CN118043066A

CN118043066A - Co-expression of constructs and immunostimulatory compounds

Info

Publication number: CN118043066A
Application number: CN202280045137.1A
Authority: CN
Inventors: 阿涅特·布伦斯维克·弗雷德里克森; A·T·H·伯萨斯; 斯蒂因·格拉纳姆; P·迪拉德
Original assignee: Necord Therapeutics Ltd
Current assignee: Necord Therapeutics Ltd
Priority date: 2021-05-10
Filing date: 2022-05-10
Publication date: 2024-05-14

Abstract

The present invention relates to vectors, such as DNA plasmids, comprising a plurality of nucleic acid sequences engineered to be co-expressed as separate molecules. Such separate molecules include a first polypeptide and one or more immunostimulatory compounds, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit, such as a dimerization unit, and an antigenic unit comprising one or more antigens or portions thereof.

Description

Co-expression of constructs and immunostimulatory compounds

Technical Field

The present invention relates to vectors, such as DNA plasmids, comprising a plurality of target nucleic acid sequences engineered to be co-expressed as separate molecules, pharmaceutical compositions comprising such vectors, and the use of such vectors and such pharmaceutical compositions in the treatment or prevention of diseases.

Background

Both B-cell responses (humoral/antibody-mediated responses) and T-cell responses are important components of protective responses against infection by pathogens. Specific antibodies to pathogen antigens can mediate a wide range of effector functions, such as a) direct neutralization of toxins or pathogens, B) neutralization of pathogen virulence factors, c) binding and capture of pathogens in mucins, d) activation of complement to mediate phagocytic clearance, degradation or lysis of anti-pathogens, e) activation of opsonophagocytosis of neutrophils, f) induction of opsonophagocytosis of macrophages, g) activation of degranulation of Natural Killer (NK) cells to kill infected cells, h) enhancement of antigen renewal, processing and presentation of dendritic cells to T cells and B cells, i) induction of degranulation of mast cells, basophils and eosinophils in the case of parasitic infection (l.lu et al, nat Rev Immunol 18 (1), 2018,46).

In addition to these activities, T cell responses are critical to limit viral replication and infection by killing infected cells, inducing apoptosis, releasing antiviral substances, and/or inducing an increase in intracellular lysis of infected cells, which helps prevent, reduce the severity of, or cure the disease. Furthermore, effective and sustained responses to both classes of immunity often require additional support by T helper (Th 1 and Th 2) lymphocytes.

Cytotoxic T Lymphocytes (CTLs) also play an important role in, for example, intracellular pathogens (f.shepherd et al, int J Mol Sci 21,2020,6144), where MHC class I-restricted cd8+ T cells are critical for clearing bacterial infections, and are known to provide protective immunity against a variety of bacterial species. MHC class II restricted cd4+ T cells support a memory cd8+ T cell response, which is important for protective immunity against bacterial infection. Naive cd4+ T cells can differentiate a subset of cells with effector capacity, such as T helper 1 (Th 1) cells and Th2 cells. Th1 and Th2 cells, upon binding to specific T cell epitopes on the surface of Antigen Presenting Cells (APCs), provide specific soluble cytokine signals that regulate the balance between antibody and CTL immunity. Thus, effective immunization involves multiple antigen recognition events of a T helper cell to a particular pathogen's immunogenic determinant (epitope), followed by molecular recognition by B cells, CTLs, or both.

Different types of lymphocytes (B cells, CTLs, and Th cells) specifically recognize different types of epitopes of pathogens. B cell epitopes can be classified as linear epitopes or conformational epitopes, with linear epitopes typically being part of conformational B cell epitopes in natural proteins. Conformational epitopes are exposed structural features on the surface of pathogens such as viral envelopes, bacterial outer membranes or secreted bacterial toxins. T cell epitopes are short peptides of any protein of a pathogen, which need only conform to host antigen processing and MHC binding mechanisms, especially class I or class II MHC haplotype restriction mechanisms. The predicted frequency of occurrence of suitable T cell epitopes is a sequence of about 1 epitope per 200-500 amino acids, depending on the host population and pathogen.

Vaccines against pathogens comprise pathogens or parts thereof which are modified in a way that does not cause injury or disease but which ensures that when the host is faced with the infectious agent, the immune system can adequately neutralize the infectious agent before it causes any adverse effects. Vaccination has been performed for over a hundred years by one of two methods: introduction of specific antigens with which the immune system reacts directly, or introduction of live attenuated infectious agents that replicate in the host without causing disease and synthesize antigens that subsequently prime the immune system.

Recently, a completely new vaccination method has been developed. A polynucleotide sequence (DNA or RNA) encoding an antigen capable of stimulating an immune response is introduced directly into the appropriate tissue to produce the target antigen in situ. This approach has many potential advantages over traditional approaches, including stimulating both B-cell and T-cell responses, improving vaccine stability, absence of any infectious material, and relative ease of mass production.

Although cancer treatment has improved over the past few decades, particularly due to early detection and diagnosis, significantly improves survival in cancer patients, only about 60% of patient-like patients survive 5 years after diagnosis. Most of the cancer treatments used are surgery, radiation therapy and cytotoxic chemotherapy, but they all have serious side effects. Currently, antibodies directed against known cancer-associated antigens are also used for treatment.

Cancer immunotherapy (i.e., cancer vaccine) that targets cancer cells via the patient's autoimmune system has been of interest over the past few years. Such treatments may reduce or even eliminate some of the side effects associated with traditional cancer treatments.

Thus, there is a need for effective agents and medicaments that can be used both for the treatment or prevention of infectious diseases and for the treatment or prevention of cancer.

The vaccine body (vaccibody) construct is a dimeric fusion protein consisting of two polypeptides, each polypeptide comprising a targeting unit that targets antigen presenting cells, a dimerization unit, and an antigenic unit that comprises one or more antigens or portions thereof, and which is effective to generate an immune response against the antigen or portion (e.g., epitope) thereof contained in the antigenic unit upon administration to a subject (e.g., animal or human). In another embodiment, the vaccine construct is a multimeric fusion protein consisting of a plurality of polypeptides, each polypeptide comprising a targeting unit that targets antigen presenting cells, a multimerizing unit, and an antigenic unit comprising one or more antigens or portions thereof, and which has been shown to be effective to generate an immune response against the antigens or portions thereof (e.g., epitopes) contained in the antigenic unit upon administration to a subject.

The vaccine construct may be administered to a subject in the form of a polynucleotide encoding a polypeptide (e.g., a polynucleotide contained in a vector such as a DNA plasmid). Upon administration to a host cell, e.g., to a muscle cell of a subject, a polypeptide is expressed that forms a multimeric fusion protein (e.g., dimer) due to multimerization units (e.g., dimerization units).

Disclosure of Invention

The inventors have surprisingly found that the overall immune response of a vaccine body can be enhanced by co-expressing one or more immunostimulatory compounds from the same vector in which the vaccine body is expressed.

In a first aspect, the invention relates to a carrier comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerization unit, such as a dimerization unit, and an antigenic unit comprising one or more antigens or portions thereof; and

(B) One or more additional nucleic acid sequences encoding one or more immunostimulatory compounds,

Wherein the vector allows co-expression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules.

In one embodiment, the vector of the invention comprises a first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit, such as a dimerization unit, and an antigenic unit comprising one or more disease associated antigens or portions thereof. Such a carrier may be used for prophylactic or therapeutic treatment of a disease, for example for therapeutic treatment of cancer or for prophylactic or therapeutic treatment of an infectious disease, for example, by administering the composition to a subject in need of such prophylactic or therapeutic treatment, in the form of a pharmaceutical composition comprising such a carrier and a pharmaceutically acceptable carrier or diluent.

Drawings

Fig. 1: co-expression elements for vectors of the invention

IRES co-expression elements for use in the vectors of the invention are shown, the elements being inserted between two coding regions. When mRNA is produced, two ribosomes (T) can begin translation at two separate sites on the mRNA and form two proteins (a and B). A and B may be, for example, a first polypeptide and an immunostimulatory compound.

Fig. 2: co-expression elements for vectors of the invention

The 2A self-cleaving peptide co-expression element for the vector of the present invention is shown, which is inserted between two genes. After transcription, one ribosome translates the mRNA and forms two proteins (a and B). The upper panel shows how the fusion protein is formed if the 2A peptide sequence is not part of the coding sequence. A and B may be, for example, a first polypeptide and an immunostimulatory compound.

Fig. 3: co-expression elements for vectors of the invention

FIG. 3a shows a bi-directional promoter (P) co-expression element for use in the vectors of the invention, said element being located between two coding regions. An mRNA is produced and two ribosomes (T) are able to start translation in separate directions and form two proteins (A and B). A and B may be, for example, a first polypeptide and an immunostimulatory compound. FIG. 3a shows two promoters (P), i.e.the coexpression elements for the vectors of the invention, which are located before the two coding regions. Two mRNAs were produced and two ribosomes (T) were able to start translation at two different mRNAs and form two proteins (A and B). A and B may be, for example, a first polypeptide of the invention and an immunostimulatory compound.

Fig. 4: embodiments of the first polypeptide

Embodiments of a first polypeptide encoded by a first nucleic acid sequence comprised in a vector of the invention are described.

Fig. 5: expression and secretion levels of proteins encoded by DNA plasmids

Protein expression and secretion levels of the first polypeptide encoded by the DNA plasmids VB4194, VB4168, VB4169 and VB4170 are shown to be detected in the supernatant of HEK293 cells transfected with said DNA plasmids by enzyme-linked immunosorbent assay (ELISA) using a mouse a-human IgG CH3 domain capture antibody (MCA 878G) and a-human MIP-1α biotinylated capture antibody (BAF 270). Lipo negative control (=cells treated with transfection agent Lipofectamine alone, used as negative control).

Fig. 6: expression and secretion levels of proteins encoded by DNA plasmids

Shows that protein expression and secretion levels of immunostimulatory compound FLTL encoded by the DNA plasmids VB4168, VB4169 and VB4170 were detected in supernatants of HEK293 cells transfected with the DNA plasmids using a mouse α -human FLT3L capture antibody (MAB 608) and a mouse α -human FLT3L biotinylated detection antibody (BAF 308) by ELISA.

Fig. 7: expression and secretion levels of proteins encoded by DNA plasmids

Protein expression and secretion levels of the immunostimulatory compound GM-CSF encoded by the DNA plasmids VB4169 and VB4170 are shown to be detected in supernatants of HEK293 cells transfected with the DNA plasmids using a rat a-mouse GM-CSF capture antibody (MAB 415) and goat a-mouse GM-CSF biotinylation detection antibody (BAM 215) by ELISA.

Fig. 8: expression and secretion levels of proteins encoded by DNA plasmids

Protein expression and secretion levels of immunostimulatory compound CCL5 encoded by DNA plasmid VB4170 in the supernatant of HEK293 cells transfected with the DNA plasmid are shown using a rat a-mouse CCL5 capture antibody (MAB 4781) and a goat a-mouse CCL5 biotinylated detection antibody (BAF 478) by ELISA.

Fig. 9: immunogenicity of DNA plasmids

The immunogenicity of DNA plasmids VB4194, VB4168 and VB4169 in mice administered with these plasmids compared to the negative control VB1026 by measuring IFN- γ secretion (total T cell response) by T cells is shown.

Fig. 10: immunogenicity of DNA plasmids

The immunogenicity of DNA plasmids VB4194, VB4168 and VB4169 in mice administered with these plasmids compared to the negative control VB1026 by measuring TNF- α secretion (total T cell response) of T cells is shown.

Fig. 11: immunogenicity of DNA plasmids

The immunogenicity of DNA plasmids VB4194, VB4168 and VB4169 in mice administered with these plasmids compared to negative control VB1026 by measuring IFN- γ+tnf- α co-secretion (total T cell response) of T cells is shown.

Fig. 12: immunogenicity of DNA plasmids

The immunogenicity of DNA plasmids VB4194, VB4168 and VB4169 in mice administered with these plasmids compared to negative control VB1026 by measuring IFN- γ secretion by cd8+ T cells (cd4+ T cell depletion samples) is shown.

Fig. 13: immunogenicity of DNA plasmids

The immunogenicity of DNA plasmids VB4194, VB4168 and VB4169 in mice administered with these plasmids compared to negative control VB1026 by measuring TNF- α secretion by cd8+ T cells (cd4+ T cell depletion samples) is shown.

Fig. 14: immunogenicity of DNA plasmids

The immunogenicity of DNA plasmids VB4194, VB4168 and VB4169 in mice administered with these plasmids compared to negative control VB1026 by measuring IFN- γ+tnf- α secretion by cd8+ T cells (cd4+ T cell depletion samples) is shown.

Fig. 15: expression and secretion levels of proteins encoded by DNA plasmids

Shows the detection of the protein expression and secretion levels of the first polypeptide encoded by the DNA plasmid VB4202 in the supernatant of HEK293 cells transfected with said DNA plasmid using a mouse a-human IgG CH3 domain capture antibody (MCA 878G) and a-human MIP-1α biotinylated capture antibody (BAF 270) by ELISA. Lipo (=cells treated with transfection agent Lipofectamine alone) was used as negative control.

Fig. 16: expression and secretion levels of proteins encoded by DNA plasmids

Protein expression and secretion levels of the immunostimulatory compound GM-CSF encoded by the DNA plasmid VB4202 are detected in supernatants (1:1000 dilution) of HEK293 cells transfected with the DNA plasmid using a rat a-mouse GM-CSF capture antibody (MAB 415) and a goat a-mouse GM-CSF biotinylation detection antibody (BAM 215) by ELISA.

Fig. 17: immunogenicity of DNA plasmids

The immunogenicity of DNA plasmids VB4194 and VB4202 in mice administered with these plasmids compared to the negative control VB1026 by measuring IFN- γ secretion (total T cell response) by T cells is shown.

Fig. 18: immunogenicity of DNA plasmids

Flow cytometry evaluation is shown for assessing Dendritic Cell (DC) responses at the single cell level in mice administered with DNA plasmids VB1026, VB4194 and VB 4202. The figure shows a gating strategy for defining DCs. A. All events were checked using the time parameter to exclude any fluid inconsistencies. B. Side Scatter (SSC) height and area parameters are used to further exclude double peaks (doublet). C. Forward Scatter (FSC) height and area parameters are used to exclude double peaks. D. Dead cells, neutrophils and T cells were excluded and further analyzed using cd45+ immune cells. E. MHCII expressing cells were gated and used for further analysis. F. B cells and plasma cell-like (p) DCs were excluded from the analysis. G. DC was defined as cd24+. H. All DCs were classified as monocyte-derived (mo) DCs and classical (c) DCs based on CD11b and CD64 expression. I. Classical DCs are classified as cDC1 and cDC2 according to the expression of XCR1 and CD172a markers.

Fig. 19: immunogenicity of DNA plasmids

The proportion of viable cd45+ cells at the site of administration 1,2 and 4 days after intramuscular administration of DNA plasmid VB4202 compared to DNA plasmid VB4194 (comparison), VB1026 (negative control) is shown. EP-free vacc refers to the results of a group of mice that have not been administered with a plasmid and that have not been subjected to electroporation treatment.

Fig. 20: immunogenicity of DNA plasmids

The DC proportion in live cd45+ cells at the site of administration 1,2 and 4 days after intramuscular administration of DNA plasmid VB4202 compared to DNA plasmid VB4194 (comparison), VB1026 (negative control) is shown. EP-free vacc refers to the results of a group of mice that have not been administered with a plasmid and that have not been subjected to electroporation treatment.

Fig. 21: immunogenicity of DNA plasmids

The proportion of cd45+ cells in live cd45+ cells at the site of administration 1,2 and 4 days after intramuscular administration of DNA plasmid VB4202 compared to DNA plasmid VB4194 (comparison), VB1026 (negative control) is shown. EP-free vacc refers to the results of a group of mice that have not been administered with a plasmid and that have not been subjected to electroporation treatment.

Fig. 22: immunogenicity of DNA plasmids

The proportion of mocc cells in live cd45+ cells at the site of administration 1,2 and 4 days after intramuscular administration of DNA plasmid VB4202 compared to DNA plasmid VB4194 (comparison), VB1026 (negative control) is shown. EP-free vacc refers to the results of a group of mice that have not been administered with a plasmid and that have not been subjected to electroporation treatment.

Fig. 23: expression and secretion levels of proteins encoded by DNA plasmids

Protein expression and secretion levels of the first polypeptide encoded by the DNA plasmids VB1020, VB4195 and VB4196 are shown to be detected in supernatants of HEK293 cells transfected with said DNA plasmids using a mouse a-human IgG CH3 domain capture antibody (MCA 878G) and a-human MIP-1α biotinylated capture antibody (BAF 270) by ELISA. Lipofectamine (=cells treated with transfection agent Lipofectamine alone) was used as negative control.

Fig. 24: expression and secretion levels of proteins encoded by DNA plasmids

Protein expression and secretion levels of the immunostimulatory compound FLT3L encoded by the DNA plasmids VB4195 and VB4196 are detected in supernatants (1:500 dilution) of HEK293 cells transfected with the DNA plasmids using a mouse α -human FLT3L capture antibody (MAB 608) and a mouse α -human FLT3L biotinylated detection antibody (BAF 308) by ELISA. Lipofectamine (=cells treated with transfection agent Lipofectamine alone, used as negative control), supernatant from cells treated with Lipofectamine alone, without dilution, was used in ELISA.

Fig. 25: expression and secretion levels of proteins encoded by DNA plasmids

Protein expression and secretion levels of the immunostimulatory compound GM-CSF encoded by the DNA plasmid VB4196 are detected in the supernatant (1:500 dilution) of HEK293 cells transfected with the DNA plasmid using a rat a-mouse GM-CSF capture antibody (MAB 415) and a goat a-mouse GM-CSF biotinylation detection antibody (BAM 215) by ELISA. Lipofectamine (=cells treated with transfection agent Lipofectamine alone, used as negative control), supernatant from cells treated with Lipofectamine alone, without dilution, was used in ELISA.

Fig. 26: expression and secretion of intact proteins encoded by DNA plasmids

Western blot of non-reduced (left) and reduced (right) supernatant samples of Expi293F cells transfected with DNA plasmids VB1020, VB4195 and VB 4196. An antibody: goat a-human MIP-1α (BAF 270). And (2) secondary antibody: donkey is against goat, dylight550 (SA 5-10087). Chemidoc channels Dyight 550 and 650 (for protein standards). The black lanes contain samples that are not relevant to the present application.

Fig. 27: expression and secretion of intact proteins encoded by DNA plasmids

Western blotting of reduced supernatant samples (lanes 1-4) and deglycosylated supernatant samples (lanes 5-6) of the Expi293F cells transfected with DNA plasmids VB1020, VB4195 and VB 4196. Left: an anti-goat a-human FLT3L (BAF 308). And (2) secondary antibody: donkey is against goat, dylight 550 (SA 5-10087). Right: an anti-goat a-mouse GM-CSF (BAF 415). And (2) secondary antibody: donkey is against goat, dylight 550 (SA 5-10087). Chemidoc channels Dyight 550 and 650 (for protein standards). The black lanes contain samples that are not relevant to the present application.

Fig. 28: expression and secretion levels of proteins encoded by DNA plasmids

Protein expression and secretion levels of the first polypeptide encoded by the DNA plasmids VB1020 and VB4204 are shown to be detected in supernatants (1:10 dilution) of HEK293 cells transfected with said DNA plasmids using a mouse a-human IgG CH3 domain capture antibody (MCA 878G) and a-human MIP-1α biotinylated capture antibody (BAF 270) by ELISA. Lipo (=cells treated with transfection agent Lipofectamine alone) was used as negative control.

Fig. 29: expression and secretion levels of proteins encoded by DNA plasmids

Protein expression and secretion levels of the immunostimulatory compound GM-CSF encoded by the DNA plasmid VB4204 are detected in HEK293 cell supernatants (1:1000 dilution) transfected with the DNA plasmid using a rat a-mouse GM-CSF capture antibody (MAB 415) and goat a-mouse GM-CSF biotinylated detection antibody (BAM 215) by ELISA. Lipo (=cells treated with transfection agent Lipofectamine alone) was used as negative control.

Fig. 30: immunogenicity of DNA plasmids

The immunogenicity of DNA plasmids VB1020 and VB4204 in mice administered with these plasmids, and the immunogenicity of co-administered DNA plasmids VB1020 and pGM-CSF, compared to negative control VB1026, by measuring IFN- γ secretion (total T cell response) by T cells is shown.

Fig. 31: immunogenicity of DNA plasmids

The percentage of IFN-. Gamma., TNF-. Alpha.or CD8+ T cells co-secreting TNF-. Alpha.and IFN-. Gamma.in spleen cells of mice administered with the DNA plasmids VB1020 and VB4204 compared to the negative control VB1026 is shown.

Fig. 32: expression and secretion levels of proteins encoded by DNA plasmids

Protein expression and secretion levels of the first polypeptide encoded by the DNA plasmids VB1020 and VB4205 are shown to be detected in supernatants (1:10 dilution) of HEK293 cells transfected with said DNA plasmids using a mouse a-human IgG CH3 domain capture antibody (MCA 878G) and a-human MIP-1α biotinylated capture antibody (BAF 270) by ELISA. Lipo (=cells treated with transfection agent Lipofectamine alone) was used as negative control.

Fig. 33: expression and secretion levels of proteins encoded by DNA plasmids

Protein expression and secretion levels of immunostimulatory compound CCL5 encoded by DNA plasmid VB4205 are detected in supernatants (1:500 dilution) of HEK293 cells transfected with the DNA plasmid using a rat a-mouse CCL5 capture antibody (MAB 4781) and goat a-mouse CCL5 biotinylated detection antibody (BAF 478) by ELISA. Lipo (=cells treated with transfection agent Lipofectamine alone) was used as negative control.

