasmids. Western blot of non-reduced (left) and reduced (right) supernatants of Expi293F cells transfected with DNA plasmids VB1020, VB4195, and VB4196. Primary antibody: goat a-human MIP-1α (BAF270). Secondary antibody: donkey anti-goat, Dylight 550 (SA5-10087). Chemidoc channels Dylight 550 and 650 for protein standards. Black lanes contain samples not related to this application. [Figure 26-2]Expression and secretion of intact proteins encoded by DNA plasmids. Western blot of non-reduced (left) and reduced (right) supernatants of Expi293F cells transfected with DNA plasmids VB1020, VB4195, and VB4196. Primary antibody: goat a-human MIP-1α (BAF270). Secondary antibody: donkey anti-goat, Dylight 550 (SA5-10087). Chemidoc channels Dylight 550 and 650 for protein standards. Black lanes contain samples not related to this application. [Figure 27-1] Expression and secretion of intact proteins encoded by DNA plasmids. Western blot of reduced (lanes 1-4) and deglycosylated (lanes 5-6) supernatant samples from Expi293F cells transfected with DNA plasmids VB1020, VB4195 and VB4196. Left: Primary antibody goat a-human FLT3L (BAF308). Secondary antibody: donkey anti-goat, Dylight 550 (SA5-10087). Right: Primary antibody goat a-mouse GM-CSF (BAF415). Secondary antibody: donkey anti-goat, Dylight 550 (SA5-10087). Chemidoc channels Dylight 550 and 650 for protein standards. Black lanes contain samples not related to this application. [Figure 27-2] Expression and secretion of intact proteins encoded by DNA plasmids. Western blot of reduced (lanes 1-4) and deglycosylated (lanes 5-6) supernatant samples from Expi293F cells transfected with DNA plasmids VB1020, VB4195 and VB4196. Left: Primary antibody goat a-human FLT3L (BAF308). Secondary antibody: donkey anti-goat, Dylight 550 (SA5-10087). Right: Primary antibody goat a-mouse GM-CSF (BAF415). Secondary antibody: donkey anti-goat, Dylight 550 (SA5-10087). Chemidoc channels Dylight 550 and 650 for protein standards. Black lanes contain samples not related to this application. [Figure 28] Expression and secretion levels of proteins encoded by DNA plasmids Figure 1 shows the protein expression and secretion levels of the first polypeptide encoded by DNA plasmids VB1020 and VB4204 detected in the supernatants (1:10 dilution) of HEK293 cells transfected with said DNA plasmids by ELISA using mouse a-human IgG CH3 domain capture Ab (MCA878G) and a-human MIP-1α biotinylated capture Ab (BAF270). Lipo (=only cells treated with the transfection agent Lipofectamine serving as a negative control). [Figure 29] Expression and secretion levels of DNA plasmid-encoded proteins. Protein expression and secretion levels of the immunostimulatory compound GM-CSF encoded by DNA plasmid VB4204 detected in the supernatants (1:1000 dilution) of HEK293 cells transfected with the DNA plasmids by ELISA using rat a-mouse GM-CSF capture Ab (MAB415) and goat a-mouse GM-CSF biotinylated detection Ab (BAM215). Lipo (=only cells treated with the transfection agent Lipofectamine serving as a negative control). [Diagram 30] Immunogenicity of DNA Plasmids The immunogenicity of DNA plasmids VB1020 and VB4204 when administered to mice, and the immunogenicity of DNA plasmids VB1020 and VB4204 when co-administered with pGM-CSF, is shown by measuring IFN-γ secretion from T cells (total T cell response) in comparison with the negative control VB1026. [Diagram 31] Immunogenicity of DNA Plasmids The percentage of CD8+ T cells secreting IFN-γ, TNF-α, or TNF-α and IFN-γ co-secreting in spleen cells from mice administered DNA plasmids VB1020 and VB4204 is shown compared to the negative control VB1026. [Diagram 32]Expression and secretion levels of proteins encoded by DNA plasmids Figure 1 shows the protein expression and secretion levels of the first polypeptide encoded by DNA plasmids VB1020 and VB4205 detected in the supernatants (1:10 dilution) of HEK293 cells transfected with said DNA plasmids by ELISA using mouse a-human IgG CH3 domain capture Ab (MCA878G) and a-human MIP-1α biotinylated capture Ab (BAF270). Lipo (=only cells treated with the transfection agent Lipofectamine serving as a negative control). [Diagram 33] Expression and secretion levels of DNA plasmid-encoded proteins. Protein expression and secretion levels of the immunostimulatory compound CCL5 encoded by DNA plasmid VB4205 detected in the supernatants (1:500 dilution) of HEK293 cells transfected with the DNA plasmids by ELISA using rat a-mouse CCL5 capture Ab (MAB4781) and goat a-mouse CCL5 biotinylated detection Ab (BAF478). Lipo (= only cells treated with the transfection agent Lipofectamine serving as a negative control). [Diagram 34] Immunogenicity of DNA Plasmids The immunogenicity of DNA plasmids VB1020 and VB4205 is shown in comparison to the negative control VB1026 by measuring IFN-γ secretion from T cells (total T cell response) in mice administered these plasmids. [Diagram 35] Immunogenicity of DNA Plasmids BALB / c mice (n=8 / group) were inoculated with 1x105 CT26 tumor cells on DO and subsequently administered (co-injected) DNA plasmids VB4194, VB4208, VB4202, and pGM-CSF + VB4194 on D4 and D11. Growth of CT26 tumors is shown compared to the negative control VB1026. [Diagram 36]Immunogenicity of DNA Plasmids. Survival rates of BALB / c mice (n=8 / group) inoculated with 1x105 CT26 tumor cells on DO and subsequently administered DNA plasmids VB4194, VB4208, VB4202, and pGM-CSF + VB4194 (co-injection) on D4 and D11 are shown compared to the negative control VB1026. [Figure 37] Expression and secretion levels of proteins encoded by DNA plasmids. Secretion of the first polypeptide encoded by DNA plasmids VB2060, TECH001-CV021, TECH001-CV022 and TECH001-CV023 was detected by ELISA in the supernatants of Expi293F cells transfected with said DNA plasmids. Supernatants were diluted 1:1500 and ELISA was performed using mouse a-human IgG CH3 domain capture Ab (MCA878G) and a-human MIP-1α biotinylated capture Ab (BAF270). Expifect (= only cells treated with the transfection agent Expifectamine serving as negative control). [Figure 38] Expression and secretion levels of proteins encoded by DNA plasmids Figure 1 shows the protein expression and secretion levels of the immunostimulatory compounds GM-CSF (38a: capture Ab MAB608, detection Ab BAF308), IL-12 (38b: capture Ab MAB419, detection Ab BAF419), and IL-21 (38c: capture Ab AF594, detection Ab BAF594) encoded by DNA plasmids TECH001-CV021, TECH001-CV022, and TECH001-CV023, respectively, detected by ELISA in the supernatants of Expi293F cells transfected with the DNA plasmids. [Figure 39]Expression and secretion of intact proteins encoded by DNA plasmids. Western blot showing secretion of initial polypeptide. Reduced supernatant samples of transfection control, VB2060, TECH001-CV021, TECH001-CV022 and TECH001-CV023. Primary antibody: goat anti-human MIP-1α (AF270). Secondary antibody: donkey anti-goat, Dylight 800 (SA5-10092). Chemidoc channels Dylight 800 and 650 for protein standards. [Diagram 40] Expression and secretion of intact proteins encoded by DNA plasmids. Western blot showing secretion of the immunostimulatory compound GM-CSF encoded by TECH001-CV021. Transfection control, VB2060 and reduced supernatant samples of TECH001-CV021. Primary antibody: goat anti-mouse GM-CSF (BAF415). Secondary antibody: donkey anti-goat, Dylight 800 (SA5-10092). Chemidoc channels Dylight 800 and 650 for protein standards. [Figure 41-1] Expression and secretion of intact proteins encoded by DNA plasmids. Western blot showing secretion of the immunostimulatory compound IL-12 encoded by TECH001-CV022. Reduced (left panel) and non-reduced (right panel) supernatant samples of transfection control, VB2060 and TECH001-CV022. Primary antibody: goat anti-mouse IL-12 (BAF419). Secondary antibody: donkey anti-goat, Dylight 800 (SA5-10092). Chemidoc channels Dylight 800 and 650 for protein standards. [Figure 41-2]Expression and secretion of intact proteins encoded by DNA plasmids. Western blot showing secretion of the immunostimulatory compound IL-12 encoded by TECH001-CV022. Reduced (left panel) and non-reduced (right panel) supernatant samples of transfection control, VB2060 and TECH001-CV022. Primary antibody: goat anti-mouse IL-12 (BAF419). Secondary antibody: donkey anti-goat, Dylight 800 (SA5-10092). Chemidoc channels Dylight 800 and 650 for protein standards. [Diagram 42] Expression and secretion of intact proteins encoded by DNA plasmids. Western blot showing secretion of the immunostimulatory compound IL-21 encoded by TECH001-CV023. Transfection control, VB2060 and reduced supernatant samples of TECH001-CV023. Primary antibody: goat anti-mouse IL-12 (BAF594). Secondary antibody: donkey anti-goat, Dylight 800 (SA5-10092). Chemidoc channels Dylight 800 and 650 for protein standards. [Diagram 43] Immunogenicity of DNA Plasmids The immunogenicity of DNA plasmids VB2060, TECH001-CV021, TECH001-CV022 and TECH001-CV023 is shown in mice administered these plasmids, compared to the negative control VB1026, by measuring total IgG antibodies binding to the RBD protein. Individual mice and mean ± SEM are shown. *(p<0.05), **(p<0.01), two-tailed Mann-Whitney test. [Diagram 44]Immunogenicity of DNA Plasmids A) The immunogenicity of DNA plasmids VB2060, TECH001-CV021, TECH001-CV022 and TECH001-CV023 is shown in mice administered these plasmids, as compared to the negative control VB1026, as measured by IFN-γ secretion from T cells (total T cell response). B) The immunogenicity of DNA plasmids VB2060, TECH001-CV021, TECH001-CV022 and TECH001-CV023 is shown in mice administered these plasmids, as compared to the negative control VB1026, as measured by IFN-γ secretion from CD8+ T cells (CD4+ T cell depleted samples). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description of the Invention The first polypeptide and / or multimeric protein is also referred to herein as a "construct." The first polypeptide / multimeric protein described herein is generally an immunogenic construct.

[0018] An "immunogenic construct" is one that elicits an immune response, particularly when administered to a subject in a form suitable for administration and in an amount effective to elicit an immune response (i.e., an immunologically effective amount).

[0019] A "subject" is an animal, such as a mouse, or a human, preferably a human. As used herein, the terms "mouse", "murine" and "m" are used interchangeably to denote or refer to a mouse. The terms human and "h" are used interchangeably herein to denote or refer to a human. A subject may be a patient, i.e., a human suffering from a disease in need of therapeutic treatment, or a subject suffering from, for example, an infectious disease in need of prophylactic treatment, or a subject suspected of suffering from a disease. As used herein, the terms "subject" and "individual" are used interchangeably.

[0020] A "disease" is an abnormal medical condition, usually accompanied by particular signs and symptoms in a subject.

[0021] An "infectious disease" is a disease caused by one or more pathogens, including viruses, bacteria, fungi, and parasites.

[0022] "Cancer" refers to a broad group of different diseases characterized by the uncontrolled growth of abnormal cells in the body. "Cancer" or "cancerous tissue" includes tumors, and as used herein, encompasses both solid tumors and tumor cells found in bodily fluids such as blood, including metastatic cancers. Unregulated cell division and growth results in the formation of malignant tumors that can invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Distant tumors after metastasis can be said to "originate" from the pre-metastatic tumor.

[0023] "Treatment" refers to prophylactic or therapeutic treatment.

[0024] "Prophylactic treatment" refers to treatment administered to a subject who does not (or does not yet) show signs or symptoms of a disease or who only shows early signs or symptoms of a disease, and is administered with the intent of preventing or reducing the risk of developing a disease and / or symptoms associated with the disease. Prophylactic treatment functions as a preventative treatment against a disease or as a treatment that inhibits or reduces the further development or aggravation of a disease and / or its associated symptoms. As used herein, the terms prophylactic treatment, prophylaxis, and prevention are used interchangeably.

[0025] "Therapeutic treatment" refers to a treatment administered to a subject who exhibits symptoms or signs of a disease, which treatment is administered to the subject with the intent of reducing or eliminating those signs or symptoms, or slowing or halting the progression of the disease.

[0026] As used herein, "T cell epitope" refers to a portion or region of an antigen that comprises a single individual T cell epitope, or multiple T cell epitopes, e.g. multiple minimal T cell epitopes such as a hotspot.

[0027] A "nucleotide sequence" is a sequence made up of nucleotides. The terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably herein.

[0028] The one or more immunostimulatory compounds enhance the effect of the first polypeptide / multimeric protein. An advantage of the present invention is that by co-expressing the first polypeptide and one or more immunostimulatory compounds from a single vector, e.g., a DNA plasmid, only such a single vector needs to be administered to the subject. Thus, there is no need to produce and administer additional vectors encoding immunostimulatory compounds or to co-administer such compounds in the form of proteins or peptides to enhance the effect of the first polypeptide / multimeric protein, reducing production costs and streamlining drug production. In addition, administering a single formulation makes it easier for patients to accept the treatment and easier for medical personnel to handle the formulation, e.g., reconstitute and administer to patients.

[0029] Furthermore, without wishing to be bound by theory, co-expression also has significant advantages at the cellular level. When transfecting with a vector, the vector comes into contact with various cells. Whether it is successfully taken up and transcription and translation are functionally initiated is a somewhat random process. When transfecting with two different vectors, there is no control over which cells express the proteins encoded by these vectors. In transfections aimed at secreting proteins into the bloodstream, this spatial distribution is not a concern. In the present invention, the vectors of the present invention produce different proteins. When a vector encoding a construct is administered intramuscularly, the construct is secreted from the muscle cells and delivered to adjacent antigen-presenting cells. Since the immunostimulatory compound is expressed in and secreted from the same muscle cells, it can stimulate the same antigen-presenting cells and thereby act directly on said antigen-presenting cells. As an example, if the antigen-presenting cells are dendritic cells, the immunostimulatory compound can promote the attraction, activation, and maturation of dendritic cells.

[0030] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0031] vector A vector of the present invention may be any molecule suitable for carrying an exogenous nucleic acid sequence, such as DNA or RNA, into a cell and expressing it there, ie, an expression vector.

[0032] In one embodiment, the vector is a DNA vector, such as a DNA plasmid, or a DNA viral vector, such as a DNA viral vector selected from the group consisting of adenovirus, vaccinia virus, adeno-associated virus, cytomegalovirus, and Sendai virus.

[0033] In another embodiment, the vector is an RNA vector, such as an RNA plasmid, or an RNA viral vector, such as a retroviral vector, e.g., a retroviral vector selected from the group consisting of an alphavirus, a lentivirus, a Moloney murine leukemia virus, and a rhabdovirus.

[0034] In a preferred embodiment, the vector is a DNA vector, more preferably a DNA plasmid.

[0035] DNA Plasmids A plasmid is a small extrachromosomal DNA molecule present in a cell, physically separated from chromosomal DNA and capable of replicating independently. Plasmids are small circular double-stranded DNA molecules present mainly in bacteria, but can also be found in archaea and eukaryotes. Artificial plasmids are widely used as vectors in molecular cloning, responsible for delivering recombinant DNA sequences and ensuring high expression within the host organism. Plasmids contain several important features, including features for selecting cells containing the plasmid, such as antibiotic resistance genes, an origin of replication, a multiple cloning site (MCS), and a promoter to drive the expression of the inserted gene of interest.

[0036] Generally, a promoter is a sequence that can attract initiation factors and polymerase to the promoter, resulting in the transcription of the gene. Promoters are located upstream of the DNA near the transcription start site of the gene. Promoters are approximately 100-1000 base pairs in length. The nature of the promoter usually depends on the gene and transcript, and the type and class of RNA polymerase that is recruited to that site. When RNA polymerase reads the DNA of the plasmid, an RNA molecule is transcribed. After processing, when the ribosome translates the mRNA into a protein, the mRNA is translated many times, resulting in many copies of the protein encoded by the gene of interest. Generally, the ribosome facilitates the reading by binding a complementary tRNA anticodon sequence to the codon of the mRNA. The tRNA carries a specific amino acid and becomes a chain of polypeptides when the mRNA passes through the ribosome and is "read". Translation proceeds in three stages: initiation, elongation, and termination. Through the process of translation, a polypeptide is folded into an active protein and either carries out its function within the cell or is exported outside the cell and carries out its function elsewhere, sometimes after a significant number of post-translational modifications.

[0037] When a protein is transported outside the cell, the signal peptide guides the protein to the endoplasmic reticulum, where the signal peptide is cleaved and translation is terminated, after which the protein is transported to the periplasm.

[0038] The DNA plasmids of the present invention are not limited to any particular plasmid, and one of skill in the art will understand that any plasmid having an appropriate backbone can be selected and engineered by methods known in the art to constitute the elements and units of the present disclosure.

[0039] Coexpression The vector of the present disclosure co-expresses multiple proteins. Such vectors (and plasmids) are also called multicistronic or polycistronic vectors (and multicistronic or polycistronic plasmids). Those skilled in the art know how to design vectors to contain sequences encoding these multiple proteins, and can choose different means and use different techniques known in the art to ensure that these proteins are co-expressed from one vector as separate proteins.

[0040] Thus, one skilled in the art can construct a vector of the invention that co-expresses different proteins, ie, a first polypeptide and one or more immunostimulatory compounds.

[0041] In a preferred embodiment, the vector of the invention comprises one or more co-expression elements, i.e., nucleic acid sequences allowing the co-expression of a first polypeptide and one or more immunostimulatory compounds from the same vector.

[0042] In one embodiment of the disclosure, the vector comprises a co-expression element (or more than one co-expression element) such that the first polypeptide and one or more immunostimulatory compounds are transcribed onto a single transcription product but are independently translated into the first polypeptide and one or more immunostimulatory compounds. Thus, the presence of the co-expression element ultimately results in the production of separate translation products.

[0043] IRES In one embodiment of the present disclosure, the co-expression element is an IRES element, the concept of which is illustrated in FIG. 1. Internal ribosome entry site, abbreviated as IRES, is an RNA element that allows translation initiation in a cap-independent manner as part of the larger process of protein synthesis. In eukaryotic translation, initiation usually occurs at the 5' end of the mRNA molecule, because the assembly of the initiation complex requires recognition of the 5' cap. By placing an IRES element between two coding regions, the initiation complex can assemble at this site, allowing translation of the downstream coding region. Thus, in one embodiment of the present disclosure, the vector contains an IRES, and one transcript is produced from the vector, which is then translated into separate proteins.

[0044] The IRES element allows the co-expression of the first polypeptide and one or more immunostimulatory compounds under the control of the same promoter. The promoter directs the transcription of a single mRNA containing the coding regions of the nucleic acid sequence encoding the first polypeptide and the nucleic acid sequence encoding one or more immunostimulatory compounds. If more than one immunostimulatory compound is expressed from the vector of the invention, the vector of the invention must have an IRES element upstream of each nucleic acid sequence encoding an immunostimulatory compound. Alternatively, other types of co-expression elements may be used when more than one immunostimulatory compound is expressed from the vector of the invention.

[0045] The IRES element used in the vectors of the invention may be derived from a viral genome or from a cellular mRNA.Vectors containing IRES elements, such as DNA plasmids, are commercially available.

[0046] 2A Self-cleaving peptide In another embodiment of the present disclosure, the co-expression element is a nucleic acid sequence encoding a 2A self-cleaving peptide (or abbreviated "2A peptide"), the concept of which is depicted in FIG.

[0047] In the context of this application, the terms "2A self-cleaving peptide" and "2A peptide" are used for peptides encoded by a nucleic acid sequence that, when located between two coding regions, transcribes the two coding regions as a single transcript, but whose translation results in two separate peptide chains. Generally, when ribosomes translate mRNA, amino acids are covalently linked from N-terminus to C-terminus. The presence of a nucleic acid sequence encoding a 2A self-cleaving peptide causes the ribosome to skip synthesis of a peptide bond at the C-terminus of the 2A peptide, resulting in two separate peptide chains. 2A self-cleaving peptides are typically 18-22 amino acids long and often contain the consensus sequence DXEXNPGP (SEQ ID NO:50), where X can be any amino acid.

[0048] In one embodiment of the invention, the ribosome skips the peptide bond between the glycine and proline residues found at the C-terminus of the 2A self-cleaving peptide, meaning that the upstream gene product has a few extra amino acid residues at its end, while the downstream gene product starts with a proline.

[0049] In one embodiment, the 2A self-cleaving peptide is a sequence of 18-22 amino acids in length that contains the consensus sequence DXEXNPGP (SEQ ID NO:50), where X can be any amino acid.

[0050] Thus, the 2A self-cleaving peptide also allows for the co-expression of a first polypeptide and one or more immunostimulatory compounds under the control of the same promoter. As with the IRES element, when more than one immunostimulatory compound is expressed from the vector of the invention, a nucleic acid sequence encoding a 2A peptide must be present in the vector upstream of each nucleic acid sequence encoding an immunostimulatory compound. As an example, the vector comprises a first nucleic acid sequence encoding a first polypeptide, a second nucleic acid sequence encoding a first immunostimulatory compound, and a third nucleic acid sequence encoding a second immunostimulatory compound. The vector may comprise a nucleic acid sequence encoding a T2A peptide between the first and second nucleic acid sequences, and a nucleic acid sequence encoding a P2A peptide between the second and third nucleic acid sequences. Alternatively, when more than one immunostimulatory compound is expressed from the vector of the invention, another type of co-expression element may be used.

[0051] In a further embodiment, the 2A autocleaving peptide is a 2A peptide selected from the group consisting of a T2A peptide, a P2A peptide, an E2A peptide, and an F2A peptide.

[0052] In one embodiment, the T2A peptide has an amino acid sequence identical to a T2A sequence listed in Table 1 or 2. In a further embodiment, the amino acid sequence DVEENPGP (SEQ ID NO:50) is present, but the remainder of the T2A amino acid sequence has 80%-100% sequence identity, e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the T2A amino acid sequence in Table 1. In another embodiment, the T2A peptide has the amino acid sequence of SEQ ID NO:9.

[0053] In one embodiment, the P2A peptide has an amino acid sequence identical to a P2A sequence listed in Table 1 or 2. In a further embodiment, the sequence DVEENPGP (SEQ ID NO:50) is present, but the remainder of the P2A amino acid sequence has 80% to 100% sequence identity, e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the P2A amino acid sequence of Table 1. In another embodiment, the P2A peptide has the amino acid sequence of SEQ ID NO:11.

[0054] In one embodiment, the E2A peptide has an amino acid sequence identical to an E2A sequence listed in Table 1 or 2. In a further embodiment, the sequence DVESNPGP (SEQ ID NO: 173) is present, but the remainder of the E2A amino acid sequence has 80% to 100% sequence identity, e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the E2A amino acid sequence of Table 1. In another embodiment, the E2A peptide has the amino acid sequence of SEQ ID NO: 14.

[0055] In one embodiment, the F2A peptide has an amino acid sequence that is identical to an F2A sequence listed in Table 1 or 2. In a further embodiment, the sequence DVESNPGP (SEQ ID NO: 173) is present, but the remainder of the F2A amino acid sequence has 80% to 100% sequence identity, e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the F2A amino acid sequence of Table 1. In another embodiment, the F2A peptide has the amino acid sequence of SEQ ID NO:51. [Table 1]

[0056] For example, it is generally known that the cleavage and expression efficiency of 2A-peptides can be adjusted to be increased by inserting a GSG sequence before the N-terminus of the wild-type sequence, as shown in Table 2. [Table 2]

[0057] In another embodiment, the vector of the present invention comprises both an IRES element and a nucleic acid sequence encoding a 2A peptide. As an example, the vector comprises a first nucleic acid sequence encoding a first polypeptide, a second nucleic acid sequence encoding a first immunostimulatory compound, and a third nucleic acid sequence encoding a second immunostimulatory compound. The vector may comprise an IRES element between the first and second nucleic acid sequences, and a nucleic acid sequence encoding a 2A peptide between the second and third nucleic acid sequences. Alternatively, the vector may comprise a nucleic acid sequence encoding a 2A peptide between the first and second nucleic acid sequences, and an IRES element between the second and third nucleic acid sequences. Additional nucleic acid sequences encoding additional immunostimulatory compounds may also be included in the vector.

[0058] In another embodiment, the vector of the present invention comprises a nucleic acid sequence encoding two 2A peptides as a contiguous sequence of two 2A peptides.As an example, the vector comprises a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid encoding an immunostimulatory compound.The vector may comprise a nucleic acid sequence encoding two 2A peptides as a contiguous sequence between the first nucleic acid sequence and the second nucleic acid sequence.

[0059] Bidirectional promoter In one embodiment of the present disclosure, the vector comprises a co-expression element (or more than one co-expression element) that causes the first polypeptide and one or more immunostimulatory compounds to be transcribed as separate transcription products, resulting in separate transcription products and therefore separate proteins.

[0060] In one embodiment of the present disclosure, the co-expression element is a bidirectional promoter, the concept of which is illustrated in Figure 3a. A bidirectional promoter is typically a short (e.g., <1 kbp) intergenic region of DNA between the 5' ends of the genes of a bidirectional gene pair. A "bidirectional gene pair" refers to two adjacent genes whose 5' ends face each other and are encoded on opposite strands.

[0061] In one embodiment of the present disclosure, the bidirectional promoter is a back-to-back arrangement of a CAG promoter with four CMV enhancers (Sladitschek HL, Neveu PA et al., PLoS One 11(5), e0155177, 2016).

[0062] In one embodiment of the present disclosure, the bidirectional promoter is RPBSA (Kevin He et al., Int. J. Mol. Sci. 21(23), 9256, 2020).

[0063] In one embodiment of the present disclosure, the bidirectional promoter is a back-to-back configuration of mouse Pgk1 and human eukaryotic translation elongation factor 1α1 promoters (Golding & Mann, Gene Therapy 18, 817-826, 2011).

[0064] In one embodiment, a vector of the invention is a plasmid comprising a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding an immunostimulatory compound as a bidirectional gene pair comprising a bidirectional promoter between their 5' ends.

[0065] Multiple promoters In another embodiment of the present disclosure, the co-expression elements are different promoters, i.e., the vector is, for example, a plasmid containing a separate promoter for each of the nucleic acid sequences encoding the first polypeptide and one or more immunostimulatory compounds, i.e., a plasmid for separate transcription of each of the first polypeptide and one or more immunostimulatory compounds.

[0066] In one embodiment, each of the nucleic acid sequences has a different promoter, and the concept is also shown in Figure 3b.In one embodiment, all nucleic acid sequences have the same promoter to achieve equal molecular expression.In another embodiment, one nucleic acid sequence has a stronger promoter than the other nucleic acid sequence; that is, the nucleic acid sequence with a stronger promoter is more likely to be expressed at a higher level than the other nucleic acid sequence.

[0067] A large number of promoters are known in the art and are suitable for incorporation into the plasmids of the present invention. In one embodiment of the present disclosure, the promoter is derived from cytomegalovirus, such as the CMV promoter.

[0068] In one embodiment, the vector of the invention comprises one or more co-expression elements, preferably a co-expression element selected from the group consisting of an IRES element, a 2A peptide, a bidirectional promoter and a promoter.

[0069] The vectors of the invention may contain any kind of combination of co-expression elements.

[0070] As an example, a vector of the invention is a DNA plasmid comprising a first nucleic acid sequence encoding a first polypeptide, a second nucleic acid sequence encoding a first immunostimulatory compound, and a third nucleic acid sequence encoding a second immunostimulatory compound. In one embodiment, the DNA plasmid comprises an IRES and a 2A peptide that allow for co-expression of the first polypeptide and the first and second immunostimulatory compounds (under the control of a promoter). In another embodiment, the DNA plasmid comprises a bidirectional promoter and another promoter.

[0071] The skilled artisan will know that the terms first, second and third nucleic acid sequences as in the above example do not mean that the plasmid of the present invention comprises nucleic acid sequences in the order of the first, second and third nucleic acid sequences. The second nucleic acid sequence may be downstream or upstream of the first or third nucleic acid sequence, the third nucleic acid sequence may be downstream or upstream of the first or second nucleic acid sequence, and the first nucleic acid sequence may be upstream or downstream of the second or third nucleic acid sequence. In another embodiment, the first and second nucleic acid sequences may be in opposite orientations on the same DNA strand, such as the first and third nucleic acid sequences, or the second and third nucleic acid sequences. In a further embodiment, the nucleic acid sequences encoding the first polypeptide and the immunostimulatory compound may be on opposite DNA strands.

[0072] immunostimulatory compounds The vectors of the invention comprise one or more nucleic acid sequences encoding one or more immunostimulatory compounds.

[0073] In one embodiment of the disclosure, the immunostimulatory compound is a compound that affects antigen presenting cells, hi another embodiment, the immunostimulatory compound is a compound that stimulates antigen presenting cells.

[0074] Antigen-presenting cells (APCs) are cells that display antigens on their surface in complexes with major histocompatibility complexes (MHC), a process known as antigen presentation. T cells recognize these complexes using the T cell receptor (TCR). APCs process antigens and present them to T cells.