Fig. 34: immunogenicity of DNA plasmids

The immunogenicity of DNA plasmids VB1020 and VB4205 in mice administered with these plasmids compared to the negative control VB1026 by measuring IFN- γ secretion (total T cell response) by T cells is shown.

Fig. 35: immunogenicity of DNA plasmids

CT26 tumor growth in BALB/c mice (n=8/group) vaccinated with 1x10 ⁵ CT26 tumor cells at D0 followed by administration of DNA plasmids VB4194, VB4208, VB4202 and pGM-csf+vb4194 (co-injection) at D4 and D11 compared to negative control VB1026 is shown.

Fig. 36: immunogenicity of DNA plasmids

The probability of survival of BALB/c mice (n=8/group) vaccinated with 1x10 ⁵ CT26 tumor cells at D0 followed by administration of DNA plasmids VB4194, VB4208, VB4202 and pGM-csf+vb4194 (co-injection) at D4 and D11 compared to the negative control VB1026 is shown.

Fig. 37: expression and secretion levels of proteins encoded by DNA plasmids

Secretion of the first polypeptide encoded by the DNA plasmid was shown to be detected by ELISA in supernatants of Expi293F cells transfected with DNA plasmids VB2060, TECH001-CV021, TECH001-CV022 and TECH001-CV 023. Supernatants were diluted 1:1500 and ELISA was performed using mouse a-human IgG CH3 domain capture antibody (MCA 878G) and a-human MIP-1α biotinylated capture antibody (BAF 270). Expifect (=cells treated with transfection agent Expifectamine alone, used as negative control).

Fig. 38: expression and secretion levels of proteins encoded by DNA plasmids

Protein expression and secretion levels of the immunostimulatory compounds GM-CSF (38 a: capture antibody MAB608, detection antibody BAF 308), IL-12 (38 b: capture antibody MAB419, detection antibody BAF 419) and IL-21 (38 c: capture antibody AF594, detection antibody BAF 594) encoded by the DNA plasmids are shown to be detected by ELISA in supernatants of the Expi293F cells transfected with the DNA plasmids TECH001-CV021, TECH001-CV022 and TECH001-CV023, respectively.

Fig. 39: expression and secretion of intact proteins encoded by DNA plasmids

Western blot shows secretion of the first polypeptide. Reduced supernatant samples from transfection control, VB2060, TECH001-CV021, TECH001-CV022 and TECH001-CV 023. An antibody: goat anti-human MIP-1 a (AF 270). And (2) secondary antibody: donkey is against goat, dylight 800 (SA 5-10092). Chemidoc channels Dyight 800 and 650 (for protein standards).

Fig. 40: expression and secretion of intact proteins encoded by DNA plasmids

Western blot shows secretion of the immunostimulatory compound GM-CSF encoded by TECH001-CV 021. Reduced supernatant samples from transfection control, VB2060 and TECH001-CV 021. An antibody: goat anti-mouse GM-CSF (BAF 415). And (2) secondary antibody: donkey is against goat, dylight 800 (SA 5-10092). Chemidoc channels Dyight 800 and 650 (for protein standards).

Fig. 41: expression and secretion of intact proteins encoded by DNA plasmids

Western blot shows secretion of the immunostimulatory compound IL-12 encoded by TECH001-CV 022. Reduced supernatant samples (left panel) and non-reduced supernatant samples (right panel) from transfection control, VB2060 and TECH001-CV 022. An antibody: goat anti-mouse IL-12 (BAF 419). And (2) secondary antibody: donkey is against goat, dylight 800 (SA 5-10092). Chemidoc channels Dyight 800 and 650 (for protein standards).

Fig. 42: expression and secretion of intact proteins encoded by DNA plasmids

Western blot shows secretion of the immunostimulatory compound IL-21 encoded by TECH001-CV 023. Reduced supernatant samples from transfection control, VB2060 and TECH001-CV 023. An antibody: goat anti-mouse IL-12 (BAF 594). And (2) secondary antibody: donkey is against goat, dylight 800 (SA 5-10092). Chemidoc channels Dyight 800 and 650 (for protein standards).

Fig. 43: immunogenicity of DNA plasmids

The immunogenicity of DNA plasmids VB2060, TECH001-CV021, TECH001-CV022 and TECH001-CV023 in mice administered with these plasmids is shown by measuring total IgG antibodies that bind RBD protein, as compared to negative control VB 1026. Individual mice and mean ± SEM are shown, 5 mice per group. * (p < 0.05) ×p < 0.01), two-tailed Mann-Whitney test.

Fig. 44: immunogenicity of DNA plasmids

A) The immunogenicity of DNA plasmids VB2060, TECH001-CV021, TECH001-CV022 and TECH001-CV023 in mice administered with these plasmids compared to negative control VB1026 by measuring IFN- γ secretion (total T cell response) of T cells is shown. B) The immunogenicity of DNA plasmids VB2060, TECH001-CV021, TECH001-CV022 and TECH001-CV023 in mice administered with these plasmids compared to negative control VB1026 by measuring IFN- γ secretion by cd8+ T cells (samples of cd4+ T cell depletion) is shown.

Detailed Description

The first polypeptide and/or multimeric protein is also referred to herein as a "construct". The first polypeptide/multimeric protein described herein is typically an immunogenic construct.

An "immunogenic construct" is a construct 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).

A "subject" is an animal, such as a mouse or a human, preferably a human. The terms "mouse," "mouse," and "m" are used interchangeably herein to refer to or refer to a mouse. The terms "person" and "h" are used interchangeably herein to refer to a person or to a person. The subject may be a patient, i.e. a person suffering from a disease requiring therapeutic treatment, or it may be a subject in need of prophylactic treatment, e.g. a subject in need of prophylactic treatment in order to avoid infection with an infectious disease, or it may be a subject suspected of suffering from a disease. The terms "subject" and "individual" are used interchangeably herein.

A "disease" is an abnormal medical condition, often associated with specific signs and symptoms in a subject suffering from the disease.

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

"Cancer" refers to a broad class of various diseases characterized by uncontrolled growth of abnormal cells in the body. "cancer" or "cancerous tissue" includes tumors, and as used herein, encompasses solid tumors as well as tumor cells found in bodily fluids such as blood, and includes metastatic cancers. Uncontrolled cell division and growth leads to the formation of malignant tumors that can invade adjacent tissues and can also metastasize to distant sites of the body through the lymphatic system or blood flow. After transfer, the distant tumor may be referred to as a "derived from" the pre-transferred tumor.

"Treatment" is prophylactic or therapeutic treatment.

"Prophylactic treatment" is a treatment administered to a subject that does not (or has not) exhibit signs or symptoms of a disease, or that exhibits only early signs or symptoms of a disease, with the aim of preventing or reducing the risk of developing a disease and/or symptoms associated with a disease. Prophylactic treatment may act as a prophylactic treatment against a disease, or as a treatment to inhibit or reduce further development or enhancement of a disease and/or a symptom associated therewith. The terms prophylactic treatment, prevention and prevention are used interchangeably herein.

"Therapeutic treatment" is a treatment administered to a subject exhibiting symptoms or signs of a disease, wherein the treatment is administered to the subject for the purpose of reducing or eliminating those signs or symptoms, or for the purpose of delaying or stopping disease progression.

"T cell epitope" as used herein refers to a discrete single T cell epitope or an antigenic portion or region containing multiple T cell epitopes (e.g., multiple minimal T cell epitopes, e.g., hot spots).

A "nucleotide sequence" is a sequence consisting of nucleotides. The terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably herein.

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 the one or more immunostimulatory compounds from a single vector (e.g., a DNA plasmid), only such a single vector need be administered to a subject. Thus, in order to enhance the effect of the first polypeptide/multimeric protein, there is no need to produce and administer additional vectors encoding immunostimulatory compounds or co-administer such compounds in the form of proteins or peptides, thereby reducing production costs and streamlining drug production. Administration of a single drug product may also help to increase patient acceptance of treatment and to make handling of the drug product (e.g., reconstitution and administration to a patient) easier for a healthcare professional.

Furthermore, without wishing to be bound by theory, co-expression may also have significant advantages at the cellular level. When transfected with the vector, the vector will hit a variety of cells. Successful uptake and initiation of transcriptional and translational functions is, to some extent, a stochastic process. When transfected with two different vectors, there is no control over in which cells the proteins encoded by these vectors will be expressed. This spatial distribution is not a problem for transfection which aims to secrete proteins into the blood stream. In the present invention, the vectors of the present invention produce different proteins. When the vector encoding the 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 cell, it can stimulate the same antigen presenting cell, thereby directly affecting the same. For example, if the antigen presenting cells are dendritic cells, the immunostimulatory compound may promote the attraction, activation and maturation of the dendritic cells.

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

Carrier body

The vector of the present invention may be any molecule suitable for carrying an exogenous nucleic acid sequence (e.g., DNA or RNA) into a cell to enable its expression in the cell, i.e., an expression vector.

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.

In another embodiment, the vector is an RNA vector, such as an RNA plasmid or an RNA viral vector, such as a retroviral vector selected from the group consisting of alphaviruses, lentiviruses, moloney murine leukemia virus, and rhabdoviruses (rhabdoviruses).

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

DNA plasmid

Plasmids are a small extrachromosomal DNA molecule within a cell, physically separated from chromosomal DNA, and capable of independent replication. Plasmids exist in bacteria mostly in the form of small circular double-stranded DNA molecules; however, plasmids are sometimes present in archaebacteria and eukaryotes. Artificial plasmids are widely used as vectors in molecular cloning for delivery and to ensure high expression of recombinant DNA sequences in host organisms. Plasmids contain several important features, including features for selecting cells containing the plasmid (e.g., antibiotic resistance genes), origins of replication, multiple Cloning Sites (MCSs), and promoters for driving expression of the inserted gene of interest.

In general, a promoter is a sequence that attracts initiation factors and polymerase to the promoter to cause transcription of a gene. The promoter is located near the transcription initiation site of the gene upstream of the DNA. The length of the promoter is about 100-1000 base pairs. The nature of the promoter generally depends on the gene and transcription product, and the type or class of RNA polymerase recruited to the site. When the RNA polymerase reads the plasmid DNA, the RNA molecule is transcribed. After processing, when the ribosome is translating the mRNA into a protein, the mRNA will be able to be translated multiple times, producing many copies of the protein encoded by the gene of interest. In general, ribosomes facilitate decoding by inducing the binding of complementary tRNA anticodon sequences to mRNA codons. tRNA carries specific amino acids that link together to form a polypeptide when mRNA passes through and is "read" by a ribosome. Translation proceeds in three stages: initiation, extension, and termination. Following the translation process, the polypeptide folds into an active protein and performs its function in the cell, or is exported from the cell and performs its function elsewhere, sometimes after a significant number of post-translational modifications.

When the protein is finally exported outside the cell, the signal peptide directs the protein into the endoplasmic reticulum, where the signal peptide is cleaved, and the protein is transferred to the periphery of the cell after translation is terminated.

The DNA plasmids of the present invention are not limited to any particular plasmid, and the skilled artisan will appreciate that any plasmid having a suitable backbone may be selected and engineered by methods known in the art to comprise the elements and units of the present disclosure.

Co-expression

The vectors of the present disclosure co-express a plurality of proteins. Such vectors (and plasmids) are also referred to as polycistronic vectors (and polycistronic plasmids). The skilled artisan knows how to engineer the vector to contain sequences encoding these several proteins, and can select different tools and use different techniques known in the art to ensure that these proteins are co-expressed as separate proteins from one vector.

Thus, the skilled artisan can construct the vectors of the invention to co-express different proteins, namely the first polypeptide and one or more immunostimulatory compounds.

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

In one embodiment of the disclosure, the vector comprises a co-expression element (or more than one co-expression element) that results in transcription of the first polypeptide and the one or more immunostimulatory compounds on a single transcript, but independent translation into the first polypeptide and the one or more immunostimulatory compounds. Thus, the presence of the coexpression element results in the final production of an individual translation product.

IRES

In one embodiment of the present disclosure, the co-expression element is an IRES element, the concept of which is shown in fig. 1. An internal ribosome entry site (abbreviated IRES) is an RNA element that allows translation to be initiated in a cap-independent manner, which is part of a larger protein synthesis process. In eukaryotic translation, initiation typically occurs at the 5 'end of the mRNA molecule, as 5' cap recognition is required for assembly of the initiation complex. By placing an IRES element between two coding regions, the starting complex can be assembled at this site and translation of the downstream coding region allowed. Thus, in one embodiment of the present disclosure, the vector comprises an IRES and one transcript is produced from the vector, which is subsequently translated into a separate protein.

The IRES element allows co-expression of the first polypeptide and the one or more immunostimulatory compounds under the control of the same promoter. The promoter directs transcription of a single mRNA comprising the nucleic acid sequence encoding the first polypeptide and the coding region of the nucleic acid sequence encoding the one or more immunostimulatory compounds. If more than one immunostimulatory compound is expressed from the vector of the invention, then an IRES element needs to be present in the vector of the invention upstream of each nucleic acid sequence encoding the immunostimulatory compound. Or if more than one immunostimulatory compound is expressed from the vectors of the invention, another type of co-expression element may be used.

IRES elements for use in vectors of the invention may be derived from viral genomes or cellular mRNA. Vectors, such as DNA plasmids, comprising IRES elements are commercially available.

2A self-cleaving peptides

In another embodiment of the present disclosure, the coexpression element is a nucleic acid sequence encoding a 2A self-cleaving peptide (or short "2A peptide") as conceptually shown in fig. 2.

In the context of the present application, the terms "2A self-cleaving peptide" and "2A peptide" are used to refer to a peptide encoded by a nucleic acid sequence that, when located between two coding regions, results in transcription of the two coding regions into a single transcript, but it translates into two separate peptide chains. Generally, in ribosome translation of mRNA, amino acids are covalently bonded N-terminal to C-terminal. The presence of the nucleic acid sequence encoding the 2A self-cleaving peptide results in two separate peptide chains because the ribosome skips the synthesis of a peptide bond at the C-terminus of the 2A peptide. The 2A self-cleaving peptide is typically 18-22 amino acids in length and typically comprises a consensus sequence DXEXNPGP (SEQ ID NO: 50), wherein X may be any amino acid.

In one embodiment of the invention, the ribosome skips the peptide bond between glycine and proline residues present on the C-terminus of the 2A self-cleaving peptide, meaning that the upstream gene product will have some additional amino acid residues added to the terminus, while the downstream gene product will start with proline.

In one embodiment, the 2A self-cleaving peptide is a 18-22 amino acid long sequence comprising a consensus sequence DXEXNPGP (SEQ ID NO: 50), wherein X may be any amino acid.

Thus, the 2A self-cleaving peptide also allows for co-expression of the first polypeptide and the one or more immunostimulatory compounds under the control of the same promoter. As with IRES elements, if more than one immunostimulatory compound is expressed from the vector of the invention, then the nucleic acid sequence encoding the 2A peptide needs to be present in the vector upstream of each nucleic acid sequence encoding the 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 located between the first and second nucleic acid sequences and a nucleic acid sequence encoding a P2A peptide located between the second and third nucleic acid sequences. Or if more than one immunostimulatory compound is expressed from the vectors of the invention, another type of co-expression element may be used.

In another embodiment, the 2A self-cleaving peptide is a 2A-peptide selected from the group consisting of a T2A peptide, a P2A peptide, an E2A peptide, and an F2A peptide.

In one embodiment, the T2A peptide has the same amino acid sequences as those T2A sequences listed in table 1 or table 2. In another embodiment, amino acid sequence DVEENPGP (SEQ ID NO: 50) is present, but the remainder of the T2A 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 T2A amino acid sequence of Table 1. In another embodiment, the T2A peptide has the amino acid sequence of SEQ ID NO. 9.

In one embodiment, the P2A peptide has the same amino acid sequences as those P2A sequences listed in table 1 or table 2. In another embodiment, 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.

In one embodiment, the E2A peptide has the same amino acid sequences as those E2A sequences listed in table1 or table 2. In another embodiment, 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 Table1. In another embodiment, the E2A peptide has the amino acid sequence of SEQ ID NO. 14.

In one embodiment, the F2A peptide has the same amino acid sequence as those F2A sequences listed in table 1 or table 2. In another embodiment, 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

Name of the name	Sequence(s)	SEQ ID NO.
			T2A	EGRGSLLTCGDVEENPGP	SEQ ID NO:9
P2A	ATNFSLLKQAGDVEENPGP	SEQ ID NO:11
			E2A	QCTNYALLKLAGDVESNPGP	SEQ ID NO:14
F2A	VKQTLNFDLLKLAGDVESNPGP	SEQ ID NO:51

It is well known that the efficiency of 2A-peptide can be regulated to increase its efficiency of cleavage and expression, for example by inserting a GSG sequence before the N-terminus of the wild type sequence, as shown in Table 2.

TABLE 2

Name of the name	Sequence(s)	SEQ ID NO.
			T2A	GSGEGRGSLLTCGDVEENPGP	SEQ ID NO:52
P2A	GSGATNFSLLKQAGDVEENPGP	SEQ ID NO:53
			E2A	GSGQCTNYALLKLAGDVESNPGP	SEQ ID NO:54
F2A	GSGVKQTLNFDLLKLAGDVESNPGP	SEQ ID NO:55

In another embodiment, the vector of the invention contains 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 located 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 located between the first and second nucleic acids and an IRES element located between the second and third nucleic acid sequences. Additional nucleic acid sequences encoding additional immunostimulatory compounds may be contained in the vector in the same manner.

In another embodiment, the vector of the invention contains a nucleic acid sequence encoding two 2A peptides in a continuous sequence consisting 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 in a continuous sequence between the first and second nucleic acid sequences.

Bidirectional promoter

In one embodiment of the disclosure, the vector comprises a co-expression element (or more than one co-expression element) that results in the first polypeptide and the one or more immunostimulatory compounds being transcribed into separate transcripts, which results in separate transcripts, thereby producing separate proteins.

In one embodiment of the present disclosure, the coexpression element is a bi-directional promoter, the concept of which is shown in FIG. 3 a. Bidirectional promoters are typically short (e.g., <1 kbp) intergenic DNA regions between the 5' ends of genes in a bidirectional gene pair. "bidirectional gene pair" refers to two adjacent genes encoded on opposite strands with their 5' ends toward each other.

In One embodiment of the disclosure, the bi-directional promoter is a back-to-back arrangement of the CAG promoter and four CMV enhancers (SLADITSCHEK HL, neveu PA et al, PLoS One 11 (5), e0155177, 2016).

In one embodiment of the disclosure, the bi-directional promoter is RPBSA (Kevin He et al, int.j.mol. Sci.21 (23), 9256,2020).

In one embodiment of the disclosure, the bi-directional promoter is in the back-to-back configuration of the mouse Pgk1 and human eukaryotic translation elongation factor 1α1 promoters (Golding & Mann, GENE THERAPY 18,817-826,2011).

In one embodiment, the 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.

Multiple promoters

In another embodiment of the disclosure, the co-expression element is a variety of promoters, i.e., the vector is a plasmid comprising, for example, a separate promoter for each nucleic acid sequence encoding the first polypeptide and the one or more immunostimulatory compounds (i.e., for separate transcription of each of the first polypeptide and the one or more immunostimulatory compounds).

In one embodiment, each of the nucleic acid sequences will have a different promoter, the concept also illustrated in FIG. 3 b. In one embodiment, all nucleic acid sequences have the same promoter to achieve equimolecular expression. In an alternative embodiment, one nucleic acid sequence has a stronger promoter than the other nucleic acid sequence; that is, nucleic acid sequences with stronger promoters may be expressed at higher levels than other nucleic acid sequences.

Many promoters are known in the art and are suitable for inclusion in the plasmids of the invention. In one embodiment of the disclosure, the promoter is derived from a cytomegalovirus, such as a CMV promoter.

In one embodiment, the vector of the invention comprises one or more co-expression elements, preferably co-expression elements selected from the group consisting of IRES elements, 2A peptides, bi-directional promoters and promoters.

Vectors of the invention may comprise all of the various combinations of co-expression elements.

As an example, the 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, which allows for co-expression of the first polypeptide (under the control of a promoter) and the first and second immunostimulatory compounds. In another embodiment, the DNA plasmid comprises a bi-directional promoter and another promoter.

The skilled person will appreciate that the terms first, second and third nucleic acid sequences as in the above examples do not mean that the plasmid of the invention comprises the 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, as may the first and third or the second and third nucleic acid sequences. In further embodiments, the nucleic acid sequences encoding the first polypeptide and the immunostimulatory compound may be located on opposite DNA strands.

Immunostimulatory compounds

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

In one embodiment of the present disclosure, the immunostimulatory compound is a compound that affects antigen presenting cells. In another embodiment, the immunostimulatory compound is a compound that stimulates antigen presenting cells.

Antigen Presenting Cells (APCs) are cells that display antigens on their surface that are complexed with Major Histocompatibility Complex (MHC), a process known as antigen presentation. T cells can use their T Cell Receptor (TCR) to recognize these complexes. APCs process antigens and present them to T cells.

Almost all cell types present antigen in some way. Professional APCs include macrophages (such as langerhans cells), B cells and dendritic cells, presenting foreign antigens to helper T cells (CD 4 ⁺) via MHC class II, whereas virally infected cells (or cancer cells) present antigens derived from the inside of the cells to cytotoxic T cells (CD 8 ⁺) via MHC class I. In addition to the MHC protein family, antigen presentation is dependent on APC and other professional signaling molecules on the surface of T cells.

"MHC" stands for "major histocompatibility complex". MHC molecules are mainly of two classes: 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.