[0075] Almost all cell types are capable of presenting antigens in some way. Professional APCs, including macrophages such as Langerhans cells, B cells, and dendritic cells, present foreign antigens to helper T cells (CD4+) via MHC class II, whereas virus-infected cells (or cancer cells) can present antigens derived from their internal cell interior to cytotoxic T cells (CD8+) via MHC class I. In addition to proteins of the MHC family, antigen presentation depends on other specialized signaling molecules on the surface of both APCs and T cells.

[0076] "MHC" is an abbreviation for "Major Histocompatibility Complex". There are two major classes of MHC molecules: MHC class I and MHC class II. The terms MHC class I and MHC class II are used interchangeably herein with HLA class I and HLA class II. HLA (human leukocyte antigen) is the major histocompatibility complex in humans.

[0077] APCs are important for effective adaptive immunity because the function of cytotoxic T cells and helper T cells depends on them. Antigen presentation enables the specificity of adaptive immunity and contributes to immune responses against intracellular and extracellular pathogens. They are also involved in defense against tumors.

[0078] In one embodiment of the disclosure, the APC is selected from the group consisting of dendritic cells, macrophages, Langerhans cells, B cells and neutrophils, and the immunostimulatory compound is a compound that affects such cells, such as stimulating such cells.

[0079] When APCs are affected, eg, stimulated, the stimulus results in the attraction, activation, maturation, and / or proliferation of APCs.

[0080] In one embodiment of the disclosure, the one or more immunostimulatory compounds promote the attraction and / or activation and / or maturation and / or proliferation of antigen presenting cells, e.g., promote the growth and / or expansion of antigen presenting cells.

[0081] In one embodiment of the disclosure, the immunostimulatory compound comprises a cytokine, a chemokine, a growth factor, a ligand that binds to a member of the TNF receptor superfamily, or a ligand that binds to a pattern recognition receptor (PRR).

[0082] In one embodiment, the vector of the invention is a plasmid, e.g., a DNA plasmid. It can be administered to a subject in need thereof, e.g., by intramuscular administration, from which the encoded compound is expressed and secreted. The efficacy of the first polypeptide (secreted in the form of a multimeric protein, e.g., a dimeric protein) can be enhanced by co-expression of an immunostimulatory compound that attracts APCs to the injection site / muscle cells where the multimeric protein and the immunostimulatory compound are secreted. Attraction of APCs results in a stronger and accelerated immune response: the multimeric protein is not only diluted in the bloodstream, but is also delivered and taken up by APCs, resulting in a locally higher number of APCs that can present one or more antigens contained in the multimeric protein to other relevant immune cells.

[0083] Immunostimulatory compounds that promote APC attraction Thus, in one embodiment, the immunostimulatory compound promotes the attraction of APCs.

[0084] APC attraction may be measured by methods known in the art, including in vitro transwell or migration assays, by measuring surface markers in vivo on muscle cells administered the vectors of the invention by flow cytometry, or by changes in gene expression patterns, for example by RT-qPCR, Nanostring or RNA sequencing.

[0085] One type of molecule that can attract APCs is the chemokines, a group of small cytokines, or signaling proteins, secreted by cells that derive their name from their ability to induce directional chemotaxis in nearby responsive cells, i.e., chemotactic cytokines.

[0086] In one embodiment of the disclosure, the immunostimulatory compound is a chemokine.

[0087] In another embodiment, the immunostimulatory compound may interact with the following surface molecules on APCs: CCR1 (CC motif chemokine receptor 1), CCR3 (CC motif chemokine receptor 3), CCR4 (CC motif chemokine receptor 4), CCR5 (CC motif chemokine receptor 5), CCR6 (CC motif chemokine receptor 6), CCR7 (C motif chemokine receptor 7), CCR8 (CC motif chemokine receptor 8) or XCR1 (XC motif chemokine receptor 1). In a preferred embodiment, the immunostimulatory compound may interact with the aforementioned surface molecules on human APCs.

[0088] In yet another embodiment of the present disclosure, the immunostimulatory compound is selected from the list consisting of macrophage inflammatory protein alpha and its isoforms, including mouse CCL3 (or MIP-1α), and the human isoforms hCCL3, hCCL3L1, hCCL3L2 and hCCL3L3, preferably human MIP-1α (also called hMIP-1α variant, LD78β or CCL3L1), RANTES (CCL5), preferably human CCL5, e.g., human CCL5 having the amino acid sequence of SEQ ID NO: 43, chemokine ligand 4 (CCL4), preferably human CCL4, chemokine ligand 20 (CCL20), preferably human CCL20, chemokine ligand 19 (CCL19), preferably human CCL19, chemokine ligand 21 (CCL21), preferably human CCL21, and chemokine motif ligand 1 or 2 (XCL1 or XCL2), preferably human XCL1 or human XCL2.

[0089] The activation process is a series of events that drive quiescent APCs towards a more differentiated and / or mature state. APCs are activated directly by interaction with pathogens or encounter with foreign antigens, and indirectly by compounds (e.g., inflammatory mediators) produced and released by other cell types that recognize such molecules. APCs are then activated through a series of cellular processes that play a key role in mounting an effective immune response against foreign antigens. For example, maturation of dendritic cells is characterized by a decrease in phagocytic capacity, enhanced antigen processing and presentation, improved ability to migrate to lymphoid tissues, and an increased capacity to stimulate B and T cells.

[0090] Immunostimulatory compounds that promote APC activation and / or maturation In one embodiment of the disclosure, the immunostimulatory compound promotes the activation and / or maturation of APCs.

[0091] A variety of techniques known in the art are available for measuring APC activation, such as comparing cytokine profiles before and after activation as measured by ELISpot or FluoroSpot, determining global changes in gene expression, and analyzing expressed proteins (e.g. activation markers) by a variety of techniques such as FACS, ELISA, WB and PCR / sequencing (qPCR (TaqMan array), Nanostring and RNA-seq).

[0092] In one embodiment of the present disclosure, the immunostimulatory compound is capable of interacting with a surface molecule on an APC selected from the group consisting of receptors of the TNF receptor superfamily, including CD40 (cluster of differentiation 40), CD137 (4-1BB), CD27, RANK, and ICOS (CD278). In a preferred embodiment, the immunostimulatory compound is capable of interacting with the aforementioned surface molecules on human APCs.

[0093] Such immunostimulatory compounds may be selected from the list consisting of CD40L (CD40 ligand, CD154), CD137L (4-1BBL, 4-1BB ligand), CD70, ICOSL (CD275) and RANKL. In a preferred embodiment, the immunostimulatory compound is selected from the group consisting of hCD40L, hCD137L, hCD70, hICOSL and hRANKL.

[0094] In another embodiment of the disclosure, the immunostimulatory compound is a cytokine selected from the group consisting of IL-2, preferably human IL-2, IL-10, preferably human IL-10, IL-12, preferably human IL-12, such as human IL-12 comprising the amino acid sequence of SEQ ID NO:45 and 47, IL-21, preferably human IL-21, such as human IL-21 comprising the amino acid sequence of SEQ ID NO:49, TNFα, preferably human TNFα, IFNγ, preferably human IFNγ and IL-1β, preferably human IL-1β.

[0095] In yet another embodiment of the present disclosure, the immunostimulatory compound is an immune signaling molecule such as MyD88 and TRIF, preferably human MyD88 and human TRIF, which activate APCs via TLR receptors present on their surface.

[0096] In yet another embodiment of the present disclosure, the immunostimulatory compound is a sensor of viral infection, such as, for example, RIG-1 or MDA-5, preferably human RIG-1 or human MDA-5.

[0097] In yet another embodiment of the disclosure, the immunostimulatory compound interacts with a pattern recognition receptor on APCs, such as a Toll-like receptor, including TLR2, TLR4, or TLR5. In a preferred embodiment, the immunostimulatory compound interacts with the aforementioned receptors on human APCs.

[0098] In one embodiment, such immunostimulatory compounds are selected from the list consisting of pathogen-associated molecular patterns (PAMPs) such as flagellin, HMGB1, heat shock proteins (HSPs), protein damage-associated molecular patterns (DAMPs) such as calrecticulin and annexin A1. In a preferred embodiment, such immunostimulatory compounds are selected from the list consisting of human pathogen-associated molecular patterns (PAMPs), human protein damage-associated molecular patterns (DAMPs) such as human HMGB1, human heat shock proteins (HSPs), human calrecticulin and human annexin A1. PAMPs / DAMPs include those that can be included as nucleic acid sequences in the vectors of the invention and expressed as functional proteins that may contain functional groups introduced by post-translational modifications. The aforementioned molecules activate the following receptors on APCs: RAGE, TLR4, TLR9 and TIM-3 (for HMGB1), FPR (for annexin A1), SREC1, LOX1 and CD91 (for HSPs). In a preferred embodiment, the immunostimulatory compound in turn activates the aforementioned receptors on human APCs.

[0099] Immunostimulatory compounds that promote the growth and / or expansion of APCs During an immune response, activated APCs undergo rapid proliferation to fight infection and disease. Cell proliferation is the process by which cells grow (increase in mass and size) and divide to produce two daughter cells. Growth factors bind to receptors on the cell surface and stimulate the cell to proliferate. Cell proliferation leads to an exponential increase in cell numbers and is therefore a mechanism for rapidly expanding a cell population. In the following, the terms "expansion" and "proliferation" are used interchangeably.

[0100] In one embodiment, the immunostimulatory compound promotes the growth and / or expansion of APCs.

[0101] Cell proliferation can be measured by various techniques known in the art, for example, MTT / MTS assay, protein translation measurement, or labeling with CFSE. Methods well known in the art are performed, for example, by measuring the metabolic activity of a cell population, which reflects the state of cell proliferation. Furthermore, since the ATP content in cells is tightly controlled, detection of ATP can also provide information on cell proliferation. Dead or dying cells contain almost no ATP, so there is a strict linear relationship between the ATP concentration in cell lysates or extracts and the number of cells. ATP detection using bioluminescent luciferase and its substrate luciferin can give very sensitive results. In the presence of ATP, luciferase emits light, and the intensity of the light emission is proportional to the ATP concentration. Furthermore, certain antigens are present only in proliferating cells, whereas non-proliferating cells lack these antigens. Cell proliferation can be detected by utilizing specific monoclonal antibodies. For example, in human cells, the Ki-67 antibody recognizes the protein of the same name, which is expressed during all active phases of the cell cycle but is absent in quiescent (resting) cells. Traditionally, radiolabeled 3H-thymine has been used as an indicator of proliferation. 3H-thymine is incubated with cells for several hours to overnight. Newly proliferating cells incorporate the radiolabel into their DNA, which can be detected after extraction in a scintillation counter.

[0102] In one embodiment of the disclosure, the immunostimulatory compound may interact with the following surface molecules on APCs: GM-CSF receptor (granulocyte-macrophage colony-stimulating factor receptor, CD116), FLT-3R (fms-like tyrosine kinase 3, CD135), IL-15R or IL-4R. In a preferred embodiment, the immunostimulatory compound interacts with the aforementioned surface molecules on human APCs.

[0103] In one embodiment of the disclosure, the immunostimulatory compound is a growth factor, e.g., GM-CSF (granulocyte-macrophage colony-stimulating factor), preferably human GM-CSF, e.g., human GM-CSF having the amino acid sequence of SEQ ID NO: 41, FLT-3L (herein the terms FLT-3L and FLT3L are used interchangeably), e.g., human FLT-3L, preferably human FLT-3L having the amino acid sequence of SEQ ID NO: 10, IL-15, preferably human IL-15, or IL-4, preferably human IL-14.

[0104] In another embodiment, the immunostimulatory compound is one or more selected from Table 3 below. In a preferred embodiment, the immunostimulatory compounds listed in Table 3 are human immunostimulatory compounds that interact with a receptor listed in Table 3 present on human APCs: [Table 3]

[0105] In one embodiment of the disclosure, the vector comprises a nucleic acid sequence encoding 2, 3, 4, 5, 6, 7 or 8 immunostimulatory compounds. In another embodiment, the vector comprises a nucleic acid sequence encoding 2 to 6 immunostimulatory compounds, i.e., 2 or 3 or 4 or 5 or 6 immunostimulatory compounds. The immunostimulatory compounds may be the same or different, preferably different.

[0106] In preferred embodiments, different immunostimulatory compounds also affect APCs differently, stimulating the immune system at a number of different levels, thereby maximizing the therapeutic or prophylactic effect of the first polypeptide.

[0107] As an example, in one embodiment, the vector comprises nucleic acids encoding three different immunostimulatory compounds, the first one being an immunostimulatory compound that promotes DC attraction (e.g., XCL1), the second one being an immunostimulatory compound that promotes DC growth (e.g., FLT3L), and the third one being an immunostimulatory compound that promotes DC activation (e.g., CD40L). In one embodiment, such vectors may be used for the treatment and / or prevention of infectious diseases, or for the treatment of cancer. The selection of a particular immunostimulatory compound also depends on the targeting unit configured in the first polypeptide, because the targeting unit targets APCs and may affect APCs in a similar manner as the immunostimulatory compound, e.g., attracting or activating APCs.

[0108] First Nucleic Acid Sequence The vector of the present disclosure comprises a first nucleic acid sequence, i.e., DNA or RNA, including genomic DNA, cDNA and mRNA, either double-stranded or single-stranded, encoding a first polypeptide. In one embodiment, the first nucleic acid sequence is DNA. In another embodiment, the first nucleic acid sequence is optimized for the species of the subject to which it is administered. When administered to humans, in one embodiment, the first nucleic acid sequence is optimized for human codons.

[0109] The first nucleic acid sequence encodes a first polypeptide, which comprises a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, which comprises one or more antigens or portions thereof, e.g., one or more disease-associated antigens or portions thereof. When administered to a subject, the first polypeptide is expressed and, due to the presence of the multimerization unit, forms a multimeric protein and induces an immune response against the antigen or portions thereof, e.g., epitopes, comprised in the antigen unit, resulting in activation of the subject's immune system.

[0110] Structures such as a first polypeptide and a dimeric or multimeric protein comprising the first polypeptide are known in the art (e.g., WO 2004 / 076489A1, WO 2011 / 161244A1, WO 2017 / 118695A1 and WO 2022 / 013277A1, the disclosures of all of which are incorporated herein by reference), and a person skilled in the art can select a targeting unit, a multimerization unit and an antigen unit that target antigen-presenting cells according to the envisaged use of the vector and the desired results after its administration.

[0111] The first polypeptide has an N-terminal start and a C-terminal end (as shown in FIG. 4). The elements and units of the first polypeptide - the targeting unit (TU), the multimerization unit, for example the dimerization unit (DimU) in this FIG. 4, and the antigen unit - can be arranged in the first polypeptide such that the antigen unit is located at the C-terminus of the first polypeptide (FIG. 4a) or at the N-terminal start of the first polypeptide (FIG. 4b). Preferably, the antigen unit is located at the C-terminus of the first polypeptide. A unit linker (UL) may link the multimerization unit, such as the dimerization unit, and the antigen unit. FIG. 4 shows an antigen unit with four neoepitopes (neo1, neo2, neo3, neo4), which are separated by linkers (SUL1, SUL2, SUL3). Another way to explain the arrangement of neoepitopes neo1-neo4 is that they are arranged in three antigen subunits, each consisting of a neoepitope and a subunit linker (SUL1, SUL2, SUL3), and a terminal neoepitope (neo4) that is closest to the C-terminus or N-terminus start of the first polypeptide. In the figure, the subunits are indicated by square brackets. Thus, an antigen unit containing n neoepitopes contains n-1 subunits, each subunit containing a neoepitope and a subunit linker. As described herein, the four neoepitopes may be the same or different, and the three linkers / subunit linkers may be the same or different. The order and orientation of the above units and elements of the first polypeptide are the same in the multimeric protein and the first nucleic acid sequence encoding the first polypeptide. The first polypeptide as shown in FIG. 4 may be for use as an anti-cancer vaccine, for example, a personalized anti-cancer vaccine, as described herein.

[0112] In the following, the various units and elements of the first polypeptide are described in detail, which are present in the first nucleic acid sequence as nucleic acid sequences encoding the units / elements, whereas they are present in the first polypeptide or multimeric protein as amino acid sequences. For ease of reading, in the following, the units / elements are described mainly in the context of the first polypeptide / multimeric protein, i.e. on the basis of amino acid sequences.

[0113] Targeting Unit The first polypeptide encoded by the first nucleic acid contained in the vector of the present invention contains a targeting unit that targets APCs. APCs include dendritic cells (DCs) and subsets thereof.

[0114] The term "targeting unit" as used herein refers to a unit that delivers a polypeptide / multimeric protein to an antigen-presenting cell for MHC class II restricted presentation to CD4+ T cells or to provide cross-presentation to CD8+ T cells via MHC class I restriction.

[0115] Due to the presence of the targeting unit, the multimeric protein attracts DCs, neutrophils, and other immune cells. Thus, the multimeric protein not only targets the antigen units constructed therein to specific cells, but also promotes a response amplification effect (adjuvant effect) by recruiting specific immune cells to the vector administration site.

[0116] The targeting unit is designed to target the multimeric protein to a surface molecule expressed on APCs, such as a molecule expressed on any or many types of APCs, or alternatively, a molecule expressed only on a subset of APCs, such as a subset of DCs.

[0117] Examples of such surface molecules on APCs include HLA, cluster of differentiation 14 (CD14), cluster of differentiation 40 (CD40), CLEC9A, chemokine receptors and Toll-like receptors (TLRs). Chemokine receptors include CC motif chemokine receptor 1 (CCR1), CC motif chemokine receptor 3 (CCR3), CC motif chemokine receptor 4 (CCR4), CC motif chemokine receptor 5 (CCR5), CC motif chemokine receptor 6 (CCR6), CC motif chemokine receptor 7 (CCR7), CC motif chemokine receptor 8 (CCR8) and XCR1. Toll-like receptors include TLR-2, TLR-4 and TLR-5. In one embodiment, the targeting unit is or includes a moiety that interacts with these surface molecules. In a preferred embodiment, the aforementioned surface molecules are present on human APCs.

[0118] Thus, in one embodiment, the targeting unit comprises or consists of antibody binding regions such as antibody variable domains (VL and VH) with specificity for MHC / HLA, CD14, CD40, CLEC9A or Toll-like receptors, preferably hCD14, hCD40, hCLEC9A or human Toll-like receptors. In another embodiment, the targeting unit comprises or consists of synthetic or natural ligands. Examples include soluble CD40 ligand (CD40L), preferably hCD40L, natural ligands such as chemokines, preferably their human forms, such as chemokine ligand 5, also called CC motif ligand 5 (CCL5 or RANTES), preferably hCCL5, e.g. hCCL5 of SEQ ID NO: 43, mouse CCL3 (or MIP-1α), and the human isoforms hCCL3, hCCL3L1, hCCL3L2 and hCCL3L3, chemokine ligand 4 (CCL4) and its isoforms. macrophage inflammatory protein alpha and its isoforms, including forms CCL4L, preferably hCCL4 and hCCL4L, chemokine ligand 19 (CCL19), preferably hCCL19, chemokine ligand 20 (CCL20), preferably hCCL20, chemokine ligand 21 (CCL21), preferably hCCL21, chemokine motif ligand 1 or 2 (XCL1 or XCL2), preferably hXCL1 or hXCL2, and bacterial antigens such as, for example, flagellin.

[0119] In one embodiment, the targeting unit has affinity for an MHC class II protein. Thus, in one embodiment, the targeting unit comprises or consists of an antibody binding region, such as an antibody variable domain (VL and VH) with specificity for an MHC class II protein selected from the group consisting of anti-HLA-DP, anti-HLA-DR and anti-pan-HLA class II.

[0120] In another embodiment, the targeting unit has affinity for a surface molecule selected from the group consisting of CD14, CD40, TLR-2, TLR-4 and TLR-5, preferably for a surface molecule selected from the group consisting of hCD14, hCD40, hTLR-2, hTLR-4 and hTLR-5. Thus, in one embodiment the targeting unit comprises or consists of antibody binding regions such as antibody variable domains (VL and VH) with specificity for CD14, CD40, TLR-2, TLR-4 or TLR-5, preferably for hCD14, hCD40, hTLR-2, hTLR-4 or hTLR-5, e.g. with specificity for anti-hCD14, anti-hCD40, anti-hTLR-2, anti-hTLR-4 or anti-hTLR-5.

[0121] In yet another embodiment, the targeting unit comprises or consists of a flagellin with affinity for TLR-5, such as hTLR-5. In yet another embodiment, the targeting unit comprises or consists of an antibody binding region with specificity for CLEC9A, such as anti-CLEC9A or a variant thereof, such as anti-CLEC9A Fv, or the targeting unit comprises or consists of a CLEC9 ligand, such as a CLEC9 ligand comprising or consisting of a nucleic acid sequence having SEQ ID NO: 115 or an amino acid sequence encoded by said nucleic acid sequence. In a preferred embodiment, the targeting unit comprises or consists of an antibody binding region with specificity for hCLEC9A, such as anti-hCLEC9A or a variant thereof, such as anti-hCLEC9A Fv, or the targeting unit comprises or consists of a human CLEC9 ligand.

[0122] Preferably, the targeting unit has affinity for a chemokine receptor selected from CCR1, CCR3, CCR5 and CCR7, more preferably for a chemokine receptor selected from CCR1, CCR3 and CCR5.In a further preferred embodiment, the targeting unit has affinity for a chemokine receptor selected from hCCR1, hCCR3, hCCR5 and hCCR7, more preferably for a chemokine receptor selected from hCCR1, hCCR3 and hCCR5.

[0123] In one embodiment, the targeting unit has affinity for the chemokine receptor CCR7, preferably the human chemokine receptor CCR7. In another embodiment, the targeting unit comprises or consists of CCL19, e.g., CCL19 comprising or consisting of the nucleotide sequence of SEQ ID NO: 121 or the amino acid sequence encoded by said nucleotide sequence, or CCL21, e.g., the human form of CCL19 or CCL21.

[0124] Preferably, the targeting comprises or consists of the chemokine human macrophage inflammatory protein alpha (also called human MIP-1α (hMIP-1α) variant, LD78β or CCL3L1), which binds to its cognate receptors, including CCR1, CCR3 and CCR5, expressed on the cell surface of APCs. Once the targeting unit binds to its cognate receptor, the multimeric protein is internalized by the APC, the protein is degraded into small peptides, which are loaded onto MHC molecules and presented to CD4+ and CD8+ T cells to induce a specific immune response. Once stimulated, with the help of activated CD4+ T cells, CD8+ T cells target and kill cells expressing the same antigen, for example cancer cells expressing the same antigen.

[0125] In another embodiment, both T and B cell responses are induced. This also allows for an antibody response, i.e., antibodies that neutralize the virus when it is circulating, for example by binding to viral surface proteins and inhibiting the virus from entering host cells.

[0126] In a preferred embodiment, the targeting unit comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence 24-93 of SEQ ID NO:1, for example comprising amino acid sequence 26-93 of SEQ ID NO:1, or comprising amino acid sequence 28-93 of SEQ ID NO:1.

[0127] In a further preferred embodiment, the targeting unit comprises an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence 24-93 of SEQ ID NO: 1. In a further preferred embodiment, the targeting unit comprises the amino acid sequence 24-93 of SEQ ID NO: 1.

[0128] In a more preferred embodiment, the targeting unit consists of an amino acid sequence having at least 80% sequence identity to amino acid sequence 24-93 of SEQ ID NO:1, for example consisting of amino acid sequence 26-93 of SEQ ID NO:1 or consisting of amino acid sequence 28-93 of SEQ ID NO:1.

[0129] In a further preferred embodiment, the targeting unit consists of an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence 24-93 of SEQ ID NO: 1. In yet another preferred embodiment, the targeting unit consists of the amino acid sequence 24-93 of SEQ ID NO: 1.

[0130] In one preferred embodiment, the targeting unit comprises the amino acid sequence 24-93 of SEQ ID NO:1, except that up to 6 amino acids, such as up to 5 amino acids, such as up to 4 amino acids, such as up to 3 amino acids, such as up to 2 amino acids, or up to 1 amino acid, etc. have been substituted, deleted or inserted. An embodiment of such a targeting unit is one that comprises the amino acid sequence 26-93 of SEQ ID NO:1, or one that comprises the amino acid sequence 28-93 of SEQ ID NO:1.

[0131] In another preferred embodiment, the targeting unit consists of the amino acid sequence 24-93 of SEQ ID NO:1, except that up to 6 amino acids, such as up to 5 amino acids, such as up to 4 amino acids, such as up to 3 amino acids, such as up to 2 amino acids, or such as up to 1 amino acid, have been substituted, deleted or inserted. An embodiment of such a targeting unit is one that consists of the amino acid sequence 26-93 of SEQ ID NO:1, or one that consists of the amino acid sequence 28-93 of SEQ ID NO:1.

[0132] In a further preferred embodiment, the targeting unit comprises a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the nucleic acid sequence having SEQ ID NO: 25. In a further preferred embodiment, the targeting unit comprises the nucleic acid sequence of SEQ ID NO: 25.

[0133] In a more preferred embodiment, the targeting unit consists of a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence having SEQ ID NO:25.

[0134] In a further preferred embodiment, the targeting unit consists of a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 25. In yet another preferred embodiment, the targeting unit has the nucleic acid sequence of SEQ ID NO: 25.

[0135] In one embodiment, the specific selection and / or combination of the targeting unit and the immunostimulatory compound in the vector of the present invention is, for example, hMIP-1α or CCL3 as the targeting unit and CCL4, GM-CSF, FLT3L and / or IFNα as the immunostimulatory compound. In another embodiment, the specific selection and / or combination is, for example, hMIP-1α or CCL3 as the targeting unit and CCL5, GM-CSF, FLT3L and / or IFNα as the immunostimulatory compound. In yet another embodiment, the specific selection and / or combination is, for example, CCL5 as the targeting unit and XCL1, GM-CSF, FLT3L and / or IFNα as the immunostimulatory compound. In yet another embodiment, the specific selection and / or combination is, for example, hMIP-1α or CCL3 as the targeting unit and IL-4, GM-CSF, CD40L and / or TNFα as the immunostimulatory compound. In yet another embodiment, the particular selection and / or combination is, for example, the selection of hMIP-1α or CCL3 as the targeting unit and IL-4, GM-CSF, IL-1β and / or TNFα as the immunostimulatory compound. In yet another embodiment, the particular selection and / or combination is, for example, the selection of hMIP-1α or CCL3 as the targeting unit and IL-4, GM-CSF, IL-1β and / or IFNγ as the immunostimulatory compound. In yet another embodiment, the particular selection and / or combination is, for example, the selection of CCL5 as the targeting unit and CCL7, GM-CSF, FLT3L and / or IFNα as the immunostimulatory compound. In yet another embodiment, the particular selection and / or combination is, for example, the selection of hMIP-1α or CCL3 as the targeting unit and 4-1BBL, GM-CSF, FLT3L and / or IFNα as the immunostimulatory compound.In yet another embodiment, the particular selection and / or combination is, for example, the selection of hMIP-1α or CCL3 as the targeting unit and the selection of CD40L, GM-CSF, FLT3L and / or IFNα as the immunostimulatory compound. In yet another embodiment, the particular selection and / or combination is, for example, the selection of hMIP-1α or CCL3 as the targeting unit and the selection of CD205, GM-CSF, FLT3L and / or IFNα as the immunostimulatory compound. In yet another embodiment, the particular selection and / or combination is, for example, the selection of CCL5 as the targeting unit and the selection of 4-1BBL, GM-CSF, FLT3L and / or IFNα as the immunostimulatory compound. In yet another embodiment, the particular selection and / or combination is, for example, the selection of CCL5 as the targeting unit and the selection of CD40L, GM-CSF, FLT3L and / or IFNα as the immunostimulatory compound. In yet another embodiment, the particular selection and / or combination is, for example, the selection of anti-CD205 as the targeting unit and CCL5, GM-CSF, FLT3L and / or IFNα as the immunostimulatory compound. In yet another embodiment, the particular selection and / or combination is, for example, the selection of hMIP-1α or CCL3 as the targeting unit and CCL4, GM-CSF, FLT3L and / or MyD88 as the immunostimulatory compound. In yet another embodiment, the particular selection and / or combination is, for example, the selection of hMIP-1α or CCL3 as the targeting unit and TRIF, GM-CSF, FLT3L and / or MyD88 as the immunostimulatory compound. In yet another embodiment, the particular selection and / or combination is, for example, the selection of hMIP-1α or CCL3 as the targeting unit and GM-CSF, IL-12, IL-21 and / or CD40L as the stimulatory compound. In yet another embodiment, the particular selection and / or combination is, for example, the selection of CD11c as the targeting unit and hMIP-1α or CCL3, IFNγ, GM-CSF and / or FLT3L as the immunostimulatory compound.In yet another embodiment, the particular selection and / or combination is, for example, selecting CD11c as the targeting unit and selecting hMIP-1α or CCL3, TNFα, GM-CSF and / or FLT3L as the immunostimulatory compound. In yet another embodiment, the particular selection and / or combination is, for example, selecting CLEC9A as the targeting unit and selecting CCL5, XCL1, GM-CSF and / or FLT3L as the immunostimulatory compound. In yet another embodiment, the selection and / or combination is, for example, selecting CD11c as the targeting unit and selecting CCL5, XCL1, GM-CSF and / or FLT3L as the immunostimulatory compound. In yet another embodiment, the particular selection and / or combination is, for example, selecting CADM1 as the targeting unit and selecting CCL5, XCL1, GM-CSF and / or FLT3L as the immunostimulatory compound. In yet another embodiment, the particular selection and / or combination is, for example, the selection of CCL19 as the targeting unit and GM-CSF, IL-12, IL-21 and / or CD40L as the immunostimulatory compounds. In yet another embodiment, the particular selection and / or combination is, for example, the selection of CCL19 as the targeting unit and GM-CSF, CCL3L, XCL1 and / or CCL5 as the immunostimulatory compounds.

[0136] In preferred embodiments, the targeting units and immunostimulatory compounds listed in the previous paragraphs are human proteins.

[0137] Multimerization Unit / Dimerization Unit The first polypeptide encoded by the first nucleic acid contained in the vector of the present invention comprises a multimerization unit, such as a dimerization unit.

[0138] The term "multimerization unit" as used herein refers to the sequence of nucleotides or amino acids between the antigen unit and the targeting unit. In addition to linking the antigen unit and the targeting unit, the multimerization unit facilitates the multimerization / joining of multiple polypeptides, such as two, three, four or more polypeptides, into a multimeric protein, such as a dimeric protein, a trimeric protein or a tetrameric protein. Furthermore, the multimerization unit provides flexibility to the multimeric protein so that the targeting unit can optimally bind to a surface molecule on the APC, even if it is separated by a distance. The multimerization unit can be any unit that meets one or more of these requirements.

[0139] Multimerization units that promote multimerization / binding of more than two polypeptides In one embodiment, the multimerization unit is a trimerization unit, such as a trimerization unit derived from collagen, such as a trimerization domain derived from human collagen, such as the XVIII trimerization domain derived from human collagen (see, for example, A. Alvarez-Cienfuegos et al., Sci Rep 6, 28643 (2016)) or the human collagen XV trimerization domain. Thus, in one embodiment, the multimerization unit is a trimerization unit comprising or consisting of a nucleic acid sequence having SEQ ID NO: 116, or an amino acid sequence encoded by said nucleic acid sequence. In another embodiment, the trimerization unit is the C-terminal domain of T4 fibritin. Thus, in one embodiment, the multimerization unit is a trimerization unit comprising or consisting of an amino acid sequence having SEQ ID NO: 56.

[0140] In another embodiment, the multimerization unit is a tetramerization unit, e.g. a domain derived from p53, optionally further comprising a hinge region as described below. Thus, in one embodiment, the multimerization unit is a tetramerization unit comprising or consisting of a nucleic acid sequence having SEQ ID NO: 57, or an amino acid sequence encoded by said nucleic acid sequence, optionally further comprising a hinge region as described below.

[0141] Dimerization Unit The term "dimerization unit" as used herein refers to a sequence of nucleotides or amino acids between the antigen unit and the targeting unit. In addition to linking the antigen unit and the targeting unit, the dimerization unit promotes the dimerization / binding of two monomeric polypeptides into a dimeric protein. Furthermore, the dimerization unit provides flexibility to the dimeric protein, allowing the targeting unit to optimally bind to a surface molecule on an APC, even if they are far apart. The dimerization unit can be any unit that meets these requirements.

[0142] Thus, in one embodiment, the first polypeptide comprises a dimerization unit comprising a hinge region. In another embodiment, the dimerization unit comprises a hinge region and another domain that promotes dimerization. In yet another embodiment, the dimerization unit comprises a hinge region, a dimerization unit linker, and another domain that promotes dimerization, said dimerization unit linker connecting the hinge region and the another domain that promotes dimerization. In one embodiment, the dimerization unit linker is a glycine-serine rich linker, preferably GGGSSGGGSG (SEQ ID NO: 134), i.e. the dimerization unit comprises a glycine-serine rich dimerization unit linker, preferably the dimerization unit linker GGGSSGGGSG (SEQ ID NO: 134).

[0143] The term "hinge region" refers to an amino acid sequence contained in a dimerization unit that contributes to the joining of two polypeptides, i.e., promotes the formation of a dimeric protein. In the context of a multimerization unit that promotes the multimerization / joining of more than two polypeptides, the term "hinge region" refers to an amino acid sequence that is composed of such a multimerization unit that contributes to the joining of two or more polypeptides, for example, three or four polypeptides, and / or functions as a flexible spacer, allowing two targeting units of a multimeric protein to simultaneously bind to multiple surface molecules on an APC, even if they are located at variable distances.

[0144] Moreover, the hinge region functions as a flexible spacer, allowing the two targeting units of the dimeric protein to bind simultaneously to two surface molecules on the APC, even if they are located at variable distances. The hinge region may be from Ig, such as from IgG, e.g., IgG1 or IgG2 or IgG3. In one embodiment, the hinge region is from IgM, and comprises or consists of, e.g., a nucleotide sequence having SEQ ID NO: 119 or an amino acid sequence encoded by said nucleic acid sequence. The hinge region may contribute to dimerization through the formation of a covalent bond, e.g., a disulfide bridge between cysteines. Thus, in one embodiment, the hinge region has the ability to form one or more covalent bonds. Preferably, the covalent bond is a disulfide bond.

[0145] In one embodiment the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4 (human hinge region 1 and human hinge region 4), preferably hinge exon h1 and hinge exon h4 from IgG3, more preferably hinge exon h1 and hinge exon h4 having an amino acid sequence of at least 80% sequence identity to amino acid sequence 94-120 of SEQ ID NO:1.

[0146] In a preferred embodiment, the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4 having at least 85% sequence identity to amino acid sequence 94-120 of SEQ ID NO:1, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, such as at least 98% or such as at least 99% sequence identity.

[0147] In a preferred embodiment, the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4 having the amino acid sequence 94-120 of SEQ ID NO:1.

[0148] In a preferred embodiment, the dimerization unit comprises or consists of the amino acid sequence 94-120 of SEQ ID NO: 1, with the proviso that up to 4 amino acids, such as up to 3 amino acids, such as up to 2 amino acids, or for example up to 1 amino acid, are substituted, deleted or inserted.

[0149] In a preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence having SEQ ID NO:26.

[0150] In a further preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 85% sequence identity to a nucleic acid sequence having SEQ ID NO: 26, such as at least 86%, or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity.

[0151] In a further preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO:26.

[0152] In another embodiment, the dimerization unit comprises another domain that promotes dimerization, said another domain being an immunoglobulin domain, for example an immunoglobulin constant domain (C domain), such as a CH1 domain, a CH2 domain or a carboxy-terminal C domain (i.e., a CH3 domain), or a sequence substantially identical to such a C domain or a variant thereof. Preferably, the other domain that promotes dimerization is a carboxy-terminal C domain from IgG. More preferably, the other domain that promotes dimerization is a carboxy-terminal C domain from IgG3.

[0153] In one embodiment, the dimerization unit comprises or consists of a carboxy-terminal C domain from IgG3 having an amino acid sequence having at least 80% sequence identity to amino acid sequence 131-237 of SEQ ID NO:1.

[0154] In a preferred embodiment, the dimerization unit comprises or consists of a carboxy-terminal C domain derived from IgG3 having an amino acid sequence having at least 85% sequence identity to amino acid sequence 131 to 237 of SEQ ID NO: 1, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, for example at least 97%, such as at least 98% or such as at least 99% sequence identity.

[0155] In a preferred embodiment, the dimerization unit comprises or consists of the carboxy-terminal C domain from IgG3 having the amino acid sequence 131-237 of SEQ ID NO:1.

[0156] In a preferred embodiment, the dimerization unit comprises or consists of the amino acid sequence 131-237 of SEQ ID NO: 1, with the proviso that up to 16 amino acids have been substituted, deleted or inserted, for example up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid.

[0157] In a preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence having SEQ ID NO:27.

[0158] In a further preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 85% sequence identity, such as at least 86%, or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to a nucleic acid sequence having SEQ ID NO: 27. In a further preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO: 27.

[0159] Immunoglobulin domains contribute to dimerization through non-covalent interactions, such as hydrophobic interactions. Thus, in one embodiment, an immunoglobulin domain has the ability to form a dimer through non-covalent interactions. Preferably, the non-covalent interactions are hydrophobic interactions.

[0160] If a dimerization unit comprises a CH3 domain, it preferably does not comprise a CH2 domain, and vice versa.

[0161] In a preferred embodiment, the dimerization unit comprises a hinge exon h1, a hinge exon h4, a dimerization unit linker and a CH3 domain of human IgG3. In a further preferred embodiment, the dimerization unit comprises a polypeptide consisting of a hinge exon h1, a hinge exon h4, a dimerization unit linker and a CH3 domain of human IgG3. In another preferred embodiment, the dimerization unit comprises a polypeptide consisting of a hinge exon h1, a hinge exon h4, a dimerization unit linker and a CH3 domain of human IgG3.

[0162] In one embodiment, the dimerization unit comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence 94-237 of SEQ ID NO:1.

[0163] In a preferred embodiment, the dimerization unit comprises an amino acid sequence having at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98% or such as at least 99% sequence identity to the amino acid sequence 94-237 of SEQ ID NO:1.

[0164] In a further preferred embodiment, the dimerization unit comprises the amino acid sequence 94-237 of SEQ ID NO:1.

[0165] In a more preferred embodiment, the dimerization unit consists of an amino acid sequence having at least 80%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, for example at least 97%, for example at least 98% or such as at least 99% sequence identity to the amino acid sequence 94 to 237 of SEQ ID NO:1.

[0166] In a further preferred embodiment, the dimerization unit consists of the amino acid sequence 94-237 of SEQ ID NO:1.

[0167] In a preferred embodiment, the dimerization unit comprises or consists of the amino acid sequence 94 to 237 of SEQ ID NO: 1, with the proviso that up to 28, for example up to 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids have been substituted, deleted or inserted.

[0168] In a preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence having SEQ ID NO:28.

[0169] In a further preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 85%, such as at least 86%, or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to a nucleic acid sequence having SEQ ID NO:28.

[0170] In a further preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO:28.

[0171] In the first polypeptide encoded by the first nucleic acid sequence, the multimerization unit, e.g., the dimerization unit, may have any orientation with respect to the antigen unit and the targeting unit. In one embodiment, the antigen unit is linked to the C-terminus of the multimerization / dimerization unit (e.g., via a unit linker), and the targeting unit is linked to the N-terminus of the multimerization / dimerization unit. In another embodiment, the antigen unit is linked to the N-terminus of the multimerization / dimerization unit (e.g., via a unit linker), and the targeting unit is linked to the C-terminus of the multimerization / dimerization unit. It is preferred that the antigen unit is linked to the C-terminus of the multimerization / dimerization unit, e.g., via a linker, preferably via a unit linker, and the targeting unit is linked to the N-terminus of the multimerization / dimerization unit.

[0172] Antigen Unit In general, the antigenic unit contained in the first polypeptide / multimeric protein can comprise any type of antigen or part thereof, for example an antigen or part thereof associated with a disease. Examples include one or more cancer antigens or parts thereof, or one or more antigens or parts thereof associated with an infectious disease, i.e. a disease caused by a pathogen, including viruses, bacteria, fungi and parasites.

[0173] As used herein, the term "disease-associated antigen" or "disease-related antigen" is used to indicate that the antigen or a portion thereof contained in the antigen unit plays a role and is relevant to a particular disease for which the vector of the present invention containing the antigen unit is designed to be used. In one example, the antigen unit contains one or more cancer antigens or a portion thereof, and the vector containing such an antigen unit is designed to be used for the treatment of cancer. In another example, the antigen unit is one or more infectious antigens or a portion thereof, such as an antigen derived from a pathogen, and the vector containing such an antigen unit is designed to be used for the treatment of an infectious disease caused by or involving such a pathogen.

[0174] "Part" refers to a part / fragment of the antigen, ie a part / fragment of the amino acid sequence of the antigen or the nucleotide sequence encoding same, e.g. an epitope.

[0175] In one embodiment, the antigenic unit comprises one T cell epitope, in another embodiment, the antigenic unit comprises one or more T cell epitopes, i.e. multiple T cell epitopes.

[0176] T cell epitopes suitable for inclusion in an antigenic unit may be those known in the art, i.e. those that have been studied, proposed and / or verified to be involved and relevant to a disease and have been published, for example in the scientific literature.

[0177] In one embodiment, the antigen unit comprises a T cell epitope having a length of 7 to 150 amino acids, preferably 7 to 100 amino acids, for example, 9 or 10 to 100 amino acids, or 15 to 100 amino acids, or 9 to 60 amino acids, or 9 to 30 amino acids, or 15 to 60 amino acids, or 15 to 30 amino acids, or 20 to 75 amino acids, or 25 to 50 amino acids.

[0178] In one embodiment, the antigenic unit comprised in the first polypeptide / multimeric protein comprises one or more antigens or parts thereof associated with an infectious disease, such as an antigen derived from a pathogen.

[0179] Such antigens are either known or predicted in the art, i.e., have been studied, proposed and / or verified to be involved or associated with certain infectious diseases and have been published, e.g., in the scientific literature.

[0180] In another embodiment, the antigenic unit comprised in the first polypeptide / multimeric protein comprises one or more antigens or portions thereof associated with cancer, such as cancer antigens, such as neoantigens and shared cancer antigens.

[0181] Antigenic units of individualized first polypeptide In one embodiment, the first polypeptide encoded by the first nucleic acid contained in the vector of the present invention comprises an antigen unit, which is specifically designed only for the patient treated with such vector. Thus, the antigen unit of such a first polypeptide comprises one or more patient-specific cancer antigens or parts thereof, such antigens including neoantigens or patient-presented shared cancer antigens.

[0182] "Patient-presented shared cancer antigen" as used herein refers to a shared cancer antigen or shared tumor antigen that has been confirmed to be present on the patient's tumor cells.

[0183] "Neoantigen," as used herein, refers to a cancer antigen or tumor antigen found in a patient's tumor cells that has one or more mutations compared to normal (i.e., healthy, non-cancerous) cells of the same patient.

[0184] The term "patient-presented shared cancer epitope," as used herein, refers to an amino acid sequence contained in a patient-presented shared cancer antigen, or a nucleic acid sequence encoding the same, that is known to be immunogenic or predicted to be immunogenic.

[0185] As used herein, the term "neoepitope or patient-specific cancer epitope" refers to an amino acid sequence contained in a neoantigen or patient-specific cancer antigen, or a nucleic acid sequence encoding the same, which contains one or more mutations predicted to be immunogenic.

[0186] Thus, in one embodiment, the present invention provides a method for producing a composition comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, the antigen unit comprising one or more patient-specific cancer antigens or portions thereof, e.g., one or more patient-presented shared cancer antigens or portions thereof and / or one or more neo-antigens or portions thereof; and (b) providing a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector enables the first polypeptide and the one or more immunostimulatory compounds to be coexpressed as separate molecules.

[0187] In one embodiment, the antigen unit comprises one or more patient-presented shared cancer antigens or portions thereof, such as one patient-presented shared cancer antigen or one or more portions of such patient-presented shared cancer antigens, such as one or more epitopes, or multiple patient-presented shared cancer antigens or one or more portions of such multiple patient-presented shared cancer antigens, such as one or more epitopes.

[0188] As used herein, the term "several" is used interchangeably with the terms "multiple," "a plurality," and "more than one."

[0189] In another embodiment, the antigen unit comprises one or more neo-antigens or parts thereof, such as one neo-antigen or one or more parts of such neo-antigens, such as one or more neo-epitopes, or several neo-antigens or one or more parts of several such neo-antigens, such as one or more neo-epitopes.

[0190] In yet another embodiment, the antigen unit comprises any combination of the above embodiments, i.e. any combination of one or more patient-presented shared cancer antigens or parts thereof with one or more neo-antigens or parts thereof as above.

[0191] Antigenic units of individualized polypeptides comprising one or more neo-antigens or portions thereof Cancer develops when one or a few cells from a patient's normal tissues initiate abnormal and uncontrollable cell proliferation due to mutations. Although cancer cells are mutated, most of their genome is intact and identical to the rest of the patient's cells. One approach to attack tumors is based on the knowledge that every tumor of every patient is unique. Patient-specific mutations lead to the expression of patient-specific mutant proteins, i.e. neo-antigens, which are unique to a particular patient. These neo-antigens are not identical to any proteins of the patient's normal cells. Such neo-antigens are therefore suitable as targets for therapeutic pharmaceutical compositions comprising the vectors of the invention, which are produced specifically only for the patient in question, i.e. personalized anti-cancer vaccines.

[0192] A mutation can be any mutation that results in a change in at least one amino acid. Thus, a mutation can be any of the following: -Non-synonymous mutation resulting in an amino acid change - A mutation that causes a frameshift, thereby resulting in an entirely different open reading frame in the post-mutation orientation -Read-through mutations in which the stop codon is altered or deleted, resulting in a longer protein with a tumor-specific epitope -Splice variants leading to specific tumor-specific protein sequences - A chromosomal rearrangement resulting in a chimeric protein with a tumor-specific epitope at the junction of the two proteins. If the mutation is due to a chromosomal rearrangement, the tumor-specific epitope results from at least one amino acid change or from the combination of two in-frame coding sequences.

[0193] In one embodiment, the antigen unit comprises one or more neo-antigens or parts thereof, such as one or more parts of one neo-antigen or one or more parts of multiple neo-antigens, preferably one or more neo-epitopes, more preferably multiple neo-epitopes. Such neo-epitopes may be selected for inclusion in the antigen unit according to the expected therapeutic effect, see WO 2017 / 118695A1, the disclosure of which is incorporated herein by reference.

[0194] Thus, in one embodiment, the present invention provides a method for the preparation of a method for treating a pulmonary artery disease comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, the antigen unit comprising one or more neo-antigens or a portion thereof; and (b) providing a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0195] In one embodiment, the antigen unit comprises one or more parts of one neo-antigen, or one or more parts of multiple neo-antigens, preferably one or more neo-epitopes. In a preferred embodiment, in the antigen unit, the neo-epitopes are separated by a linker. Another way to describe all neo-epitopes separated by a linker is that all but the terminal neo-epitopes, i.e., the neo-epitopes at the N-terminal start of the first polypeptide or at the C-terminal end of the first polypeptide, are located in antigen subunits, each subunit comprising a neo-epitope and a subunit linker. Separation of the neo-epitopes by a linker allows each neo-epitope to be optimally presented to the immune system.

[0196] Thus, an antigen unit comprising n neoepitopes comprises n-1 antigen subunits, each of which comprises a neoepitope and a subunit linker, and further comprises a terminal neoepitope. In one embodiment, n is an integer from 1 to 50, e.g., 3 to 50, or 15 to 40, or 10 to 30, or 10 to 25, or 10 to 20, or 15 to 30, or 15 to 25, or 15 to 20. In a preferred embodiment, the antigen subunit comprises a neoepitope and a subunit linker.

[0197] Thus, in a preferred embodiment, the present invention provides a method for the preparation of a method for the treatment of a pulmonary artery disease comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, the antigen unit comprising (i) n-1 antigen subunits, each subunit comprising a neoepitope and a subunit linker, and (ii) a terminal neoepitope, where n is the number of neoepitopes in the antigen unit, and n is an integer from 1 to 50; and (b) providing a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector enables co-expression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0198] The neoepitope preferably has a length suitable for presentation by HLA molecules. Thus, in a preferred embodiment, the neoepitope has a length of 7 to 30 amino acids. More preferred are neoepitopes having a length of 7 to 10 amino acids, or 13 to 30 amino acids, for example 20 to 30 amino acids, for example 27 amino acids.

[0199] Preferably, the antigenic unit comprises multiple neoepitopes. In one embodiment, the antigenic unit comprises multiple different neoepitopes. In another embodiment, the antigenic unit comprises multiple copies of the same neoepitope. In yet another embodiment, the antigenic unit comprises multiple different neoepitopes and multiple copies of the same neoepitope.

[0200] Therefore, a preferred approach is to include as many neoepitopes as possible in the antigen unit (i.e., different and / or multiple copies of the same neoepitope) in order to efficiently attack the cancer while not compromising the ability to activate T cells against the neoepitopes due to dilution of the desired T cell effect. Furthermore, to ensure that all neoepitopes are efficiently loaded into the same antigen-presenting cell, the nucleotide sequence encoding all neoepitopes is composed of a contiguous polynucleotide chain, so that instead of expressing each neoepitope as an individual peptide, a protein containing all the neoepitopes is expressed.

[0201] To design the antigen unit, the patient's tumor exome is analyzed to identify neo-antigens, and preferably the most immunogenic neo-epitope sequences from one or more neo-antigens are selected for inclusion in the antigen unit.

[0202] In one embodiment, the antigen unit comprises at least one neoepitope. Preferably, the antigen unit comprises at least three neoepitopes, more preferably at least five neoepitopes, such as seven neoepitopes. In another more preferred embodiment, the antigen unit comprises at least 10 neoepitopes. In another more preferred embodiment, the antigen unit comprises at least 15 neoepitopes, such as at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 neoepitopes.

[0203] Antigen units comprising one or more neoepitopes are described in detail in WO2017 / 118695A1. Any of such antigen units can be used as the antigen unit in the first polypeptide encoded in the vector of the present invention for use in personalized anti-cancer therapy.

[0204] Antigenic units of personalized polypeptides comprising one or more patient-presented shared cancer antigens or portions thereof Shared tumor antigens are expressed in many tumors, and are expressed between patients with the same cancer type or between patients and cancer types. For example, HPV16 antigen is a viral antigen that is expressed in about 50% of patients with head and neck squamous cell carcinoma, but also in patients with other cancers, such as cervical cancer and vulvar squamous cell carcinoma. Many of these shared antigens have been previously characterized and / or known as immunogenic, i.e., their immunogenicity has been confirmed by suitable methods and the results have been published, for example, in scientific publications. Others have already been predicted, for example, by algorithms known in the art, to be presented on certain HLA class I or class II alleles, and their predicted immunogenicity has been published, for example, in scientific publications, without confirming their immunogenicity by suitable methods.

[0205] In one embodiment, the antigenic unit comprises one or more patient-presented shared cancer antigens or portions thereof, e.g., patient-presented shared cancer epitopes, that are known to be immunogenic, have known expression patterns, and / or are known or previously predicted to bind to specific HLA class I and class II molecules.

[0206] T cells specific for patient-presented cancer antigens can migrate to tumors and influence the tumor microenvironment, further increasing the likelihood that tumor-specific T cells can attack cancer.

[0207] Thus, in one embodiment, the present invention provides a method for the preparation of a method for treating a pulmonary artery disease comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, the antigen unit comprising one or more patient-presented shared cancer antigens or a portion thereof; and (b) providing a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0208] Some patient-presented shared cancer antigens are proteins that contain an amino acid sequence that contains one or more mutations, i.e., patient-presented shared cancer epitopes that are known to be immunogenic or predicted to be immunogenic. Other patient-presented shared cancer antigens are proteins that do not contain mutations, e.g., overexpressed cellular proteins.

[0209] In one embodiment, the patient-presented shared cancer antigen is selected from the group consisting of overexpressed cellular proteins, aberrantly expressed cellular proteins, cancer-testis antigens, viral antigens, differentiation antigens, mutated oncogenes and mutated tumor suppressor genes, carcinoembryonic antigens, shared fusion antigens, shared intron-bearing antigens, dark matter antigens, and shared antigens due to spliceosomal or frameshift mutations.

[0210] In one embodiment, the patient-presented shared cancer antigen is an overexpressed or aberrantly expressed human cellular protein, i.e., a cellular protein found at increased levels in tumors compared to normal healthy cells or tissues. Examples of such overexpressed or aberrantly expressed cellular proteins include tumor proteins D52, Her-2 / neu, hTERT (telomerase), and survivin.

[0211] In another embodiment, the patient-presented shared cancer antigen is a cancer-testis antigen that is normally expressed in male germ cells of the testis, but not in adult somatic tissues. In some cases, such antigens are also expressed in the ovaries and chorionic villi. In malignant tumors, this genetic regulation is disrupted, resulting in antigen expression in a subset of tumors of various types. Examples of cancer-testis antigens include MAGE-A, MAGE-B, GAGE, PAGE-1, SSX, HOM-MEL-40 (SSX2), NY-ESO-1, LAGE-1, and SCP-1.

[0212] In yet another embodiment, the patient-presented shared cancer antigen is a differentiation antigen, such as tyrosinase.

[0213] In yet another embodiment, the patient-presented shared antigen is a viral antigen. Examples of viral antigens include human papillomavirus (HPV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Merkel cell polyomavirus (MCV or MCPyV), human cytomegalovirus (HCMV), and human T-lymphotropic virus (HTLV).

[0214] In yet another embodiment, the patient-presented shared cancer antigen is a mutated cancer gene. Examples of mutated cancer genes include KRAS, CALR, and TRP-2.

[0215] In yet another embodiment, the patient-presented shared cancer antigen is a mutated tumor suppressor gene. Examples include mutated p53, mutated pRB, mutated BCL2, and mutated SWI / SNF.

[0216] In yet another embodiment, the patient-presented shared cancer antigen is an oncofetal antigen, such as alpha-fetoprotein or a carcinoembryonic antigen.

[0217] In yet another embodiment, the patient-presented shared antigen is a shared intron-retained antigen, or a shared antigen with a frameshift mutation, such as CDX2 or CALR.

[0218] In yet another embodiment, the patient-presented shared antigen is a shared antigen resulting from a spliceosomal mutation. An example is an antigen caused by a mutation such as the SF3B1 mutation.

[0219] In general, immune tolerance is likely to occur for any cancer antigen at the time a patient is afflicted with cancer. Anti-cancer vaccines must specifically induce an immune response against the antigen incorporated in the vaccine. In one embodiment, the first polypeptide encoded by the plasmid functions as an anti-cancer vaccine. Peripheral immune tolerance to the selected antigen may be weak or strong. By incorporating such patient-presented shared cancer antigens or one or more portions thereof into an antigen unit - alone or together with other patient-presented shared cancer antigens or portions thereof and / or neo-antigens or neo-epitopes - the polypeptides constituting such antigen units induce immune responses that are strong and broad enough to affect the tumor microenvironment and change the patient's immune response against the tumor from a suppressive / tolerant type to a pro-inflammatory type. This helps to break tolerance to some other antigens, thus resulting in considerable clinical benefit for the patient. The above-mentioned concept is also called tipping the cancer immunity set point.

[0220] In one embodiment, the antigenic unit comprises one or more patient-presented shared cancer antigens or parts thereof which are human cellular proteins, preferably overexpressed or aberrantly expressed human cellular proteins or differentiation antigens.

[0221] Patient-presented shared cancer antigens can be detected in the patient's tissues or body fluids by methods known in the art, such as: - Sequencing of the patient's genome or exome, optionally searching, e.g. using custom-made software, in the whole genome / exome-seq data, e.g. to identify mutated oncogenes or mutated tumor suppressor genes; - Immunohistochemistry of the patient's tumor tissue to detect the presence of the mutant protein; - RT-PCR to detect, for example, the presence of known mutations in viral antigens or cancer genes; - ELISA using antibodies against mutant tumor proteins in serum samples; - RNA-seq of tumor tissues compared to healthy tissues to detect expression / overexpression of shared cancer antigens; -Searching with bespoke software etc. in raw RNA-seq data to identify intron-bearing antigens; -Searching whole genome sequencing data using custom software to identify transposable elements that are elements of dark matter antigens; -Detection of short repeats in raw whole-exome / RNA-seq data, e.g. to identify dark matter antigens; - RNA-seq data, e.g., to identify shared viral antigens; and -Compare RNA-seq of patient tumor samples against the patient's own healthy tissue or against a cohort / database (e.g. TCGA) for consensus transcript expression such as GTEX / HPA gene expression data.

[0222] In a preferred embodiment, the antigenic unit comprises a shared cancer antigen present in one or more patients, or a portion of such an antigen, that is known to be immunogenic, e.g., has previously been described to elicit an immune response in other patients, or is predicted to bind to the patient's HLA class I and / or class II alleles.

[0223] In one embodiment, the antigenic unit comprises one or more patient-presented shared cancer epitopes. In a preferred embodiment, such epitopes have a length suitable for presentation by the patient's HLA alleles.

[0224] In one embodiment, the antigen unit comprises one or more patient-presented shared cancer epitopes having a length suitable for specific presentation by HLA class I or HLA class II. In one embodiment, the epitopes have a length of 7-11 amino acids for HLA class I presentation. In another embodiment, the epitopes have a length of 13-30 amino acids for HLA class II presentation.

[0225] In one embodiment, the antigenic unit comprises one or more patient-presented shared cancer epitopes having a length of 7 to 30 amino acids, such as 7 to 10 amino acids (such as 7, 8, 9, or 10 amino acids) or 13 to 30 amino acids (such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids), for example, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.

[0226] The antigen unit may comprise one or more patient-presented shared cancer antigens in full length or one or more portions thereof.

[0227] In one embodiment, the antigen unit comprises one full-length patient-presented shared cancer antigen, hi another embodiment, the antigen unit comprises multiple patient-presented shared cancer antigens, each of which is full-length.

[0228] In yet another embodiment, the antigen unit comprises one or more portions of a patient-presented shared cancer antigen, such as one or more patient-presented shared cancer epitopes.In yet another embodiment, the antigen unit comprises one or more portions of a plurality of patient-presented shared cancer antigens, such as one or more epitopes of a plurality of patient-presented shared cancer antigens.