APCs are critical for an effective adaptive immune response because both cytotoxic T cell and helper T cell functions are dependent on APCs. Antigen presentation produces specificity for adaptive immunity and contributes to immune responses against intracellular and extracellular pathogens. It also participates in the defense against tumors.

In one embodiment of the present disclosure, the APCs are selected from dendritic cells, macrophages, langerhans cells, B cells, and neutrophils, and the immunostimulatory compound is a compound that affects such cells (e.g., stimulates such cells).

When an APC is affected, e.g., stimulated, the stimulation can result in attraction, activation, maturation, and/or proliferation of the APC.

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

In one embodiment of the present disclosure, the immunostimulatory compound comprises a cytokine, chemokine, growth factor, ligand that binds to a TNF receptor superfamily member, or ligand that binds to a Pattern Recognition Receptor (PRR).

In one embodiment, the vector of the invention is a plasmid, such as a DNA plasmid. It may be administered to a subject in need thereof, for example, by intramuscular administration, and the encoded compound expressed and secreted from the muscle cells. The efficacy of the first polypeptide (secreted in the form of a multimeric protein, e.g., a dimeric protein) may be enhanced by co-expression of an immunostimulatory compound that attracts APCs to the injection site/myocyte, from which the multimeric protein and the immunostimulatory compound are secreted. Attraction of APCs can lead to a stronger and accelerated immune response: the multimeric proteins are delivered to and absorbed by APCs, rather than merely diluted in the blood stream, and with a locally higher number of APCs, one or more antigens contained in the multimeric proteins can be presented to other related immune cells.

Immunostimulatory compounds that promote APC attraction

Thus, in one embodiment, the immunostimulatory compound is a compound that promotes APC attraction.

APC attraction can be measured by methods known in the art, including in vitro cross-well assays or migration assays, in vivo surface markers of muscle cells administered with the vectors of the invention measured by flow cytometry, or changes in gene expression patterns measured by, for example, RT-qPCR, nanostring or RNA sequencing.

One class of molecules capable of attracting APCs are chemokines. Chemokines are a class of small cytokines or signaling proteins secreted by cells. Their name derives from their ability to induce directional chemotaxis in nearby responsive cells, i.e. they are chemotactic cytokines.

In one embodiment of the present disclosure, the immunostimulatory compound is a chemokine.

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

In another embodiment of the present disclosure, the immunostimulatory compound is selected from macrophage inflammatory protein α and isoforms thereof, including mouse CCL3 (or MIP-1α) and isoforms hCCL3, hCCL3L1, hCCL3L2, and hCCL3L3, preferably human MIP-1α (hMIP-1α variant, also referred to as LD78 β or CCL3L 1); RANTES (CCL 5), preferably human CCL5, e.g., human CCL5 having the amino acid sequence of SEQ ID No. 43; chemokine ligand 4 (CCL 4), preferably human CCL4; chemokine ligand 20 (CCL 20), preferably human CCL20; chemokine ligand 19 (CCL 19), preferably human CCL19; chemokine ligand 21 (CCL 21), preferably human CCL21; and chemokine motif ligand 1 or 2 (XCL 1 or XCL 2), preferably human XCL1 or human XCL2.

The activation process is a series of events that drive resting APCs toward a more differentiated and/or mature state. APC is activated directly by interaction with/encounter with a pathogen, foreign antigen, and indirectly by recognition of compounds (e.g., inflammatory mediators) produced and released by other cell types of such molecules. APC then undergoes a series of cellular processes ending in its activation, which plays an important role in triggering an effective immune response to foreign antigens. For example, dendritic cell maturation is characterized by reduced phagocytic capacity, enhanced antigen processing and presentation, improved migration to lymphoid tissues, and increased ability to stimulate B cells and T cells.

Immunostimulatory compounds that promote APC activation and/or maturation

In one embodiment of the present disclosure, the immunostimulatory compound is a compound that promotes APC activation and/or maturation.

Different techniques known in the art can be used to measure activation of APCs, such as comparing cytokine profiles before and after activation as measured by ELISpot or FluoroSpot, determining overall changes in gene expression, and analyzing expressed proteins (e.g., activation markers) by various techniques, such as FACS, ELISA, WB and PCR/sequencing methods (qPCR (TaqMan array), nanostring, and RNA-seq).

In one embodiment of the present disclosure, the immunostimulatory compound may interact with a surface molecule on the APC selected from the group consisting of: receptors of the TNF receptor superfamily, including CD40 (cluster of differentiation 40), CD137 (4-1 BB), CD27, RANK and ICOS (CD 278). In a preferred embodiment, the immunostimulatory compound may interact with a surface molecule on the aforementioned human APC.

Such immunostimulatory compounds may be selected from the group consisting of CD40L (CD 40 ligand, CD 154), CD137L (4-1 BBL, 4-1BB ligand), CD70, ICOSL (CD 275) and RANKL. In a preferred embodiment, the immunostimulatory compound is selected from hCD40L, hCD137L, hCD, hICOSL and hRANKL.

In another embodiment of the present 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, for example comprising the amino acid sequences of SEQ ID NOs 45 and 47 human IL-12; IL-21, preferably human IL-21, e.g. human IL-21 comprising the amino acid sequence of SEQ ID NO. 49; TNFa, preferably human TNFa; ifnγ, preferably human ifnγ; and IL-1β, preferably human IL-1β.

In another embodiment of the disclosure, the immunostimulatory compound is an immune signaling molecule, such as MyD88 and TRIF, preferably, such as human MyD88 and human TRIF, that activates APC through TLR receptors present on the surface of the APC.

In another embodiment of the present disclosure, the immunostimulatory compounds are viral infection sensing agents, such as RIG-1 and MDA-5, preferably human RIG-1 and human MDA-5.

In yet another embodiment of the present disclosure, the immunostimulatory compound is a compound that interacts with a pattern recognition receptor (e.g., a Toll-like receptor, including TLR2, TLR4, or TLR 5) on an APC. In a preferred embodiment, the immunostimulatory compound interacts with a receptor on the aforementioned human APC.

In one embodiment, such immunostimulatory compounds are selected from pathogen-associated molecular patterns (PAMPs) (e.g., flagellins), protein damage-associated molecular patterns (DAMP) (e.g., HMGB 1), heat Shock Proteins (HSP), calreticulin, and annexin A1. In a preferred embodiment, such immunostimulatory compounds are selected from the group consisting of human pathogen-associated molecular patterns (PAMPs), human protein damage-associated molecular patterns (DAMP) (e.g., human HMGB 1), human Heat Shock Proteins (HSP), human calreticulin, and human annexin A1.PAMP/DAMP include those that can be included as nucleic acid sequences in the vectors of the invention and are to be expressed as functional proteins that can include functional groups introduced by post-translational modification. The above molecules in turn activate the following receptors on APC: RAGE, TLR4, TLR9 and TIM-3 (for HMGB 1), FPR (for annexin A1), SREC1, LOX1 and CD91 (for HSP). In a preferred embodiment, the immunostimulatory compound in turn activates a receptor on the human APC.

Immunostimulatory compounds that promote APC growth and/or amplification

During the immune response, activated APCs will rapidly expand to combat infection or disease. Cell proliferation is the process by which cells grow (increase in mass and size) and divide to produce two daughter cells. Growth factors stimulate cells by binding to receptors on the cell surface, resulting in cell proliferation. Cell proliferation results in an exponential increase in cell number and is therefore a rapid mechanism for expanding cell populations. Hereinafter, the terms "amplification" and "proliferation" are used interchangeably.

In one embodiment, the immunostimulatory compound is a compound that promotes APC growth and/or amplification.

Cell proliferation may be measured by different techniques known in the art, for example by MTT/MTS assay, measuring protein translation or by labeling with CFSE. Methods well known in the art are performed, for example, by measuring the metabolic activity of a cell population, which will reflect the state of cell proliferation. In addition, since the intracellular ATP content is tightly controlled, detection of ATP may also provide information about cell proliferation. There is a strict linear relationship between the concentration of ATP measured in cell lysates or extracts and the number of cells, with little or no ATP in dead cells or cells that are about to die. ATP detection using bioluminescent luciferase and its substrate luciferin can provide very sensitive results. If ATP is present, the luciferase will emit light with the intensity of light being proportional to the concentration of ATP. Furthermore, some antigens are only present in proliferating cells, and non-proliferating cells lack these antigens. Specific monoclonal antibodies can be used to detect cell proliferation. For example, in human cells, the Ki-67 antibody can recognize homonymous proteins that are expressed at all active phases of the cell cycle but not in resting (resting) cells. Traditionally, radiolabeled 3H-thymine has been used to measure proliferation. It is incubated with the cells for several hours or overnight. The newly proliferated cells will incorporate a radiolabel into their DNA, which can be detected by scintillation counting after DNA extraction.

In one embodiment of the present disclosure, the immunostimulatory compound may interact with the following surface molecules on the APC: GM-CSF-receptor (granulocyte-macrophage colony stimulating factor receptor, CD 116), FLT-3R (fms-like tyrosine kinase 3, CD 135), IL-15R, or IL-4R. In a preferred embodiment, the immunostimulatory compound interacts with the surface molecules described above on the human APC.

In one embodiment of the present disclosure, the immunostimulatory compound is a growth factor, such as GM-CSF (granulocyte-macrophage colony stimulating factor), preferably human GM-CSF, such as a peptide having the amino acid sequence of SEQ ID NO:41, human GM-CSF of the amino acid sequence of seq id no; 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.

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 receptors listed in table 3 present on human APCs.

TABLE 3 Table 3

In one embodiment of the present 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 or3 or 4 or 5 or 6 immunostimulatory compounds). The immunostimulatory compounds may be the same or different, preferably different.

In a preferred embodiment, the different immunostimulatory compounds also affect APCs in a different manner to stimulate the immune system at a number of different levels and thereby maximize the therapeutic or prophylactic effect of the first polypeptide.

For example, in one embodiment, the vector comprises nucleic acids encoding 3 different immunostimulatory compounds, the first being an immunostimulatory compound that promotes DC attraction (e.g., XCL 1), the second being an immunostimulatory compound that promotes DC growth (e.g., FLT 3L), and the third being an immunostimulatory compound that promotes DC activation (e.g., CD 40L). In one embodiment, such vectors may be used to treat and/or prevent infectious diseases or to treat cancer. The choice of the particular immunostimulatory compound will also depend on the targeting unit contained in the first polypeptide, as the targeting unit targets the APC and can affect the APC in a similar manner as the immunostimulatory compound, e.g., attract or activate the APC.

First nucleic acid sequence

The vectors of the present disclosure comprise a first nucleic acid sequence, i.e., DNA or RNA, including genomic DNA, cDNA, and mRNA, which is double-stranded or single-stranded and encodes 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 subject species to which it is administered. For administration to humans, in one embodiment, the first nucleic acid sequence is human codon optimized.

The first nucleic acid sequence encodes a first polypeptide comprising a targeting unit that targets an antigen presenting cell, a multimerizing unit, such as a dimerization unit, and an antigenic unit comprising one or more antigens or portions thereof (e.g., one or more disease-associated antigens or portions thereof). Upon administration to a subject, the first polypeptide is expressed and, due to the presence of multimerization units, multimeric proteins are formed which elicit an immune response against the antigen or portion thereof (e.g., epitope) contained in the antigenic unit, resulting in activation of the immune system of the subject.

Structures such as the first polypeptide and the dimeric or multimeric proteins comprising the first polypeptide are known in the art (e.g. WO 2004/076489A1, WO 2011/161244A1, WO 2017/118695A1 and WO 2022/013777 A1, all of which disclosures are included herein by reference), and the skilled person can select targeting units, multimerizing units and antigenic units targeting antigen presenting cells according to the envisaged use of the vector and the desired outcome after administration thereof.

The first polypeptide has an initial N-terminus and a terminal C-terminus (as shown in fig. 4). The elements and units of the first polypeptide, i.e., the Targeting Unit (TU), the multimerizing unit, e.g., the dimerization unit (DimU) and the antigenic unit in fig. 4, may be arranged in the first polypeptide such that the antigenic unit is located at the terminal C-terminus of the first polypeptide (fig. 4 a) or at the initial N-terminus of the first polypeptide (fig. 4 b). Preferably, the antigenic unit is located at the terminal C-terminus of the first polypeptide. The Unit Linker (UL) may link multimerization units such as dimerization units and antigenic units. FIG. 4 shows an antigenic unit with 4 neo-epitopes (neo 1, neo2, neo3, neo 4) separated by a linker (SUL 1, SUL2, SUL 3). Another way to describe the arrangement of neo-epitopes neo1-neo4 is that these neo-epitopes are arranged in 3 antigenic subunits, each comprising a neo-epitope and subunit linker (SUL 1, SUL2, SUL 3), and a terminal neo-epitope (neo 4), which is closest to the terminating C-terminus or the starting N-terminus of the first polypeptide. Subunits are indicated in brackets in the figures. Thus, an antigenic unit comprising n neoepitopes comprises n-1 subunits, each subunit comprising a neoepitope and a subunit linker. As described herein, the 4 neo-epitopes may be the same or different neo-epitopes, and the 3 linker/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 in the first nucleic acid sequence encoding the first polypeptide. The first polypeptide as shown in fig. 4 may be used as an anti-cancer vaccine, e.g., a personalized anti-cancer vaccine, as described herein.

Various units and elements of the first polypeptide will be discussed in detail below. They are present as coding units/elements in the first nucleic acid sequence, while they are present as amino acid sequences in the first polypeptide or multimeric protein. For ease of reading, in the following the units/elements are mainly explained with respect to the first polypeptide/multimeric protein, i.e. based on their amino acid sequence.

Targeting unit

The first polypeptide encoded by the first nucleic acid comprised in the vector of the invention comprises a targeting unit that targets APCs. APCs include Dendritic Cells (DCs) and subpopulations thereof.

The term "targeting unit" as used herein refers to a unit that delivers a polypeptide/multimeric protein to antigen presenting cells for MHC class II restricted presentation to cd4+ T cells or for providing cross-presentation to cd8+ T cells by MHC class I restriction.

Due to the presence of the targeting unit, multimeric proteins will attract DCs, neutrophils and other immune cells. Thus, multimeric proteins not only target the antigenic units contained therein to specific cells, but also promote a response amplifying effect (adjuvant effect) by recruiting specific immune cells to the site of administration of the vector.

The targeting unit is designed to target the multimeric protein to a surface molecule expressed on an APC, e.g. a molecule expressed on any one or more types of APCs or a molecule only on a subset of APCs (e.g. on a DC subset).

Examples of such surface molecules on APCs include HLA, cluster of differentiation 14 (CD 14), cluster of differentiation 40 (CD 40), CLEC9A, chemokine receptors, and Toll-like receptors (TLRs). Chemokine receptors include C-C motif chemokine receptor 1 (CCR 1), C-C motif chemokine receptor 3 (CCR 3), C-C motif chemokine receptor 4 (CCR 4), C-C motif chemokine receptor 5 (CCR 5), C-C motif chemokine receptor 6 (CCR 6), C-C motif chemokine receptor 7 (CCR 7), C-C motif chemokine receptor 8 (CCR 8), and XCR1. Toll-like receptors include TLR-2, TLR-4 and TLR-5. In one embodiment, the targeting unit is or comprises a moiety that interacts with these surface molecules. In a preferred embodiment, the surface molecule is present on a human APC.

Thus, in one embodiment, the targeting unit comprises or consists of an antibody binding region, e.g. antibody variable domains (VL and VH), which are specific for MHC/HLA, CD14, CD40, CLEC9A or Toll-like receptor, preferably hCD14, hCD40, hCLEC a or human Toll-like receptor. In another embodiment, the targeting unit comprises or consists of a synthetic or natural ligand. Examples include soluble CD40 ligand (CD 40L), preferably hCD40L, natural ligands such as chemokines, preferably e.g. in their human form, e.g. chemokine ligand 5, also known as C-C motif ligand 5 (CCL 5 or RANTES), preferably hCCL, e.g. having the amino acid sequence of SEQ ID NO:43, including mouse CCL3 (or MIP-1α) and isoforms hCCL, hCCL3L1, hCCL L2 and hCCL L3, chemokine ligand 4 (CCL 4) and its isoforms CCL4L, preferably hCCL and hCCL L, chemokine ligand 19 (CCL 19), preferably hCCL, chemokine ligand 20 (CCL 20), preferably hCCL20, chemokine ligand 21 (CCL 21), preferably hCCL21, chemokine motif ligand 1 or 2 (XCL 1 or XCL 2), preferably hXCL1 or hXCL2, and bacterial antigens such as flagellin.

In one embodiment, the targeting unit has affinity for MHC class II proteins. Thus, in one embodiment, the targeting unit comprises or consists of an antibody binding region, e.g. antibody variable domains (VL and VH), which are specific for MHC class II proteins selected from the group consisting of anti-HLA-DP, anti-HLA-DR and anti-pan-HLA class II.

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 an antibody binding region, e.g. an antibody variable domain (VL and VH), which is specific for CD14, CD40, TLR-2, TLR-4 or TLR-5, e.g. anti-CD 14, anti-CD 40, anti-TLR-2, anti-TLR-4 or anti-TLR-5, preferably for hCD14, hCD40, hTLR-2, hTLR-4 or hTLR-5, e.g. anti-hCD 14, anti-hCD 40, anti-hTLR-2, anti-hTLR-4 or anti-hTLR-5.

In yet another embodiment, the targeting unit comprises or consists of flagellin, which has affinity for TLR-5, e.g. hTLR-5. In another embodiment, the targeting unit comprises or consists of an antibody binding region, which is specific for CLEC9A, e.g. anti-CLEC 9A or a variant thereof, e.g. anti-CLEC 9A Fv, or the targeting unit comprises or consists of a CLEC9 ligand, e.g. comprising the amino acid sequence of SEQ ID NO:115 or an amino acid sequence encoded by said nucleic acid sequence or CLEC9 ligand consisting of the foregoing. In preferred embodiments, the targeting unit comprises or consists of an antibody binding region, which is specific for hCLEC a, e.g. anti hCLEC a or a variant thereof, e.g. anti hCLEC a Fv, or the targeting unit comprises or consists of a human CLEC9 ligand.

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 CCR 5. 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 hCCR 5.

In one embodiment, the targeting unit has affinity for the chemokine receptor CCR7, preferably for the human chemokine receptor CCR 7. In another embodiment, the targeting unit comprises or consists of: CCL19, for example a human form comprising or encoded by the nucleotide sequence of SEQ ID NO. 121 or a sequence of amino acids consisting of CCL19 as defined above, or CCL21, for example CCL19 or CCL 21.

Preferably, the targeting unit comprises or consists of a variant of the chemokine human macrophage inflammatory protein alpha (human MIP-1 alpha (hMIP-1 alpha), also known as LD78 beta or CCL3L 1), which binds to its cognate receptor expressed on the surface of APC cells, including CCR1, CCR3 and CCR5. Binding of the targeting unit to its cognate receptor results in internalization of the multimeric protein into the APC and degradation of the protein into small peptides that 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 will target and kill cells expressing the same antigen, e.g., cancer cells expressing such the same antigen.

In another embodiment, both a T cell response and a B cell response are induced. This also makes it possible for the antibody response, i.e. when the virus is in circulation, the antibody binds to e.g. viral surface proteins and neutralizes the virus by inhibiting its entry into the host cell.

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

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

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

In a further preferred embodiment, the targeting unit consists of an amino acid sequence having at least 85% sequence identity with the amino acid sequence of 24-93 of SEQ ID NO. 1, e.g. at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity. In a further preferred embodiment, the targeting unit consists of the amino acid sequence 24-93 of SEQ ID NO. 1.

In a preferred embodiment, the targeting unit comprises the amino acid sequence of 24-93 of SEQ ID NO.1, except that at most 6 amino acids have been substituted, deleted or inserted, e.g. at most 5 amino acids, e.g. at most 4 amino acids, e.g. at most 3 amino acids, e.g. at most 2 amino acids or e.g. at most 1 amino acid have been substituted, deleted or inserted. One embodiment of such targeting units is a targeting unit comprising the amino acid sequence of 26-93 of SEQ ID NO.1 or a targeting unit comprising the amino acid sequence of 28-93 of SEQ ID NO. 1.

In another preferred embodiment, the targeting unit consists of the amino acid sequence of 24-93 of SEQ ID NO. 1, with the difference that at most 6 amino acids have been substituted, deleted or inserted, for example at most 5 amino acids, for example at most 4 amino acids, for example at most 3 amino acids, for example at most 2 amino acids or for example at most 1 amino acid have been substituted, deleted or inserted. One embodiment of such targeting units is a targeting unit consisting of the amino acid sequence of 26-93 of SEQ ID NO. 1 or a targeting unit consisting of the amino acid sequence of 28-93 of SEQ ID NO. 1.

In a preferred embodiment, the targeting unit comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO. 25.