[0229] In yet another embodiment, the antigen unit comprises one or more full-length patient-presented shared antigens and one or more portions of one or more patient-presented shared cancer antigens. Examples include: - the antigen unit comprises one full-length patient-presented shared antigen and one or more epitopes of one patient-presented shared cancer antigen; and - the antigen unit comprises a plurality of patient-presented shared antigens, each of which is full length, and one or more epitopes of one patient-presented shared cancer antigen; and - the antigen unit comprises one full-length patient-presented shared antigen and one or more epitopes of multiple patient-presented shared cancer antigens; and The antigen unit comprises multiple patient-presented shared antigens, each of which is full length, and one or more epitopes of multiple patient-presented shared cancer antigens.

[0230] In a preferred embodiment, the epitope is already known to be immunogenic, e.g. described in the literature as being immunogenic, or already predicted to bind to the patient's HLA class I and class II alleles, preferably already predicted to bind to the patient's HLA class I alleles, e.g. as described in the literature. In another preferred embodiment, the immunogenicity of the epitope is predicted, e.g. the binding of the epitope to one or more of the patient's HLA class I and / or HLA class II molecules is predicted by methods known in the art, e.g. the methods disclosed in WO 2021 / 205027 A1, the disclosure of which is incorporated herein by reference, or the methods described herein, including those described in the section "Methods for designing antigen units of an individualized first polypeptide".

[0231] In one embodiment, the antigen unit comprises 1-10 full length patient-presented shared antigens.

[0232] In another embodiment, the antigen unit comprises 1-30 portions of one or more patient-presented shared antigens, which portions comprise multiple epitopes predicted to bind to the patient's HLA class I or class II alleles.In yet another embodiment, the antigen unit comprises 1-50 patient-presented shared cancer epitopes, preferably epitopes predicted to bind to the patient's HLA class I or class II alleles.

[0233] Antigenic units of personalized polypeptides comprising one or more patient-presented shared cancer antigens or portions thereof and one or more neo-antigens or portions thereof In a further embodiment, the antigen unit is a combination of all of the above embodiments relating to an antigen unit comprising one or more patient-presented shared cancer antigens or portions thereof and all of the above embodiments relating to an antigen unit comprising one or more neo-antigens or portions thereof.

[0234] Thus, in one embodiment, the present invention provides a method for producing a composition comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, the antigen unit comprising one or more patient-presented shared cancer antigens or portions thereof, and one or more neo-antigens or portions thereof; and (b) one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds; wherein the vector allows for the co-expression of the first polypeptide and one or more immunostimulatory compounds as separate molecules.

[0235] Antigen units comprising one or more patient-presented shared cancer antigens or parts thereof and optionally one or more neo-antigens and parts thereof are described in detail in WO 2021 / 205027A1, the contents of which are incorporated herein by reference. Any of such antigen units may be used as an antigen unit in the first polypeptide encoded by the vector of the invention for use in personalized anti-cancer therapy.

[0236] Methods for designing antigenic units of individualized first polypeptides The patient-presented shared cancer antigens and neo-antigens identified in a particular patient are preferably further processed to find those antigens that, when included in the antigen unit, make the first polypeptide most effective. The method and order of such processing depends on how the antigens were identified, i.e., the data on which such processing is based.

[0237] In one embodiment, the processing and selection of antigens contained in the antigen unit is carried out as follows: 1) Conduct a search in the literature and / or one or more databases to retrieve information and sequences about the shared cancer antigens, and preferably information about their expression patterns, immunogenicity or predicted immunogenicity, epitopes and HLA presentation. Such searches are also performed to determine whether the identified antigens are patient-presented shared cancer antigens or neo-antigens. 2) If the identified antigen is determined to be a patient-presented shared cancer antigen, its sequence is studied to identify epitopes, preferably all epitopes, that are predicted to bind to the patient's HLA class I / II alleles. This prediction can be performed using prediction tools known in the art, such as prediction software known in the art, such as NetMHCpan or similar software. 3) The most likely sequences of the patient-presented shared cancer antigen that are the most immunogenic or predicted to be the most immunogenic, i.e., predicted to bind to one or more of the patient's HLA class I / II alleles, are selected for inclusion in the antigen unit. In one embodiment, a minimal epitope is selected, for example, if only a few likely epitopes were identified in step 2, or if there are long non-immunogenic sequences between the epitopes. In another embodiment, a longer sequence is selected that includes several epitopes that bind to the patient's specific HLA alleles. In yet another embodiment, the full-length sequence of the antigen is selected for inclusion in the antigen unit. 4) The most promising portions of the neoantigenic sequences, e.g., neoepitopes, are selected for inclusion in the antigenic unit based on predicted immunogenicity and binding of such sequences to the patient's HLA class I / II alleles.

[0238] Tumor mutations are discovered by sequencing tumor and normal tissues and comparing the sequences obtained from the tumor tissue with those of the normal tissue. A variety of methods are available to detect the presence of specific mutations or alleles in the DNA or RNA of a patient. These methods include dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMan system, and various DNA "chip" technologies such as the Affymetrix SNP chip. Alternatively, mutations can be identified by direct protein sequencing.

[0239] Among the potentially hundreds to thousands of mutations in the tumor exome, the most promising sequences are selected in silico based on HLA binding prediction algorithms. The intention is to identify all relevant epitopes, rank and score them, and then determine which sequences to include in the antigenic unit. Methods known in the art that are suitable for scoring, ranking and selecting neoepitopes include those disclosed in WO 2020 / 065023A1 and WO 2020 / 221 / 783A1.

[0240] Moreover, any suitable algorithm may be used for such scoring or ranking, such as: Free software for analyzing peptide-MHC binding (IEDB and NetMHCpan) can be downloaded from the following sites: www.iedb.org / www.cbs.dtu.dk / services / NetMHC / Advanced commercial software to predict optimal sequences for vaccine design is available here: www.oncoimmunity.com / omictools.com / t-cell-epitopes-category github.com / griffithlab / pVAC-Seq crdd.osdd.net / raghava / cancertope / help.php www.epivax.com / tag / neoantigen /

[0241] Each mutation is scored for its antigenicity and the most antigenic neoepitopes are selected and optimally positioned on the antigenic unit.

[0242] Antigenic units of a non-individualized first polypeptide An antigenic unit of a first polypeptide comprising one or more shared cancer antigens or portions thereof. The non-individualized or "off-the-self" vector encoding the first polypeptide (also referred to as the first polypeptide comprising the shared cancer antigen) comprises a polynucleotide sequence encoding an antigen unit comprising one or more shared cancer antigens or portions thereof.

[0243] "Shared cancer antigen" or "shared tumor antigen" is used herein to refer to an antigen that is reported to be expressed in many tumors across patients with the same cancer type, or across patients and cancer types.

[0244] The term "shared cancer epitope" is used herein to refer to an amino acid sequence contained in a shared cancer antigen that is known or predicted to be immunogenic.

[0245] In one embodiment, the antigenic unit non-individualized first polypeptide for use in the treatment of cancer comprises one or more shared cancer antigens or portions thereof, e.g., shared cancer epitopes, that are known to be immunogenic, have known expression patterns, and / or are known or previously predicted to bind to specific HLA class I and class II molecules.

[0246] Thus, in one embodiment, the present invention provides a method for producing a composition comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, the antigen unit comprising one or more shared cancer antigens or a portion thereof; and (b) one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds; wherein the vector is capable of co-expressing the first polypeptide and one or more immunostimulatory compounds as separate molecules.

[0247] Some shared cancer antigens are proteins that contain amino acid sequences that contain one or more mutations, i.e., shared cancer epitopes that are known or predicted to be immunogenic. Other shared cancer antigens are proteins that do not contain mutations, e.g., overexpressed cellular proteins.

[0248] In one embodiment, the shared cancer antigen is selected from the group consisting of overexpressed cellular proteins, aberrantly expressed cellular proteins, cancer-testis antigens, viral antigens, differentiation antigens, mutated oncogenes and mutated tumor suppressor genes, carcinoembryonic antigens, shared fusion antigens, shared intron-retained antigens, dark matter antigens, and shared antigens caused by spliceosomal or frameshift mutations.

[0249] In one embodiment, the shared cancer antigen is an overexpressed or aberrantly expressed human cellular protein, i.e., a cellular protein found at increased levels in tumors compared to normal healthy cells or tissues. Examples of such overexpressed or aberrantly expressed cellular proteins include tumor proteins D52, Her-2 / neu, hTERT (telomerase), and survivin.

[0250] In another embodiment, the shared cancer antigen is a cancer-testis antigen that is normally expressed in male germ cells of the testis, but not in adult somatic tissues. In some cases, such antigens are also expressed in the ovaries and chorionic villi. In malignant tumors, this genetic regulation is disrupted, resulting in the antigen being expressed in a subset of tumors of various types. Examples of cancer-testis antigens include MAGE-A, MAGE-B, GAGE, PAGE-1, SSX, HOM-MEL-40 (SSX2), NY-ESO-1, LAGE-1, and SCP-1.

[0251] In yet another embodiment, the shared cancer antigen is a differentiation antigen, such as tyrosinase.

[0252] In yet another embodiment, the shared antigen is a viral antigen. Examples of viral antigens include human papillomavirus (HPV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Merkel cell polyomavirus (MCV or MCPyV), human cytomegalovirus (HCMV), and human T-lymphotropic virus (HTLV).

[0253] In yet another embodiment, the shared cancer antigen is a mutated cancer gene. Examples of mutated cancer genes include KRAS, CALR, and TRP-2.

[0254] In yet another embodiment, the shared cancer antigen is a mutated tumor suppressor gene. Examples include mutated p53, mutated pRB, mutated BCL2, mutated SWI / SNF.

[0255] In yet another embodiment, the shared cancer antigen is a carcinoembryonic antigen, such as alpha-fetoprotein or a carcinoembryonic antigen.

[0256] In yet another embodiment, the shared antigen is a shared intron-retained antigen or a shared antigen with a frameshift mutation, such as CDX2 or CALR.

[0257] In yet another embodiment, the shared antigen is a shared antigen caused by a spliceosomal mutation. An example is an antigen caused by a mutation such as a SF3B1 mutation.

[0258] Further examples of shared cancer antigens include scFvs derived from monoclonal Igs produced by myelomas or lymphomas, also called myeloma / lymphoma M components in patients with B cell lymphoma or multiple myeloma, HIV-derived sequences, e.g., gpl20 or sequences from Gag, tyrosinase-related protein (TRP)-1, melanoma antigens, prostate-specific antigens and idiotypes, HPV antigens selected from the list consisting of E1, E2, E6, E7, L1 and L2, e.g., E6 and / or E7 of HPV16 and / or HPV18.

[0259] Any shared cancer antigen sequence of sufficient length that contains a specific epitope can be used as an antigen unit.Thus, in one embodiment, the antigen unit comprises at least 7 amino acid sequences, for example, at least 8 amino acid sequences, and in the nucleic acid sequence encoding such antigen unit, at least 21 nucleotides, for example, at least 24 nucleotides, corresponding thereto.

[0260] In yet another embodiment, the antigen unit comprises one or more portions of a shared cancer antigen, such as one or more shared cancer epitopes. In yet another embodiment, the antigen unit comprises one or more portions of multiple shared cancer antigens, such as one or more epitopes of multiple shared cancer antigens. In yet another embodiment, the antigen unit comprises one or more full-length shared antigens and one or more portions of one or more shared cancer antigens. Examples include: an antigenic unit comprising one full-length shared antigen and one or more epitopes of one shared cancer antigen; and - an antigen unit comprising multiple shared cancer antigens, each of which is full length and comprises one or more epitopes of one shared cancer antigen; and - an antigenic unit comprising one full-length shared antigen and one or more epitopes of multiple shared cancer antigens; and An antigen unit comprising multiple shared cancer antigens, each of which is full length and comprises one or more epitopes of the multiple shared cancer antigens.

[0261] Examples of polypeptides comprising shared antigens for HPV are disclosed in WO 2013 / 092875A1, the contents of which are incorporated herein by reference.

[0262] Method for designing an antigenic unit of a first polypeptide that contains a shared cancer antigen Additionally, in the case of vectors encoding a first polypeptide that contains a shared cancer antigen, the antigen unit is designed to contain sequences that are likely to render the polypeptide effective in a variety of patients, e.g., patients with certain types of cancer.

[0263] In one embodiment, the selection of antigens to be included in the antigen unit is performed by searching the literature and / or one or more databases for information and sequences of shared cancer antigens, preferably their expression patterns, immunogenicity or predicted immunogenicity, epitopes and / or HLA presentation. Epitopes known or predicted to bind to various HLA class I / II alleles in many patients are then identified, or epitopes that bind to a specific subset of HLA class I / II alleles that are dominant in a particular cancer indication and / or a particular patient population across different cancer indications are identified. Preferably, the most promising, i.e., the most immunogenic or predicted to be most immunogenic, sequences of shared cancer antigens are selected for inclusion in the antigen unit.

[0264] An antigenic unit of a first polypeptide comprising one or more infectious antigens or a portion thereof. In another embodiment of the invention, the first polypeptide encoded by the first nucleic acid comprised in the vector of the invention comprises an antigenic unit designed for the treatment of an infectious disease, and the vector / first polypeptide is for use in the treatment of an infectious disease.

[0265] In one embodiment, the antigenic unit comprised in the first polypeptide comprises one or more antigens or parts thereof associated with an infectious disease, i.e. one or more infectious antigens, i.e. antigens or parts thereof derived from a pathogen.

[0266] "Infectious disease" as used herein refers to a condition caused by or in the etiology of which a pathogen is involved. Examples of the latter include parasitic eggs, which do not cause disease themselves but develop into disease-causing larvae.

[0267] "Pathogens" include viruses, bacteria, fungi, and parasites.

[0268] The antigens described in this section are "infectious antigens", i.e., antigens derived from a pathogen, i.e., antigens that are comprised (or naturally found) in proteins of a pathogen that causes or is involved in causing disease. The terms "infectious antigen" and "pathogen-derived antigen" may be used interchangeably herein.

[0269] Thus, in one embodiment, the present invention provides a method for producing a composition comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, the antigen unit comprising one or more infectious antigens or a portion thereof; and (b) one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds; wherein said vector is capable of co-expressing the first polypeptide and one or more immunostimulatory compounds as separate molecules.

[0270] In another embodiment, the present invention provides a method for producing a composition comprising the steps of: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, the antigen unit comprising one or more antigens or portions of such antigens from one or more pathogens; and (b) A vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of coexpressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0271] In the above embodiments, the antigen unit comprises one or more antigens from a pathogen, or parts of such antigens, and is for example composed of one antigen from a pathogen or more than one antigen from a pathogen, i.e. multiple antigens from a pathogen, for example antigens contained in the same or different proteins of such a pathogen.

[0272] In one embodiment, the antigenic unit comprises one or more antigens or parts of such antigens derived from multiple pathogens. In one embodiment, the multiple pathogens are multiple different pathogens. In that context, "different pathogens" may be, for example, different viruses or bacteria, or different strains of the same virus or bacteria, or the same strain but with one or more mutations.

[0273] Vectors containing one or more antigens or parts thereof from multiple pathogens may be used in pan-vaccines, e.g. vaccines targeting different (seasonal) viruses, e.g. a pan-vaccine could target betacoronavirus and influenza, or different strains of a betacoronavirus or different mutations of the same strain.

[0274] Examples of infectious antigens / antigens derived from pathogens are those derived from bacteria, e.g. tuberculosis antigens or OMP31 from brucellosis, or those derived from viruses, e.g. sequences derived from HIV, e.g. sequences derived from gp120, glycoprotein D, hemagglutinin, nucleoprotein, influenza virus antigens such as M2 from HSV-2, antigens derived from HPV, e.g. E1, E2, E6, E7, L1 or L2, such as E6 and E7 of HPV16 or HPV18.

[0275] In one embodiment, the antigenic unit comprises one or more betacoronavirus antigens or portions thereof.

[0276] Betacoronavirus refers to a genus in the subfamily Orthocoronaviridae. Betacoronaviruses are enveloped, positive-sense, single-stranded RNA viruses. Within the genus, four lineages are commonly recognized: lineage A (subgenus Embecovirus), lineage B (subgenus Sarbecovirus), lineage C (Merbecovirus), and lineage D (Nobecovirus). Betacoronaviruses include the following viruses that have caused epidemics / pandemics in humans or have the potential to infect humans: SARS-CoV, which causes Severe Acute Respiratory Syndrome (SARS), MERS-CoV, which causes Middle East Respiratory Syndrome (MERS), SARS-CoV-2, which causes Coronavirus Disease 2019 (Covid-19), HCoV-OC43, and HCoV-HKU1. SARS-CoV and SARS-CoV-2 belong to lineage B (subgenus Sarbecovirus), MERS-CoV belongs to lineage C (Merbecovirus), and HCoV-OC43 and HCoV-HKU1 belong to lineage A (subgenus Envecovirus).

[0277] In one embodiment, the antigen is the spike protein or a portion thereof of SARS-CoV or SARS-CoV-2.

[0278] In one embodiment of the invention, the antigen may be a T cell epitope which is part of the sequence of spike protein or membrane protein or envelope protein or nucleocapsid protein or ORF1a / b or ORF3a protein, in another embodiment the T cell epitope is part of the following genes / proteins: NCAP, AP3A, spike, ORF1a / b, ORF3a, VME1 and VEMP.

[0279] In some embodiments, the antigenic unit of the vector of the present invention comprises one or more antigens or portions thereof derived from one or more pathogens selected from the list consisting of influenza virus, herpes simplex virus, CMV, HPV, HBV, Brucella, HIV, HSV-2 and Mycobacterium tuberculosis.

[0280] The vectors of the invention used to treat infectious diseases are ideal for fighting pandemics and epidemics, as they are capable of inducing a rapid and strong immune response. Such vectors are designed to induce an antigenic effect by including in the antigenic unit full length or parts of one or more infectious antigens, which may be, for example, selected T cell epitopes, or a combination thereof.

[0281] In one embodiment, the targeting unit of such a first polypeptide is anti-pan HLA class II or human MIP-1α and the immune response is raised through B cells and / or T cells. In one embodiment, the vector may be used in a prophylactic or therapeutic setting, or in both a prophylactic and therapeutic setting.

[0282] An antigenic unit of a first polypeptide comprising one or more T cell epitopes derived from one or more pathogens. In one embodiment, the antigenic unit of the vector / first polypeptide for use in treating an infectious disease comprises at least one T cell epitope from one or more pathogens. Such T cell epitopes are comprised (or naturally found) in the proteins of the pathogens. Conserved parts of the genomes of many pathogens contain T cell epitopes that can initiate an immune response.

[0283] Thus, one aspect of the present invention is a method for producing a method for treating a pulmonary circulation comprising the steps of: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, the antigen unit comprising at least one T cell epitope from one or more infectious antigens; and (b) A vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0284] In one embodiment, the present invention provides a method for producing a composition comprising the steps of: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, the antigen unit comprising at least one T cell epitope derived from one or more pathogens; and (b) A vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0285] In some embodiments, the antigenic unit comprises at least one T cell epitope of a pathogen, i.e. one T cell epitope of a pathogen, or more than one T cell epitope of a pathogen, i.e. multiple T cell epitopes of a pathogen. In one embodiment, the multiple T cell epitopes are of the same pathogen, i.e. (naturally) comprised in the same or different proteins of the pathogen. In other embodiments, the multiple T cell epitopes are of different pathogens, i.e. (naturally) comprised in proteins of different pathogens.

[0286] At least one T cell epitope contained in an antigen unit has a length of 7 to about 200 amino acids, and longer T cell epitopes may contain a minimal T cell epitope hotspot. A "minimum epitope hotspot" is a region that contains several minimal T cell epitopes (e.g., having a length of 7 to 15 amino acids) predicted to be presented by different HLA alleles, covering a wide range of the world population.

[0287] In some embodiments, the antigen unit comprises at least one T cell epitope having a length of 7 to 150 amino acids, preferably 7 to 100 amino acids, for example, about 10 to about 100 amino acids, or about 15 to about 100 amino acids, or about 20 to about 75 amino acids, or about 25 to about 50 amino acids.

[0288] A T cell epitope having a length of about 60 to 200 amino acids can be divided into shorter sequences and included in an antigen unit separated by a linker, such as a linker as described herein. For example, a T cell epitope having a length of 150 amino acids can be divided into three sequences of 50 amino acids each and included in an antigen unit, with the three sequences separated from each other by a linker.

[0289] In one embodiment, the antigen unit comprises multiple T cell epitopes separated from each other by a linker, e.g. a linker as referred to herein, e.g. a linker as discussed in the "Linkers in Antigen Units" section herein.

[0290] In some embodiments, at least one T cell epitope has a length suitable for presentation by MHC. Thus, in some embodiments, the antigen unit comprises at least one T cell epitope having a length suitable for specific presentation on MHC class I or MHC class II. In some embodiments, at least one T cell epitope has a length of 7-11 amino acids for MHC class I presentation. In other embodiments, at least one T cell epitope has a length of about 15 amino acids for MHC class II presentation.

[0291] The number of T cell epitopes contained in an antigen unit varies and depends on other elements contained in the antigen unit, such as the length and number of linkers.

[0292] In some embodiments, the antigenic unit comprises 1 to 10 T cell epitopes, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 or 10 T cell epitopes, or 11 to 20 T cell epitopes, such as 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 T cell epitopes, or 21 to 30 T cell epitopes, such as 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 2, 23, 24, 25, 26, 27, 28, 29 or 30 T cell epitopes, or 31 to 40 T cell epitopes, for example 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 T cell epitopes, or 41 to 50 T cell epitopes, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 T cell epitopes.

[0293] In a preferred embodiment, the at least one T cell epitope is a conserved region of the pathogen, ie, conserved among several subgenera, species or strains of the respective pathogen.

[0294] T cell epitopes may be contained in proteins of the pathogen, i.e. surface proteins but also internal proteins such as the viral nucleocapsid protein, the viral replicase polyprotein, or other structural or non-structural proteins.

[0295] Vectors containing antigenic units that contain T cell epitopes derived from conserved regions of a pathogen provide protection against multiple species / strains of the pathogen. Such vectors also provide protection against multiple variants of the pathogen, which is important for the efficacy of such vectors / first polypeptides against future mutated pathogens. Viruses are known to mutate, resulting in, for example, viral antigenic drift or antigenic shift. The presence of conserved regions across viral genera suggests that these conserved regions are necessary to maintain essential structure and function. By raising an immune response against the conserved regions, patients receiving the plasmid will also be protected against future mutant (and therefore novel) strains.

[0296] Thus, in one embodiment of the present invention, the antigenic units are designed to induce a cell-mediated immune response through activation of T cells against T cell epitopes derived from infectious antigens / pathogens contained in such antigenic units, which are recognised by T cells when the epitopes are processed and presented in complex with MHC molecules.

[0297] In one embodiment, the T cell epitopes are known in the art, e.g., have been studied and described in the literature, e.g., are known to be immunogenic, e.g., their immunogenicity has been confirmed by suitable methods and the results have been published, e.g., in a scientific publication. In one embodiment, the antigenic unit comprises multiple T cell epitopes known to be immunogenic.

[0298] For example, useful T cell epitopes known in the art are directed to infection by SARS-CoV2 in humans and can be found in Grifoni et al., Cell Host Microbe. 2021 Jul 14; 29(7): 1076-1092. Thus, such T cell epitopes can be included in the antigenic unit of a vector for use in treating SARS-CoV2 in humans. Other examples of such T cell epitopes are the T cell epitope having the sequence CTELKLSDY (SEQ ID NO: 82) of the nucleoprotein from influenza A virus, the T cell epitope having the sequence NLVPMVATV (SEQ ID NO: 83) of the 65 kDa phosphoprotein from human herpesvirus 5 (human cytomegalovirus), and the T cell epitope having the sequence KLVANNTRL (SEQ ID NO: 84) of the diacylglycerol acyltransferase / mycolyltransferase Ag85B from Mycobacterium tuberculosis.

[0299] As an example, the at least one T cell epitope may be from a region of human papillomavirus (HPV), such as HPV16 or HPV18, such as at least one T cell epitope contained in an HPV antigen from the group consisting of E1, E2, E6, E7, L1 and L2, such as E6 and / or E7 of HPV16 and / or HPV18. By including such a T cell epitope in the vector of the present disclosure, a pharmaceutical composition comprising such a vector can provide protection against HPV. HPV infection is involved in certain cancers, such as squamous cell carcinoma of the head and neck, cervical cancer, and vulvar squamous cell carcinoma. In fact, HPV16 viral antigens are expressed in about 50% of the cancer patients.

[0300] As another example, the at least one T cell epitope may be derived from a region of a human influenza virus, such as human influenza virus A, human influenza virus B, human influenza virus C, and human influenza virus D. As an example, the human influenza virus may be a specific hemagglutinin (HA) subtype, such as H1, H2, H3, and / or a specific neuraminidase (NA) subtype, such as N1 or N5. As an example, the human influenza virus may be an H1N1 subtype. Thus, such T cell epitopes may be included in the antigenic unit of the vector of the present disclosure for use in treating influenza infection.

[0301] In another embodiment, the T cell epitopes are predicted to be immunogenic, e.g., selected based on their predicted ability to bind to HLA class I / II alleles. In one embodiment, the antigen unit comprises multiple T cell epitopes, e.g., multiple T cell epitopes predicted to bind to HLA class I / II alleles, separated from each other by linkers as discussed herein, e.g., linkers as discussed in the "Linkers in Antigen Units" section herein. The T cell epitopes are selected in silico based on HLA binding prediction algorithms. After identifying all relevant epitopes, the epitopes are ranked according to their ability to bind to HLA class I / II alleles, and the epitopes predicted to bind best are selected for inclusion in the antigen unit.

[0302] Suitable HLA binding algorithms are known in the art.

[0303] In yet another embodiment, the antigenic unit comprises multiple T cell epitopes, some of which are known to be immunogenic and others predicted to be immunogenic, in one embodiment, the T cell epitopes are separated from each other by linkers, e.g., linkers as referred to herein, e.g., as discussed in the "Linkers in Antigenic Units" section herein.

[0304] Antigenic units comprising T cell epitopes for use in vectors for the prophylactic and therapeutic treatment of betacoronavirus infections and generally applicable methods for selecting T cell epitopes for vectors of the invention for use in the prophylactic and therapeutic treatment of infectious diseases are disclosed in detail in WO2021 / 219897A1, the disclosure of which is incorporated herein by reference.

[0305] An antigenic unit of a first polypeptide comprising one or more full-length infectious antigens or portions thereof, or one or more B-cell epitopes from one or more pathogens. In another aspect of the invention, the subject is, e.g., a human individual, a healthy individual, and the vector of the invention is used prophylactically, e.g., to prevent disease. Typically, the vector is used to induce immunity in an individual in which it is desired to prophylactically raise neutralizing antibodies against a pathogen, e.g., to prevent infection.

[0306] In one embodiment, the vector of the invention encodes a first polypeptide comprising an antigenic unit comprising at least one infectious antigen that is a full-length protein of a pathogen or a portion of such a protein. Thus, in one embodiment, the at least one infectious antigen is a full-length surface protein or a portion thereof, such as a full-length viral surface protein or bacterial surface protein or a full-length surface protein of another pathogen.

[0307] In other embodiments, the infectious antigen is a full-length bacterial protein that is secreted by the bacteria, for example, secreted into the cytoplasm of the infectious agent.

[0308] In other embodiments, the antigenic unit comprises more than one infectious antigen or parts of more than one infectious antigen, for example multiple full length infectious antigens.

[0309] In yet another embodiment, the antigenic unit comprises one or more antigens or portions of such antigens from multiple pathogens, such as multiple full length infectious antigens from multiple pathogens, in one embodiment the multiple pathogens are multiple different pathogens.

[0310] In one embodiment, such a protein of a pathogen is selected from a betacoronavirus protein, e.g., selected from the group consisting of an envelope protein, a spike protein, a membrane protein, and, if the betacoronavirus is an envelope virus, a spike-like protein hemagglutinin esterase.

[0311] In other embodiments, the antigenic unit comprises a portion of an infectious antigen. The RBD domain of the spike protein of SARS-CoV-2 or the head or stem domain of the hemagglutinin of influenza viruses are examples of portions of infectious antigens.

[0312] In other embodiments, an antigenic unit comprises multiple portions of one infectious antigen. In other embodiments, an antigenic unit comprises multiple portions of infectious antigens, for example, one portion of infectious antigen 1, one portion of infectious antigen 2, and one portion of infectious antigen 3. In other embodiments, an antigenic unit comprises several portions of multiple infectious antigens, for example, two portions of infectious antigen 1 and three portions of infectious antigen 2. Infectious antigens 1, 2, and 3 may be derived from one pathogen or from multiple different pathogens.

[0313] When more than one infectious antigen is comprised in an antigen unit, or more than one portion of one or more infectious antigens, the antigens or portions thereof may be separated by a linker, e.g. by a linker as mentioned herein, e.g. by a linker as mentioned herein under the section "Linkers in antigen units".

[0314] The infectious antigen(s) or parts thereof contain conformational B cell epitopes, but may also contain linear B cell epitopes and / or T cell epitopes. In contrast to the T cell epitopes discussed in the previous section of this specification, these T cell epitopes are not isolated but are presented to the immune system in their natural environment, i.e. flanked by amino acid residues present in the antigen.

[0315] Thus, in one embodiment, the present invention provides a method for producing a composition comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, the antigen unit comprising one or more full-length infectious antigens or a portion thereof; and (b) providing a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0316] In another embodiment, the present invention provides a method for producing a composition comprising the steps of: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit, the antigen unit comprising one or more full-length antigens from one or more pathogens, or a portion of such full-length antigens; and (b) providing a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0317] In one embodiment, the antigenic unit comprises at least a B cell epitope derived from a pathogen, e.g. comprised in a full length protein of the pathogen, e.g. comprised in any of the aforementioned proteins, and preferably comprises a plurality of B cell epitopes derived from a pathogen, e.g. comprised in a full length protein of the pathogen, e.g. comprised in any of the aforementioned proteins, e.g. comprised in a full length surface protein of the pathogen, e.g. comprised in any of the aforementioned proteins. At least one B cell epitope may be a linear or a conformational B cell epitope.

[0318] In yet another embodiment, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit that comprises at least one B-cell epitope from one or more pathogens; and (b) providing a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector enables the first polypeptide and the one or more immunostimulatory compounds to be coexpressed as separate molecules.

[0319] That is, once administered, a first polypeptide encoded by a first nucleic acid constructed in a vector of the present invention as described above, which comprises an antigen unit containing one or more full-length infectious antigens or a portion of such antigens, induces a B cell response and a T cell response and can be used prophylactically or therapeutically.

[0320] Such antigens may be selected for inclusion in the antigen unit according to the predicted therapeutic effect, see WO2021 / 219897A1, the disclosure of which is incorporated herein by reference.

[0321] An antigenic unit of a first polypeptide comprising one or more pathogen-derived B cell epitopes and T cell epitopes. In one embodiment, the first polypeptide encoded by the first nucleic acid contained in the vector of the invention induces a T cell response and a B cell response when administered to a subject. In a pandemic or epidemic situation, it is time inefficient to first diagnose an individual to determine whether he or she is primarily in need of a B cell response or a T cell response and whether preventive or therapeutic treatment is most medically necessary. It is difficult to determine whether or not someone is infected due to the lack of (sufficient) relevant tests. It is therefore important to be able to prevent and treat at the same time. By combining both full length infectious antigens or parts of infectious antigens, or several B cell epitopes present in the infectious antigen, and T cell epitopes, such as conserved T cell epitopes, both strong humoral and cellular responses are elicited when the vector is administered. This response can be more humoral or more cellular depending on the targeting unit selected.

[0322] Thus, one aspect of the present invention is a method for producing a medicament comprising the steps of: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit, a multimerization unit, such as a dimerization unit, and an antigenic unit, the antigenic unit comprising (i) one or more full-length infectious antigens or a portion of such antigens, and (ii) at least one T cell epitope from the one or more infectious antigens; and (b) A vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of coexpressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0323] In one embodiment, the present invention provides a method for producing a composition comprising the steps of: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit, a multimerization unit, such as a dimerization unit, and an antigenic unit, the antigenic unit comprising (i) one or more full-length antigens or portions of such antigens, and (ii) at least one T cell epitope, wherein the one or more antigens and the at least one T cell epitope are derived from one or more pathogens; and (b) A vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0324] Such combinations of T cell epitopes and infectious antigens or portions thereof may be selected for inclusion in the antigenic unit according to the predicted immunogenicity of the T cell epitope or by selecting T cell epitopes known in the art, as described in WO2021 / 219897A1, the contents of which are incorporated herein by reference.

[0325] In one embodiment, the present invention provides a method for producing a composition comprising the steps of: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit, a multimerization unit, such as a dimerization unit, and an antigenic unit, the antigenic unit comprising (i) one or more B cell epitopes from one or more infectious antigens, and (ii) at least one T cell epitope from one or more infectious antigens; and (b) A vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of coexpressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0326] In yet another embodiment, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit, a multimerization unit, such as a dimerization unit, and an antigenic unit, the antigenic unit comprising (i) one or more B cell epitopes and (ii) at least one T cell epitope, the one or more B cell epitopes and the at least one T cell epitope being derived from one or more pathogens; and (b) A vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0327] In one embodiment, the full-length infectious antigen / part thereof and at least one T cell epitope are arranged in the antigen unit as follows: at least one T cell epitope is arranged in a subunit that is linked to the multimerization unit by a first linker, such as a unit linker. If multiple T cell epitopes are present in the subunit, the T cell epitopes are preferably separated by a subunit linker. Furthermore, the subunit is separated from one or more full-length infectious antigens or parts thereof by a second linker. Thus, the subunit with the T cell epitope is closest to the multimerization unit, and the infectious antigen or parts thereof constitute the termini of the polypeptide.

[0328] Thus, in one embodiment, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit, a multimerization unit, such as a dimerization unit, and an antigenic unit, the antigenic unit comprising (i) one or more full-length infectious antigens or portions of such antigens, and (ii) one or more T cell epitopes, wherein the one or more antigens and the one or more T cell epitopes are derived from a pathogen; and (b) a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules; and If more than one T cell epitope is contained in a subunit, the antigenic unit comprises subunits that contain T cell epitopes separated from each other by a subunit linker; and The subunits are linked into a multimerization unit by a first linker, such as a unit linker, and are separated from one or more full-length infectious antigens or portions of such antigens by a second linker.

[0329] The subunit linker, first linker / unit linker and second linker may be linkers as referred to herein, for example, linkers as referred to herein in the sections "Linkers of antigen units" and "Unit linkers".

[0330] Further embodiments of the antigen unit The following generally applies to the antigen unit in the first polypeptide encoded by the first nucleic acid contained in the vector of the present invention.

[0331] As used herein, the term antigen is used for neoantigen, neoepitope, patient-presented shared cancer antigen, part of a patient-presented shared cancer antigen, e.g., patient-presented shared cancer epitope, shared cancer antigen, part of a shared cancer antigen, e.g., a shared cancer epitope, an infectious antigen or part thereof, or a T cell epitope of an infectious antigen.

[0332] In one embodiment, the antigen unit contains only one copy of each antigen, hi another embodiment, the antigen unit contains multiple copies of one or more antigens.

[0333] In one embodiment, the antigen unit contains only one copy of each antigen, so that, for example, if 10 types of antigens are contained in the antigen unit, a vector containing the antigen unit can induce an immune response against all 10 types of antigens, thereby efficiently attacking cancer.

[0334] In another embodiment, the antigen unit comprises at least two copies of a particular antigen, e.g., a particular neoepitope, in order to enhance the immune response to the antigen, e.g., when only a few neoepitopes have been identified in a particular patient that are predicted to be sufficiently immunogenic / binding to the patient's HLA alleles. In such a patient, when one or more patient-presented shared cancer antigens have been identified in addition to a few neoepitopes, it is preferable to include such one or more patient-presented shared cancer antigens or parts thereof in the antigen unit, rather than including multiple copies of the same neoepitope in the antigen unit.

[0335] The length of an antigen unit is determined by the length and number of antigens contained therein.

[0336] In one embodiment, the antigen unit contains up to 3500 amino acids, for example, 60 to 3500 amino acids, for example, about 80 or about 100 or about 150 amino acids to about 3000 amino acids, for example, about 200 to about 2500 amino acids, for example, about 300 to about 2000 amino acids, or about 400 to about 1500 amino acids, or about 500 to about 1000 amino acids.

[0337] To enhance the immune response, particularly in the case of a first polypeptide comprising a neo-antigen, the antigen may be arranged into antigen subunits as described in the following paragraphs.

[0338] An antigen unit can be described as a polypeptide that starts at the N-terminus and ends at the C-terminus. The antigen unit is connected to a multimerization unit, such as a dimerization unit, for example, via a linker, preferably via a unit linker. The antigen unit is connected to the COOH-terminus or NH2 It is preferred that the antigenic unit is at the COOH-terminus of the first polypeptide.

[0339] In one embodiment, the antigens, preferably epitopes, are arranged in order from most to least antigenic in the direction from the N-terminal start to the C-terminal end of the antigen unit, or, preferably, in cases where there is a large difference in hydrophilicity / hydrophobicity between the antigens, the most hydrophobic antigen is arranged substantially in the center of the antigen unit and the most hydrophilic antigen is arranged at the N-terminal start and / or C-terminal end of the antigen unit.

[0340] Since exact central location of the antigen unit is only possible if the antigen unit contains an odd number of antigens, the term "substantially" in this context refers to an antigen unit containing an even number of antigens, with the most hydrophobic antigen being positioned as close to the middle as possible.

[0341] In one example, an antigenic unit may contain five antigenic subunits, each of which contains a different epitope, e.g., a different neoepitope, arranged as follows: 1, 2, 3*, 4, 5: 1, 2, 3*, 4, and 5 are different neoepitopes, - is the subunit linker, and * indicates the most hydrophobic neoepitope located in the center of the antigen unit.

[0342] In another example, the antigenic unit comprises six antigenic subunits, each subunit comprising a different epitope, e.g., a different neoepitope, arranged as follows: 1-2-3*-4-5-6, or arranged as follows: 1-2-4-3*-5-6; where 1, 2, 3*, 4, 5 and 6 are each a different neoepitope, - is a subunit linker, and * indicates the most hydrophobic neoepitope located substantially in the center of the antigenic unit.

[0343] Alternatively, the antigen subunits may be arranged in alternating hydrophilic and hydrophobic antigens.

[0344] Optionally, GC-rich sequences encoding antigens (e.g., GC-rich sequences encoding neoepitopes or epitopes) are positioned to avoid GC clusters. In one embodiment, GC-rich sequences encoding antigens are positioned such that there is at least one non-GC-rich sequence between them.

[0345] In one embodiment, the antigen unit comprises one or more linkers. In another embodiment, the antigen unit comprises multiple antigens, e.g. multiple epitopes, e.g. neoepitopes, where the antigens are separated by linkers. In yet another embodiment, the antigen unit comprises multiple antigens, where each antigen is separated from the other antigens by a linker. An alternative way of describing each antigen being separated from the other antigens by a linker is that all but the terminal antigens, i.e. the antigens at the N-terminal beginning or C-terminal end of the polypeptide (i.e. the antigens located at the end of the antigen unit that are not linked to a multimerization unit), are arranged in an antigenic subunit, where each subunit consists of or comprises an antigen, e.g. neoepitopes, and a subunit linker.

[0346] Thus, an antigen unit containing n antigens contains n-1 antigen subunits, each of which contains an antigen and a subunit linker, and further contains a terminal antigen. In one embodiment, n is an integer of 1 to 50, for example, 3 to 50, or 15 to 40, or 10 to 30, or 10 to 25, or 10 to 20, or 15 to 30, or 15 to 25, or 15 to 20.

[0347] The antigens are separated by a linker so that each antigen is optimally presented to the immune system.

[0348] In one embodiment, the antigen unit comprises a B cell epitope and a T cell epitope, e.g., a full-length infectious antigen or a portion thereof and one or more T cell epitopes contained in a protein of a pathogen, and the antigen unit is designed such that the T cell epitope is located closest to the multimerization unit and the infectious antigen is located at the end of the antigen unit. The T cell epitope is preferably separated by a linker, and the infectious antigen is preferably separated by a linker from the "subunit" containing the T cell epitope. Such antigen unit designs are disclosed in PCT / EP2022 / 061819, the disclosure of which is incorporated herein by reference.

[0349] Linker contained in antigen unit The antigen unit may include a linker, for example, a linker that separates the antigens contained therein, such as neoantigens, neoepitopes, patient-presented shared cancer antigens or parts thereof, such as patient-presented shared cancer epitopes, shared cancer antigens or parts thereof, such as shared cancer epitopes, infectious antigens or parts thereof, or T cell epitopes of infectious antigens.As mentioned above, all antigens, such as neoepitopes, may be arranged in subunits, separated from each other by linkers.In the following, the terms subunit linker and linker are used interchangeably, and both refer to the linker in the antigen unit.

[0350] In one embodiment, the linker is designed to be non-immunogenic. The linker may be a rigid linker, meaning that it does not allow the two amino acid sequences it links to move substantially freely relative to each other. Alternatively, it may be a flexible linker, i.e., a linker that allows the two amino acid sequences it links to move substantially freely relative to each other. Both types of linkers are useful. In one embodiment, the linker is a flexible linker, which allows the antigen to be presented to T cells in an optimal manner, even if the antigen unit is composed of multiple antigens.

[0351] In one embodiment, the subunit linker is a peptide of 4 to 40 amino acids, such as 35, 30, 25 or 20 amino acids, such as 5 to 20 amino acids or 5 to 15 amino acids or 8 to 20 amino acids or 8 to 15 amino acids, 10 to 15 amino acids or 8 to 12 amino acids. In another embodiment, the subunit linker consists of 10 amino acids.

[0352] In one embodiment, for example in antigen units containing neoepitopes, the subunit linkers are identical for all antigen subunits. However, if one or more of the antigens contain a sequence similar to that of the linker, it may be advantageous to replace adjacent subunit linkers with linkers of different sequences. Also, linkers of different sequences can be used if the antigen-subunit linker junction is predicted to constitute an immunogenic epitope itself.

[0353] In one embodiment, the subunit linker is a flexible linker, preferably one that comprises small, non-polar (e.g., glycine, alanine or leucine) or polar (e.g., serine or threonine) amino acids. The small size of these amino acids allows flexibility and allows for mobility of the linked amino acid sequence. The incorporation of serine or threonine can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing unfavorable interactions between the linker and the antigen. In one embodiment, the flexible linker is a serine (S) and / or glycine (G) rich linker, i.e., a linker that comprises several serine residues and / or several glycine residues. Preferred examples are GGGGS (SEQ ID NO: 58), GGGSS (SEQ ID NO: 59), GGGSG (SEQ ID NO: 60), GGSGG (SEQ ID NO: 61), SGSSGS (SEQ ID NO: 62), or several variants thereof, such as GGGGSGGGS (SEQ ID NO: 17), (GGGGS)m (SEQ ID NO: 64), (GGGS)m (SEQ ID NO: 65), (GGSGG)m (SEQ ID NO: 66), (GGGSG)m (SEQ ID NO: 67) or (SGSSGS)m (SEQ ID NO: 68), where m is an integer from 1 to 5, such as 1, 2, 3, 4, or 5. In a preferred embodiment, m is 2. In another preferred embodiment, the serine and / or glycine rich linker further comprises at least one leucine (L) residue, such as at least one or at least two or at least three leucine residues, such as 1, 2, 3 or 4 leucine residues.

[0354] In one embodiment, the subunit linker comprises, or consists of, LGGGS (SEQ ID NO:69), GLGGS (SEQ ID NO:70), GGLGS (SEQ ID NO:71), GGGLS (SEQ ID NO:72) or GGGGL (SEQ ID NO:73). In another embodiment, the subunit linker comprises, or consists of, LGGSG (SEQ ID NO:74), GLGSG (SEQ ID NO:75), GGLSG (SEQ ID NO:76), GGGLG (SEQ ID NO:77) or GGGSL (SEQ ID NO:78). In yet another embodiment, the subunit linker comprises, or consists of, LGGSS (SEQ ID NO:79), GLGSS (SEQ ID NO:80) or GGLSS (SEQ ID NO:81).

[0355] In yet another embodiment, the subunit linker comprises, or consists of, LGLGS (SEQ ID NO:85), GLGLS (SEQ ID NO:86), GLLGS (SEQ ID NO:87), LGGLS (SEQ ID NO:88) or GLGGL (SEQ ID NO:89). In yet another embodiment, the subunit linker comprises, or consists of, LGLSG (SEQ ID NO:90), GLLSG (SEQ ID NO:91), GGLSL (SEQ ID NO:92), GGLLG (SEQ ID NO:93) or GLGSL (SEQ ID NO:94). In yet another embodiment, the subunit linker comprises, or consists of, LGLSS (SEQ ID NO:95) or GGLLS (SEQ ID NO:96).

[0356] In another embodiment, the subunit linker is a serine-glycine linker having a length of 10 amino acids and containing one or two leucine residues.

[0357] In one embodiment, the subunit linker comprises, or consists of, LGGGSGGGS (SEQ ID NO:97), GLGGSGGGS (SEQ ID NO:98), GGLGSGGGS (SEQ ID NO:99), GGGLSGGGGS (SEQ ID NO:100), or GGGGLGGGS (SEQ ID NO:101). In another embodiment, the subunit linker comprises, or consists of, LGGSGGGGSG (SEQ ID NO:102), GLGSGGGGSG (SEQ ID NO:103), GGGLSGGGGSG (SEQ ID NO:104), GGGGLGGGSG (SEQ ID NO:105), or GGGSLGGGSG (SEQ ID NO:106). In yet another embodiment, the subunit linker comprises, or consists of, LGGSSGGGSS (SEQ ID NO:107), GLGSSGGGSS (SEQ ID NO:108), GGLSGGGSS (SEQ ID NO:109), GGGLSGGGSS (SEQ ID NO:110), or GGGSLGGGSS (SEQ ID NO:111).

[0358] In further embodiments, the subunit linker comprises, or consists of, LGGGSLGGGS (SEQ ID NO:112), GLGGSGLGGS (SEQ ID NO:113), GGLGSGGLGS (SEQ ID NO:114), GGGLSGGGLS (SEQ ID NO:115), or GGGGLGGGL (SEQ ID NO:116). In another embodiment, the subunit linker comprises, or consists of, LGGSGLGGSG (SEQ ID NO:117), GLGSGGLGSG (SEQ ID NO:118), GGLSGGGLSG (SEQ ID NO:119), GGGLGGLG (SEQ ID NO:120), or GGGSLGGGSL (SEQ ID NO:121). In yet another embodiment, the subunit linker comprises, or consists of, LGGSSLGGSS (SEQ ID NO:122), GLGSSGLGSS (SEQ ID NO:123), or GGLSSGGLSS (SEQ ID NO:124).

[0359] In yet another embodiment, the subunit linker comprises, or consists of, GSGGGA (SEQ ID NO: 125), GSGGAGSGGGA (SEQ ID NO: 126), GSGGAGSGGAGSGGGA (SEQ ID NO: 127), GSGGAGSGGAGSGGAGSGGGA (SEQ ID NO: 128), or GENLYFQSGG (SEQ ID NO: 129). In yet another embodiment, the subunit linker comprises, or consists of, SGGGSSGGGS (SEQ ID NO: 130), SSGGGSSGGGG (SEQ ID NO: 131), GGSGGGGSGG (SEQ ID NO: 132), GSGSGSGSGS (SEQ ID NO: 133), GGGSSGGGSG (SEQ ID NO: 134) (amino acids 121-130 of SEQ ID NO: 1), GGGSSS (SEQ ID NO: 135), GGGSSGGSSGGSS (SEQ ID NO: 136), or GLGGLAAA (SEQ ID NO: 137).

[0360] In another embodiment, the subunit linker is a rigid linker. Such rigid linkers can be useful to efficiently separate (larger) antigens and prevent them from interfering with each other. In one embodiment, the subunit linker comprises or consists of KPEPKPAPAPKP (SEQ ID NO: 138), AEAAAKEAAAKA (SEQ ID NO: 139), (EAAAK)m (SEQ ID NO: 140), PSRLEEELRRRLTEP (SEQ ID NO: 141) or SACYCELS (SEQ ID NO: 142).

[0361] In yet another embodiment, the subunit linker comprises, or consists of, TQKSLSLSPGKGLGGL (SEQ ID NO: 143). In yet another embodiment, the subunit linker comprises, or consists of, SLSLSPGKGLGGL (SEQ ID NO: 144).

[0362] In yet another embodiment, the subunit linker comprises, or consists of, GGSAGGSGSGGSSGASGTGTAGTGSGSGTGSG (SEQ ID NO: 145); or GGSGGGSEGGGSEGGGGGSGGS (SEQ ID NO: 146), or ELKTPLGDTTHT (SEQ ID NO: 147) (amino acids 94 to 105 of SEQ ID NO: 1), or EPKSCDTPPPCPRCP (SEQ ID NO: 148) (amino acids 106 to 120 of SEQ ID NO: 1).

[0363] In yet another embodiment, the subunit linker is a cleavable linker, e.g., a linker that contains one or more recognition sites for endopeptidases, e.g., furin, caspases, cathepsins, etc. Cleavable linkers may be introduced to release free functional protein domains (e.g., encoded by larger antigens), which may overcome steric hindrance between such domains or other drawbacks due to interference of such domains, such as reduced biological activity, altered biological distribution, etc.

[0364] Examples of suitable linkers are disclosed in paragraphs

[0098] to

[0099] and the sequences described in WO 2020 / 176797A1, paragraphs

[0135] to

[0139] of US 2019 / 0022202A1, WO 2017 / 118695 A1 and WO 2021 / 219897A1, all of which are incorporated herein by reference.

[0365] Unit Linker The antigen unit is preferably linked to the multimerization unit by a unit linker. Thus, in one embodiment, the first nucleic acid sequence contained in the vector of the present invention encodes a first polypeptide comprising a unit linker that links the antigen unit to the multimerization unit.

[0366] The unit linker may include a restriction site to facilitate construction of the first nucleic acid sequence. In one embodiment, the unit linker is GLGGL (SEQ ID NO: 89) or GLSGL (SEQ ID NO: 149). In another embodiment, the unit linker comprises or consists of GGGGS (SEQ ID NO: 58), GGGGSGGGS (SEQ ID NO: 17), (GGGGS)m (SEQ ID NO: 64), EAAAK (SEQ ID NO: 150), (EAAAK)m (SEQ ID NO: 140), (EAAAK)mGS (SEQ ID NO: 151), (EAAAK)mGS (SEQ ID NO: 63), GPSRLEEELRRRLTEPG (SEQ ID NO: 152), AAY, or HEYGAEALERAG (SEQ ID NO: 153).

[0367] Signal peptide In one embodiment of the present disclosure, at least one of the first nucleic acid sequences or one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds also encodes a signal peptide. The signal peptide is located at either the N-terminus or C-terminus of the targeting unit, depending on the orientation of the targeting unit in the first polypeptide. Furthermore, the signal peptide is located at the N-terminus of the immunostimulatory compound. The signal peptide is designed to allow secretion of the first polypeptide / immunostimulatory compound from a cell containing the vector of the present invention. Preferably, each of the first nucleic acid sequence and the additional nucleic acid sequences encoding one or more immunostimulatory compounds also encodes a signal peptide. Preferably, the signal peptide is naturally present at the N-terminus of any of the targeting units or immunostimulatory compounds described herein.

[0368] Any suitable signal peptide can be used. An example of a suitable peptide is an Ig VH signal peptide, preferably a human Ig VH signal peptide such as SEQ ID NO: 2, preferably when the targeting unit is an antibody or a part thereof, e.g. an scFv. In one embodiment, the signal peptide is the native leader sequence of the protein that is the targeting unit, i.e. a signal peptide that is naturally present at the N-terminus of any of the proteins encoded by the vector of the invention as a targeting unit. In another embodiment, the signal peptide is the native leader sequence of the immunostimulatory compound, i.e. a signal peptide that is naturally present at the N-terminus of the protein that is the immunostimulatory compound.

[0369] Examples of signal peptides are human TPA signal peptide, e.g., SEQ ID NO: 3, human MIP1-α signal peptide, e.g., amino acid sequence 1-23 of SEQ ID NO: 1, human GM-CSF signal peptide, e.g., amino acid sequence of SEQ ID NO: 40, human CCL5 signal peptide, e.g., amino acid sequence of SEQ ID NO: 42, human IL-12A signal peptide, e.g., amino acid sequence of SEQ ID NO: 44, human IL-12B signal peptide, e.g., amino acid sequence of SEQ ID NO: 46, or human IL-21 signal peptide, e.g., amino acid sequence of SEQ ID NO: 48.

[0370] In a preferred embodiment, the vector of the present invention comprises a first nucleotide sequence encoding a first polypeptide and further encoding a signal peptide having an amino acid sequence having at least 85%, for example at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence 1 to 23 of SEQ ID NO:1.

[0371] In another preferred embodiment, the vector of the invention comprises a first nucleotide sequence further encoding a first polypeptide and a signal peptide comprising amino acid sequence 1-23 of SEQ ID NO: 1, with the proviso that up to three amino acids, such as up to two amino acids, or for example up to one amino acid, have been substituted, deleted or inserted.

[0372] In another preferred embodiment, the vector of the present invention comprises a first nucleotide sequence encoding a first polypeptide and further encoding a signal peptide having amino acid sequence 1-23 of SEQ ID NO:1.

[0373] In a more preferred embodiment, the vector of the present invention comprises a first nucleotide sequence encoding a first polypeptide and further encoding a signal peptide having an amino acid sequence having at least 85%, for example at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence 1 to 23 of SEQ ID NO:1.

[0374] In another preferred embodiment, the vector of the present invention comprises a first nucleotide sequence encoding a first polypeptide and encoding a signal peptide consisting of amino acid sequence 1 to 23 of SEQ ID NO:1, except that up to three amino acids have been substituted, deleted or inserted, such as up to two amino acids, or up to one amino acid.

[0375] In another preferred embodiment, a vector of the present invention comprises a first nucleotide sequence encoding a first polypeptide and further encoding a signal peptide having amino acid sequence 1-23 of SEQ ID NO:1.

[0376] In one preferred embodiment, the vector of the present invention comprises a first nucleotide sequence encoding a first polypeptide and further encoding a signal peptide, wherein said nucleotide sequence of said signal peptide has at least 80% sequence identity to a nucleic acid sequence having SEQ ID NO:29.

[0377] In a further preferred embodiment, the vector of the invention comprises a first nucleotide sequence encoding a first polypeptide and further encoding a signal peptide, wherein said nucleotide sequence of said signal peptide has at least 85% sequence identity, such as at least 86%, or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to a nucleic acid sequence having SEQ ID NO:29.

[0378] In a further preferred embodiment, the vector of the present invention comprises a first nucleotide sequence encoding a first polypeptide and further encoding a signal peptide, wherein said nucleotide sequence of said signal peptide is SEQ ID NO:29.

[0379] Sequence identity Sequence identity may be determined as follows: a high sequence identity indicates a high probability that the second sequence is derived from the first sequence. Amino acid sequence identity requires identical amino acid sequences between the two aligned sequences. Thus, a candidate sequence that shares 70% amino acid identity with a reference sequence requires that after alignment, 70% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence. Identity can be determined by computer analysis, such as, but not limited to, the ClustalW computer alignment program (Higgins D., Thompson J., Gibson T., Thompson JD, Higgins DG, Gibson TJ,1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment by sequence weighting, position-specific gap penalties, and weight matrix selection. Nucleic Acids Res. 22:4673-4680) and the default parameters proposed therein. The program is used with default settings to align the mature (bioactive) portion of the query and the reference polypeptide. The number of perfectly conserved residues is counted and divided by the length of the reference polypeptide, while ignoring any tags or fusion protein sequences that form part of the query sequence in the alignment and subsequent determination of sequence identity.

[0380] The ClustalW algorithm can also be used to align nucleotide sequences. Sequence identity can be calculated in a similar manner as shown for amino acid sequences.

[0381] Another preferred mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated in the ALIGN program (version 2.0), which is part of the FASTA sequence alignment software package (Pearson WR, Methods Mol Biol, 2000, 132:185-219). Align calculates sequence identity based on a global alignment. Align0 does not impose a penalty for gaps at the end of the sequence. When using the ALIGN and Align0 programs for comparing amino acid sequences, it is advisable to use the BLOSUM50 substitution matrix and a gap opening / extension penalty of -12 / -2.

[0382] Variants of amino acid sequences can be prepared by introducing appropriate changes into the nucleotide sequence encoding the first polypeptide and / or one or more immunostimulatory compounds or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues in the amino acid sequence. The terms substitution / substitution, deletion / deletion and insertion / insertion used herein with respect to amino acid sequences and sequence identity are well known and clear to those skilled in the art. Any combination of deletion, insertion and substitution can be made to arrive at the final first polypeptide and / or one or more immunostimulatory compounds, provided that the final protein has the desired properties. For example, deletion, insertion or substitution of amino acid residues may result in a silent change, resulting in a functionally equivalent polypeptide / immunostimulatory compound.

[0383] Deliberate amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues, so long as the desired properties of the protein in question are retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0384] Included herein are conservative substitutions, i.e., like substitutions such as basic to basic, acidic to acidic, polar to polar, etc., and non-conservative substitutions, i.e., substitutions from one class of residue to another, or involving the inclusion of unnatural amino acids such as ornithine, diaminobutyric acid ornithine, norleucine, ornithine, pyrylalanine, thienylalanine, naphthylalanine, and phenylglycine. Conservative substitutions that may be made are, for example, made within the groups of basic amino acids (arginine, lysine, histidine), acidic amino acids (glutamic acid, aspartic acid), aliphatic amino acids (alanine, valine, leucine, isoleucine), polar amino acids (glutamine, asparagine, serine, threonine), aromatic amino acids (phenylalanine, tryptophan, tyrosine), hydroxyl amino acids (serine, threonine), large amino acids (phenylalanine, tryptophan), and small amino acids (glycine, alanine).