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

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

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

In one embodiment, a specific selection and/or combination of targeting units and immunostimulatory compounds in the vectors of the invention is, for example, selection of hMIP-1 alpha or CCL3 as targeting unit and selection of CCL4, GM-CSF, FLT3L and/or ifnα as immunostimulatory compounds. In another embodiment, a specific selection and/or combination is, for example, selection of hMIP-1 a or CCL3 as the targeting unit and selection of CCL5, GM-CSF, FLT3L, and/or ifnα as the immunostimulatory compound. In another embodiment, a specific selection and/or combination is, for example, selecting CCL5 as the targeting unit and selecting XCL1, GM-CSF, FLT3L, and/or ifnα as the immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selection of hMIP-1 alpha or CCL3 as the targeting unit and IL-4, GM-CSF, CD40L, and/or TNF alpha as the immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selection of hMIP-1 alpha or CCL3 as the targeting unit and IL-4, GM-CSF, IL-1 beta, and/or TNF alpha as the immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selection of hMIP-1 alpha or CCL3 as targeting unit and IL-4, GM-CSF, IL-1 beta and/or IFN gamma as immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selecting CCL5 as the targeting unit and CCL7, GM-CSF, FLT3L, and/or ifnα as the immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selection of hMIP-1 alpha or CCL3 as the targeting unit and selection of 4-1BBL, GM-CSF, FLT3L, and/or IFN alpha as the immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selection of hMIP-1 alpha or CCL3 as the targeting unit and selection of CD40L, GM-CSF, FLT3L and/or IFN alpha as the immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selection of hMIP-1 alpha or CCL3 as targeting unit and selection of CD205, GM-CSF, FLT3L, and/or IFN alpha as immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selecting CCL5 as the targeting unit and 4-1BBL, GM-CSF, FLT3L, and/or IFNα as the immunostimulatory compound. In yet another embodiment, a specific 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, a specific selection and/or combination is, for example, selecting anti-CD 205 as the targeting unit and CCL5, GM-CSF, FLT3L, and/or ifnα as the immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selection of hMIP-1 alpha or CCL3 as the targeting unit and CCL4, GM-CSF, FLT3L, and/or MyD88 as the immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selection of hMIP-1 alpha or CCL3 as targeting unit and selection of TRIF, GM-CSF, FLT3L, and/or MyD88 as immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selection of hMIP-1 alpha or CCL3 as the targeting unit and selection of GM-CSF, IL-12, IL-21, and/or CD40L as the stimulating compound. In yet another embodiment, a specific selection and/or combination is, for example, selecting CD11c as the targeting unit and hMIP-1α or CCL3, IFNγ, GM-CSF and/or FLT3L as the immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selecting CD11c as the targeting unit and hMIP-1α or CCL3, TNF α, GM-CSF and/or FLT3L as the immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selecting CLEC9A as the targeting unit and 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 CCL5, XCL1, GM-CSF and/or FLT3L as the immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selecting CADM1 as the targeting unit and CCL5, XCL1, GM-CSF and/or FLT3L as the immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selecting CCL19 as the targeting unit and selecting GM-CSF, IL-12, IL-21, and/or CD40L as the immunostimulatory compound. In yet another embodiment, a specific selection and/or combination is, for example, selecting CCL19 as the targeting unit and GM-CSF, CCL3L, XCL1, and/or CCL5 as the immunostimulatory compound.

In a preferred embodiment, the targeting unit and immunostimulatory compound listed in the preceding paragraph are human proteins.

Multimerization unit/dimerization unit

The first polypeptide encoded by the first nucleic acid comprised in the vector of the invention comprises multimerization units, e.g. dimerization units.

The term "multimerization unit" as used herein refers to a nucleotide or amino acid sequence between an antigenic unit and a targeting unit. In addition to linking the antigenic unit and the targeting unit, the multimerization unit also facilitates multimerization/linking of multiple polypeptides (e.g., two, three, four, or more polypeptides) into multimeric proteins, such as dimeric, trimeric, or tetrameric proteins. Furthermore, the multimerization units also provide flexibility to the multimeric protein to allow optimal binding of the targeting units to surface molecules on APCs even if they are located at different distances. The multimerization unit may be any unit that meets one or more of these requirements.

Multimerization units that promote multimerization/ligation of more than two polypeptides

In one embodiment, the multimerization unit is a trimerization unit, e.g., a collagen-derived trimerization unit, e.g., a human collagen-derived trimerization domain, e.g., a human collagen-derived XVIII trimerization domain (see, e.g., A.Alvarez-Cienfuegos et al, sci Rep 6,28643 (2016)) or a human collagen XV trimerization domain. Thus, in one embodiment, the multimerization unit is a trimerization unit comprising a polypeptide having the amino acid sequence of SEQ ID NO:116 or an amino acid sequence encoded by said nucleic acid sequence, or a combination thereof. In another embodiment, the trimerization unit is the C-terminal domain of T4 fibrin. Thus, in one embodiment, the multimerization unit is a trimerization unit comprising or consisting of the amino acid sequence having SEQ ID NO: 56.

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 the nucleic acid sequence having SEQ ID NO. 57 or an amino acid sequence encoded by the nucleic acid sequence, optionally further comprising a hinge region as described below.

Dimerization unit

The term "dimerization unit" as used herein refers to a nucleotide or amino acid sequence between an antigenic unit and a targeting unit. In addition to linking the antigenic unit and the targeting unit, the dimerization unit also facilitates dimerization/linking of the two monomeric polypeptides into a dimeric protein. Furthermore, the dimerization unit also provides flexibility to the dimeric protein to allow optimal binding of the targeting unit to surface molecules on the APC even though they are located at different distances. The dimerization unit may be any unit that meets these requirements.

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 facilitates dimerization. In yet another embodiment, the dimerization unit comprises a hinge region, a dimerization unit linker and another domain that facilitates dimerization, wherein the dimerization unit linker connects the hinge region and the another domain that facilitates 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, and preferably is dimerization unit linker GGGSSGGGSG (SEQ ID NO: 134).

The term "hinge region" refers to an amino acid sequence contained in a dimerization unit that facilitates joining two polypeptides, i.e., facilitates the formation of a dimeric protein. Where multimerization/ligation of more than two polypeptides is facilitated, the term "hinge region" refers to an amino acid sequence contained in such multimerization units that facilitates ligation of more than two polypeptides (e.g., three or four polypeptides) and/or serves as a flexible spacer allowing two targeting units of a multimeric protein to bind simultaneously to multiple surface molecules on an APC even though they are located at different distances.

Furthermore, the hinge region acts as a flexible spacer allowing two targeting units of the dimeric protein to bind simultaneously to two surface molecules on APC even though they are located at different distances. The hinge region may be Ig derived, e.g. derived from IgG, e.g. IgG1 or IgG2 or IgG3. In one embodiment, the hinge region is derived from IgM, e.g., comprises a polypeptide having the amino acid sequence of SEQ ID NO:119 or an amino acid sequence encoded by said nucleic acid sequence, or a combination thereof. The hinge region may promote dimerization by forming covalent bonds such as disulfide bridges 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 bridge.

In one embodiment, the dimerization unit comprises 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 an amino acid sequence having at least 80% sequence identity to the amino acid sequence of 94-120 of SEQ ID No. 1; or consist of, it.

In preferred embodiments, the dimerization unit comprises hinge exon h1 and hinge exon h4, which have an amino acid sequence having at least 85% sequence identity with the amino acid sequence of 94-120 of SEQ ID NO. 1, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity; or consists of the hinge exon h1 and the hinge exon h 4.

In a preferred embodiment, the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4 having the amino acid sequences 94 to 120 of SEQ ID NO. 1.

In a preferred embodiment, the dimerization unit comprises or consists of the amino acid sequence 94-120 of SEQ ID NO. 1, with the difference that at most 4 amino acids have been substituted, deleted or inserted, for example at most 3 amino acids, for example at most 2 amino acids or for example at most 1 amino acid have been substituted, deleted or inserted.

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

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

In another preferred embodiment, the dimerization unit comprises the amino acid sequence of SEQ ID NO:26, or consists of a nucleic acid sequence of seq id no.

In another embodiment, the dimerization unit comprises another domain that promotes dimerization, the other domain being an immunoglobulin domain, such as 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 derived from IgG. More preferably, the other domain that promotes dimerization is a carboxy-terminal C domain derived from IgG 3.

In one embodiment, the dimerization unit comprises a carboxy-terminal C domain derived from IgG3 having an amino acid sequence having at least 80% sequence identity to the amino acid sequence of 131-237 of SEQ ID NO. 1; or consists of said carboxy terminal C domain derived from IgG 3.

In preferred embodiments, the dimerization unit comprises a carboxy-terminal C domain derived from IgG3 having an amino acid sequence having at least 85% sequence identity to the amino acid sequence of 131-237 of SEQ ID No. 1, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99% sequence identity; or consists of said carboxy terminal C domain derived from IgG 3.

In a preferred embodiment, the dimerization unit comprises a carboxy-terminal C domain derived from IgG3 having the amino acid sequence of 131-237 of SEQ ID NO. 1; or consists of said carboxy terminal C domain derived from IgG 3.

In a preferred embodiment, the dimerization unit comprises or consists of the amino acid sequence of 131-237 of SEQ ID NO.1, with the exception 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 has been substituted, deleted or inserted.

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

In a further preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 85% sequence identity with the nucleic acid sequence of SEQ ID NO. 27, e.g., at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity. In another preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO. 27.

Immunoglobulin domains promote dimerization through non-covalent interactions such as hydrophobic interactions. Thus, in one embodiment, the immunoglobulin domain has the ability to form dimers through non-covalent interactions. Preferably, the non-covalent interactions are hydrophobic interactions.

Preferably, if the dimerization unit comprises a CH3 domain, it does not comprise a CH2 domain and vice versa.

In a preferred embodiment, the dimerization unit comprises hinge exon h1, hinge exon h4, dimerization unit linker and the CH3 domain of human IgG 3. In a further preferred embodiment, the dimerization unit comprises a polypeptide consisting of hinge exon h1, hinge exon h4, a dimerization unit linker and the CH3 domain of human IgG 3. In another preferred embodiment, the dimerization unit consists of a polypeptide consisting of hinge exon h1, hinge exon h4, dimerization unit linker and the CH3 domain of human IgG 3.

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

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

In an even more preferred embodiment, the dimerization unit comprises the amino acid sequence of 94-237 of SEQ ID NO. 1.

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

In an even more preferred embodiment, the dimerization unit consists of the amino acid sequence of 94-237 of SEQ ID NO. 1.

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

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

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

In another preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO. 28.

In the first polypeptide encoded by the first nucleic acid sequence, the multimerization units, e.g., dimerization units, may have any orientation relative to the antigenic units and targeting units. In one embodiment, the antigenic unit is attached to the C-terminus of the multimerization/dimerization unit (e.g., via a unit linker), while the targeting unit is attached to the N-terminus of the multimerization/dimerization unit. In another embodiment, the antigenic unit is attached to the N-terminus of the multimerization/dimerization unit (e.g., via a unit linker), and the targeting unit is attached to the C-terminus of the multimerization/dimerization unit. Preferably, the antigenic 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.

Antigenic unit

In general, the antigenic units comprised in the first polypeptide/multimeric protein may comprise any type of antigen or part thereof, for example an antigen associated with a disease or part thereof. Examples include one or more cancer antigens or portions thereof or one or more antigens associated with infectious diseases or portions thereof, i.e., diseases caused by pathogens (including viruses, bacteria, fungi, and parasites).

"Disease-associated antigen" or "disease-associated antigen" is used herein to describe an antigen or portion thereof comprised in an antigenic unit that functions in and has relevance to certain diseases for which the vectors of the present invention comprising such antigenic unit are designed to treat. As an example, an antigenic unit comprises one or more cancer antigens or parts thereof, and a vector comprising such an antigenic unit is designed for the treatment of cancer. In another example, the antigenic unit comprises one or more infectious antigens or parts thereof, e.g. antigens derived from a pathogen, and the vector comprising such antigenic unit is designed for the treatment of an infectious disease caused by such pathogen or an infectious disease in which such pathogen is involved.

"Part" refers to a part/fragment of an antigen, i.e. the amino acid sequence of an antigen or a part/fragment of a nucleotide sequence encoding it, e.g. an epitope.

In one embodiment, the antigenic unit comprises a T cell epitope. In another embodiment, the antigenic unit comprises more than one T cell epitope, i.e. a plurality of T cell epitopes.

T cell epitopes suitable for inclusion in an antigenic unit may be known in the art, i.e. have been studied, proposed and/or validated as being involved in and associated with a certain disease and disclosed in e.g. the scientific literature.

In one embodiment, the antigenic unit comprises a T cell epitope of 7 to 150 amino acids in length, preferably a T cell epitope of 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.

In one embodiment, the antigenic units comprised in the first polypeptide/multimeric protein comprise one or more antigens associated with an infectious disease or a portion thereof, e.g. antigens derived from a pathogen.

Such antigens may be known in the art or predicted, i.e. have been studied, proposed and/or validated as being involved in and associated with a certain infectious disease and disclosed in e.g. the scientific literature.

In another embodiment, the antigenic unit comprised in the first polypeptide/multimeric protein comprises one or more antigens associated with cancer or parts thereof, for example a cancer antigen such as a neoantigen or a consensus cancer antigen.

Individualizing antigenic units of a first polypeptide

In one embodiment, the first polypeptide encoded by the first nucleic acid comprised in the vector of the invention comprises an antigenic unit, which is specifically designed and is directed only to the patient to be treated with such a vector. Thus, the antigenic unit of such a first polypeptide comprises one or more patient-specific cancer antigens or parts thereof, such antigens comprising a neoantigen or a consensus cancer antigen present in the patient.

"Patient-present consensus cancer antigen" is used herein to describe a consensus cancer antigen or consensus tumor antigen that has been identified as being present in a patient's tumor cells.

"Neoantigen" is used herein to describe a cancer antigen or tumor antigen found in a tumor cell of a patient that contains one or more mutations compared to normal (i.e., healthy, non-cancerous) cells of the same patient.

"Patient-present consensus cancer epitope" is used herein to describe an amino acid sequence contained in a patient-present consensus cancer antigen or a nucleic acid sequence encoding the same, which is known to be immunogenic or predicted to be immunogenic.

"Neoepitope or patient-specific cancer epitope" is used herein to describe 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 that are predicted to be immunogenic.

Accordingly, in one embodiment, the present invention provides a vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit, e.g., a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more patient-specific cancer antigens or portions thereof, e.g., one or more patient-present consensus cancer antigens or portions thereof, and/or one or more neoantigens or portions thereof; and

In one embodiment, the antigenic unit comprises one or more patient-present consensus cancer antigen or a portion thereof, e.g., one patient-present consensus cancer antigen or one or more portion of such patient-present consensus cancer antigen, e.g., one or more epitope, or a plurality of patient-present consensus cancer antigen or one or more portion of such plurality of patient-present consensus cancer antigen, e.g., one or more epitope.

The term "several" is used interchangeably herein with the terms "plurality," a plurality, "and" more than one.

In another embodiment, the antigenic unit comprises one or more neoantigens or parts thereof, e.g. one neoantigen or one or more parts of such neoantigen, e.g. one or more neoepitopes or several neoantigens or one or more parts of such several neoantigens, e.g. one or more neoepitopes.

In another embodiment, the antigenic unit comprises any combination of the preceding embodiments, i.e., any combination of the consensus cancer antigen or portion thereof and any combination of one or more neoantigens or portions thereof present in one or more patients as described above.

Antigenic units of personalized polypeptides comprising one or more neoantigens or parts thereof

Cancers develop from uncontrolled proliferation of one or more cells in the normal tissue of a patient that begin to be abnormal due to mutations. Although cancer cells are mutated, a substantial portion of the genome is intact and identical to the rest of the cells in the patient. One approach to attacking tumors is based on the following knowledge: any tumor of any patient is unique: patient-specific mutations result in the expression of patient-specific muteins, i.e., neoantigens unique to a particular patient. These novel antigens are not identical to any protein in the normal cells of the patient. Thus, such novel antigens are suitable targets for therapeutic pharmaceutical compositions comprising the vectors of the invention, which are specifically manufactured only for the patient in question, i.e. personalized anti-cancer vaccines.

The mutation may be any mutation resulting in a change of at least one amino acid. Thus, the mutation may be one of the following mutations:

Non-synonymous mutations leading to amino acid changes,

Mutations that lead to frame shifts and thus to completely different open reading frames in the direction after mutation,

Read-through mutations with modified or deleted stop codons resulting in longer proteins with tumor specific epitopes

Splice mutations, which result in unique tumor specific protein sequences

Chromosomal rearrangements that result in chimeric proteins with tumor-specific epitopes at the junction of the two proteins. When the mutation is due to chromosomal rearrangement, the tumor-specific epitope may be generated due to a change in at least one amino acid or a combination of two in-frame coding sequences.

In one embodiment, the antigenic unit comprises one or more neoantigens or parts thereof, e.g. one or more parts of one neoantigen or one or more parts of several neoantigens, preferably one or more neoepitopes, more preferably several neoepitopes. Such neo-epitopes may be selected for inclusion in antigenic units according to their predicted therapeutic efficacy, see WO 2017/118695A1, the disclosure of which is incorporated herein by reference.

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerization unit, such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more neoantigens or portions thereof; and

In one embodiment, the antigenic unit comprises one or more parts of one neoantigen or one or more parts of several neoantigens, preferably one or more neoepitopes. In a preferred embodiment, the neo-epitopes are separated by linkers in the antigenic unit. Another way of describing that all neo-epitopes are separated by linkers is that all neo-epitopes except the terminal neo-epitope (i.e. the neo-epitope at the N-terminus of the first polypeptide start or at the C-terminus of the first polypeptide end) are arranged in antigen subunits, wherein each subunit comprises a neo-epitope and a subunit linker. Since the neo-epitopes are separated by linkers, each neo-epitope is presented to the immune system in an optimal manner.

Thus, an antigenic unit comprising n neoepitopes comprises n-1 antigenic subunits, wherein each subunit comprises a neoepitope and a subunit linker, and further comprises a terminal neoepitope. In one embodiment, n is an integer from 1 to 50, such as from 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 consists of a neoepitope and subunit linker.

Accordingly, in a preferred embodiment, the present invention provides a vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit, such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises (i) n-1 antigenic subunits, each subunit comprising a neoepitope and a subunit linker, and (ii) a terminal neoepitope, and wherein n is the number of neoepitopes in the antigenic unit, and n is an integer from 1 to 50; and

The neoepitope is preferably of a length suitable for presentation of the HLA molecule. Thus, in a preferred embodiment, the neoepitope is 7 to 30 amino acids in length. More preferably a neoepitope of 7 to 10 amino acids or 13 to 30 amino acids, e.g. 20 to 30 amino acids, e.g. 27 amino acids in length.

Preferably, the antigenic unit comprises a plurality of neoepitopes. In one embodiment, the antigenic unit comprises a plurality of different neoepitopes. In another embodiment, the antigenic unit comprises multiple copies of the same neoepitope. In another embodiment, the antigenic unit comprises a plurality of different neoepitopes and a plurality of copies of the same neoepitope.

Thus, a preferred approach is to include as many neo-epitopes (i.e. different neo-epitopes and/or multiple copies of the same neo-epitope) in the antigenic unit as possible to effectively attack the cancer while not compromising the ability to activate T cells against the neo-epitope due to dilution of the desired T cell effect. Furthermore, to ensure that all neoepitopes are efficiently loaded into the same antigen presenting cell, all neoepitope encoding nucleotide sequences are contained in a continuous polynucleotide chain, resulting in expression of proteins containing all neoepitopes, rather than expressing each neoepitope as a separate peptide.

To design antigenic units, the patient's tumor exome is analyzed to identify new antigens. Preferably, the sequence of the most immunogenic neoepitope from one or more neoantigens is selected for inclusion into the antigenic unit.

In one embodiment, the antigenic unit comprises at least 1 neoepitope. Preferably, the antigenic unit comprises at least 3 neo-epitopes, more preferably at least 5 neo-epitopes, e.g. 7 neo-epitopes. In another more preferred embodiment, the antigenic unit comprises at least 10 neoepitopes. In another more preferred embodiment, the antigenic unit comprises at least 15 neo-epitopes, e.g. at least 20 or at least 25 or at least 30 or at least 35 or at least 40 or at least 45 neo-epitopes.

Antigenic units comprising one or more neoepitopes are described in detail in WO 2017/118695 A1. Any such antigenic unit may be used as an antigenic unit in the first polypeptide encoded in the vector of the invention for use in personalized anti-cancer therapy.

Antigenic units comprising a personalized polypeptide of a consensus cancer antigen or portion thereof present in one or more patients

A common tumor antigen is expressed by many tumors, spanning patients with the same cancer type or spanning both patient and cancer type. An example is HPV16 antigen, a viral antigen, expressed in about 50% of head and neck squamous cell carcinoma patients, and also in cervical and vulvar squamous cell carcinoma and other cancer patients. Many of these consensus antigens have previously been characterized as immunogenic and/or are known, i.e. their immunogenicity has been confirmed by suitable methods and the results have been published in, for example, scientific publications. Other consensus antigens have been predicted to be present on specific HLA class I or class II alleles, for example by algorithms known in the art, and their predicted immunogenicity has been published in, for example, scientific publications, but their immunogenicity has not been confirmed by appropriate methods.

In one embodiment, the antigenic unit comprises one or more patient-present consensus cancer antigens or portions thereof, e.g., a patient-present consensus cancer epitope known to be immunogenic has a known expression profile and/or is known or has been predicted to bind to a particular HLAI class and class II molecule.

T cells specific for the common cancer antigen present in the patient can migrate to the tumor and affect the tumor microenvironment, thereby increasing the likelihood that other tumor-specific T cells will be able to attack the cancer.

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerization unit, e.g., a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more patient-present consensus cancer antigens or portions thereof; and

Some patients present with a common cancer antigen is a protein comprising an amino acid sequence that contains one or more mutations, i.e., a common cancer epitope that is known to be immunogenic or that has been predicted to be immunogenic. Other patients present with common cancer antigens are proteins that do not contain mutations, such as over-expressed cellular proteins.

In one embodiment, the consensus cancer antigen present in the patient is selected from the group consisting of over-expressed cellular proteins, aberrantly expressed cellular proteins, cancer testis antigens, viral antigens, differentiation antigens, mutated oncogenes and mutated tumor suppressor genes, cancer embryo antigens, consensus fusion antigens, consensus intron retention antigens, dark matter antigens, and consensus antigens resulting from spliceosome mutations or frameshift mutations.