[0385] Substitution with unnatural amino acids is also possible, and examples of the substitution residues include α* and α-disubstituted* amino acids, N-alkylamino acids*, lactic acid*, halide derivatives of natural amino acids, e.g., trifluorotyrosine*, p-CI-phenylalanine*, p-Br-phenylalanine*, pI-phenylalanine*, L-allyl-glycine*, β-alanine*, La-aminobutyric acid*, Ly-aminobutyric acid*, La-aminoisobutyric acid*, Includes Le-aminocaproic acid*, 7-aminoheptanoic acid*, L-methionine sulfone*, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline*, L-thioproline*, methyl derivatives of phenylalanine (Phe), such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe(4-amino)#, L-Tyr(methyl)*, L-Phe(4-isopropyl)*, L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid)*, L-diaminopropionic acid*, and L-Phe(4-benzyl)*.

[0386] In the above paragraphs, * indicates the hydrophobic nature of the substituted residue, # indicates the hydrophilic nature of the substituted residue and #* indicates the amphipathic nature of the substituted residue. Variant amino acid sequences may contain suitable spacer groups that may be inserted between any two amino acid residues of the sequence, including alkyl groups such as methyl, ethyl or propyl groups, in addition to amino acid spacers such as glycine or β-alanine residues. In a further variation, one or more amino acid residues are present in the form of a peptoid.

[0387] Polypeptides and multimeric / dimeric proteins The vectors of the invention encode a first polypeptide as described above, which polypeptide (and one or more immunostimulatory compounds) are expressed in vivo as a result of administration of the vector to a subject.

[0388] The presence of a multimerization unit, such as a dimerization unit, results in the formation of a multimeric protein when the polypeptide is expressed.

[0389] The multimeric protein may be a homomultimer or a heteromultimer, for example, when the protein is a dimeric protein, the dimeric protein may be a homodimer, i.e., a dimeric protein in which two polypeptide chains are identical and therefore contain the same unit and therefore antigen sequence, or the dimeric protein may be a heterodimer containing two polypeptide chains, where polypeptide chain 1 has a different antigen sequence in its antigen unit than polypeptide chain 2. The latter may be relevant when the number of antigens contained in the antigen unit exceeds the upper size limit of the antigen unit. It is preferred that the multimeric protein is a homomultimeric protein.

[0390] Vector and host cell production Vectors of the present invention are generally vectors suitable for transfecting a host cell to a) express a first polypeptide encoded by a first nucleic acid sequence and form a multimeric protein composed of a plurality of such first polypeptides, and b) express one or more immunostimulatory compounds encoded by further nucleic acid sequences.

[0391] In one embodiment, the host cell comprising a vector of the invention is a cell in cell culture, e.g., a bacterial cell, and the protein encoded by the vector is expressed in vitro, hi another embodiment, the host cell comprising a vector of the invention is a cell of a subject, and the protein encoded by the vector is expressed in said subject, i.e., in vivo, as a result of administration of the vector to the subject.

[0392] Suitable host cells for in vitro transfection include prokaryotic cells, yeast cells, insect cells, or higher eukaryotic cells. Suitable host cells for in vivo transfection are, for example, muscle cells.

[0393] In one embodiment, the vector allows easy exchange of the various units described above, particularly antigen units in the case of individualized antigen units.

[0394] In one embodiment, the vector is a pUMVC4a vector or a vector comprising the NTC9385R vector backbone. The antigen unit may be exchanged with an antigen unit cassette restricted by an SfiI restriction enzyme cassette, the 5' site of which incorporates a nucleotide sequence encoding the GLGGL (SEQ ID NO: 89) / GLSGL (SEQ ID NO: 149) unit linker, and the 3' site of which is included after the stop codon in the vector.

[0395] Methods for engineering and producing vectors of the invention, e.g., expression vectors such as DNA and RNA plasmids or viral vectors, are well known and one of skill in the art would be able to engineer / produce the vectors of the invention using such known methods. In addition, various commercial manufacturers offer services for the design and production of vectors.

[0396] In one aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit that comprises one or more antigens or portions thereof; and (b) a method for producing a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules, the method comprising: a) transfecting cells in vitro with the vector; b) culturing the cells; c) optionally, lysing the cells to liberate the vector from the cells; and d) recovering and optionally purifying the vector.

[0397] In one embodiment, the one or more antigens or portions thereof are disease associated antigens or portions thereof.

[0398] Pharmaceutical Compositions In one embodiment of the disclosure, the vector, such as a DNA plasmid, is used as a pharmaceutical agent.

[0399] Thus, in one embodiment of the present disclosure, the vector is provided in a pharmaceutical composition comprising the vector and a pharma- ceutically acceptable carrier or diluent.

[0400] Thus, in one aspect, the disclosure provides a pharmaceutical composition comprising: (i) a pharma- ceutically acceptable carrier or diluent; and (ii) one of the following: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit that comprises one or more antigens or portions thereof; and (b) A pharmaceutical composition comprising a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0401] In one embodiment, the one or more antigens or portions thereof are disease associated antigens or portions thereof.

[0402] Suitable pharma- ceutically acceptable carriers or diluents include, but are not limited to, saline, buffered saline such as PBS, dextrose, water, glycerol, ethanol, isotonic aqueous buffer, and combinations thereof.

[0403] In one embodiment, the pharma- ceutically acceptable carrier or diluent is an aqueous buffer. In another embodiment, the aqueous buffer is Tyrode's buffer, e.g., 140 mM NaCl, 6 mM KCl, 3 mM CaCl 2 , 2 mM MgCl 2 , Tyrode's buffer containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) pH 7.4, and 10 mM glucose.

[0404] The pharmaceutical composition may also include a molecule that facilitates transfection of a host cell, ie, a transfection agent.

[0405] In some specific embodiments, the pharmaceutical compositions comprise a pharma- ceutically acceptable amphiphilic block copolymer comprising blocks of poly(ethylene oxide) and polypropylene oxide).

[0406] As used herein, an "amphiphilic block copolymer" is a linear or branched copolymer comprising or consisting of blocks of poly(ethylene oxide) ("PEO") and blocks of poly(propylene oxide) ("PPO"). Typical examples of useful PEO-PPO amphiphilic block copolymers have the general structures PEO-PPO-PEO (poloxamer), PPO PEO PPO, (PEO PPO-)4ED (poloxamine), and (PPO PEO-)4ED (reverse poloxamine), where "ED" is an ethylenediaminyl group.

[0407] A "poloxamer" is a linear amphiphilic block copolymer of one block of poly(ethylene oxide), one block of poly(propylene oxide), and one block of PEO, i.e., a structure of the formula EOa-POb-EOa, where EO is ethylene oxide, PO is propylene oxide, a is an integer between 2 and 130, and b is an integer between 15 and 67. Poloxamers are conventionally named with a three-digit identifier, where the first two digits multiplied by 100 indicate the approximate molecular weight of the PPO content, and the last digit multiplied by 10 indicates the approximate percentage of PEO content. For example, "poloxamer 188" refers to a polymer containing a PPO block of molecular weight about 1800 (corresponding to a PPO with b of about 31) and about 80% (w / w) PEO (corresponding to a of about 82). However, this value is known to vary to some extent, and commercial products such as research grade Lutrol® F68 and clinical grade Kolliphor® P188, both of which are poloxamer 188, vary widely in molecular weight (7,680-9,510) according to the manufacturer's data sheets, with the values ​​of a and b given for these particular products being approximately 79 and 28, respectively. This reflects the heterogeneous nature of the block copolymers, and means that the values ​​of a and b are averages found in the final formulation.

[0408] "Poloxamines" or "sequential poloxamines" (sold under the name Tetronic®) are X-shaped block copolymers having four PEO-PPO arms linked to a central ethylenediamine moiety via bonds between the free OH groups of the PEO-PPO arms and the primary amine groups of the ethylenediamine moiety. Reverse poloxamines are similarly X-shaped block copolymers having four PPO-PEO arms linked to a central ethylenediamine moiety via bonds between the free OH groups of the PPO-PEO arms and the primary amine groups of the ethylenediamine moiety.

[0409] Preferred amphiphilic block copolymers are poloxamers or poloxamines. Poloxamers 407 and 188 are preferred, especially poloxamer 188. Preferred poloxamines are sequential poloxamines of formula (PEO-PPO)4-ED. Particularly preferred poloxamines are those sold under Tetronic® 904, 704 and 304, respectively. The characteristics of these poloxamines are as follows: Tetronic® 904 has a total average molecular weight of 6700, a total average weight of PPO units of 4020 and a PEO proportion of about 40%. Tetronic® 704 has a total average molecular weight of 5500, a total average weight of PPO units of 3300 and a PEO proportion of about 40%; Tetronic® 304 has a total average molecular weight of 1650, a total average weight of PPO units of 990 and a PEO proportion of about 40%.

[0410] In one embodiment, the pharmaceutical composition comprises the amphiphilic block copolymer in an amount of from 0.2% w / v to 20% w / v, e.g., from 0.2% w / v to 18% w / v, from 0.2% w / v to 16% w / v, from 0.2% w / v to 14% w / v, from 0.2% w / v to 12% w / v, from 0.2% w / v to 10% w / v, from 0.2% w / v to 8% w / v, from 0.2% w / v to 6% w / v, from 0.2% w / v to 4% w / v, from 0.4% w / v to 18% w / v, from 0.6% w / v to 18% w / v, from 0.8% w / v to 18% w / v, from 1% w / v to 18% w / v, from 2% w / v to 18% w / v, from 1% w / v to 5% w / v, or from 2% w / v to 4% w / v. Amounts in the range of 0.5% w / v to 5% w / v are particularly preferred. In another embodiment, the pharmaceutical composition comprises the amphiphilic block copolymer in an amount of 2% w / v to 5% w / v, for example about 3% w / v.

[0411] The pharmaceutical compositions can be formulated in any manner suitable for administration to a subject, such as a liquid formulation for injection, such as for intradermal or intramuscular injection.

[0412] The pharmaceutical compositions may be administered in any manner suitable for administration to a subject, for example by intradermal, intramuscular, or subcutaneous injection, or by mucosal or epithelial application, such as intranasal or oral.

[0413] In a preferred embodiment, the pharmaceutical composition is administered by intramuscular or intradermal injection.

[0414] The amount of vector, eg, a DNA plasmid, in a pharmaceutical composition may vary depending on whether the pharmaceutical composition is being administered for prophylactic or therapeutic treatment.

[0415] A pharmaceutical composition of the invention typically comprises in the range 0.1 to 10 mg, for example about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mg, or for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg of a vector, for example a DNA plasmid.

[0416] In a preferred embodiment, the pharmaceutical composition is a sterile pharmaceutical composition.

[0417] treatment In some aspects of the disclosure, the vectors, e.g., DNA plasmids, are for use in the therapeutic or prophylactic treatment of a disorder, such as a disorder in a human.

[0418] Thus, in one aspect, the present disclosure relates to a method of treating a subject having a disease or in need of prevention of said disease, said method comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more antigens or portions thereof associated with the disease; and (b) administering to the subject a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0419] In the methods of treatment, the vector is preferably administered in a therapeutically or prophylactically effective amount, which may be administered in a single dose, i.e., in a single administration, or in several doses, i.e., in a series of doses over a period of days, weeks or months.

[0420] The amount actually administered will depend on whether the treatment is prophylactic or therapeutic, the subject's age, weight, sex, medical history, pre-existing conditions, general condition, the severity of the disease being treated, and the judgment of a medical professional and may vary.

[0421] In the methods of treatment, the vectors may be administered in the form of pharmaceutical compositions and with modes of administration described herein.

[0422] Treatment methods according to the present invention may be continued for as long as the clinician overseeing the patient's care determines that the method is effective and that treatment is necessary.

[0423] In one embodiment of the present disclosure, the vector, e.g., a DNA plasmid, is for use in the treatment of cancer. Such vectors and antigen units of such vectors, including personalized and non-personalized antigen units of first polypeptides and various embodiments thereof, are described in detail herein.

[0424] Thus, in one embodiment, the present disclosure relates to a method of treating a subject having cancer, the method comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more cancer antigens or portions thereof; and (b) administering to the subject a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0425] Cancers can be solid or liquid. Examples of solid cancers are cancers that form a solid mass, such as a tumor. Examples of liquid cancers are cancers that exist in bodily fluids, such as lymphomas and blood cancers.

[0426] In one embodiment of the disclosure, the vector, e.g., a DNA plasmid, is for use in the treatment of a cancer selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, bone cancer, colorectal cancer, gastric cancer, lymphoma, malignant melanoma, liver cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer, bile duct cancer, brain cancer, cervical cancer, bladder cancer, esophageal cancer, Hodgkin's disease, and adrenocortical carcinoma.

[0427] In another embodiment of the present disclosure, the vector, e.g., a DNA plasmid, is for use in treating infectious diseases. Such vectors and antigenic units of such vectors are described in detail herein.

[0428] Thus, in one embodiment, the present disclosure relates to a method of treating a subject having or in need of prevention of an infectious disease, the method comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more antigens or portions thereof associated with said infectious disease; and (b) administering to the subject a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0429] Antigens or portions thereof associated with infectious diseases, such as antigens or portions thereof derived from pathogens, are described in detail herein.

[0430] below: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more antigens or portions thereof associated with a disease; and (b) Disclosed herein is a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector enables coexpression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules for use in treating a subject having or in need of prevention of the disease, wherein the vector is administered to the subject.

[0431] below: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more cancer antigens or portions thereof; and (b) Disclosed herein is a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing a first polypeptide and one or more immunostimulatory compounds as separate molecules for use in treating a subject having cancer, wherein the vector is administered to the subject.

[0432] below: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more antigens or portions thereof associated with an infectious disease; and (b) Disclosed herein is a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and one or more immunostimulatory compounds as separate molecules for use in treating a subject having or in need of prevention of an infectious disease, and the vector is administered to the subject.

[0433] below: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more antigens or portions thereof associated with a disease; and (b) Disclosed herein are vectors comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vectors enable coexpression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules, which are administered to a subject for the manufacture of a medicament for use in treating a subject having or in need of prevention of the disease.

[0434] below: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more cancer antigens or portions thereof; and (b) Also disclosed herein is the use of a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector enables coexpression of the first polypeptide and one or more immunostimulatory compounds as separate molecules for the manufacture of a medicament for use in treating a subject having cancer, which is administered to the subject.

[0435] below: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more antigens or portions thereof associated with an infectious disease; and (b) Also disclosed in the present specification is the use of a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and one or more immunostimulatory compounds as separate molecules for the manufacture of a medicament for use in treating a subject having or in need of prevention of an infectious disease, which is administered to the subject.

[0436] below: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more antigens or portions thereof associated with a disease; and (b) the use of a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, Here, the vector allows for the co-expression of the first polypeptide and one or more immunostimulatory compounds as separate molecules to treat a subject having the disease or in need of prevention of the disease.

[0437] below: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more cancer antigens or portions thereof; and (b) Also disclosed herein is the use of a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector enables co-expression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules to treat a subject with cancer.

[0438] below: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more antigens or portions thereof associated with an infectious disease; and (b) Also disclosed herein is the use of a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector allows for the co-expression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules to treat a subject having or in need of prevention of said infectious disease.

[0439] below: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit that comprises one or more antigens or portions thereof associated with a disease; and (b) Disclosed herein are vectors comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vectors enable the co-expression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules when used in the therapeutic or prophylactic treatment of the disease.

[0440] below: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more cancer antigens or portions thereof; and (b) Disclosed herein are vectors comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector, when used to treat cancer, enables the first polypeptide and the one or more immunostimulatory compounds to be coexpressed as separate molecules.

[0441] below: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more antigens or portions thereof associated with an infectious disease; and (b) Disclosed herein are vectors comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vectors, when used in the therapeutic or prophylactic treatment of said infectious disease, enable the first polypeptide and the one or more immunostimulatory compounds to be coexpressed as separate molecules.

[0442] below: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more antigens or portions thereof associated with a disease; and (b) Also disclosed herein is the use of a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector allows for the coexpression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules for the therapeutic or prophylactic treatment of the disease.

[0443] below: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more cancer antigens or portions thereof; and (b) Also disclosed herein is the use of a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector allows for the co-expression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules for the treatment of cancer.

[0444] below: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more antigens or portions thereof associated with an infectious disease; and (b) Also disclosed herein is the use of a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector allows for the co-expression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules for therapeutic or prophylactic treatment of the infectious disease.

[0445] Also disclosed herein is a method for administering to a subject having or in need of prevention of said disease: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more antigens or portions thereof associated with the disease; and (b) A medicament for the treatment or prevention of a disease in a subject having said disease or in need of prevention of said disease by administering a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector allows the first polypeptide and the one or more immunostimulatory compounds to be coexpressed as separate molecules.

[0446] Also disclosed herein is a method for administering to a subject having cancer: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more cancer antigens or portions thereof; and (b) A medicament for treating cancer in a subject having cancer by administering a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector is capable of co-expressing the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

[0447] Also disclosed herein is a method for treating a subject having or in need of prevention of said disease, comprising administering to a subject having or in need of prevention of said disease: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit comprising one or more antigens or portions thereof associated with the disease; and (b) A medicament for the treatment or prevention of an infectious disease by administering a vector comprising one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds, wherein the vector allows for the co-expression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules. EXAMPLES

[0448] The foregoing written description is believed to be sufficient to enable one skilled in the art to practice the present invention. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the present invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0449] Example 1: A variety of DNA plasmids have been designed that are capable of co-expressing a first polypeptide described herein and one or more immunostimulatory compounds as separate molecules.

[0450] All DNA plasmids, VB4194, VB4168, VB4169 and VB4170, contain nucleic acid sequences encoding the elements / units shown in Table 4 below: [Table 4]

[0451] The DNA plasmid further comprises nucleic acid sequences encoding the elements / units shown in Table 5 below: [Table 5]

[0452] In the following, "m", "murine" and "mouse" are used interchangeably, and "h" and human are used interchangeably.

[0453] DNA plasmids VB4194, VB4168, VB4169 and VB4170 containing eight epitopes with mutations: Exome sequencing and RNA sequencing of the mouse colon cancer cell line CT26 revealed hundreds to thousands of tumor-specific nonsynonymous mutations. Using in silico methods, we identified potential immunogenic sequences, i.e. epitopes with mutations, of which eight (Table 6) were selected for inclusion in the antigen unit of the first polypeptide encoded by the above DNA plasmid. The epitopes in the antigen unit are separated by glycine-serine linkers (GGGGSGGGS, SEQ ID NO: 17), i.e. all epitopes except the terminal epitopes are located in subunits, each of which contains one epitope and one GGGGSGGGS (SEQ ID NO: 17) linker.

[0454] Each of these DNA plasmids is a model of a DNA plasmid according to the present invention encoding an individualized first polypeptide, i.e. comprising an antigen unit comprising several patient-specific epitopes, e.g. several neoepitopes and / or several patient-presented shared cancer epitopes, where the patient-presented shared cancer antigen is a mutated patient-presented shared cancer antigen, or a model of a DNA plasmid according to the present invention encoding a non-individualized first polypeptide, i.e. comprising an antigen unit comprising multiple shared cancer epitopes, where the shared cancer antigen is a mutated shared cancer antigen.

[0455] DNA plasmids VB4168, VB4169 and VB4170 allow the co-expression of a first polypeptide as described above and in Tables 4 and 5 with the following immunostimulatory compounds as separate molecules (h=human; m=mouse): - VB4194: encodes only the first polypeptide (the same first polypeptide as VB4168, VB4169 and VB4170) and does not encode an immunostimulatory compound, serves as a comparison -VB4168:hFLT3L -VB4169: hFLTL3 and mGM-CSF -VB4170: hFLTL3, mGM-CSF and mCCL5 [Table 6]

[0456] Production of DNA plasmids The sequences of antigen units, coexpression elements and immunostimulatory compounds of all DNA plasmids disclosed in the examples were ordered from Genscript (Genscript Biotech BV, The Netherlands) and cloned into the expression vector pUMVC4a; a master plasmid containing the nucleotide sequences encoding the signal peptide, targeting unit, dimerization unit and unit linker as described in Table 4 above.

[0457] Assessing expression and secretion of DNA plasmid-encoded proteins HEK293 cells (ATCC) were transiently transfected with the above DNA plasmids. Briefly, 2x10 5 Cells / well were plated in 24-well tissue culture plates in 10% FBS growth medium and transfected with 1 μg of each DNA plasmid using Lipofectamine® 2000 reagent under conditions suggested by the manufacturer (Invitrogen, Thermo Fischer Scientific). Transfected cells were incubated at 37° C., 5% CO 2After 5 days at RT, cell supernatants were harvested for characterization of expression and secretion of the plasmid-encoded proteins by sandwich ELISA of the supernatants using mouse anti-human IgG CH3 domain antibody (capture antibody, 100 μl / well, 1 μg / ml, MCA878G, Bio-Rad) and goat anti-human MIP-1α antibody (biotinylated detection antibody, 100 μl / well, 0.2 μg / ml, BAF270, R&D systems) (Figure 5). Expression and secretion of the encoded immunostimulatory compounds FLT3L and GM-CSF were measured by sandwich ELASA using mouse anti-human FLT3L antibody (capture antibody, 100 μl / well, 0.5 μg / ml, MAB608, R&D systems) and mouse anti-human FLT3L antibody (biotinylated detection antibody, 100 μl / well, 0.1 μg / ml, BAF308, R&D Systems) (Figure 6), and rat anti-mouse GM-CSF antibody (capture antibody, 100 μl / well, 1.0 μg / ml mouse GM-CSF antibody, MAB415, R&D Systems) and goat anti-mouse GM-CSF antibody (biotinylated detection antibody, 100 μl / well, 0.2 μg / ml, BAM215, R&D Systems) (Figure 7), respectively. Expression and secretion of the encoded immunostimulatory compound CCL5 was measured by sandwich ELISA using rat anti-mouse CCL5 (capture antibody, 100 μl / well, 1.0 μg / ml, MAB4781, R&D Systems) and goat anti-mouse CCL5 (biotinylated detection antibody, 100 μl / well, 0.2 μg / ml, BAF478, R&D Systems) (Figure 8 ).

[0458] The results shown in FIG. 5 indicate that the first polypeptide / dimer protein containing the targeting unit, dimerization unit, and antigen unit encoded by VB4168, VB4169, and VB4170 was expressed and secreted from transfected HEK293 cells at a level comparable to that of the comparative VB4194. FLT3L, encoded as the second protein by VB4168, VB4169, and VB4170, was expressed and secreted at high levels from all three DNA plasmids as shown in FIG. 6. In addition, GM-CSF, encoded as the third protein by VB4169 and VB4170, was also expressed and secreted at high levels as shown in FIG. 7. CCL5, encoded as the fourth protein by VB4170, was expressed and secreted at high levels as shown in FIG. 8.

[0459] Example 2: Evaluation of the immunogenicity of DNA plasmids VB4194, VB4168 and VB4169 The immunogenicity of DNA plasmids VB4194 (comparison), VB4168 and VB4169 was determined by measuring the T cell immune response elicited in mice administered with such plasmids. As a negative control, VB1026 was included, which encodes a polypeptide having the amino acid sequence 1-237 of SEQ ID NO: 1. This DNA plasmid is identical to VB4194, but does not contain a linker or antigen unit.

[0460] For all experiments with mice, the following study design was applied: Female 6-week-old mice were obtained from Janvier Labs (France). All animals were maintained in the animal facilities of the Radium Hospital (Oslo, Norway). All animal protocols were approved by the Norwegian Food Safety Authority (Oslo, Norway). Five mice / group were used for testing constructs containing antigen units and three mice / group for negative controls.

[0461] BALB / c mice were given a single intramuscular injection of 6 μg of DNA plasmid followed by electroporation. Ten days after injection, spleens were harvested and crushed through a cell strainer to obtain single cell suspensions. For each plasmid tested, a portion of the single cell suspension was used to deplete CD4+ T cells using Dynabeads® anti-CD4 beads. Total splenocytes and CD4+ depleted splenocytes were tested for INF-γ and TNF-α production by FluoroSpot assay according to the manufacturer's protocol (Mabtech).

[0462] Spleen cells collected from mice administered with these plasmids were restimulated with peptides (Table 7 below) having the same sequences as the eight epitopes contained in VB4194, VB4168, and VB4169 shown in Table 6: [Table 7]

[0463] DNA plasmids VB4194, VB4168 and VB4169 were compared for their ability to induce T cell immune responses against the peptides in Table 7. VB1026 was included as a negative control.

[0464] As shown in Figures 9-14, mice treated with the negative control VB1026 exhibited low basal immunogenicity to the peptides in Table 7.

[0465] VB4194 induced T cell responses against all eight epitopes. VB4168, which encodes the same first polypeptide as VB4194 and also encodes FLT3L, induced stronger T cell responses than VB4194 (Figures 9-11). In addition to the expression of the first polypeptide containing the eight epitopes encoded by VB4169, co-expression of two immunostimulatory compounds, FLT3L and GM-CSF, induced stronger immune responses than VB4194 and VB4168 (Figures 9-11). The number of T cells secreting only IFN-γ (Figure 9), the number of T cells secreting only TNF-α (Figure 10), and the number of INF-γ + TNF-α co-secreting cells (Figure 11) all increased from VB4194 to VB4168 and from VB4168 to VB4169. Similarly, the numbers of CD8+ T cells secreting only IFN-γ (CD4+ T cell depleted samples) (Figure 12), CD8+ T cells secreting only TNF-α (Figure 13), and IFN-γ + TNF-α co-secreting cells (Figure 14) all increased from VB4194 to VB4168 and from VB4168 to VB4169.

[0466] These results show that a DNA plasmid according to the present invention encoding a first polypeptide and one or more immunostimulatory compounds co-expressed as separate molecules from said plasmids is capable of enhancing an antigen-specific immune response against the antigen contained in the first polypeptide compared to a DNA plasmid encoding only the first polypeptide.

[0467] Example 3: DNA plasmid VB4202 was designed and constructed to contain nucleic acid sequences encoding the elements / units listed in Table 4, and further to contain nucleic acid sequences encoding the elements listed in Table 8 below: [Table 8]

[0468] Assessment of expression and secretion of VB4202-encoded protein HEK293 cells (ATCC) were transiently transfected with the above DNA plasmids as described in Example 1. Secreted first polypeptide / dimer proteins were characterized by sandwich ELISA of the supernatants using mouse anti-human IgG CH3 domain antibody (capture antibody, 100 μl / well, 1 μg / ml, MCA878G, Bio-Rad) and goat anti-human MIP-1α antibody (biotinylated detection antibody, 100 μl / well, 0.2 μg / ml, BAF270, R&D systems) ( FIG. 15 ). Secretion of the encoded immunostimulatory compound GM-CSF was measured in supernatants diluted 1:1000 by sandwich ELISA using rat anti-mouse GM-CSF (capture antibody, 100 μl / well, 1.0 μg / ml mouse GM-CSF antibody, MAB415, R&D Systems) and goat anti-mouse GM-CSF (biotinylated detection antibody, 100 μl / well, 0.2 μg / ml, BAM215, R&D Systems) (FIG. 16).

[0469] The results shown in Figure 15 indicate that the first polypeptide / dimer protein containing the targeting unit, dimerization unit, and antigen unit encoded by VB4202 was expressed and secreted from transfected HEK293 cells. GM-CSF, encoded as the second protein by VB4202, was expressed and secreted at high levels as shown in Figure 16.

[0470] Evaluation of the immunogenicity of DNA plasmids VB4194 and VB4202 The immunogenicity of DNA plasmids VB4194 (comparison), VB1026 (negative control) and VB4202 was determined in BALB / c mice as described in Example 2.

[0471] As shown in Figure 17, no IFN-γ production was detected in response to VB1026 treatment. VB4194 induced T cell responses against all eight epitopes. VB4202, which encodes the same first polypeptide as VB4194 and additionally encodes GM-CSF, induced even stronger T cell responses than VB4194 as analyzed by IFN-γ FluoroSpot.

[0472] Flow cytometry evaluation Multiflow cytometry was used to assess APC / dendritic cell influx at the single cell level in mice treated with VB1026, VB4194, and VB4202. Female, 6-week-old BALB / c mice were obtained from Janvier Labs (France). All animals were kept in the animal facility at the University of Oslo. All animal protocols were approved by the Norwegian Food Safety Authority (Oslo, Norway). Six mice per group were used to compare VB1026, VB4202, and VB4194. An additional group of six untreated mice served as controls. Six micrograms of each DNA plasmid was administered intramuscularly into the tibialis anterior muscle, followed by electroporation. The untreated group received neither DNA plasmid nor electroporation. The tibialis anterior muscle was extracted under sterile conditions 1, 2, and 4 days after administration or in the untreated group. To obtain single cell suspensions, the muscles were first mechanically dissociated using scissors and then enzymatically digested. For enzymatic digestion, dissociated muscles were incubated in digestion medium (DMEM, collagenase A [2 mg / ml], DNase [50 U / ml]) with a stirring magnet for 1 h at 37°C. After incubation, single cell suspensions were filtered through a 70 μm filter and washed twice in PBS at 400 × g for 6 min at 4°C.

[0473] For flow cytometry analysis, single cell suspensions were first incubated with a viability dye (eFluor 780, Invitrogen) for 10 min at room temperature (RT). The viability dye was washed out with PBS (centrifugation at 400 xg for 6 min at 4 °C twice). Cells were then incubated with Fc block for 10 min at room temperature to block non-specific binding of fluorescent antibodies. After the blocking step, cells were stained with a surface marker-specific antibody pool (Table 9 below) for 30 min on ice. Stained cells were measured on a BD FACSymphony A5 flow cytometer. Flow cytometry data were analyzed using FlowJo software. A gating strategy was used to define dendritic cells (DCs) / APCs as described in the legend of Figure 18. [Table 9]

[0474] As a result, there was an increased influx of immune cells (CD45+ cells) into the muscles of mice administered VB4202 compared to the muscles of mice administered VB4194 (Figure 19).