In one embodiment, the patient is presented with a consensus cancer antigen that is an over-or abnormally expressed human cellular protein, i.e., a cellular protein that is found at elevated levels in tumors as compared to normal healthy cells and tissues. Examples of such over-or abnormally expressed cellular proteins include tumor protein D52, her-2/neu, hTERT (telomerase), and survivin (survivin).

In another embodiment, the patient presents with a consensus cancer antigen that is a cancer testis antigen that is normally expressed in male germ cells in the testis, but not in adult body tissue. In some cases, such antigens are also expressed in ovaries and trophoblasts. In malignant tumors, this gene regulation is disrupted, resulting in antigen expression in a portion of the different types of tumors. Examples of cancer testis antigens include MAGE-A, MAGE-B, GAGE, PAGE-1, SSX, HOM-MEL-40 (SSX 2), NY-ESO-1, LAGE-1 and SCP-1.

In another embodiment, the patient is presented with a consensus cancer antigen that is a differentiation antigen, such as tyrosinase.

In yet another embodiment, the patient's presence of a consensus antigen is a viral antigen. Examples of viral antigens include Human Papilloma Virus (HPV), hepatitis B Virus (HBV), epstein-barr virus (EBV), kaposi's sarcoma-associated herpesvirus (KSHV), merck's cell polyoma virus (MCV or MCPyV), human Cytomegalovirus (HCMV), and human T-lymphocyte virus (HTLV).

In yet another embodiment, the patient's common cancer antigen is a mutated oncogene. Examples of mutated oncogenes include KRAS, CALR and TRP-2.

In yet another embodiment, the patient's common cancer antigen is a mutated tumor suppressor gene. Examples include mutant p53, mutant pRB, mutant BCL2 and mutant SWI/SNF.

In another embodiment, the patient is presented with a consensus cancer antigen that is a carcinoembryonic antigen, such as alpha fetoprotein or carcinoembryonic antigen.

In yet another embodiment, the patient is presented with a consensus antigen that is a consensus intron retention antigen or a consensus antigen caused by a frameshift mutation, such as CDX2 or CALR.

In yet another embodiment, the patient's presence of a consensus antigen is a consensus antigen caused by a splice mutation. One example is an antigen caused by a mutation such as SF3B1 mut.

In general, for any cancer antigen, immune tolerance is likely to have occurred when the patient developed cancer. An anti-cancer vaccine should specifically trigger an immune response against the antigen contained in the vaccine. In one embodiment, the first polypeptide encoded by the plasmid serves as an anti-cancer vaccine. The peripheral immune tolerance against the selected antigen may be weak or strong. By incorporating such patient-present consensus cancer antigen or one or more portions thereof into an antigenic unit (alone or together with other patient-present consensus cancer antigen or portions thereof and/or neoantigen or neoepitope), polypeptides comprising such antigenic unit elicit an immune response sufficiently strong and extensive to affect the tumor microenvironment and change the patient's immune response to the tumor from an inhibitory/tolerogenic to a pro-inflammatory type. This can help break tolerance to a variety of other antigens, thereby bringing considerable clinical benefit to the patient. The above concepts may be referred to as tilting the cancer immune set point.

In one embodiment, the antigenic unit comprises one or more patient-present consensus cancer antigens or parts thereof, which are human cellular proteins, preferably over-expressed or abnormally expressed human cellular proteins or differentiation antigens.

The presence of a common cancer antigen in a patient can be detected in the patient's tissue or body fluid by methods known in the art, including:

Sequencing the patient's genome or exome and optionally searching, for example by custom software, in whole genome/exome sequencing data to identify, for example, mutated oncogenes or mutated tumor suppressor genes;

immunohistochemical analysis of patient tumor tissue, for example to detect the presence of muteins;

RT-PCR to detect, for example, the presence of known mutations in viral antigens or oncogenes;

ELISA, performed using antibodies against, for example, mutant tumor proteins in serum samples;

RNA-seq of tumor tissue and comparing with healthy tissue, e.g. to detect expression/overexpression of consensus cancer antigen;

searching, for example by custom software, in the original RNA sequence data to identify intron-retained antigens;

searching, for example by custom software, in whole genome-seq data to identify transposable elements as elements of dark matter antigens;

Detecting short repeats in the original whole-exome/RNA sequence data, for example to identify dark matter antigens;

RNA-seq data, e.g., to identify consensus viral antigens; and

Comparing the RNA-seq of the patient's tumor sample to the patient's own healthy tissue or cohort/database (e.g., TCGA) and consensus transcript expression (e.g., GTEX/HPA gene expression data).

In a preferred embodiment, the antigenic unit comprises a consensus cancer antigen or one or more portions of such antigen present in one or more patients, which is known to be immunogenic, e.g. has been described previously as eliciting an immune response in other patients, or has been predicted to bind to HLA class I and/or class II alleles of a patient.

In one embodiment, the antigenic unit comprises one or more common cancer epitopes present in the patient. In a preferred embodiment, such epitopes have a length suitable for presentation by the patient's HLA allele.

In one embodiment, the antigenic unit comprises one or more patient-present consensus cancer epitopes of a length suitable for specific presentation on the HLAI class or the HLAII class. In one embodiment, the epitope has a length of 7 to 11 amino acids for HLAI class presentation. In another embodiment, the epitope has a length of 13 to 30 amino acids for HLA class II presentation.

In one embodiment, the antigenic unit comprises a consensus cancer epitope present in one or more patients, e.g., 7 to 30 amino acids in length, e.g., 7 to 10 amino acids (e.g., 7, 8, 9, or 10 amino acids) or 13 to 30 amino acids (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids), e.g., 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

The antigenic unit may comprise the consensus cancer antigen or one or more portions thereof present in one or more patients in full length.

In one embodiment, the antigenic unit comprises a consensus cancer antigen present for a full-length patient. In another embodiment, the antigenic unit comprises a consensus cancer antigen present for a plurality of patients, each of which is full length.

In yet another embodiment, the antigenic unit comprises one or more portions of a common cancer antigen present in a patient, e.g., one or more common cancer epitopes present in a patient. In yet another embodiment, the antigenic unit comprises one or more portions of a common cancer antigen present in a plurality of patients, e.g., one or more epitopes of a common cancer antigen present in a plurality of patients.

In yet another embodiment, the antigenic unit comprises one or more portions of the common antigen present in one or more full length patients and the common cancer antigen present in one or more patients. Examples include:

The antigenic unit comprises one or more epitopes of a common antigen present in a full-length patient and a common cancer antigen present in a patient; and

An antigenic unit comprising a plurality of patient-present consensus cancer antigens (each of which is full length) and one or more epitopes of a patient-present consensus cancer antigen; and

-The antigenic unit comprises one full length patient-present consensus antigen and one or more epitopes of a plurality of patient-present consensus cancer antigens; and

The antigenic unit comprises a plurality of patient-present consensus cancer antigens (each of which is full length) and one or more epitopes of the plurality of patient-present consensus cancer antigens.

In a preferred embodiment, the aforementioned epitope is known to be immunogenic, e.g. has been described in the literature as being immunogenic, or has been predicted to bind to a patient's HLAI class and class II allele, e.g. as described in the literature, preferably has been predicted to bind to a patient's HLAI class II allele. In another preferred embodiment, the immunogenicity of the above-described epitope is predicted by methods known in the art, e.g., predicting binding of the epitope to one or more HLAI-like and/or HLAII-like molecules of the patient, as disclosed in WO 2021/205027 A1 (incorporated herein by reference), or as described herein, including those described in the section "methods for designing antigenic units of an individualized first polypeptide".

In one embodiment, the antigenic unit comprises 1 to 10 total length of the patient's consensus antigen present.

In another embodiment, the antigenic unit comprises 1 to 30 portions of a consensus antigen present in one or more patients, wherein the portions comprise a plurality of epitopes predicted to bind to HLA class I or class II alleles of the patient. In another embodiment, the antigenic unit comprises a consensus cancer epitope present in 1 to 50 patients, preferably an epitope predicted to bind to a patient's HLAI or class II allele.

Antigenic units comprising one or more patient-present consensus cancer antigen or portion thereof and one or more neoantigen or portion thereof personalized polypeptides

In further embodiments, the antigenic units are a combination of all of the foregoing embodiments directed to an antigenic unit comprising one or more common cancer antigens or portions thereof present in a patient and all of the foregoing embodiments directed to an antigenic unit comprising one or more neoantigens or portions thereof.

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit, such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more consensus cancer antigens or portions thereof present in a patient and one or more neoantigens or portions thereof; and

Antigenic units comprising one or more patient-present consensus cancer antigens or parts thereof, and optionally one or more neoantigens and parts thereof, are described in detail in WO2021/205027A1, the contents of which are incorporated herein by reference. Any such antigenic unit may be used as the antigenic unit in the first polypeptide encoded in the vector of the invention for use in personalized anti-cancer therapy.

Method for designing an antigenic unit of an individualized first polypeptide

The presence of the common cancer antigen and the neoantigen of the patient identified in a particular patient is preferably further processed to find those antigens that, when included in the antigenic unit, make the first polypeptide most effective. The manner and order in which such treatments are performed depends on how the antigen is identified, i.e. the data forming the basis for such treatments.

In one embodiment, the treatment and selection of the antigen to be included in the antigenic unit is performed as follows:

1) Searches are performed in the literature and/or one or more databases to find information and sequences about the consensus cancer antigen, and preferably information about its expression pattern, immunogenicity or predicted immunogenicity, epitopes and HLA presentation. Such searches are also conducted to determine whether the identified antigen is a consensus cancer antigen or a neoantigen present in the patient.

2) If it is determined that the identified antigen is a consensus cancer antigen present in the patient, its sequence is studied to identify epitopes, preferably all epitopes, predicted to bind to the patient's HLA class I/II allele. The prediction may be performed by using prediction tools known in the art, such as prediction software known in the art, e.g., NETMHCPAN and the like.

3) The most immunogenic or most promising sequences for predicting the most immunogenic cancer antigens shared by patients, i.e. those sequences that show predicted binding to one or more HLA class I/II alleles of a patient, are selected for inclusion in the antigenic unit. In one embodiment, the smallest epitope is selected, for example in case only a few promising epitopes are identified in step 2 or longer non-immunogenic sequence segments are present between the epitopes. In another embodiment, a longer sequence comprising multiple epitopes that bind to a particular HLA allele of a patient is selected. In yet another embodiment, the full length sequence of the antigen is selected for inclusion in the antigenic unit.

4) Based on predicted immunogenicity and binding to the patient's HLA class I/II alleles, the most promising part of the neoantigen sequence, e.g. neoepitope, is selected to be comprised in the antigenic unit.

Tumor mutations are found by sequencing tumor and normal tissue and comparing sequences obtained from tumor tissue with sequences of normal tissue. There are a variety of methods available for detecting the presence of a particular mutation or allele in a patient's DNA or RNA. Such methods include dynamic allele-specific hybridization (DASH), microplate Array Diagonal Gel Electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, taqMan systems, and various DNA "chip" techniques (e.g., affymetrix SNP chips). Alternatively, mutations can be identified by direct protein sequencing.

Of the hundreds or thousands of possible mutations in a tumor exome, the most promising sequences were selected using computer based on predictive HLA binding algorithms. The objective is to identify all relevant epitopes and to determine the sequences to be included in the antigenic units after ranking or scoring. Methods known in the art that may be suitable for scoring, ranking and selecting neoepitopes include those disclosed in WO 2020/065023A1 and WO 2020/221/783A 1.

Further, any suitable algorithm for such scoring and ranking may be used, including the following:

free software analysis of peptide-MHC binding (IEDB and NETMHCPAN) available, can be downloaded from the following website:

www.iedb.org/

www.cbs.dtu.dk/services/NetMHC/；

commercially available high-level software for predicting the optimal sequence for vaccine design can be found in the following:

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/

Each mutation is scored for its antigenicity, the most antigenic neoepitope is selected, and optimally aligned in antigenic units.

Antigenic units of non-individualised first polypeptides

An antigenic unit comprising one or more first polypeptides sharing a cancer antigen or portion thereof

A non-personalized or "off-the-shelf vector encoding a first polypeptide (also referred to as a first polypeptide comprising a consensus cancer antigen) comprises a polynucleotide sequence encoding an antigenic unit comprising one or more consensus cancer antigens or portions thereof.

"Consensus cancer antigen" or "consensus tumor antigen" is used herein to describe an antigen that has been described as being expressed by a number of tumors, across patients with the same cancer type or across patients and cancer types.

"Consensus cancer epitope" is used herein to describe the amino acid sequence contained in a consensus cancer antigen that is known or predicted to be immunogenic.

In one embodiment, the antigenic unit of the non-personalized first polypeptide for use in treating cancer comprises one or more consensus cancer antigens or parts thereof, e.g. consensus cancer epitopes, which are known to be immunogenic, have a known expression pattern and/or are known or predicted to bind to specific HLAI and class II molecules.

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerization unit, e.g., a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more consensus cancer antigens or portions thereof; and

Some consensus cancer antigens are proteins comprising an amino acid sequence containing one or more mutations, i.e. consensus cancer epitopes that are known or have been predicted to be immunogenic. Other consensus cancer antigens are proteins that do not contain mutations, such as over-expressed cellular proteins.

In one embodiment, the consensus cancer antigen is selected from the group consisting of an overexpressed cellular protein, an abnormally expressed cellular protein, a cancer testis antigen, a viral antigen, a differentiation antigen, a mutated oncogene and a mutated tumor suppressor gene, a cancer embryo antigen, a consensus fusion antigen, a consensus intron retention antigen, a dark matter antigen, and a consensus antigen resulting from a splice mutation or a frameshift mutation.

In one embodiment, the consensus cancer antigen is an over-expressed or abnormally expressed human cellular protein, i.e., a cellular protein found at elevated levels in tumors as compared to normal healthy cells and tissues. Examples of such over-or abnormally expressed cellular proteins include tumor protein D52, her-2/neu, hTERT (telomerase), and survivin.

In another embodiment, the consensus cancer antigen is a cancer testis antigen that is normally expressed in male germ cells in the testis, but not in adult body tissue (somatic tissue). In some cases, such antigens are also expressed in ovaries and trophoblasts. In malignant tumors, this gene regulation is disrupted, resulting in antigen expression in a portion of the different types of tumors. Examples of cancer testis antigens include MAGE-A, MAGE-B, GAGE, PAGE-1, SSX, HOM-MEL-40 (SSX 2), NY-ESO-1, LAGE-1 and SCP-1.

In another embodiment, the consensus cancer antigen is a differentiation antigen, such as tyrosinase.

In yet another embodiment, the consensus antigen is a viral antigen. Examples of viral antigens include Human Papilloma Virus (HPV), hepatitis B Virus (HBV), epstein-barr virus (EBV), kaposi's sarcoma-associated herpesvirus (KSHV), merck's cell polyoma virus (MCV or MCPyV), human Cytomegalovirus (HCMV), and human T-lymphotropic virus (HTLV).

In yet another embodiment, the consensus cancer antigen is a mutated oncogene. Examples of mutated oncogenes include KRAS, CALR and TRP-2.

In yet another embodiment, the consensus cancer antigen is a mutated tumor suppressor gene. Examples include mutant p53, mutant pRB, mutant BCL2 and mutant SWI/SNF.

In another embodiment, the consensus cancer antigen is a carcinoembryonic antigen (oncofetal antigen), such as alpha fetoprotein or carcinoembryonic antigen (carcinoembryonic antigen).

In another embodiment, the consensus antigen is a consensus intron retention antigen or a consensus antigen caused by a frameshift mutation, such as CDX2 or CALR.

In yet another embodiment, the consensus antigen is a consensus antigen caused by a splice mutation. One example is an antigen caused by a mutation such as SF3B1 mut.

Further examples of consensus cancer antigens include scFv derived from monoclonal igs produced by myeloma or lymphoma, also known as myeloma/lymphoma M component in patients with B-cell lymphoma or multiple myeloma, HIV derived sequences such as gpl20 or Gag derived sequences, tyrosinase Related Protein (TRP) -1, melanoma antigens, prostate specific antigens and idiotypes, HPV antigens selected from E1, E2, E6, E7, L1 and L2, e.g. E6 and/or E7 of HPV16 and/or HPV 18.

Any sufficient length of consensus cancer antigen sequence including a particular epitope may be used as the antigenic unit. Thus, in one embodiment, the antigenic unit comprises an amino acid sequence of at least 7 amino acids, e.g. at least 8 amino acids (corresponding to at least about 21 nucleotides, e.g. at least 24 nucleotides, of a nucleic acid sequence encoding such an antigenic unit).

In yet another embodiment, the antigenic unit comprises one or more portions of a common cancer antigen, e.g., one or more common cancer epitopes. In yet another embodiment, the antigenic unit comprises one or more portions of a plurality of common cancer antigens, e.g., one or more epitopes of a plurality of common cancer antigens. In yet another embodiment, the antigenic unit comprises one or more consensus antigens in full length and one or more portions of one or more consensus cancer antigens. Examples include:

An antigenic unit comprising one consensus antigen and one or more epitopes of one consensus cancer antigen in full length; and

An antigenic unit comprising a plurality of consensus cancer antigens (each full length) and one or more epitopes of one consensus cancer antigen; and

An antigenic unit comprising one consensus antigen and one or more epitopes of a plurality of consensus cancer antigens in full length; and

An antigenic unit comprising a plurality of consensus cancer antigens and one or more epitopes of the plurality of consensus cancer antigens, each of which is full length.

Examples of polypeptides comprising consensus antigens against HPV are disclosed in WO 2013/092875A1, the contents of which are incorporated herein by reference.

Method for designing an antigenic unit comprising a first polypeptide sharing a cancer antigen

Furthermore, for vectors encoding a first polypeptide comprising a common cancer antigen, the antigenic units are designed to include those sequences that would render the polypeptide effective in multiple patients (e.g., patients with a certain type of cancer).

In one embodiment, the antigen to be included in the antigenic unit is selected by searching in the literature and/or one or more databases for information and sequences about the consensus cancer antigen, and preferably for information about its expression pattern, immunogenicity or predicted immunogenicity, epitopes and HLA presentation. Epitopes are then identified which are known or predicted to bind to multiple HLA class I/II alleles of many patients, or to a subset of HLA class I/II alleles, which subset predominates in a certain cancer indication and/or a certain patient population across different cancer indications (dominant). Preferably, the sequence of the most promising, i.e. most immunogenic or predicted to be the most immunogenic, consensus cancer antigen is selected for inclusion in the antigenic unit.

Antigenic units of a first polypeptide comprising one or more infectious antigens or parts thereof

In another aspect 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 use in the treatment of an infectious disease, and the vector/first polypeptide is used for the treatment of an infectious disease.

In one embodiment, the antigenic units comprised in the first polypeptide comprise one or more antigens associated with an infectious disease, or parts thereof, i.e. one or more infectious antigens, i.e. antigens derived from a pathogen, or parts thereof.

An "infectious disease" is used herein to describe a condition caused by a pathogen or a condition in which a pathogen participates in causing the condition. An example of the latter is parasite eggs, which do not cause disease by themselves, but develop into larvae causing disease.

"Pathogen" includes viruses, bacteria, fungi and parasites.

Antigens described in this section are "infectious antigens", i.e. antigens derived from a pathogen, i.e. they are comprised in (or naturally present in) a protein of a pathogen causing or involved in causing a disease. The terms "infectious antigen" and "pathogen-derived antigen" are used interchangeably herein.

Accordingly, in one embodiment, the present invention relates to a vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerization unit, such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more infectious antigens or portions thereof; and

In another embodiment, the invention relates to a vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerization unit, such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more antigens derived from one or more pathogens or portions of such antigens; and

In the above embodiments, the antigenic unit comprises one or more antigens derived from a pathogen or parts of such antigens, e.g. one antigen derived from a pathogen or more than one antigen derived from a pathogen, i.e. multiple antigens derived from a pathogen, e.g. comprised in the same or different proteins of such a pathogen.

In one embodiment, the antigenic unit comprises one or more antigens derived from multiple pathogens or portions of such antigens. In one embodiment, the plurality of pathogens is a plurality of different pathogens. In this case, the "different pathogens" may for example be different viruses or bacteria or different strains of the same virus or bacteria or it may be the same strain but contain one or more mutations.

Vectors comprising one or more antigens derived from multiple pathogens or parts thereof may be used in a universal vaccine (pan-vaccine), for example a vaccine targeting different (seasonal) viruses. For example, the flood vaccine may target betacoronaviruses and influenza viruses, or different strains of betacoronaviruses or different mutations of the same strain, for example.

Examples of infectious/pathogen-derived antigens are antigens of bacterial origin, such as tuberculosis antigen and OMP31 from brucellosis, or antigens of viral origin, such as HIV derived sequences, such as gp120 derived sequences, glycoprotein D from HSV-2, and influenza virus antigens, such as hemagglutinin, nucleoprotein and M2, and HPV derived antigens, such as E1, E2, E6, E7, L1 or L2, e.g. E6 and E7 of HPV16 or HPV 18.

In one embodiment, the antigenic unit comprises one or more than one β coronavirus antigen or portion thereof.

Beta coronaviruses represent a genus in the subfamily of the orthopoxviridae. Beta coronaviruses are enveloped, positive-sense, single-stranded RNA viruses. Within this genus, there are four lineages common: lineage a (Embecovirus subgenera), lineage B (Sarbecovirus subgenera), lineage C (Merbecovirus), and lineage D (Nobecovirus). Beta coronaviruses include the following viruses that cause/lead to human epidemics/pandemics or may 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 (subgenera Sarbecovirus), MERS-CoV belongs to lineage C (Merbecovirus), HCoV-OC43 and HCoV-HKU1 belong to lineage A (subgenera Embecovirus).

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

In one embodiment of the invention, the antigen may be a T cell epitope which is part of the sequence of a spike protein or a membrane protein or an envelope protein or a nucleocapsid protein or an 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.

In some embodiments, the antigenic units of the vectors of the invention comprise one or more antigens derived from one or more pathogens selected from the group consisting of influenza virus, herpes simplex virus, CMV, HPV, HBV, brucella, HIV, HSV-2, and mycobacterium tuberculosis, or portions thereof.

The vector of the present invention for the treatment of infectious diseases is an ideal vector against pandemics and epidemics because it can induce a rapid, strong immune response. Such vectors are designed to induce an antigenic effect by incorporating into the antigenic unit, or by a combination thereof, the full length or portion of one or more infectious antigens, which may be, for example, a selected T cell epitope.

In one embodiment, the targeting unit of such a first polypeptide is anti-pan-HLA class II or human MIP-1α, and will elicit an immune response by B cells and/or T cells. In one embodiment, the carrier may be used in a prophylactic or therapeutic environment or both.

Antigenic units comprising a first polypeptide comprising one or more T cell epitopes 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 in (or naturally present in) the proteins of the pathogen. The part of the genome that is conserved among many pathogens comprises T cell epitopes that are capable of initiating an immune response.

Accordingly, one aspect of the invention relates to a carrier comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit, such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises at least one T cell epitope from one or more infectious antigens; and

In one embodiment, the invention relates to a vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit, such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises at least one T cell epitope derived from one or more pathogens; and

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

The at least one T cell epitope comprised in the antigenic unit is 7 to about 200 amino acids in length, the longer T cell epitope possibly comprising a hot spot of the smallest T cell epitope. A "hot spot of a minimum epitope" is a region containing multiple minimum T cell epitopes (e.g., having a length of 7-15 amino acids) that are predicted to be presented by different HLA alleles to cover a broad population of the world.

In some embodiments, the antigenic unit comprises at least one T cell epitope that is 7 to 150 amino acids in length, 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.

T cell epitopes of about 60 to 200 amino acids in length can be divided into shorter sequences and contained in antigenic units separated by linkers (e.g., linkers as described herein). For example, a T cell epitope of 150 amino acids in length can be divided into 3 sequences of 50 amino acids each and contained in an antigenic unit, with the 3 sequences separated from each other by a linker.

In one embodiment, the antigenic unit comprises a plurality of T cell epitopes separated from each other by a linker, such as the linkers discussed herein, e.g., the linkers discussed in the section "linker in antigenic unit" herein.

In some embodiments, at least one T cell epitope has a length suitable for presentation by MHC. Thus, in some embodiments, the antigenic 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, the at least one T cell epitope has a length of 7 to 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.

The number of T cell epitopes in an antigenic unit can vary and depends on the length and number of other elements (e.g., linkers) contained in the antigenic unit.

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

In a preferred embodiment, the at least one T cell epitope is derived from a conserved region of the pathogen, i.e. conserved among several subgenera, species or strains of the respective pathogen.

T cell epitopes can be comprised in any pathogen protein, i.e. in surface proteins but also in internal proteins, such as viral nucleocapsid proteins or viral replicase polyproteins, or in other structural and non-structural proteins.

Vectors comprising antigenic units containing T cell epitopes from conserved regions of pathogens will provide protection against multiple species/strains of pathogens. Such a vector will also provide protection against multiple variants of the pathogen, which is important for the efficacy of such a vector/first polypeptide against future mutated pathogens. Viruses are known to mutate, e.g., undergo viral antigen drift or antigen shift. The conserved regions found across the genus virons make these conserved regions likely necessary to maintain basic structure or function, so future mutations are expected to occur in less conserved regions. By increasing the immune response against the conserved regions, individuals treated with the plasmid will also be protected against future (and thus new) mutants.

Thus, in one embodiment of the invention, the antigenic unit is designed to elicit a cell-mediated immune response against/from T cell epitopes of the pathogen comprised in the infectious antigen by activating T cells. When an epitope is processed and presented in complex with an MHC molecule, T cells recognize the epitope.

In one embodiment, T cell epitopes are known in the art, e.g. epitopes that have been studied and described in the literature, e.g. epitopes that are known to be immunogenic, e.g. whose immunogenicity has been confirmed by suitable methods and the results have been published in e.g. scientific publications. In one embodiment, the antigenic unit comprises a plurality of T cell epitopes known to be immunogenic.

For example, useful T Cell epitopes known in the art are those in humans that are directed against SARS-CoV2 infection, as seen in Grifoni et al, cell Host microbe.2021Jul 14;29 (7):1076-1092. Thus, such T cell epitopes can be included in the antigenic unit of the vector for the treatment of human SARS-CoV2. Another example of such T cell epitopes are a T cell epitope with sequence CTELKLSDY (SEQ ID NO: 82) from the nucleoprotein of influenza A virus, a T cell epitope with sequence NLVPMVATV (SEQ ID NO: 83) from the 65kDa phosphoprotein of human herpes virus 5 (human cytomegalovirus), and a T cell epitope with sequence KLVANNTRL (SEQ ID NO: 84) from the diacylglycerol acyltransferase/mycobacterial acyltransferase of Mycobacterium tuberculosis (mycolyltransferase) Ag 85B.

As an example, the at least one T cell epitope may be from a region of Human Papillomavirus (HPV), e.g. from HPV16 or HPV18, e.g. at least one T cell epitope comprised in HPV antigens from E1, E2, E6, E7, L1 and L2 (e.g. E6 and/or E7 of HPV16 and/or HPV 18). By including such T cell epitopes in the vectors of the present disclosure, pharmaceutical compositions comprising such vectors may provide protection against HPV. HPV infection is involved in certain cancers, such as head and neck squamous cell carcinoma, cervical cancer, and vulvar squamous cell carcinoma. In fact, HPV16 virus antigen is expressed in about 50% of patients with the cancer.

As another example, the at least one T cell epitope may be from a region of human influenza virus such as human influenza virus a, human influenza virus B, human influenza virus C, and human influenza virus D. By way of example, human influenza viruses may be of a particular Hemagglutinin (HA) subtype, such as H1, H2, and H3, and/or of a particular Neuraminidase (NA) subtype, such as N1 or N5. As an example, human influenza virus may be an H1N1 subtype. Thus, such T cell epitopes may be included in the antigenic units of the vectors of the present disclosure for the treatment of influenza infection.

In another embodiment, the T cell epitope is predicted to be immunogenic, e.g., selected based on the predicted ability to bind to HLA class I/class II alleles. In one embodiment, the antigenic unit comprises a plurality of T cell epitopes, e.g., a plurality of T cell epitopes separated from each other by a linker, e.g., a linker as discussed herein, e.g., a linker as discussed in the section "linker in antigenic unit" herein that is predicted to bind to an HLA class I/II allele. T cell epitopes were selected on the computer based on HLA binding prediction algorithm. After all relevant epitopes are identified, the epitopes are ranked according to their ability to bind to HLA class I/II alleles, and the epitope predicted to have the best binding is selected for inclusion in the antigenic unit.

Suitable HLA binding algorithms are known in the art.

In another embodiment, the antigenic unit comprises a plurality of T cell epitopes, some of which are known to be immunogenic, while others are predicted to be immunogenic. In one embodiment, the T cell epitopes are separated from each other by a linker, such as the linkers discussed herein, e.g., the linkers discussed in the section "linker in antigenic unit" herein.

The antigenic unit comprising a T cell epitope in a vector for prophylactic and therapeutic treatment of a β coronavirus infection, and the widely applicable method of selecting a T cell epitope of the vector of the invention for prophylactic and therapeutic treatment of an infectious disease, are disclosed in detail in WO 2021/219897A1, the disclosure of which is incorporated herein by reference.

Antigenic units of a first polypeptide comprising one or more full-length infectious antigens or parts thereof or one or more B cell epitopes from one or more pathogens

In another aspect of the invention, the subject, e.g., a human individual, is a healthy individual, and the vector of the invention is for prophylactic use, e.g., to prevent a disease. Typically, the vector will be used to induce immunity in individuals in need of neutralizing antibodies against the pathogen in a prophylactic setting, such as to prevent infection.

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 the protein. Thus, in one embodiment, the at least one infectious antigen is a full-length surface protein or a portion thereof, e.g., a full-length viral surface protein or a bacterial surface protein or a full-length surface protein of any other pathogen.

In other embodiments, the infectious antigen is a full length bacterial protein secreted by the bacteria, e.g., into the cytoplasm of the infected subject.

In other embodiments, the antigenic unit comprises more than one infectious antigen or a portion of more than one infectious antigen, e.g., a plurality of full length infectious antigens.

In another embodiment, the antigenic unit comprises one or more antigens derived from a plurality of pathogens or portions of such antigens, e.g., a plurality of full length infectious antigens from a plurality of pathogens. In one embodiment, the plurality of pathogens is a plurality of different pathogens.

In one embodiment, such pathogen proteins are selected from the group consisting of beta coronavirus proteins, for example selected from the group consisting of envelope proteins, spike proteins, membrane proteins and spike-like proteins hemagglutinin esterases (if the beta coronavirus is Embecovirus).

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 neck domain of influenza virus hemagglutinin are examples of portions of infectious antigens.

In other embodiments, the antigenic unit comprises several portions of one infectious antigen. In other embodiments, the antigenic unit comprises one portion of several infectious antigens, e.g. one portion of infectious antigen 1 and one portion of infectious antigen 2 and 1 portion of infectious antigen 3. In other embodiments, the antigenic unit comprises several portions of several infectious antigens, e.g. 2 portions of infectious antigen 1 and 3 portions of infectious antigen 2. Infectious antigens 1,2 and 3 may be derived from one pathogen or from a plurality of different pathogens.

If more than one infectious antigen is contained in an antigenic unit, or more than 1 portion of one or more infectious antigens, the antigens or portions thereof may be separated by a linker, such as by a linker as discussed herein, such as the one discussed in the section "linker in antigenic unit" herein.

The one or more infectious antigens or portions thereof comprise conformational B-cell epitopes, but may also comprise linear B-cell epitopes and/or T-cell epitopes. In contrast to the T cell epitopes discussed in the previous section herein, these T cell epitopes are not isolated, but rather are presented to the immune system in their natural environment, i.e. they are flanked by amino acid residues present in the antigen.

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerization unit, e.g., a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more full-length infectious antigens or portions thereof; and

In another embodiment, the invention provides a vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit, such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more full-length antigens derived from one or more pathogens, or portions of such full-length antigens; and

In one embodiment, the antigenic unit comprises at least a B cell epitope derived from a pathogen, such as a full length protein derived from a pathogen, such as a full length surface protein, such as comprised in any of the foregoing proteins, and preferably comprises several B cell epitopes derived from a pathogen, such as comprised in a full length protein of a pathogen, such as a full length surface protein, such as comprised in any of the foregoing proteins. The at least one B cell epitope may be a linear or conformational B cell epitope.

In another embodiment, the invention provides a vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit, such as a dimerization unit, and an antigenic unit comprising at least one B cell epitope derived from one or more pathogens; and

After administration, the first polypeptide encoded by the first nucleic acid comprised in the vector of the invention as described above (i.e. a vector comprising antigenic units comprising one or more infectious full length antigens or parts of such antigens) elicits a B cell response and a T cell response and is useful for prophylaxis or therapy.

Such antigens may be selected for inclusion in the antigenic unit according to their predicted therapeutic efficacy, see WO2021/219897A1, the disclosure of which is incorporated herein by reference.

Antigenic units comprising a first polypeptide comprising a B cell epitope and a T cell epitope from one or more pathogens

In one embodiment, the first polypeptide encoded by the first nucleic acid comprised in the vector of the invention elicits a T cell response and a B cell response upon administration to a subject. In the case of pandemics or epidemics, there is not enough time to first diagnose an individual to determine if he or she is in need of a B or T cell response primarily, nor is it known whether prophylactic or therapeutic treatment is the highest medical need. Or it may be difficult to determine if an individual is infected due to a lack of (sufficient) suitable detection. Therefore, it is important to be able to perform both protection and treatment simultaneously. By combining a full length infectious antigen or a portion of an infectious antigen or several B-cell epitopes present in an infectious antigen with T-cell epitopes (e.g. conserved T-cell epitopes), a strong humoral and cellular response is elicited upon administration of the vector. The response may be more biased towards a humoral response or more biased towards a cellular response, depending on the targeting unit selected.

Accordingly, one aspect of the invention relates to a carrier comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit, a multimerization unit, such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises (i) one or more full-length infectious antigens or portions of such antigens, and (ii) at least one T cell epitope from one or more infectious antigens; and

In one embodiment, the invention relates to a vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit, a multimerization unit, e.g., a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises (i) one or more full-length antigens or portions of such antigens, (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

Such combinations of T cell epitopes and infectious antigens or parts 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, see WO2021/219897A1, the disclosure of which is incorporated herein by reference.

In one embodiment, the invention relates to a carrier comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit, a multimerization unit, e.g., a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises (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

In another embodiment, the invention relates to a vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit, a multimerization unit, e.g., a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises (i) one or more B cell epitopes, and (ii) at least one T cell epitope, wherein the one or more B cell epitopes and the at least one T cell epitope are derived from one or more pathogens; and

In one embodiment, the full-length infectious antigen/portion thereof and the at least one T cell epitope are arranged in an antigenic unit as follows: the at least one T cell epitope is arranged in subunits that are linked to the multimerization unit by a first linker, such as a unit linker. If there are multiple T cell epitopes in the subunit, the T cell epitopes are preferably separated by subunit linkers. In addition, the subunit is separated from the one or more full-length infectious antigens or portions thereof by a second linker. Thus, subunits with T cell epitopes are closest to the multimerization unit, while infectious antigens or parts thereof constitute the ends of the polypeptide.

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit, a multimerization unit, such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises (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

Wherein the vector allows co-expression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules; and

Wherein the antigenic unit comprises subunits comprising T cell epitopes separated from each other by subunit linkers, provided that more than one T cell epitope is comprised in said subunits; and

Wherein the subunits are linked to the multimerization unit by a first linker, e.g., a unit linker, and are separated from the one or more full-length infectious antigens or portions of such antigens by a second linker.

The subunit linker, the first linker/unit linker and the second linker may be linkers discussed herein, e.g., linkers discussed in the "antigenic unit" and "unit linker" sections herein.

Other embodiments of the antigenic units

In general, the following applies to antigenic units in a first polypeptide encoded by a first nucleic acid comprised in a vector of the invention.

The term antigen is used in this section of the application for a neoantigen, a neoepitope, a consensus cancer antigen present in a patient, a portion of a consensus cancer antigen present in a patient (e.g., a consensus cancer epitope present in a patient), a consensus cancer antigen, a portion of a consensus cancer antigen (e.g., a consensus cancer epitope), an infectious antigen or portion thereof, or a T cell epitope of an infectious antigen.

In one embodiment, the antigenic unit comprises only one copy of each antigen. In another embodiment, the antigenic unit comprises multiple copies of one or more antigens.

In one embodiment, the antigenic unit comprises only one copy of each antigen, such that when, for example, 10 different antigens are included in the antigenic unit, a vector comprising the antigenic unit may elicit an immune response against all 10 different antigens, thereby attacking cancer efficiency.

In another embodiment, an antigenic unit may comprise at least two copies of a particular antigen (e.g., a particular neoepitope) if, for example, only a few neoepitopes predicted to be sufficiently immunogenic/predicted to bind to the patient's HLA allele can be identified in a particular patient in order to enhance the immune response against the antigen. If in such patients, in addition to such a few neo-epitopes, one or more patient-present consensus cancer antigens are identified, it is preferred to include such one or more patient-present consensus cancer antigens or parts thereof into the antigenic unit, rather than including multiple copies of the same neo-epitope.

The length of the antigenic unit is determined by the length of the antigen contained therein and its number.

In one embodiment, the antigenic unit comprises up to 3500 amino acids, for example from 60 to 3500 amino acids, for example from about 80 or about 100 or about 150 amino acids to about 3000 amino acids, for example from about 200 to about 2500 amino acids, for example from about 300 to about 2000 amino acids or from about 400 to about 1500 amino acids or from about 500 to about 1000 amino acids.

In order to enhance the immune response, in particular to a first polypeptide comprising a neoantigen, the antigen may be arranged in antigen subunits as described in the following paragraphs.

An antigenic unit can be described as a polypeptide having an initial N-terminus and a terminal C-terminus. The antigenic units are linked to multimerisation units such as dimerization units, for example via linkers, preferably via a unit linker. The antigenic unit is located at the COOH-terminus or NH 2-terminus of the first polypeptide. Preferably, the antigenic unit is located at the COOH terminus of the first polypeptide.

In one embodiment, the antigens (preferably epitopes) are arranged in a sequence from more antigenic to less antigenic along the direction from the beginning N-terminus of the antigenic unit to the ending C-terminus of the antigenic unit. Or particularly if the hydrophilicity/hydrophobicity variation between antigens is large, it is preferred that the most hydrophobic antigen is located substantially in the middle of the antigenic unit and that the most hydrophilic antigen is located at the initial N-terminus and/or the terminal C-terminus of the antigenic unit.

Since it is only possible to locate truly in the middle of an antigenic unit when the antigenic unit contains an odd number of antigens, the term "substantially" herein refers to an antigenic unit containing an even number of antigens, wherein the most hydrophobic antigen is located as close to the middle as possible.

For example, an antigenic unit comprises 5 antigenic subunits, each comprising a different epitope, e.g. a different neoepitope, arranged as follows: 1-2-3 x-4-5; wherein 1, 2,3, 4 and 5 are each different neo-epitopes, -are subunit linkers, ×represent the most hydrophobic neo-epitope located in the middle of the antigenic unit.

In another example, the antigenic unit comprises 6 antigenic subunits, each comprising a different epitope, e.g. a different neoepitope, arranged as follows: 1-2-3 x-4-5-6, or alternatively, as follows: 1-2-4-3 x-5-6; wherein 1,2, 3, 4, 5 and 6 are each different neo-epitopes, -are subunit linkers, representing the most hydrophobic neo-epitope located substantially in the middle of the antigenic unit.

Or the antigen subunits may be arranged such that they alternate between hydrophilic and hydrophobic antigens.

Optionally, GC-rich antigen coding sequences (e.g., GC-rich neoepitopes or epitope coding sequences) are arranged in a manner that avoids GC clusters. In one embodiment, the GC-rich antigen coding sequences are arranged such that there is at least one non-GC-rich sequence between them.

In one embodiment, the antigenic unit comprises one or more linkers. In another embodiment, the antigenic unit comprises a plurality of antigens, such as a plurality of epitopes, such as neo-epitopes, wherein the antigens are separated by a linker. In another embodiment, the antigenic unit comprises a plurality of antigens, wherein each antigen is separated from the other antigens by a linker. Another way of describing the separation of each antigen from the other antigens by a linker is that all antigens except the terminal antigen, i.e. the antigen located at the initial N-terminus of the polypeptide or at the terminal C-terminus of the polypeptide (i.e. the antigen located at the terminal end of the antigenic unit but not linked to the multimerizing unit), are arranged in antigen subunits, wherein each subunit comprises or consists of an antigen such as a neoepitope and subunit linker.

Thus, an antigenic unit comprising n antigens comprises n-1 antigenic subunits, wherein each subunit comprises an antigen and a subunit linker, and further comprises a terminal antigen. In one embodiment, wherein n is an integer from 1 to 50, such as from 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.

Because the linker separates the antigens, each antigen is presented to the immune system in an optimal manner.

In one embodiment, the antigenic unit comprises a B cell epitope and a T cell epitope, such as a full length infectious antigen or a portion thereof and one or more T cell epitopes comprised in a pathogen protein, and the antigenic unit is designed such that the T cell epitope is arranged closest to the multimerization unit and the infectious antigen is located at the end of the antigenic unit. The T cell epitopes are preferably separated by a linker, and the infectious antigen is preferably separated from a "subunit" comprising T cell epitopes by a linker. The design of such aforementioned antigenic units is disclosed in PCT/EP2022/061819, the disclosure of which is incorporated herein by reference.

Linkers contained in antigenic units

The antigenic unit may comprise a linker, e.g. a linker separating the antigens comprised therein, e.g. a neoantigen, a neoepitope, a consensus cancer antigen present by the patient or a part thereof (e.g. a consensus cancer epitope present by the patient), a consensus cancer antigen or a part thereof (e.g. a consensus cancer epitope), an infectious antigen or a part thereof or a T cell epitope of an infectious antigen. As described above, all antigens, e.g. neoepitopes, may be separated from each other by linkers and arranged in subunits. Hereinafter, the terms subunit linker and linker are used interchangeably and both represent linkers in an antigenic unit.

In one embodiment, the linker is designed to be non-immunogenic. The linker may be a rigid linker, meaning that it does not allow for a substantial free movement of the two amino acid sequences to which it is attached relative to each other. Or it may be a flexible linker, i.e. a linker allowing the two amino acid sequences to which it is attached to move substantially freely relative to each other. Both types of joints are available. In one embodiment, the linker is a flexible linker that allows for optimal presentation of antigen to T cells even though the antigenic unit contains a large amount of antigen.

In one embodiment, the subunit linker is a peptide consisting of 4 to 40 amino acids, e.g., 35, 30, 25 or 20 amino acids, e.g., 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.

In one embodiment, for example in an antigenic unit comprising a neoepitope, the subunit linker is the same in all antigenic subunits. However, if one or more antigens comprise a sequence similar to the linker sequence, it may be advantageous to replace adjacent subunit linkers with linkers of a different sequence. In addition, if the antigen-subunit linker linkage itself is predicted to constitute an immunogenic epitope, a linker of a different sequence may be used.