[0475] The proportion of DCs in the CD45+ cell population present in muscle was higher in mice treated with VB4202 compared to mice treated with VB4194 (Figure 20). Furthermore, both cDC1 (Figure 21) and moDC (Figure 22) populations were increased in muscle of mice treated with VB4202 compared to mice treated with VB4194.

[0476] In summary, these results show that a DNA plasmid according to the present invention encoding a first polypeptide and one or more immunostimulatory compounds co-expressed from a plasmid as separate molecules, when administered intramuscularly, can promote the influx of dendritic cells to the site of administration, ultimately further contributing to an increased antigen-specific immune response against the antigen contained in the first polypeptide, compared to a DNA plasmid encoding only said first polypeptide.

[0477] Example 4: The following DNA plasmids were designed and constructed: All DNA plasmids, VB1020, VB4195, VB4196, contain nucleic acid sequences encoding the elements / units listed in Table 4 and further contain nucleic acid sequences encoding the elements / units listed in Table 10 below: [Table 10]

[0478] DNA plasmids VB1020, VB4195 and VB4196 contain a nucleic acid sequence encoding a first polypeptide comprising an antigenic unit comprising human papillomavirus 16 (HPV16) antigens E7 and E6.

[0479] Each of these DNA plasmids is a model of a DNA plasmid according to the invention encoding a non-individualized first polypeptide for use in the treatment of cancer, i.e. comprising an antigen unit comprising a plurality of shared cancer antigens, said shared cancer antigen being a viral shared cancer antigen (here an antigen from HPV16, which causes certain cancers), or a model of a DNA plasmid according to the invention encoding a first polypeptide for use in the treatment of an infectious disease, i.e. comprising an antigen unit comprising an antigen from a pathogen (here an antigen from HPV16).

[0480] The DNA plasmids VB4195 and VB4196 allow the co-expression, as separate molecules, of the first polypeptide described above and the following immunostimulatory compounds: - VB1020: Encoding only the first polypeptide and no immunostimulatory compound, serves as a comparison. -VB4195:hFLT3L -VB4169: hFLTL3 and mGM-CSF

[0481] Assessing expression and secretion of DNA plasmid-encoded proteins HEK293 cells were obtained from ATCC and transiently transfected with VB1020 (control), VB4195 or VB4196 as described in Example 1.

[0482] Secreted proteins encoded by DNA plasmids were characterized by sandwich ELISA of the supernatants using mouse anti-human IgG CH3 domain antibody (capture antibody, 100 μl / well, 1 μg / ml, MCA878G, Bio-Rad) and goat anti-human MIP-1α antibody (biotinylated detection antibody, 100 μl / well, 0.2 μg / ml, R&D systems, BAF270).

[0483] The secretion amount of FLT3L, encoded as the second protein of VB4195 and VB4196, in the cell culture supernatant (diluted 1:500) was measured by sandwich ELISA using mouse anti-human FLT3L antibody (capture antibody, 100 μl / well, 0.5 μg / ml, MAB608, R&D Systems) and mouse anti-human FLT3L antibody (biotinylated detection antibody, 100 μl / well, 0.1 μg / ml, BAF308, R&D Systems). Secretion of GM-CSF in cell culture supernatants (diluted 1:500), encoded as the third protein of VB4196, was measured by sandwich ELISA using rat anti-mouse GM-CSF (capture antibody, 100 μl / well, 1.0 μg / ml mouse GM-CSF antibody, MAB415, R&D Systems) and goat anti-mouse GM-CSF (biotinylated detection antibody, 100 μl / well, 0.2 μg / ml, BAM215, R&D Systems).

[0484] The results shown in Figure 23 indicate that the first polypeptide / dimer protein containing a targeting unit, a dimerization unit and an antigen unit encoded by VB4195 and VB4196 was successfully expressed and secreted from transfected HEK293 cells. FLT3L, encoded by VB4195 and VB4196 as the second protein, was expressed and secreted at high levels from both plasmids as shown in Figure 24. Furthermore, GM-CSF, encoded by VB4196 as the third protein, was also expressed and secreted at high levels as shown in Figure 25.

[0485] Characterization of intact proteins expressed from VB4195 and VB4196 Western blot analysis was performed on supernatant samples from transfected Expi293F cells to further characterize the proteins encoded by VB4195 and VB4196. VB1020, which encodes a first polypeptide identical to VB4195 and VB4196, was included as a comparison.

[0486] Briefly, Expi293F cells (3x10 6 Cells / ml, 1.6 ml) were seeded into 6-well culture plates. The plates were transfected with 1 μg / ml of plasmid DNA using ExpiFectamine 293 Reagent (Thermo Fisher Sci.), and the plates were incubated in a humidified CO 2 Cell incubator (8% CO 2The plates were incubated on an orbital shaker (19 mm diameter, 125 rpm) in a 37°C oven. After 18 h of incubation, ExpiFectamine 293 Transfection Enhancer (Thermo Fisher Sci.) was added to each well. The plates were further incubated for 28 h, and then the supernatants were harvested. 70 μl of the supernatant from transfected Expi293F cells was mixed with 25 μl of 4x Laemmli sample buffer (Bio-Rad), 5 μl of DTT (Thermo Fisher Sci.), or 5 μl of ultrapure water to prepare samples for reducing and non-reducing conditions, respectively. Furthermore, the Expi293F supernatant was deglycosylated by mixing 64 μl of the sample with 16 μl of PNGase F buffer (NEB) and incubated at 80°C for 2 min. After cooling, 4 μl Rapid PNGase F enzyme (NEB) was added and incubated at 50°C for 10 min. Deglycosylated samples were further mixed with 30 μl 4x Laemmli buffer and 6 μl DTT. Samples (reduced, non-reduced, or deglycosylated) were heated at 70°C for 10 min and loaded onto 4%-20% Criterion TGX Stain-Free precast gels (Bio-Rad). SDS-PAGE was performed in 1x Tris / Glycine / SDS running buffer (Bio-Rad) using Precision Plus Protein All Blue Prestained protein standard (Bio-Rad). Proteins were transferred from the gel to EtOH-activated low-fluorescence (LF) 0.45 μm PVDF membranes (Bio-Rad) using the Tran-Blot Turbo semi-dry transfer system (Bio-Rad). PVDF membranes were blocked with EveryBlot buffer (Bio-Rad) for 5 min and probed with goat anti-human MIP-1α (BAF270, R&D Systems), goat anti-mouse GM-CSF (BAF415, R&D Systems), or goat anti-human FLT3L (BAF308, R&D Systems) to detect the first polypeptide / dimer protein, GM-CSF, or FLT3L, respectively.The specificity of the primary antibodies was confirmed by initial testing using the respective recombinant proteins as probes. The membranes were incubated with fluorochrome-conjugated secondary antibodies for 1 hour at room temperature, then washed and dried. Images were acquired using a ChemiDoc® MP Imaging System (Setting Dylight 550 and 650, Auto Optimal). Western blot analysis confirmed the ELISA results demonstrating that VB4195 expresses two proteins: the first polypeptide / dimer protein (Figure 26), and FLT3L (Figure 27). VB4196 expressed three proteins: the first polypeptide / dimer protein (Figure 26), FLT3L, and GM-CSF (Figure 27). The P2A sequence used to isolate the nucleic acid sequences encoding the FLT3L and GM-CSF proteins of VB4196 appears to be glycosylated, resulting in a shift in the size of the proteins observed in the Western blot. The PNGaseF deglycosylation protocol reduced this size shift. Furthermore, the P2A peptide left a 21 amino acid tail at the C-terminus of FLT3L, which was observed to have a size shift of approximately 2.2 kDa on Western blots. Importantly, no additional bands were observed when probing membranes with anti-FLT3L and anti-GM-CSF, indicating successful ribosome skipping at the P2A and T2A sequences, resulting in the expression of multiple separate proteins from a single DNA plasmid.

[0487] Taken together, the ELISA and Western blot data indicate that an intact dimeric protein containing a targeting unit, a dimerization unit and an antigen unit can be co-expressed from a DNA plasmid with one or more other proteins (immunostimulatory compounds) by using different 2A peptides as co-expression elements.

[0488] Example 5: A DNA plasmid, VB4204, has been designed and constructed that contains nucleic acid sequences encoding the elements / units listed in Table 4, and further contains nucleic acid sequences encoding the elements / units listed in Table 11 below: [Table 11]

[0489] Furthermore, a DNA plasmid pGM-CSF was designed and generated by cloning the natural leader sequence of mouse GM-CSF (SEQ ID NO: 12) and the sequence of mouse GM-CSF (SEQ ID NO: 13) into the expression vector pUMVC4a.

[0490] Assessment of expression and secretion of VB4204-encoded protein HEK293 cells were obtained from ATCC and transiently transfected with VB4204 or VB1020 (control) as described in Example 1.

[0491] Secreted proteins encoded by VB4204 or VB1020 were characterized by sandwich ELISA of supernatants (1:10 dilution) using mouse anti-human IgG CH3 domain antibody (capture antibody, 100 μl / well, 1 μg / ml, MCA878G, Bio-Rad) and goat anti-human MIP-1α antibody (biotinylated detection antibody, 100 μl / well, 0.2 μg / ml, R&D systems, BAF270).

[0492] Secretion of GM-CSF, encoded as the second protein of VB4204, was measured by sandwich ELISA of cell culture supernatants (1:1000 dilution) using rat anti-mouse GM-CSF (capture antibody, 100 μl / well, 1.0 μg / ml mouse GM-CSF antibody, MAB415, R&D Systems) and goat anti-mouse GM-CSF (biotinylated detection antibody, 100 μl / well, 0.2 μg / ml, BAM215, R&D Systems).

[0493] The results shown in Figure 28 indicate that the first polypeptide / dimer protein encoded by VB4204, which includes a targeting unit, a dimerization unit, and an antigen unit, is well expressed and secreted from transfected HEK293 cells. Furthermore, GM-CSF, which is encoded as a separate second protein by VB4204, is also expressed and secreted at high levels as shown in Figure 29.

[0494] Immunogenicity evaluation of VB4204 (1) The immunogenicity of VB4204, VB1020 (comparison) and VB1026 (negative control) was determined in C57BL / 6 mice as described in Example 2, except that CD4+ T cell depleted splenocyte data was not generated. T cell responses in splenocytes were then tested for INF-γ production in a FluoroSpot assay. In addition, the immunogenicity of co-injected DNA plasmids (6 μg total DNA) of VB1020 (encoding the same polypeptide as VB4204 but not GM-CSF) and pGM-CSF (encoding GM-CSF but not the polypeptide of VB4204) was determined as described in this paragraph.

[0495] Peptides corresponding to the E6 and E7 antigens listed in Table 12 below were used to restimulate splenocytes harvested from mice administered VB1020, VB4204, VB1026 and (VB1020+pGM-CSF). [Table 12]

[0496] VB1020 (first polypeptide only), VB4204 (first polypeptide and GM-CSF), and co-injection of VB1020 with pGM-CSF were compared for their ability to elicit T cell immune responses against the peptides in Table 12. VB1026 was included as a negative control.

[0497] As shown in FIG. 30, no IFN-γ production was detected in response to VB1026 administration.

[0498] Furthermore, VB1020 induced strong T cell responses to the peptides in Table 12, while VB4202 induced even stronger T cell responses than VB1020. Furthermore, VB4202 also induced stronger T cell responses than those induced by co-injection of VB1020+pGM-CSF (Figure 30).

[0499] The immunogenicity of VB4204, VB1020 (comparison) and VB1026 (negative control) was determined by flow cytometry. C57BL / 6 mice were treated as described in Example 2, spleen cells were pooled per group and restimulated with single peptides corresponding to the HPV16 E7 (49-57) antigen listed in Table 12 for 1 h at RT, after which monensin and brefeldin were added to each well to inhibit endocytosis. Cells were further incubated at 37°C for 15 h. After restimulation, cells were harvested for flow cytometry analysis. Briefly, single cell suspensions were first incubated with dead cell labeling dye (eFluor 780, Invitrogen) for 10 min at RT. The dead cell labeling dye was washed out with PBS (centrifugation at 400 xg for 6 min at 4°C twice). Cells were then incubated with Fc block for 10 min to block non-specific binding to fluorescent antibodies. After a blocking step, cells were stained with surface marker-specific antibody pools (Table 9) for 30 min on ice. Antibodies were washed off with PBS (twice centrifuged at 400 xg for 6 min at 4°C) and cells were incubated with fixation / permeabilization solution (60 min at 4°C). Cells were centrifuged, washed and then resuspended in 100 μl of antibody mix in permeabilization buffer and incubated for 30 min at 4°C. Stained cells were analyzed on a BD FACSymphony A5 flow cytometer. Flow cytometry data were analyzed using FlowJo software.

[0500] In Table 12, DNA plasmid VB4202 was compared for its ability to induce a T cell immune response against the single peptide HPV16 E7(49-57). VB1020 was included as a comparison and VB1026 was included as a negative control.

[0501] As shown in FIG. 31, no production of IFN-γ or TNF-α was detected in response to VB1026 treatment.

[0502] The numbers of CD8+ T cells secreting only IFN-γ, only TNF-α (CD4+ T cell depleted samples), and the numbers of IFN-γ+TNF-α co-secreting cells were all increased from VB1020 to VB4202 (Figure 31).

[0503] These results demonstrate that a DNA plasmid of the present invention encoding a first polypeptide and an immunostimulatory compound co-expressed as separate molecules from the plasmids can enhance antigen-specific T cell responses against an antigen contained in the first polypeptide compared to a DNA plasmid encoding the first polypeptide alone and compared to coinjection of the same DNA plasmid encoding the first polypeptide with a plasmid encoding the same immunostimulatory compound.

[0504] Example 6: A DNA plasmid, VB4205, was designed and constructed that contains nucleic acid sequences encoding the elements / units listed in Table 4, and further contains nucleic acid sequences encoding the elements / units listed in Table 13 below: [Table 13]

[0505] Assessment of expression and secretion of VB4205-encoded protein HEK293 cells were obtained from ATCC and transiently transfected with VB4205 or VB1020 (comparison) as described in Example 1. Secreted proteins encoded by VB4205 or VB1020 were characterized by sandwich ELISA (1:10 dilution) of supernatants with mouse anti-human IgG CH3 domain antibody (capture antibody, 100 μl / well, 1 μg / ml, MCA878G, Bio-Rad) and goat anti-human MIP-1α antibody (biotinylated detection antibody, 100 μl / well, 0.2 μg / ml, R&D systems, BAF270).

[0506] Secretion of CCL5, encoded as the second protein of VB4205, was measured in the supernatants (1:1000 dilution) by sandwich ELISA using rat anti-mouse CCL5 (capture antibody, 100 μl / well, 1.0 μg / ml, MAB4781, R&D Systems) and goat anti-mouse CCL5 (biotinylated detection antibody, 100 μl / well, 0.2 μg / ml, BAF478, R&D Systems).

[0507] The results shown in Figure 32 indicate that the first polypeptide / dimer protein encoded by VB4205, which includes a targeting unit, a dimerization unit and an antigen unit, is well expressed and secreted from transfected HEK293 cells. Furthermore, the second protein, CCL5, encoded by VB4205, is expressed and secreted at high levels as shown in Figure 33.

[0508] Assessment of immunogenicity of VB4205 The immunogenicity of VB4205 was determined in C57BL / 6 mice and compared with that of VB1020 (comparison) and VB1026 (negative control) as described in Example 5(1).

[0509] VB1020 (first polypeptide only) and VB4205 (first polypeptide and CCL5) were compared for their ability to elicit a T cell immune response against the peptides in Table 12.

[0510] No IFN-γ production was detected in response to administration of VB1026, as shown in Figure 34. VB1020 induced strong T cell responses to the peptides in Table 12, while VB4205 induced even stronger T cell responses compared to VB1020.

[0511] These results also demonstrate that the DNA plasmid of the present invention encoding a first polypeptide and an immunostimulatory compound co-expressed as separate molecules from the plasmid can enhance antigen-specific T cell responses against the antigen contained in the first polypeptide, compared to a DNA plasmid encoding only the first polypeptide.

[0512] Example 7: DNA plasmids VB1026, VB4208, VB4194, VB4202 and pGM-CSF having nucleic acid sequences encoding the elements / units listed in Table 14 were designed and constructed as described herein: [Table 14]

[0513] The DNA plasmids encode the following proteins: - VB4202 encodes a first polypeptide as described above and the immunostimulatory compound mGM-CSF as separate molecules and can be co-expressed. - VB4194: encodes only a first polypeptide containing an antigenic unit containing the CT26 epitope, and does not encode an immunostimulatory compound, serves as a comparison - VB1026: encodes a polypeptide having an amino acid sequence of 1-237 of SEQ ID NO: 1, which is identical to the first polypeptide encoded by VB4194, but does not contain a linker or antigen unit. This serves as a negative control. - VB4208 (SEQ ID NO: 24): encodes the first polypeptide without an antigenic unit, i.e. without encoding the CT26 epitope, and mGM-CSF as separate molecules. Serves as a negative control. - pGM-CSF: encoding mGM-CSF, for comparison.

[0514] Treatment of CT26 tumor-challenged mice The antitumor effect of VB4202 was evaluated in a CT26 tumor challenge. VB4202 was compared to VB4194, which encodes the same first polypeptide as VB4202. Furthermore, the antitumor effect of VB4202 was compared to co-injection of VB4194 and pGM-CSF. VB1026 and VB4208 were included as negative controls.

[0515] Each group AF contained 8 BALB / c mice, with 1x10 5 Mice were inoculated with CT26 tumor cells on day (D) 0 by injecting 1000 x 1000 tumor cells. Mice were administered DNA plasmids and respective amounts listed in Table 15 intramuscularly in the right leg on days 4 and 11. Due to variability in plasmid size between the VB4194 and pGM-CSF plasmids, a second coinjection group (Group F) was included in which the amount of each plasmid was adjusted to match the plasmid copy number of the single plasmid injections in Group D (Table 16) to ensure comparable protein levels were expressed. [Table 15] [Table 16]

[0516] Tumor size was measured using calipers. Tumors were measured in two dimensions, length and width, with height being equal to width. Tumor volume was calculated using the formula: tumor volume = length (mm) x width (mm) x height (mm) / 2000. Treatment was terminated on day 32.

[0517] Tumors in the VB4194-treated group (group C), VB4202-treated group (group D), and VB4194 and pGM-CSF co-injection groups (groups E and F) grew slower than those in the VB1026 and VB4208 negative control groups (groups A and B, respectively).

[0518] The group administered VB4202, which co-expresses the same first polypeptide as VB4194 and GM-CSF (Group D), showed a reduced tumor growth rate compared to the group administered VB4194 alone (Group C). Furthermore, administration of VB4202 resulted in a reduced tumor growth rate compared to two co-injection groups administered 10 μg total of VB4194 and pGM-CSF (E) or adjusted to the equivalent copy number (F). Such co-injection resulted in a tumor growth rate comparable to that of VB4194 alone.

[0519] These results (shown in FIG. 35) indicate that co-expression of GM-CSF from VB4202, which encodes the same first polypeptide as VB4194, further enhanced the tumor growth inhibition effect of VB4194. As shown in FIG. 36, tumor growth inhibition was accompanied by increased survival of animals treated with VB4202. The antitumor effect was mediated by an antigen-specific immune response, as shown by comparing VB4202 with the negative controls VB1026 and VB4208. Furthermore, VB4202 provided stronger antitumor efficacy than that observed when VB4194 and pGM-CSF were co-injected as two separate plasmids.

[0520] Example 8: DNA plasmids TECH001-CV021, TECH001-CV022 and TECH001-CV023 were designed and constructed that contain nucleic acid sequences encoding the elements / units listed in Table 4, and further contain nucleic acid sequences encoding the elements / units listed in Table 17 below: [Table 17]

[0521] DNA plasmids TECH001-CV021, TECH001-CV022 and TECH001-CV023 contain a nucleic acid sequence encoding a first polypeptide comprising an antigen unit that includes the SARS-CoV-2 receptor binding domain (RBD) antigen.

[0522] Each of these DNA plasmids is a model of a DNA plasmid according to the invention that encodes an antigenic unit comprising a first polypeptide, i.e. an antigen derived from a pathogen (here an antigen derived from SARS-CoV-2), for use in treating an infectious disease.

[0523] DNA plasmids TECH001-CV021, TECH001-CV022 and TECH001-CV023 allow for the co-expression of the above-mentioned first polypeptide and the following immunostimulatory compound as separate molecules: - VB2060: Encoding only the first polypeptide and no immunostimulatory compound, serves as a comparison. -TECH001-CV021: mGM-CSF -TECH001-CV022: mIL-12 -TECH001-CV023: mIL-21

[0524] IL-12 is a heterodimeric cytokine encoded by two genes, IL-12A (p35) and IL-12B (p40). An active heterodimer (called p70) and a homodimer of p40 are formed following protein synthesis.

[0525] Assessing expression and secretion of DNA plasmid-encoded proteins Briefly, Expi293F cells (2x10 6 The cells / ml (1 ml) were seeded into a 96-well culture plate. The plate was transfected with 0.64 μg / ml of plasmid DNA using ExpiFectamine 293 reagent (Thermo Fisher Sci.), and the plate was incubated in a humidified CO 2 Cell incubator (8% CO 2The plates were incubated in an orbital shaker (3 mm diameter, 900 rpm) at 37° C. The plates were incubated for 72 hours, after which the supernatants were harvested.

[0526] The secreted first polypeptide / dimeric protein in the supernatant (1:1500 dilution) was characterized by sandwich ELISA using mouse anti-human IgG CH3 domain antibody (capture antibody, 100 μl / well, 1 μg / ml, MCA878G, Bio-Rad) and goat anti-human MIP-1α antibody (biotinylated detection antibody, 100 μl / well, 0.2 μg / ml, BAF270, R&D systems) (Figure 37).

[0527] Protein expression and secretion of the immunostimulatory compounds GM-CSF, IL-12, and IL-21 in the supernatants were analyzed using anti-mouse GM-CSF antibody (rat anti-mouse GM-CSF capture antibody, 100 μl / well, 1.0 μg / ml mouse GM-CSF antibody, MAB415, R&D Systems; goat anti-mouse GM-CSF biotinylated detection antibody, 100 μl / well, 0. 2 μg / ml, BAM215, R&D Systems) (Figure 38a), anti-mouse IL-12 antibody (rat anti-mouse IL-12 capture antibody, 100 μl / well, 1.0 μg / ml, MAB419 R&D systems; goat anti-mouse IL-12 biotinylated detection antibody, 100 μl / well, 0. 4 μg / ml, BAF419, R&D systems) (Figure 38b). 38b), and anti-mouse IL-21 antibody (goat anti-mouse IL-21 capture antibody, 100 μl / well, 0.1 μg / ml, AF594, R&D systems; goat anti-mouse IL-21 biotinylated detection antibody, 100 μl / well, 0.4 μg / ml, BAF594, R&D systems) (Figure 38c).

[0528] The results shown in Figure 37 indicate that the first polypeptide / dimer protein containing a targeting unit, a dimerization unit and an antigen unit encoded by TECH001-CV021, TECH001-CV022 and TECH001-CV023 was expressed and secreted from transfected Expi293F cells. GM-CSF, IL-12 and IL-21 encoded as the second protein by TECH001-CV021, TECH001-CV022 and TECH001-CV023, respectively, were also expressed and secreted at high levels as shown in Figure 38a-c.

[0529] Characterization of intact proteins expressed from TECH001-CV021, TECH001-CV022, and TECH001-CV023 Western blot (WB) analysis was performed on supernatant samples from transfected Expi293F cells to characterize the proteins encoded by TECH001-CV021, TECH001-CV022, and TECH001-CV023. VB2060, which encodes the same first polypeptide as the DNA plasmid described above, was included as a comparison.

[0530] Samples were prepared for reducing and non-reducing conditions by mixing 70 μl of supernatant from transfected Expi293F cells with 25 μl of 4x Laemmli sample buffer (Bio-Rad), 5 μl of DTT (Thermo Fisher Sci.), or 5 μl of ultrapure water. Samples (reduced or non-reduced) were heated at 70°C for 10 min, and then 20 μL per lane was loaded onto 4%–20% Criterion TGX Stain-Free precast gels (Bio-Rad). SDS-PAGE was performed in 1x Tris / Glycine / SDS running buffer (Bio-Rad) using Precision Plus Protein All Blue Prestained and Unstained protein standards (Bio-Rad). Proteins were transferred from the gel to EtOH-activated low-fluorescence (LF) 0.45 μm PVDF membranes (Bio-Rad) using the Tran-Blot Turbo semi-dry transfer system (Bio-Rad). The PVDF membranes were blocked with EveryBlot buffer (Bio-Rad) for 5 min and probed with goat anti-human MIP-1α (AF270, R&D Systems), goat anti-mouse GM-CSF (BAF415, R&D Systems), goat anti-mouse IL-12 (BAF419, R&D Systems), or goat anti-mouse IL-21 (BAF594, R&D Systems) to detect the first polypeptide, GM-CSF, IL-12, and IL-21, respectively. The membranes were washed and incubated with fluorochrome-conjugated anti-goat secondary antibodies for 1 h at room temperature, followed by washing and drying (rinsing with ethanol). Images were acquired using a ChemiDoc® MP Imaging System (Dylight 650 and 800, Auto Optimal settings). Expifectamine-treated cells (transfection control) were included in each gel as a negative control.

[0531] WB analysis confirmed the ELISA results and demonstrated expression of intact first polypeptide from all four DNA plasmids: TECH001-CV021, TECH001-CV022, TECH001-CV023, and VB2060 (Figure 39). TECH001-CV021 also expressed GM-CSF (heterogeneously glycosylated) (Figure 40). Figure 41 shows WB analysis of TECH001-CV022 probed with goat anti-mouse IL-12 under reducing (left panel) and non-reducing (right panel) conditions. In addition to expressing the first polypeptide, TECH001-CV022 expressed glycosylated IL-12B (p40) and IL-12A (p35) (Figure 41, left panel). Previous studies have reported that cells secreting bioactive IL-12 (p70 heterodimer) also secrete free p40 (monomer) (Jalah et al., J Biol Chem Vol 288, No.9, 6763-6776, 2013). Indeed, both IL-12 p70 heterodimer and p40 monomer bands were detected under non-reducing conditions (Figure 41, right panel). In addition to the first polypeptide, TECH001-CV023 expressed IL-21 (Figure 42). Importantly, no additional bands were observed with anti-GM-CSF, anti-IL-12 and anti-IL-21 probe membranes, indicating successful ribosome skipping at the T2A sequence, resulting in the expression of multiple separate proteins from a single DNA plasmid.

[0532] Taken together, the ELISA and Western blot data demonstrated that an intact first polypeptide comprising a targeting unit, a dimerization unit and an antigen unit can be co-expressed from a DNA plasmid with one or more immunostimulatory compounds by using the same 2A peptide as a co-expression element.

[0533] Immunogenicity evaluation of TECH001-CV021, TECH001-CV022, and TECH001-CV023 The immunogenicity of TECH001-CV021, TECH001-CV022 and TECH001-CV023 was measured and compared with that of VB2060 and VB1026 (negative control).

[0534] Female, 6-week-old BALB / c mice were obtained from Janvier Labs (France). All animals were maintained in the animal facility at the University of Oslo (Oslo, Norway). All animal protocols were approved by the Norwegian Food Safety Authority (Oslo, Norway). Five animals / group were used for the studies TECH001-CV021, TECH001-CV022, TECH001-CV023 and VB2060, and three animals / group for the negative control.

[0535] Each tibialis anterior muscle was intramuscularly injected with 1 μg of DNA plasmid (2 × 25 μl, 20 μg / ml), followed by electroporation using the AgilePulse in vivo electroporation system (BTX, USA).

[0536] Assessment of the humoral immune response in mice against the SARS-CoV-2 RBD. Serum from mice injected with the DNA plasmid was collected 13 days after injection and tested for anti-RBD IgG antibodies that bind to the RBD protein (Wuhan variant).

[0537] Briefly, blood was collected from the saphenous vein of vaccinated mice. The clotted blood was centrifuged twice (1000g, 15 min) and serum was collected and transferred to clean tubes. Humoral immune responses were evaluated with an ELISA assay to detect total IgG in serum binding to the RBD (aa319-542) of SARS-CoV2 (Wuhan variant). ELISA plates (MaxiSorp Nunc-Immuno plates) were coated with 1 μg / ml recombinant RBD-His protein antigen in PBS overnight at 4 °C. The plates were blocked with 4% BSA in PBS for 1 h at room temperature. Then, serial dilutions of mouse sera (diluted in 0.1% BSA in PBS) were added and incubated for 2 h at 37 °C. The plates were washed three times and incubated with anti-mouse total IgG-HRP antibody (Southern Biotech) diluted 1:50,000 in 0.1% BSA in PBS and incubated for 1 h at 37 °C. After a final wash, plates were developed with TMB substrate (Merck, cat. CL07-1000). Plates were read within 30 min at 450 nm wavelength using a SPARK® Multimode Microplate Reader (Tecan). Binding antibody endpoint titers were calculated as the reciprocal of the highest dilution that gave a signal above the cutoff value. Binding antigens tested included SARS-CoV-2 antigen: RBD (Sino Biological 40592-V08H; SEQ ID NO: 30).