In one embodiment, the subunit linker is a flexible linker, preferably a flexible linker comprising a small non-polar (e.g., glycine, alanine, or leucine) or polar (e.g., serine or threonine) amino acid. The small size of these amino acids provides flexibility and allows mobility of the attached amino acid sequences. The incorporation of serine or threonine can reduce adverse interactions between the linker and the antigen by maintaining the stability of the linker in aqueous solution through hydrogen bonding with water molecules. In one embodiment, the flexible linker is a serine (S) and/or glycine (G) rich linker, i.e. a linker comprising a plurality of serine and/or glycine residues. A preferred example is 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 variants thereof, e.g. GGGGSGGGGS(SEQ ID NO:17)、(GGGGS)m(SEQ ID NO:64)、(GGGSS)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, e.g. 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, e.g. at least 1 or at least 2 or at least 3 leucine residues, e.g. 1,2,3 or 4 leucine residues.

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).

In 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 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).

In another embodiment, the subunit linker is a serine-glycine linker that is 10 amino acids in length and comprises 1 or 2 leucine residues.

In one embodiment, the subunit linker comprises or consists of LGGGSGGGGS(SEQ ID NO:97)、GLGGSGGGGS(SEQ ID NO:98)、GGLGSGGGGS(SEQ ID NO:99)、GGGLSGGGGS(SEQ ID NO:100) or GGGGLGGGGS (SEQ ID NO: 101). In another embodiment, the subunit linker comprises or consists of LGGSGGGGSG(SEQ ID NO:102)、GLGSGGGGSG(SEQ ID NO:103)、GGLSGGGGSG(SEQ ID NO:104)、GGGLGGGGSG(SEQ ID NO:105) or GGGSLGGGSG (SEQ ID NO: 106). In another embodiment, the subunit linker comprises or consists of LGGSSGGGSS(SEQ ID NO:107)、GLGSSGGGSS(SEQ ID NO:108)、GGLSSGGGSS(SEQ ID NO:109)、GGGLSGGGSS(SEQ ID NO:110) or GGGSLGGGSS (SEQ ID NO: 111).

In a further embodiment, 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 GGGGLGGGGL (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)、GGGLGGGGLG(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).

In yet another embodiment, the subunit linker comprises or consists of GSGGGA(SEQ ID NO:125)、GSGGGAGSGGGA(SEQ ID NO:126)、GSGGGAGSGGGAGSGGGA(SEQ ID NO:127)、GSGGGAGSGGGAGSGGGAGSGGGA(SEQ ID NO:128) or GENLYFQSGG (SEQ ID NO: 129). In another embodiment, the subunit linker comprises or consists of amino acids 121-130 of SGGGSSGGGS(SEQ ID NO:130)、SSGGGSSGGG(SEQ ID NO:131)、GGSGGGGSGG(SEQ ID NO:132)、GSGSGSGSGS(SEQ ID NO:133)、GGGSSGGGSG(SEQ ID NO:134)(SEQ ID NO:1, GGGSSS (SEQ ID NO: 135), GGGSSGGGSSGGGSS (SEQ ID NO: 136) or GLGGLAAA (SEQ ID NO: 137).

In another embodiment, the subunit linker is a rigid linker. Such rigid linkers can be used to effectively 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).

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).

In another embodiment, the subunit linker comprises or consists of GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 145) or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 146) or ELKTPLGDTTHT (SEQ ID NO: 147) (amino acids 94-105 of SEQ ID NO: 1) or EPKSCDTPPPCPRCP (SEQ ID NO: 148) (amino acids 106-120 of SEQ ID NO: 1).

In yet another embodiment, the subunit linker is a cleavable linker, e.g., a linker comprising one or more endopeptidase recognition sites, e.g., furin, caspase, cathepsin, and the like. Cleavable linkers can be introduced to release free functional protein domains (e.g., encoded by larger antigens), which can overcome steric hindrance between these domains or other drawbacks due to interference of these domains, such as reduced bioactivity, altered biodistribution.

Examples of suitable linkers are disclosed in paragraphs [0098] - [0099] and the cited sequences of 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.

Unit joint

The antigenic units are linked to the multimerization units, preferably via a unit linker. Thus, in one embodiment, a first nucleic acid sequence comprised in a vector of the invention encodes a first polypeptide comprising a unit linker linking the antigenic unit and the multimerization unit.

The unit adaptor may comprise 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)、GGGGSGGGGS(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)、(EAAK)mGS(SEQ ID NO:63)、GPSRLEEELRRRLTEPG(SEQ ID NO:152)、AAY or HEYGAEALERAG (SEQ ID NO: 153).

Signal peptides

In one embodiment of the present disclosure, at least one of the first nucleic acid sequence or the one or more additional nucleic acid sequences encoding one or more immunostimulatory compounds further encodes a signal peptide. The signal peptide is located at the N-terminus of the targeting unit or at the C-terminus of the targeting unit, depending on the orientation of the targeting unit in the first polypeptide. In addition, 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 comprising the vector of the invention. Preferably, the first nucleic acid sequence and each further nucleic acid sequence encoding one or more immunostimulatory compounds further encode a signal peptide. Preferably, the signal peptide is a signal peptide naturally occurring at the N-terminus of any targeting unit or immunostimulatory compound described herein.

Any suitable signal peptide may be used. Preferably, if the targeting unit is an antibody or a portion thereof (e.g. scFv), examples of suitable peptides are Ig VH signal peptides, preferably human Ig VH signal peptides, e.g. SEQ ID NO:2. in one embodiment, the signal peptide is the native leader sequence of the protein as the targeting unit, i.e., the signal peptide naturally occurring at the N-terminus of any protein encoded in the vector of the invention as the targeting unit. In another embodiment, the signal peptide is the natural leader sequence of the immunostimulatory compound, i.e., the signal peptide naturally occurring at the N-terminus of the protein as the immunostimulatory compound.

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

In a preferred embodiment, the vector of the invention comprises a first nucleotide sequence encoding a first polypeptide and further encoding a signal peptide comprising an amino acid sequence having at least 85% sequence identity with the amino acid sequence of 1-23 of SEQ ID No. 1, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% identity.

In another preferred embodiment, the vector of the invention comprises a first nucleotide sequence encoding a first polypeptide and further encoding a signal peptide comprising the amino acid sequence 1-23 of SEQ ID NO. 1, with the difference that at most 3 amino acids have been substituted, deleted or inserted, such as at most 2 amino acids or such as at most 1 amino acid have been substituted, deleted or inserted.

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

In a more preferred embodiment, the vector of the invention comprises a first nucleotide sequence encoding a first polypeptide and further encoding a signal peptide consisting of a sequence identical to SEQ ID NO:1, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99% identical.

In another preferred embodiment, the vector of the invention comprises a first nucleotide sequence encoding a first polypeptide and further encoding a signal peptide consisting of the amino acid sequence 1-23 of SEQ ID NO.1, with the difference that at most 3 amino acids have been substituted, deleted or inserted, such as at most 2 amino acids or such as at most 1 amino acid have been substituted, deleted or inserted.

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

In a 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 hybridizes with SEQ ID NO:29 has at least 80% sequence identity. .

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 hybridizes with the nucleotide sequence of SEQ ID NO:29, e.g., at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.

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 is SEQ ID NO. 29.

Sequence identity

Sequence identity can be determined as follows: a high level of sequence identity indicates the likelihood that the second sequence is derived from the first sequence. Amino acid sequence identity requires that there be amino acid sequence identity between two aligned sequences. Thus, a candidate sequence sharing 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 may be determined by means of computer analysis such as, but not limited to, the ClustalW computer alignment program (Higgins D.,Thompson J.,Gibson T.,Thompson J.D.,Higgins D.G.,Gibson T.J.,1994.CLUSTAL W:improving the sensitivity of progressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice.Nucleic Acids Res.22:4673-4680), and default parameters suggested therein. Using this procedure and its default settings, the mature (biologically active) portions of the query and reference polypeptides can be aligned. The number of fully conserved residues is counted and divided by the length of the reference polypeptide. In this process, any tag or fusion protein sequences that form part of the query sequence are ignored in the alignment and subsequent sequence identity determination.

The ClustalW algorithm can be similarly used to align nucleotide sequences. Sequence identity can be calculated in a similar manner as shown for the amino acid sequence.

Another preferred mathematical algorithm for comparing sequences is algorithm MYERS AND MILLER, CABIOS (1989). This algorithm was incorporated into the ALIGN program (version 2.0) which was part of the FASTA sequence alignment software package (Pearson WR, methods Mol Biol,2000, 132:185-219). Align calculates sequence identity based on global alignment. Align0 does not penalty gaps at the end of the sequence. When amino acid sequences are compared using the ALIGN and ALIGN0 programs, the BLOSUM50 substitution matrix is preferably used with a gap opening/extension penalty of-12/-2.

Amino acid sequence variants may be prepared by introducing appropriate alterations into the nucleotide sequence encoding the first polypeptide and/or one or more immunostimulatory compounds or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence. The terms substitution/substitution, deletion/deletion and insertion/insertion as used herein with respect to amino acid sequence and sequence identity are well known and clear to those skilled in the art. Any combination of deletions, insertions and substitutions may be made to obtain the final first polypeptide and/or one or more immunostimulatory compounds, provided that the final protein has the desired characteristics. For example, deletions, insertions or substitutions of amino acid residues may produce a silent change and result in a functionally equivalent polypeptide/immunostimulatory compound.

Artificial amino acid substitutions may be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as 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; amino acids having uncharged polar head groups with similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions, i.e., equivalent (like-for-like) substitutions, such as basic substitution basic, acidic substitution acidic, polar substitution polar, etc., are contemplated herein, as well as non-conservative substitutions, i.e., from one type of residue to another or alternatively include the incorporation of unnatural amino acids, such as ornithine, diaminobutyrate, norleucine, ornithine, pyridylalanine (PYRIYLALANINE), thienylalanine, naphthylalanine, and phenylglycine. Conservative substitutions that may be made within, for example, the following groups: basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), aliphatic amino acids (alanine, valine, leucine, isoleucine), polar amino acids (glutamine, asparagine, serine, threonine), aromatic amino acids (phenylalanine, tryptophan, tyrosine), hydroxy amino acids (serine, threonine), large amino acids (phenylalanine, tryptophan) and small amino acids (glycine, alanine).

Substitutions may also be made by unnatural amino acids, and the substitution residues include: α and α -disubstituted amino acids, N-alkylamino acids, lactic acid, halide derivatives of natural amino acids such as trifluorotyrosine, p-chloro-phenylalanine, p-bromo-phenylalanine, p-L-phenylalanine, L-allyl-glycine, β -alanine, L-a-aminobutyric acid, L-y-aminobutyric acid, L-a-aminoisobutyric acid, L-e-aminocaproic acid, 7-aminoheptanoic acid, L-methionine sulfone, L-norleucine, L-norvaline, methyl derivatives of p-nitro-L-phenylalanine, L-hydroxyproline, L-thioproline, phenylalanine (Phe) such as 4-methyl-Phe, pentamethyl-Phe, L-Phe (4-amino), L-Tyr (methyl), L-Phe (4-isopropyl) L-3, 3-tetrahydrophe (3, 3-phenylic acid) and 4-benzyl-amino-3-isopropyl-3, 3-amino-propanic acid.

In the above paragraph, # denotes the hydrophobicity of the substituted residue, while # denotes the hydrophilicity of the substituted residue, and # denotes the amphipathic nature of the substituted residue. The variant amino acid sequence may include a suitable spacer group that may be inserted between any two amino acid residues of the sequence, including alkyl groups such as methyl, ethyl or propyl in addition to amino acid spacers such as glycine or β -alanine residues. Another form of variation involves the presence of one or more amino acid residues in the peptidomimetic form.

Polypeptides and multimeric/dimeric proteins

The vector of the invention encodes a first polypeptide as described above. As a result of administering the vector to the subject, the polypeptide (and the one or more immunostimulatory compounds) are expressed in vivo.

Due to the presence of multimerization units, e.g. dimerization units, the polypeptides form multimeric proteins upon expression.

The multimeric protein may be a homomultimer or a heteromultimer, e.g., if the protein is a dimeric protein, the dimeric protein may be a homodimer, i.e., a dimeric protein in which the two polypeptide chains are identical and thus comprise identical units and thus comprise identical antigen sequences, or the dimeric protein may be a heterodimer comprising two polypeptide chains, wherein polypeptide chain 1 comprises an antigen sequence in its antigenic unit that is different from polypeptide 2. The latter may be the case if the amount of antigen contained in an antigenic unit exceeds the upper size limit of the antigenic unit. Preferably, the multimeric protein is a homomultimeric protein.

Production of vectors and host cells

The vectors of the invention are generally vectors suitable for transfection of host cells and are suitable for a) expression of a first polypeptide encoded by a first nucleic acid sequence and formation of a multimeric protein consisting of a plurality of such first polypeptides, and b) expression of the one or more immunostimulatory compounds encoded by a further nucleic acid sequence, respectively.

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

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

In one embodiment, the vector allows for easy exchange of the various units described above, particularly in the case of personalized antigenic units.

In one embodiment, the vector is a pUMVC4a vector or a vector comprising an NTC9385R vector backbone. The antigenic unit may be exchanged with an antigenic unit cassette limited by a SfiI restriction enzyme cassette, wherein the 5 'site is incorporated into the nucleotide sequence encoding the GLGGL (SEQ ID NO: 89)/GLSGL (SEQ ID NO: 149) unit linker and the 3' site is comprised after the stop codon in the vector.

Engineering and production methods for vectors of the invention, such as expression vectors (e.g., DNA and RNA plasmids) or viral vectors, are well known and the skilled artisan is able to engineer/produce vectors of the invention using such known methods. In addition, various commercial manufacturers offer carrier design and production services.

In one aspect, the present disclosure relates to a method of producing a vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerization unit, e.g., a dimerization unit, and an antigenic unit comprising one or more antigens or portions thereof; and

Wherein the vector allows co-expression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules, the method comprising:

a) Transfecting the cells with a vector in vitro;

b) Culturing the cells;

c) Optionally, lysing the cells to release the carrier from the cells; and

D) The vector is collected and optionally purified.

In one embodiment, the one or more antigens or portions thereof are disease-associated antigens or portions thereof.

Pharmaceutical composition

In one embodiment of the present disclosure, a vector, such as a DNA plasmid, is used as a medicament.

Thus, in one embodiment of the invention, the carrier is provided in the form of a pharmaceutical composition comprising the carrier and a pharmaceutically acceptable carrier or diluent.

Thus, in one aspect, the present disclosure relates to a pharmaceutical composition comprising (i) a pharmaceutically acceptable carrier or diluent and (ii) a carrier comprising:

Suitable pharmaceutically acceptable carriers or diluents include, but are not limited to, saline, buffered saline such as PBS, dextrose, water, glycerol, ethanol, isotonic aqueous buffers, and combinations thereof.

In one embodiment, the pharmaceutically acceptable carrier or diluent is an aqueous buffer. In another embodiment, the aqueous buffer is a Tyrode buffer, e.g., a Tyrode buffer comprising 140mM NaCl, 6mM KCl, 3mM CaCl2, 2mM MgCl2, 10mM 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (Hepes) pH7.4, and 10mM glucose.

The pharmaceutical composition may comprise a molecule that is easy to transfect by a host cell, i.e. a transfection agent.

In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable amphiphilic block copolymer comprising blocks of poly (ethylene oxide) and poly (propylene oxide).

As used herein, an "amphiphilic block copolymer" is a linear or branched copolymer comprising or consisting of poly (ethylene oxide) ("PEO") blocks and poly (propylene oxide) ("PPO") blocks. Typical examples of useful PEO-PPO amphiphilic block copolymers have the following general structure: PEO-PPO-PEO (poloxamer), PPO PEO PPO, (PEO PPO-) 4ED (poloxamine)) and (PPO PEO-) 4ED (reverse poloxamine), wherein "ED" is ethylenediamido.

"Poloxamer" is a linear amphiphilic block copolymer consisting of a block of poly (ethylene oxide) coupled to a block of poly (propylene oxide) coupled to a block of PEO, i.e., the structure of formula EOa-POb-EOa, wherein EO is ethylene oxide, PO is propylene oxide, a is an integer from 2 to 130, and b is an integer from 15 to 67. Poloxamers are typically named using a 3-digit numerical identifier, where the first 2 digits multiplied by 100 provide an approximate molecular weight of the PPO content, and the last digit multiplied by 10 represents an approximate percentage of PEO content. For example, "poloxamer 188" refers to a polymer comprising a PPO block of molecular weight about 1800 (corresponding to b of about 31 PPO) and about 80% (w/w) PEO (corresponding to a of about 82). However, these values are known to vary to some extent, and are of research gradeF68 and clinical grade/>Commercial products such as P188 (poloxamer 188 according to the manufacturer's data sheet) exhibit large molecular weight variations (between 7,680 and 9,510), and the values of a and b provided for these specific products are about 79 and 28, respectively. This reflects the heterogeneity of the block copolymer, indicating that the values of a and b are the average values in the most formulation.

"Poloxamine" or "sequential Poloxamine" (under the trade nameCommercially available) is an X-block copolymer that carries four PEO-PPO arms attached to a central ethylenediamine moiety through bonds between free OH groups contained in the PEO-PPO-arms and primary amine groups in the ethylenediamine moiety. Reverse poloxamine is also an X-block copolymer that carries four PPO-PEO arms attached to a central ethylenediamine moiety through bonds between free OH groups contained in the PPO-PEO arms and primary amine groups in ethylenediamine.

Preferred amphiphilic block copolymers are poloxamers or poloxamines. Preferred are poloxamers 407 and 188, in particular poloxamer 188. The preferred poloxamine is the sequential poloxamine of formula (PEO-PPO) 4-ED. Particularly preferred poloxamines are under the registered trade marks respectively904. 704 And 304. These poloxamines are characterized as follows: /(I)904 Has a total average molecular weight of 6700, a total average weight of ppo units of 4020, and a peo percentage of about 40%. /(I)Is 5500, the total average weight of ppo units is 3300, the peo percentage is about 40%; /(I)304 Has a total average molecular weight of 1650, a total average weight of ppo units of 990, and a peo percentage of about 40%.

In one embodiment, the pharmaceutical composition comprises the amphiphilic block copolymer in an amount of 0.2% w/v to 20% w/v, e.g. 0.2% w/v to 18% w/v, 0.2% w/v to 16% w/v, 0.2% w/v to 14% w/v, 0.2% w/v to 12% w/v, 0.2% w/v to 10% w/v, 0.2% w/v to 8% w/v, 0.2% w/v to 6% w/v, 0.2% w/v to 4% w/v, 0.4% w/v to 18% w/v, 0.6% w/v to 18% w/v, 0.8% w/v to 18% w/v, 1% w/v to 18% w/v, 2% w/v to 18% w/v, 1% w/v to 5% w/v or 2% w/v to 4% w/v. Particularly preferred is an amount in the range of 0.5% w/v to 5% w/v. 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.

The pharmaceutical composition may 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.

The pharmaceutical composition 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 administration, for example intranasal or oral.

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

The amount of vector (e.g., DNA plasmid) in the pharmaceutical composition may vary depending on whether the pharmaceutical composition is administered for prophylactic or therapeutic treatment.

The pharmaceutical compositions of the invention generally comprise a vector, e.g. a DNA plasmid, in the range of 0.1 to 10mg, e.g. about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1mg or e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10mg.

In a preferred embodiment, the pharmaceutical composition is a sterile pharmaceutical composition.

Treatment of

In some aspects of the disclosure, vectors, such as DNA plasmids, are used for therapeutic or prophylactic treatment of disorders, such as human disorders.

Thus, in one aspect, the present disclosure relates to a method of treating a subject suffering from a disease or in need of prevention of the disease, the method comprising administering to the subject a vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerization unit, e.g., a dimerization unit, and an antigenic unit comprising one or more antigens or portions thereof associated with the disease; and

In the method of treatment, the carrier is preferably administered in a therapeutically effective amount or a prophylactically effective amount. Such amounts of carrier may be administered in one administration, i.e. one dose, or in several administrations, i.e. repeated doses, i.e. a series of doses, e.g. over the course of days, weeks or months.

The actual dosage to be administered can vary and depends on whether the treatment is prophylactic or therapeutic, the age, weight, sex, medical history of the subject, the pre-existing condition and overall status, the severity of the disease being treated and the judgment of the healthcare professional.

In the methods of treatment, the carrier may be administered in the form of a pharmaceutical composition and in the manner of administration described herein.

The treatment method of the present invention may continue as long as the clinician administering patient care considers the method to be effective and in need of treatment.

In one embodiment of the present disclosure, a vector, such as a DNA plasmid, is used to treat cancer. Such vectors and antigenic units of such vectors, including both individualized and non-individualized antigenic units of the first polypeptide, and various embodiments thereof, have been described in detail herein.

Thus, in one embodiment, the present disclosure relates to a method of treating a subject having cancer, the method comprising administering to the subject a vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerization unit, such as a dimerization unit, and an antigenic unit comprising one or more cancer antigens or portions thereof; and

The cancer may be a solid cancer or a liquid cancer. An example of a solid cancer is a cancer that forms a solid tumor (e.g., a tumor). Examples of liquid cancers are cancers present in body fluids, such as lymphomas or blood cancers.

In one embodiment of the present disclosure, a vector, such as a DNA plasmid, is used to treat 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, renal cancer, cholangiocarcinoma, brain cancer, cervical cancer, bladder cancer, esophageal cancer, hodgkin's disease, and adrenocortical cancer.

In another embodiment of the present disclosure, vectors, such as DNA plasmids, are used to treat infectious diseases. Such vectors and the antigenic units of such vectors have been described in detail herein.

Thus, in one embodiment, the present disclosure relates to a method of treating a subject suffering from an infectious disease or in need of prevention of an infectious disease, the method comprising administering to the subject a vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit, e.g., a dimerization unit, and an antigenic unit comprising one or more antigens or portions thereof associated with the infectious disease; and

Antigens or portions thereof associated with infectious diseases, such as antigens derived from pathogens or portions thereof, have been described in detail herein.