[0538] The results shown in FIG. 43 show that TECH001-CV021 and TECH001-CV023, which encode a first polypeptide containing the RBD (aa319-542) derived from SARS-CoV-2 (Wuhan variant) in the antigen unit and GM-CSF and IL-21, respectively, as the second protein, induce a stronger total IgG response against the RBD than the comparative VB2060, which encodes only the first polypeptide (Mann-Whitney test, TECH001-CV021: P = 0.008, TECH001-CV023: P = 0.047). Furthermore, TECH001-CV022, which encodes the first polypeptide and two subdomains of IL-12 as the second and third proteins, also induced a stronger IgG response against the RBD than the comparative VB2060 (Mann-Whitney test, P = 0.047).

[0539] Assessment of T cell responses to SARS-CoV-2 RBD Spleens from mice injected with DNA plasmids were harvested 14 days after injection and crushed with a cell strainer to obtain a single cell suspension. Red blood cells were lysed with ammonium chloride-potassium (ACK) lysis buffer. Spleen cells were counted using a NucleoCounter NC-202 (ChemoMetec, Denmark) and 6x10 6 The cells were then resuspended to a final concentration of 6x10 cells / ml. For each plasmid tested, a portion of the single cell suspension was used to deplete CD4+ T cells using Dynabeads® anti-CD4 beads. Total splenocytes and CD4+ T cell depleted splenocytes were then diluted with 6x10 5 Cells / well were seeded and tested for INF-γ production in the FluoroSpot assay by restimulating for 22.5 hours with 2 μg / ml of the RBD peptide pool (Table 18), which contained 15mer peptides overlapping by 12 amino acids spanning a region of the RBD. [Table 18]

[0540] The results shown in FIG. 44 show that TECH001-CV021 and TECH001-CV023, which encode a first polypeptide containing the RBD (aa319-542) derived from SARS-CoV-2 (Wuhan variant) in the antigen unit and GM-CSF and IL-21 as the second protein, respectively, induced a much stronger total T cell response against the RBD (FIG. 44A) compared to the comparative VB2060, which encodes only the aforementioned first polypeptide. Furthermore, TECH001-CV021 and TECH001-CV023 induced a stronger CD8+ T cell response (CD4+ depleted spleen cell fraction) compared to VB2060 (FIG. 44B). TECH001-CV022, which encodes the aforementioned first polypeptide and two subdomains of IL-12 as the second and third proteins, also induced a much stronger total T cell response against the RBD (FIG. 44A) compared to VB2060. The increased T cell response induced by the addition of IL-12 cytokine appeared to be primarily due to increased IFN-γ secretion from CD4+ T cells, as a greater decrease in response was observed in TECH001-CV022 CD4+ T cell depleted samples compared to the TECH001-CV021 and TECH001-CV023 treated groups (Figure 44B).

[0541] In summary, the presented results show that the humoral and cellular immune responses elicited in mice against SARS-CoV-2 RBD were enhanced by co-expression of a first polypeptide / dimer protein comprising a targeting unit, a dimerization unit and an antigen unit comprising an infectious antigen (RBD derived from the pathogen SARS-CoV-2) with an immunostimulatory compound (GM-CSF, IL-12 or IL-21) compared to expression of the first polypeptide / dimer protein alone. Sequence Overview SEQ ID NO:1 M 1 QVSTAALAVLLCTMALCNQVLS 23 A 24PLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSA 93 E 94 LKTPLGDTTHT 105 E 106 PKSCDTPPPCPRCP 120 G 121 GGSSGGGSG 130 G 131 QPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK 237 SEQ ID NO:2 Signal peptide MNFGLRLIFLVLTLKGVQC SEQ ID NO:3 Signal peptide MDAMKRGLCCVLLLCGAVFVSP SEQ ID NO:4 Signal peptide of human FLT3L MTVLAPAWSPTTYLLLLLLLSSGLSG SEQ ID NO:5 VB4194 MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQ PREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLKSWIHCWKYLSVQSQLFRGSSL LFRRVGGGGSGGGGGSNNLQKYIEIYVQKINPSRLPVVIGGLLGGGGSGGGGSEVIQTSKYYMRDVIAIESAWLLELAPHGGGGSGGGGSVILPQAPSGPSYATYLQPAQAQMLTPPGGGGSGGGGSFVSPMA HYVPGIMAIESVVARFQFIVPGGGGGSGGGGSGDVKIHAHKVVLANISPYFKAMFTGNLGGGGSGGGGSTPLRKHTVHAIRKFYLEFKGSSPPPRLGGGGSGGGGSKIYEFDYHLYGQNITMIMTSVSGHLLA SEQ ID NO:6 VB4168 MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNT TPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLKSWIHCWKYLSVQSQLFRGSSLLFRRVGGGGSGGGGSNNLQKYIEIYVQKINPSRLPVVIGGLLGGGGSGGGGSEVIQTSKYYMRDVIAIESAWLLELAPHGGGGSGGGGSVILPQAPSGPSYA TYLQPAQAQMLTPPGGGGSGGGGSFVSPMAHYVPGIMAIESVVARFQFIVPGGGGGSGGGGSGDVKIHAHKVVLANISPYFKAMFTGNLGGGGSGGGGSTPLRKHTVHAIRKFYLEFKGSSPPPRLGGGGSGGGGSKIYEFDYHLYGQNITMIMTSVSGHLLAGSGEGRGSLLTCGDVEENPGP MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSRPLEATAPTAPQP SEQ ID NO:7 VB4169 MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDT PPPCPRCPGGGSSGGGSGGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHN RFTQKSLSLSPGKGLGGLKSWIHCWKYLSVQSQLFRGSLLFRRVGGGGGSGGGGSNNLQKYIEIYVQKINPSRLPVVIGGLLGGGGSGGGGSEVIQTSKYYMRDVIAIESAW LLELAPHGGGGSGGGGSVILPQAPSGPSYATYLQPAQAQMLTPPGGGGSGGGGSFVSPMAHYVPGIMAIESVVARFQFIVPGGGGGSGGGGSGDVKIHAHKVVLANISPYFKA MFTGNLGGGGSGGGGSTPLRKHTVHAIRKFYLEFKGSSPPPRLGGGGSGGGGSKIYEFDYHLYGQNITMIMTSVSGHLLAGSGEGRGSLLTCGDVEENPGPMTVLAPAWSPT TYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFV QTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPGSGATNFSLLKQAGDVEENPGPMWLQNLLFLGIVVYSLSAPTRSPITVTRP WKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQK SEQ ID NO:8 VB4170 SEQ ID NO:9 T2A EGRGSLLTCGDVEENPGP SEQ ID NO:10 Human FLT3L TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQP SEQ ID NO:11 P2A ATNFSLLKQAGDVEENPGP SEQ ID NO:12 Signal peptide mouse GM-CSF MWLQNLLFLGIVVYSLS SEQ ID NO:13 Mouse GM-CSF APTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQK SEQ ID NO:14 E2A QCTNYALLKLAGDVESNPGP SEQ ID NO:15 Signal peptide mouse CCL5 MKISAAALTIILTAAALCTPAPA SEQ ID NO:16 Mouse CCL5 SPYGSDTTPCCFAYLSLALPRAHVKEYFYTSSKCSNLAVVFVTRRNRQVCANPEKKWVQEYINYLEMS SEQ ID NO:17 Linker GGGGSGGGGS SEQ ID NO:18 VB4202 MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLKSWIHCWKYLSVQSQLFRGSSLLFRRVGGGGGSGGGGSNNLQKYIEIYVQKINPSRLPVVIGGLLGGGGSGGGGSEVIQTSKYYMRDVIAIESAWLLELAPHGG GGSGGGGSVILPQAPSGPSYATYLQPAQAQMLTPPGGGGSGGGGSFVSPMAHYVPGIMAIESVVARFQFIVPGGGGGSGGGGSGDVKIHAHKVVLANISPYFKAMFTGNLGGGGSGGGGSTPLRKHTVHAIRKFYLEFKGSSPPPRLGGGGSGGGGSKIYEFDYHLYGQNITM IMTSVSGHLLAGSGEGRGSLLTCGDVEENPGPMWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQK SEQ ID NO:19 VB1020 MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSS GGGSGGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLMHGDTPTL HEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGSGGSGMFQDPQERPRKLPQLCTELQTTIHD IILECVYCKQQLLRREVYDFARRDLCIVYRDGNPYAVRDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINRQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL SEQ ID NO:20 VB4195 MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQ PENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLMHGDTTPTLHEYMLDLQPETTDLYGYGQLNDSSEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGSSGGGSGMFQ DPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFARRDLCIVYRDGNPYAVRDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINRQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLGSGEGRGSLLTCGDVEENPGPMTVLAPA WSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQP SEQ ID NO:21 VB4196 MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPK SCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSV MHEALHNRFTQKSLSLSPGKGLGGLMHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLL MGTLGIVCPICSQKPGGGSSGGGSGMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFARRDLCIVYRDGNPYAVRDKCLKFYSKISEYRHYCYSLY GTTLEQQYNKPLCDLLIRCINRQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLGSGEGRGSLLTCGDVEENPGPMTVLAPAWSPTTYLLLLLL SSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISR LLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPGSGATNFSLLKQAGDVEENPGPMWLQNLLFLGIVVYSLSAPTRSPITVTRPWKH VEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQK SEQ ID NO:22 VB4204 MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESSGQPENNYNTTPPMLDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLMHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGI VCPICSQKPGGGSSGGGSGMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFARRDLCIVYRDGNPYAVRDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINRQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRR ETQLGSGEGRGSLLTCGDVEENPGPMWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQK SEQ ID NO:23 VB4205 MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLMHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDST LRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGSSGGGSGMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFARRDLCIVYRDGNPYAVRDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINRQKP LCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLGSGEGRGSLLTCGDVEENPGPMKISAAALTIILTAAALCTPAPASPYGSDTTPCCFAYLSLALPRAHVKEYFYTSSKCSNLAVVFVTRRNRQVCANPEKKWVQEYINYLEMS SEQ ID NO:24 VB4208 MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTV DKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLGSGEGRGSLLTCGDVEENPGPMWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQK SEQ ID NO:25 Nucleotide sequence encoding amino acids 24-93 of SEQ ID NO:1 GCACCACTTGCTGCTGACACGCCGACCGCCTGCTGCTTCAGCTACACCTCCCGACAGATTCCACAGAATTTCATAGCTGACTACTTTGAGACGAGCAGCCAGTGCTCCAAGCCCAGTGTCATCTTCCTAACCAAGAGAGGCCGGCAGGTCTGTGCTGACCCCAGTGAGGAGTGGGTCCAGAAATACGTCAGTGACCTGGAGCTGAGTGCC SEQ ID NO:26 Nucleotide sequence encoding amino acids 94-120 of SEQ ID NO:1 GAGCTCAAAACCCCACTTGGTGACACAACTCACACAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCA SEQ ID NO:27 Nucleotide sequence encoding amino acids 131-237 of SEQ ID NO:1 GGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA Sequence number 28 Nucleotide sequence encoding amino acids 94 - 237 of SEQ ID NO: 1 GAGCTCAAAACCCCACTTGGTGACACAACTCACACAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGGCGGTGGAAGCAGCGGAGGTGGAAGTGGAGGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA Sequence number 29 Nucleotide sequence encoding amino acids 1 - 23 of SEQ ID NO: 1 ATGCAGGTCTCCACTGCTGCCCTTGCCGTCCTCCTCTGCACCATGGCTCTCTGCAACCAGGTCCTCTCT SEQ ID NO:30 SARS-CoV-2 RBD (amino acids 319-542) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF SEQ ID NO:31 TECH001-CV021 MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLND LCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFGSGEG RGSLLTCGDVEENPGPMWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQK SEQ ID NO:32 TECH001-CV022 SEQ ID NO:33 TECH001-CV023 MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF TNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFGSGEGRGSL LTCGDVEENPGPMERTLVCLVVIFLGTVAHKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLDPELLSAPQDVKGHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSCDSYEKRTPKEFLERLKWLLQKMIHQHLS SEQ ID NO:34 Mouse IL-12 A signal peptide MCQSRYLLFLATLALLNHLSLA SEQ ID NO:35 Mouse IL-12 A RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA SEQ ID NO:36 Mouse IL-12 B signal peptide MCPQKLTISWFAIVLLVSPLMA SEQ ID NO:37 Mouse IL-12 B MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVT LDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS SEQ ID NO:38 Mouse IL-21 signal peptide MERTLVCLVVIFLGTVA SEQ ID NO:39 Mouse IL-21 HKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLDPELLSAPQDVKGHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSCDSYEKRTPKEFLERLKWLLQKMIHQHLS SEQ ID NO:40 Human GM-CSF signal peptide MWLQSLLLLGTVACSIS SEQ ID NO:41 Human GM-CSF APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE SEQ ID NO:42 Human CCL5 signal peptide MKVSAAALAVILIATALCAPASA SEQ ID NO:43 Human CCL5 SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMS SEQ ID NO:44 Human IL-12A signal peptide MCPARSLLLVATLVLLDHLSLA SEQ ID NO:45 Human IL-12A RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS SEQ ID NO:46 Human IL-12B signal peptide MCHQQLVISWFSLVFLASPLVA SEQ ID NO:47 Human IL-12B IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATL SAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS SEQ ID NO:48 Human IL-21 signal peptide MRSSPGNMERIVICLMVIFLGTLV SEQ ID NO:49 Human IL-21 HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS EMBODIMENT 1 (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit that comprises one or more antigens or portions thereof, e.g., one or more disease-associated antigens or portions thereof; and (b) one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds; A vector comprising: The vector can co-express the first polypeptide and one or more immunostimulatory compounds as separate molecules. EMBODIMENT 2 The vector of embodiment 1, wherein the one or more immunostimulatory compounds are compounds that affect antigen-presenting cells, including dendritic cells, macrophages, Langerhans cells, B cells and neutrophils, e.g., compounds that stimulate antigen-presenting cells, preferably, the one or more immunostimulatory compounds are compounds that affect human antigen-presenting cells, including human dendritic cells, human macrophages, human Langerhans cells, human B cells and human neutrophils. EMBODIMENT 3 The vector of embodiment 1 or 2, wherein the one or more immunostimulatory compounds promote the attraction and / or activation and / or maturation and / or proliferation, e.g., growth and / or expansion, of antigen-presenting cells. EMBODIMENT 4 The vector of any one of embodiments 1 to 3, wherein the one or more immunostimulatory compounds promote the attraction of antigen-presenting cells. EMBODIMENT 5 The vector of embodiment 4, wherein the one or more immunostimulatory compounds is a chemokine, preferably a human chemokine. EMBODIMENT 6 The vector of embodiment 5, wherein the one or more immunostimulatory compounds are capable of interacting with a surface molecule on an antigen-presenting cell selected from the group consisting of CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 and XCR1, preferably, the one or more immunostimulatory compounds are capable of interacting with a surface molecule on a human antigen-presenting cell selected from the group consisting of hCCR1, hCCR3, hCCR4, hCCR5, hCCR6, hCCR7, hCCR8 and hXCR1. EMBODIMENT 7 The vector according to embodiment 5 or 6, wherein the one or more immunostimulatory compounds are selected from the list consisting of macrophage inflammatory protein alpha including its isoforms, such as mouse CCL3, human CCL3, human CCL3L1, human CCL3L2 and human CCL3L3, CCL4, preferably hCCL4, CCL5, preferably hCCL5, CCL19, preferably hCCL19, CCL20, preferably hCCL20, CCL21, preferably hCCL21, XCL1, preferably hXCL1 and XCL2, preferably hXCL2. EMBODIMENT 8 The vector of any one of embodiments 3 to 7, wherein the one or more immunostimulatory compounds promote activation and / or maturation of antigen-presenting cells. EMBODIMENT 9 The vector described in any of embodiments 3 to 8, wherein the one or more immunostimulatory compounds are capable of interacting with a surface molecule on an antigen-presenting cell selected from the group consisting of receptors of the TNF receptor superfamily including CD40 (cluster of differentiation 40), CD137 (4-1BB), CD27, RANK, and ICOS (CD278), preferably, the one or more immunostimulatory compounds are capable of interacting with a surface molecule on a human antigen-presenting cell selected from the group consisting of receptors of the human TNF receptor superfamily including hCD40, hCD137, hCD27, hRANK, and hICOS. EMBODIMENT 10 10. The vector of embodiment 9, wherein the one or more immunostimulatory compounds are selected from the list consisting of CD40L, CD137L, CD70, RANKL and ICOSL, preferably, the one or more immunostimulatory compounds are selected from the list consisting of hCD40L, hCD137L, hCD70, hRANKL and hICOSL. EMBODIMENT 11 The vector according to any one of embodiments 3 to 8, wherein the one or more immunostimulatory compounds are cytokines selected from the group consisting of IL-2, IL-10, IL-12, IL-21, TNFα, IFNγ and IL-1β, preferably the one or more immunostimulatory compounds are human cytokines selected from the group consisting of hIL-2, IL-10, hIL-12, hIL-21, hTNFα, hIFNγ and hIL-1β. EMBODIMENT 12 The vector according to any of embodiments 3 to 8, wherein the one or more immunostimulatory compounds are a viral infection sensor such as MyD88 or TRIF, preferably a human viral infection sensor such as human MyD88 or human TRIF. EMBODIMENT 13 The vector of any one of embodiments 3 to 8, wherein the one or more immunostimulatory compounds are capable of interacting with a pattern recognition receptor on an antigen-presenting cell, such as a Toll-like receptor, including TLR2, TLR4, TLR5 and TLR9, and / or a receptor on an antigen-presenting cell selected from the group consisting of RAGE, TIM-3, FPR, SREC1, LOX1 and CD91, preferably, the one or more immunostimulatory compounds are capable of interacting with a pattern recognition receptor on a human antigen-presenting cell, such as a human Toll-like receptor, including hTLR2, hTLR4, hTLR5 and hTLR9, and / or a receptor on a human antigen-presenting cell selected from the group consisting of hRAGE, hTIM-3, hFPR, hSREC1, hLOX1 and hCD91. EMBODIMENT 14 The vector of embodiment 13, wherein the one or more immunostimulatory compounds are selected from the group consisting of pathogen-associated molecular patterns (PAMPs) such as flagellin, HMGB1, heat shock proteins (HSPs), protein damage-associated molecular patterns (DAMPs) such as callecticulin and annexin A1, preferably, the one or more immunostimulatory compounds are selected from the group consisting of human...

Claims

1. (a) a first nucleic acid sequence encoding a first polypeptide, where the first polypeptide comprises a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigen unit that comprises one or more antigens or portions thereof, e.g., one or more disease-associated antigens or portions thereof; and (b) one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds. A vector comprising: The vector is capable of co-expressing a first polypeptide and the one or more immunostimulatory compounds as separate molecules.

2. The vector of claim 1 , wherein the one or more immunostimulatory compounds promote attraction and / or activation and / or maturation and / or proliferation, e.g., growth and / or expansion, of antigen presenting cells, preferably human antigen presenting cells.

3. the one or more immunostimulatory compounds promote the attraction of antigen-presenting cells; Preferably, the one or more immunostimulatory compounds are chemokines, preferably human chemokines; More preferably, the one or more immunostimulatory compounds are capable of interacting with a surface molecule on an antigen presenting cell selected from the group consisting of CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 and XCR1, e.g., a surface molecule on a human antigen presenting cell selected from the group consisting of hCCR1, hCCR3, hCCR4, hCCR5, hCCR6, hCCR7, hCCR8 and hXCR1; Most preferably, the one or more immunostimulatory compounds are selected from the list consisting of macrophage inflammatory protein alpha including its isoforms such as, for example, murine CCL3, human CCL3, human CCL3L1, human CCL3L2 and human CCL3L3, CCL4, preferably human CCL4, CCL5, preferably human CCL5, CCL19, preferably human CCL19, CCL20, preferably human CCL20, CCL21, preferably human CCL21, XCL1, preferably human XCL1 and XCL2, preferably human XCL2.

4. the one or more immunostimulatory compounds promote activation and / or maturation of antigen-presenting cells, preferably the one or more immunostimulatory compounds are capable of interacting with a surface molecule on an antigen-presenting cell selected from the group consisting of receptors of the TNF receptor superfamily including CD40 (cluster of differentiation 40), CD137 (4-1BB), CD27, RANK, and ICOS (CD278), e.g., interacting with a surface molecule on a human antigen-presenting cell selected from the group consisting of receptors of the human TNF receptor superfamily including hCD40, hCD137, hCD27, hRANK, and hICOS; 4. The vector of claim 3, wherein the one or more immunostimulatory compounds are selected from the list consisting of CD40L, CD137L, CD70, RANKL and ICOSL, such as hCD40L, hCD137L, hCD70, hRANKL and hICOSL.

5. the one or more immunostimulatory compounds are a) cytokines selected from the group consisting of IL-2, IL-10, IL-12, IL-21, TNFα, IFNγ and IL-1β, preferably the one or more immunostimulatory compounds are human cytokines selected from the group consisting of hIL-2, IL-10, hIL-12, hIL-21, hTNFα, hIFNγ and hIL-1β; and / or b) a viral infection sensor, such as MyD88 or TRIF, preferably a human viral infection sensor, such as human MyD88 or human TRIF, and / or c) may interact with a pattern recognition receptor on an antigen presenting cell, such as a Toll-like receptor, including TLR2, TLR4, TLR5 and TLR9, and / or a receptor on an antigen presenting cell selected from the group consisting of RAGE, TIM-3, FPR, SREC1, LOX1 and CD91, preferably said one or more immunostimulatory compounds may interact with a pattern recognition receptor on a human antigen presenting cell, such as a human Toll-like receptor, including hTLR2, hTLR4, hTLR5 and hTLR9, and / or a receptor on a human antigen presenting cell selected from the group consisting of hRAGE, hTIM-3, hFPR, hSREC1, hLOX1 and hCD91, and / or d) selected from the group consisting of pathogen-associated molecular patterns (PAMPs) such as flagellin, HMGB1, heat shock proteins (HSPs), protein damage-associated molecular patterns (DAMPs) such as calrecticulin and annexin A1, preferably wherein the one or more immunostimulatory compounds are selected from the group consisting of human pathogen-associated molecular patterns (PAMPs), human protein damage-associated molecular patterns (DAMPs) such as hHMGB1, human heat shock proteins (HSPs), human calrecticulin and human annexin A1.

6. the one or more immunostimulatory compounds promote the growth and / or expansion of antigen-presenting cells; Preferably, the one or more immunostimulatory compounds are a) a growth factor, preferably a human growth factor, and / or b) capable of interacting with a surface molecule on antigen-presenting cells selected from the group consisting of GM-CSF-receptor, FLT-3R, IL-15R and IL-4R, preferably said one or more immunostimulatory compounds capable of interacting with a surface molecule on human antigen-presenting cells selected from the group consisting of hGM-CSF-receptor, hFLT-3R, hIL-15R and hIL-4R, and / or c) selected from the group consisting of GM-CSF, FLT-3L, IL-15 and IL-4, preferably, the one or more immunostimulatory compounds are selected from the group consisting of hGM-CSF, hFLT-3L, hIL-15 and hIL-4.

7. the vector comprises a plurality of further nucleic acid sequences encoding more than one immunostimulatory compound, e.g., 2, 3, 4, 5, 6, 7, or 8 immunostimulatory compounds, e.g., 2, 3, 4, 5, 6, 7, or 8 different immunostimulatory compounds; The vector of claim 6 , wherein the multiple immunostimulatory compounds are preferably different immunostimulatory compounds that differentially affect (e.g. stimulate) antigen presenting cells.

8. The vector comprises one or more a) comprising a co-expression element which causes the transcription of the first polypeptide and said one or more immunostimulatory compounds on a single transcript and their independent translation into a separate first polypeptide and separate one or more immunostimulatory compounds, and which is preferably selected from the list consisting of an IRES element or a 2A self-cleaving peptide; and / or 8. The vector of claim 7, wherein b) it comprises a co-expression element which causes transcription of the first polypeptide and the one or more immunostimulatory compounds as separate transcription products and which is preferably selected from the list consisting of a bidirectional promoter and a promoter, the vector comprising a separate promoter for each of the nucleic acid sequences encoding the first polypeptide and the one or more immunostimulatory compounds.

9. The antigen unit is a) comprising one or more neo-antigens or portions thereof, e.g., a neo-epitope, e.g., a plurality of neo-epitopes, such as a plurality of neo-epitopes separated from each other by a linker, and optionally further comprising one or more patient-presented shared cancer antigens or portions thereof, e.g., a patient-presented shared cancer epitope, e.g., one or more patient-presented shared cancer antigens or portions thereof, e.g., a patient-presented shared cancer epitope, or The vector of claim 8, comprising b) one or more shared cancer antigens or parts thereof, e.g., shared cancer epitopes.

10. the antigenic unit comprises one or more antigens from one or more pathogens and / or parts of such antigens; and When the antigenic unit comprises one or more portions of one or more antigens derived from one or more pathogens, such moieties are B-cell epitopes, such that said antigenic units contain one or more B-cell epitopes derived from one or more pathogens; or such moieties are T cell epitopes, such that the antigenic unit comprises one or more T cell epitopes derived from one or more pathogens; and The vector according to claim 8 , wherein the one or more pathogens are preferably selected from the group consisting of viruses, bacteria, fungi and parasites.

11. the targeting unit is or comprises a moiety that interacts with a surface molecule on an antigen-presenting cell, For example, the surface molecule is selected from the group consisting of MHC, HLA, CD14, CD40, CLEC9A, a chemokine receptor, e.g., CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 or XCR1, and a Toll-like receptor, e.g., TLR-2, TLR-4 or TLR-5, preferably, the surface molecule is selected from the group consisting of HLA, hCD14, hCD40, hCLEC9A, a human chemokine receptor, e.g., hCCR1, hCCR3, hCCR4, hCCR5, hCCR6, hCCR7, hCCR8 or hXCR1, and a human Toll-like receptor, e.g., hTLR-2, hTLR-4 or hTLR-5, Preferably, the targeting unit comprises or consists of soluble CD40 ligand, CCL4 and its isoforms, CCL5, CCL19, CCL20, CCL21, macrophage inflammatory protein alpha including isoforms such as mouse CCL3, human CCL3, human CCL3L1, human CCL3L2 and human CCL3L3, XCL1, XCL2, flagellin, anti-HLA-DP, anti-HLA-DR, anti-pan-HLA class II, anti-CD40, anti-TLR-2, anti-TLR-4, anti-TLR-5 or anti-CLEC9A. Preferably, said targeting unit comprises or consists of soluble hCD40 ligand, hCCL4 and its isoforms, hCCL5, hCCL19, hCCL20, hCCL21, human macrophage inflammatory protein alpha, including isoforms such as human CCL3, human CCL3L1, human CCL3L2 and human CCL3L3, hXCL1, hXCL2, anti-HLA-DP, anti-HLA-DR, anti-pan-HLA class II, anti-hCD40, anti-hTLR-2, anti-hTLR-4, anti-hTLR-5 or anti-hCLEC9, Most preferably, the vector according to claim 9 or 10, wherein said targeting unit comprises or consists of human MIP-1α (LD78β, CCL3L1).

12. the multimerization unit is selected from the group consisting of a dimerization unit, a trimerization unit, for example a trimerization unit derived from collagen, for example a trimerization domain derived from human collagen, for example the XVIII trimerization domain derived from human collagen, or the human collagen XV trimerization domain, or the C-terminal domain of T4 fibritin, and a tetramerization unit, such as a domain derived from p53; Preferably, the multimerization unit is a dimerization unit, More preferably, said dimerization unit is capable of forming one or more covalent bonds and further comprises a hinge region, preferably from an Ig, and another domain promoting dimerization, preferably said other domain is an immunoglobulin domain, more preferably an immunoglobulin constant domain, most preferably the carboxy-terminal C domain from an IgG, preferably IgG3, The vector of claim 11, wherein the dimerization unit further comprises a dimerization unit linker, such as a glycine-serine rich linker such as GGGSSGGGSG (SEQ ID NO: 134), and preferably the dimerization unit linker connects the hinge region and the other domain that promotes dimerization.

13. the first nucleic acid sequence encodes a first polypeptide further comprising a unit linker linking the antigen unit and the multimerization unit, the unit linker being a non-immunogenic linker and / or a flexible or rigid linker; and / or 13. The vector of claim 12, wherein the first nucleic acid sequence encodes a first polypeptide further comprising a signal peptide, and preferably the one or more further nucleic acid sequences also further encode a signal peptide.

14. 14. The vector of claim 13, wherein the vector is a viral vector, such as an RNA viral vector or a DNA viral vector, or a plasmid, such as an RNA plasmid or a DNA plasmid, and optionally the vector is contained within a host cell, e.g. a host cell selected from the group consisting of a prokaryotic cell, a yeast cell, an insect cell, a higher eukaryotic cell, such as a cell of animal or human origin.

15. A vector according to claim 14 for use as a medicament.

16. 15. A pharmaceutical composition comprising the vector of claim 14 and a pharma- ceutically acceptable carrier or diluent, said composition further comprising a transfection agent.

17. A pharmaceutical composition for use in treating cancer or an infectious disease, comprising the vector described in claim 14.

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