Also disclosed herein is a vector for treating a subject suffering from a disease or in need of prevention of the disease, wherein the vector is administered to the subject, the vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerization unit, e.g., a dimerization unit, and an antigenic unit comprising one or more antigens associated with a disease or a portion thereof; and

Also disclosed herein is a vector for treating a subject having cancer, wherein the vector is administered to the subject, the vector comprising:

Also disclosed herein is a vector for treating a subject suffering from an infectious disease or in need of prevention of the infectious disease, wherein the vector is administered to the subject, the vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit, e.g., a dimerization unit, and an antigenic unit comprising one or more antigens associated with an infectious disease or a portion thereof; and

Also disclosed herein is a method for treating a disease in a subject in need of prevention of the disease, wherein the method comprises administering to the subject a drug comprising:

Also disclosed herein is a method of treating a subject having cancer, wherein the method comprises administering to the subject a drug comprising:

Also disclosed herein is a method for treating a subject suffering from an infectious disease or in need of prevention of the infectious disease, wherein the method comprises administering to the subject a drug comprising:

Also disclosed herein is the use of a vector for treating a subject suffering from a disease or in need of prevention of said disease, said vector comprising:

Also disclosed herein is a use of a vector for treating a subject having cancer, the vector comprising:

Also disclosed herein is the use of a vector for treating a subject suffering from or in need of prevention of an infectious disease, the vector comprising:

Also disclosed herein is a vector comprising:

Wherein the vector allows co-expression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules for therapeutic or prophylactic treatment of the disease.

Also disclosed herein is a vector comprising:

Wherein the vector allows co-expression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules for use in the treatment of cancer.

Also disclosed herein is a vector comprising:

Wherein the vector allows co-expression of the first polypeptide and the one or more immunostimulatory compounds as separate molecules for use in the therapeutic or prophylactic treatment of the infectious disease.

Also disclosed herein is the use of a vector for the therapeutic or prophylactic treatment of a disease, the vector comprising:

Also disclosed herein is a use of a vector for treating cancer, the vector comprising:

Also disclosed herein is the use of a vector for the therapeutic or prophylactic treatment of an infectious disease, the vector comprising:

Also disclosed herein is a medicament for treating or preventing a disease by administering to a subject suffering from the disease or in need of prevention of the disease a vector comprising:

Also disclosed herein is a medicament for treating cancer in a subject having cancer by administering to the subject a vector comprising:

Also disclosed herein is a medicament for treating or preventing an infectious disease by administering to a subject suffering from the disease or in need of prevention of the disease a vector comprising:

Examples

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

Example 1:

a variety of DNA plasmids were designed that allowed the co-expression of the first polypeptide and one or more immunostimulatory compounds described herein as separate molecules.

All DNA plasmids, VB4194, VB4168, VB4169 and VB4170, comprise nucleic acid sequences encoding the elements/units listed in table 4 below:

TABLE 4 Table 4

The DNA plasmid also comprises nucleic acid sequences encoding the elements/units listed in table 5 below:

TABLE 5

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Hereinafter, "m", "mouse" and "mouse" are used interchangeably, and "h" and human are used interchangeably.

DNA plasmids VB4194, VB4168, VB4169 and VB4170 contain 8 epitopes with mutations:

The previously described exome sequencing and RNA sequencing of the mouse colon cancer cell line CT26 revealed hundreds to thousands of tumor-specific non-synonymous mutations. A computer method was used to identify potential immunogenic sequences, i.e. epitopes comprising mutations, and 8 of them (table 6) were selected to be included in the antigenic units of the first polypeptide encoded by the DNA plasmid described above. The epitopes in the antigenic units are separated by glycine-serine linkers (GGGGSGGGGS, SEQ ID NO: 17), i.e., all epitopes except the terminal epitope are arranged in subunits, each consisting of one epitope and one GGGGSGGGGS (SEQ ID NO: 17) linker.

Each of these DNA plasmids is a model of a DNA plasmid of the invention encoding an individualized first polypeptide, i.e. a DNA plasmid comprising antigenic units comprising a plurality of patient-specific epitopes, e.g. a plurality of neo-epitopes and/or a plurality of patient-present consensus cancer epitopes, wherein the patient-present consensus cancer antigen is a mutated patient-present consensus cancer antigen, or a model of a DNA plasmid of the invention encoding a non-individualized first polypeptide, i.e. a polypeptide comprising antigenic units comprising a plurality of consensus cancer epitopes, wherein the consensus cancer antigen is a mutated consensus cancer antigen.

DNA plasmids VB4168, VB4169 and VB4170 allow the first polypeptides and the following immunostimulatory compounds as described above and in tables 4 and 5 to be co-expressed as separate molecules (h=human; m=mouse):

VB4194: for comparison, only the first polypeptide (first polypeptide identical to VB4168, VB4169 and VB 4170) was encoded, but not the immunostimulatory compound

·VB4168：hFLT3L

VB4169: hFLTL3 and mGM-CSF

VB4170: hFLTL3, mGM-CSF and mCCL5

TABLE 6

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Production of DNA plasmids

The sequences of the antigenic units, co-expression elements and immunostimulatory compounds of all DNA plasmids disclosed in the examples were purchased from Genscript (GENSCRIPT BIOTECH BV, netherlands) and cloned into the expression vector pucv 4a, which is a master plasmid (MASTER PLASMID) comprising nucleotide sequences encoding the signal peptides, targeting units, dimerization units and unit junctions described in table 4 above.

Assessment of expression and secretion of proteins encoded by DNA plasmids

HEK293 cells (ATCC) were transiently transfected with the DNA plasmids described above. Briefly, 2x10 ⁵ cells/well were plated in 24-well tissue culture plates containing 10% FBS growth medium and used under conditions recommended by the manufacturer (Invitrogen, thermo FISCHER SCIENTIFIC)2000 Reagent, transfection with 1. Mu.g of the corresponding DNA plasmid. Transfected cells were then maintained at 37 ℃ for 5 days at 5% co ₂, and cell supernatants were collected and the expression and secretion of proteins encoded by the plasmids were characterized by sandwich ELISA using mouse anti-human IgG CH3 domain antibodies (capture antibody, 100 μl/well, 1 μg/ml, MCA878G, bio-Rad) and goat anti-human MIP-1 a antibodies (biotinylated detection antibody, 100 μl/well, 0.2 μg/ml, BAF270, R & D systems) (fig. 5). The expression and ELISA of the encoded immunostimulatory compounds FLT3L and GM-CSF were measured by sandwich using mouse anti-human FLT3L antibody (capture antibody, 100. Mu.l/well, 0.5. Mu.g/ml, MAB608, R & D Systems) and mouse anti-human FLT3L antibody (biotinylated detection antibody, 100. Mu.l/well, 0.1. Mu.g/ml, BAF308, R & D Systems) (FIG. 6) and rat anti-mouse GM-CSF (capture antibody, 100. Mu.l/well, 1.0. Mu.g/ml mouse GM-CSF antibody, MAB415, R & D Systems) and goat anti-mouse GM-CSF (biotinylated detection antibody, 100. Mu.l/well, 0.2. Mu.g/ml, BAM215, R & D Systems), respectively (FIG. 7). The 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 & DSystems) and goat anti-mouse CCL5 (biotinylated detection antibody, 100 μl/well, 0.2 μg/ml, BAF478, R & D Systems) (fig. 8).

The results presented in fig. 5 demonstrate that the first polypeptide/dimer protein comprising targeting, dimerization and antigenic units encoded in VB4168, VB4169 and VB4170 is expressed and secreted from transfected HEK293 cells at levels similar to VB4194 (comparison). FLT3L encoded as the second protein in VB4168, VB4169 and VB4170 was expressed and secreted at high levels from all 3 DNA plasmids as shown in fig. 6. In addition, GM-CSF encoded in VB4169 and VB4170 as the third protein was also expressed and secreted at high levels as shown in FIG. 7. CCL5 encoded in VB4170 as the fourth protein was also expressed and secreted at high levels as shown in figure 8.

Example 2:

evaluation of immunogenicity of DNA plasmids VB4194, VB4168 and VB4169

The immunogenicity of DNA plasmids VB4194 (comparative), VB4168 and VB4169 was determined by measuring the T cell immune response elicited in mice administered with these plasmids. A negative control VB1026 encoding a polypeptide having the sequence of SEQ ID NO: 1-237, and a polypeptide of amino acid sequence 1-237. The DNA plasmid is identical to VB4194 but contains neither a unit linker nor an antigenic unit.

For all mouse experiments, the following study design was used:

Female mice of 6 weeks of age were obtained from Janvier Labs (France). All animals were kept in Radium Hospital (Norway oslo) animal facilities. All animal protocols were approved by the norwegian food safety agency (norwegian oslo). 5 mice/group were used to test constructs containing antigenic units, while 3 mice/group were used for negative controls.

6 Μg of DNA plasmid was administered intramuscularly to BALB/c mice once, followed by electroporation. Spleens were collected 10 days after administration and triturated in a cell filter to obtain a single cell suspension. For each plasmid tested, a portion of the single cell suspension was subjected to cd4+ T cell depletion using Dynabeads ^TM anti-CD 4 beads. The total splenocytes and CD4+ depleted splenocytes were then tested for INF-gamma and TNF-alpha production in FluoroSpot assays according to the manufacturer's protocol (Mabtech).

Spleen cells harvested from mice administered with these plasmids were re-stimulated with peptides having the same sequence as the 8 epitopes contained in VB4194, VB4168 and VB4169 shown in table 6 (table 7 below):

TABLE 7

The ability of DNA plasmids VB4194, VB4168 and VB4169 to elicit T cell immune responses against the peptides in table 7 was compared. VB1026 was included as a negative control.

As shown in fig. 9-14, mice administered with the negative control VB1026 exhibited low basal immunogenicity against the peptides in table 7.

VB4194 induced T cell responses against all 8 epitopes. VB4168, which encodes the same first polypeptide as VB4194, and additional FLT3L induced a stronger T cell response than VB4194 (FIGS. 9-11). In addition to expressing the first polypeptide comprising the 8 epitopes, co-expression of the other two immunostimulatory compounds FLT3L and GM-CSF encoded by VB4169 induced an even stronger immune response than either VB4194 or VB4168 (fig. 9-11). The number of IFN-gamma secreting T cells (FIG. 9), TNF-alpha secreting T cells (FIG. 10), and INF-gamma + TNF-alpha co-secreting cell numbers (FIG. 11) all increased from VB4194 to VB4168 and from VB4168 to VB 4169. Likewise, the number of cd8+ T cells secreting only IFN- γ (cd4+ T cell depleting samples) (fig. 12), the number of cd8+ T cells secreting only TNF- α (cd4+ T cell depleting samples) (fig. 13), and the number of IFN- γ+ TNF- α co-secreting cells (fig. 14) all increased from VB4194 to VB4168 and from VB4168 to VB 4169.

These results indicate that DNA plasmids of the invention encoding a first polypeptide and one or more immunostimulatory compounds co-expressed as separate molecules from a plasmid can boost an antigen-specific immune response against an antigen contained in the first polypeptide compared to a DNA plasmid encoding only the first polypeptide.

Example 3:

A DNA plasmid VB4202 was designed and produced comprising a nucleic acid sequence encoding the elements/units listed in table 4 and comprising further nucleic acid sequences encoding the elements listed in table 8 below:

TABLE 8

Assessment of expression and secretion of proteins encoded by VB4202

HEK293 cells (ATCC) were transiently transfected with the DNA plasmids described above as described in example 1. The supernatant was subjected to sandwich ELISA using mouse anti-human IgG CH3 domain antibodies (capture antibody, 100 μl/well, 1 μg/ml, MCA878G, bio-Rad) and goat anti-human MIP-1α antibodies (biotinylated detection antibody, 100 μl/well, 0.2 μg/ml, BAF270, R & D systems) to characterize the secreted first polypeptide/dimer protein (fig. 15). Secretion of the encoded immunostimulatory compound GM-CSF was measured by sandwich ELISA in supernatants diluted 1:1000 using rat anti-mouse GM-CSF (capture antibody, 100. Mu.l/well, 1.0. Mu.g/ml mouse GM-CSF antibody, MAB415, R & D Systems) and goat anti-mouse GM-CSF (biotinylated detection antibody, 100. Mu.l/well, 0.2. Mu.g/ml, BAM215, R & D Systems) (FIG. 16).

The results presented in fig. 15 demonstrate that the first polypeptide/dimer protein encoded in VB4202 comprising targeting units, dimerization units and antigenic units is expressed and secreted from transfected HEK293 cells. GM-CSF encoded in VB4202 as the second protein was expressed and secreted at high levels as shown in FIG. 16.

Evaluation of immunogenicity of DNA plasmids VB4194 and VB4202

Immunogenicity of DNA plasmids VB4194 (comparative), VB1026 (negative control) and VB4202 was determined in BALB/c mice as described in example 2.

As shown in fig. 17, no IFN- γ production was detected in response to administration of VB 1026. VB4194 induced T cell responses against all 8 epitopes. Using IFN-gamma FluoroSpot analysis, VB4202 encoding the same first polypeptide as VB4194 and additional GM-CSF induced a stronger T cell response than VB 4194.

Flow cytometry evaluation

APC/dendritic cell influx at single cell level in mice administered with VB1026, VB4194 and VB4202 was assessed using multi-flow cytometry (influx). Female BALB/c mice of 6 weeks of age were obtained from Janvier Labs (France). All animals were kept in animal facilities at oslo university. All animal protocols were approved by the norwegian food safety agency (norwegian oslo). Each group of 6 mice was used to compare VB1026, VB4202 and VB4194. A group of 6 untreated mice was used as a further control. 6 μg of each DNA plasmid was administered intramuscularly into tibialis anterior, and then electroporated. Untreated groups received neither DNA plasmid nor electroporation. Tibialis anterior was extracted under sterile conditions 1,2 or 4 days after administration or in untreated groups. To obtain a single cell suspension, the muscles are first mechanically dissociated using scissors, followed by enzymatic digestion. For enzymatic digestion, the dissociated muscles were incubated with stirring magnets in digestion medium (DMEM, collagenase A [2mg/ml ], DNase [50U/ml ]) at 37℃for 1 hour. After incubation, the single cell suspension was filtered through a 70 μm filter and washed twice at 400Xg in PBS for 6 minutes at 4 ℃.

For flow cytometry analysis, single cell suspensions were first incubated with a vital dye (eFluor 780, invitrogen) for 10 minutes at Room Temperature (RT). The vital dye was rinsed off with PBS (centrifuged twice at 400Xg for 6 min each at 4 ℃). The cells were then incubated with an Fc block (Fc block) for 10 minutes at room temperature to block non-specific binding of the fluorescent antibody. Following the blocking step, the cells were stained with a pool of surface marker specific antibodies (table 9 below) for 30 minutes on ice. Stained cells were run on BD FACSymphony A 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 description of fig. 18.

TABLE 9

The results showed that the muscle of the mice administered VB4202 had increased immune cell (cd45+ cells) influx compared to the muscle of the mice administered VB4194 (fig. 19).

The proportion of DCs in the cd45+ cell population present in the muscle of mice receiving VB4202 was higher compared to mice receiving VB4194 (fig. 20). In addition, both the cDC1 population (fig. 21) and the moDC population (fig. 22) were increased in the muscle of mice receiving VB4202 compared to mice receiving VB 4194.

Taken together, these results demonstrate that when administered intramuscularly, the DNA plasmids of the invention encoding the first polypeptide and one or more immunostimulatory compounds as separate molecules co-expressed from the DNA plasmids of the invention can promote the influx of dendritic cells into the site of administration, ultimately further contributing to enhancing the antigen-specific immune response against the antigen contained in the first polypeptide, as compared to DNA plasmids encoding only the first polypeptide.

Example 4:

the following DNA plasmids were designed and produced:

all DNA plasmids VB1020, VB4195, VB4196 comprise the nucleic acid sequences encoding the elements/units listed in table 4 and further comprise the nucleic acid sequences encoding the elements/units listed in table 10 below:

Table 10

DNA plasmids VB1020, VB4195 and VB4196 comprise a nucleic acid sequence encoding a first polypeptide comprising an antigenic unit comprising human papillomavirus 16 (HPV 16) antigens E7 and E6.

Each of these DNA plasmids is a model of a DNA plasmid of the invention encoding a non-personalized first polypeptide, i.e. a DNA plasmid comprising antigenic units comprising a plurality of consensus cancer antigens, wherein the consensus cancer antigens are consensus cancer antigens of a virus (here antigens of HPV16 that cause certain types of cancer), or a model of a DNA plasmid of the invention encoding a first polypeptide, i.e. a DNA plasmid comprising antigenic units comprising antigens derived from a pathogen (here antigens derived from HPV 16), for use in the treatment of infectious diseases.

DNA plasmids VB4195 and VB4196 allow co-expression of the first polypeptide as described above and the following immunostimulatory compounds as separate molecules:

VB1020: encoding only the first polypeptide but not the immunostimulatory compound, for comparison

·VB4195：hFLT3L

VB4169: hFLTL3 and mGM-CSF

Assessment of protein expression and secretion encoded by DNA plasmids

HEK293 cells were obtained from ATCC and transiently transfected with VB1020 (comparative), VB4195 or VB4196 as described in example 1.

Secreted proteins encoded by the DNA plasmids were characterized in the supernatant by sandwich ELISA using mouse anti-human IgG CH3 domain antibodies (capture antibody, 100 μl/well, 1 μg/ml, MCA878G, bio-Rad) and goat anti-human MIP-1 a antibodies (biotinylated detection antibody, 100 μl/well, 0.2 μg/ml, R & D systems, BAF 270).

Secretion of FLT3L encoded as the second protein in VB4195 and VB4196 in cell culture supernatants (dilution 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 FLE3L antibody (biotinylated detection antibody, 100 μl/well, 0.1 μg/ml, BAF308, R & D systems). Secretion of GM-CSF encoded as the third protein in VB4196 in cell culture supernatant (dilution 1:500) was measured by sandwich ELISA using rat anti-mouse GM-CSF (capture antibody, 100. Mu.l/well, 1.0. Mu.g/ml mouse GM-CSF antibody, MAB415, R & D Systems) and goat anti-mouse GM-CSF (biotinylated detection antibody, 100. Mu.l/well, 0.2. Mu.g/ml, BAM215, R & D Systems).

The results shown in fig. 23 demonstrate that the first polypeptide/dimer protein encoded in VB4195 and VB4196 comprising targeting units, dimerization units and antigenic units is well expressed and secreted in transfected HEK293 cells. FLT3L encoded as the second protein in VB4195 and VB4196 was expressed and secreted at high levels in both plasmids as shown in fig. 24. In addition, GM-CSF encoded in VB4196 as the third protein was also expressed and secreted at high levels as shown in FIG. 25.

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 VB 4196. VB1020 encoding the same first polypeptide as VB4195 and VB4196 was included as a comparison.

Briefly, the Expi293F cells (3×10 ⁶ cells/ml, 1.6 ml) were seeded in 6-well plates. Cells were transfected with 1. Mu.g/ml plasmid DNA using ExpiFectamine 293Reagent (Thermo Fisher Sci.) and plates were incubated on an orbital shaker (diameter 19mm,125 rpm) in a humidified CO ₂ cell incubator (8% CO ₂, 37 ℃). After 18 hours of incubation, expiFectamine 293 transfection enhancer (Thermo Fisher sci.) was added to each well. Plates were incubated for a further 28 hours and then supernatants were harvested. Samples were prepared by mixing 70 μl of supernatant from transfected Expi293F cells with 25 μl of 4x Laemmli sample buffer (Bio-Rad) and 5 μl of DTT (Thermo Fisher Sci.) or 5 μl of ultrapure water for reducing and non-reducing conditions, respectively. In addition, the Expi293F supernatant was deglycosylated by mixing 64 μl of sample with 16 μl PNGase F buffer (NEB) and incubating at 80 ℃ for 2 min. After cooling, 4 μ L RAPID PNGASE F enzyme (NEB) was added and the samples incubated at 50 ℃ for 10 min. The deglycosylated samples were further mixed with 30 μl of 4x Laemmli buffer and 6 μl DTT. The sample (reduced, non-reduced or deglycosylated) was heated at 70℃for 10 min and added to 4% -20% Criterion TGX Stain-Free pre-gel (Bio-Rad). SDS-PAGE was performed with Precision Plus Protein All Blue Prestained protein standards (Bio-Rad) in 1 XTris/glycine/SDS running buffer (Bio-Rad). Proteins were transferred from the gel onto EtOH-activated Low Fluorescence (LF) 0.45 μm PVDF membrane (Bio-Rd) using a Tran-Blot-Turbo semi-dry transfer system (Bio-Rad). PVDF membranes were blocked in EveryBlot buffer (Bio-Rad) for 5 min and probed with goat anti-human MIP-1α (BAF 270, R & D Systems), goat anti-murine GM-CSF (BAF 415, R & D Systems) or goat anti-human FLT3L (BAF 308, R & D Systems), respectively, to detect the first polypeptide/dimer protein, GM-CSF or FLT3L. The specificity of the primary antibodies was confirmed in an initial test to detect their respective recombinant proteins. The membrane was incubated with the fluorochrome conjugated secondary antibody for 1 hour at RT, then washed and dried. Images were acquired using a ChemiDoc ^TM MP imaging system (set up dlight 550 and 650, auto Optimal). Western blot analysis confirmed the ELISA results, indicating that VB4195 expresses two proteins: first polypeptide/dimer protein (fig. 26) and FLT3L (fig. 27). VB4196 expresses three proteins: first polypeptide/dimer protein (FIG. 26), FLT3L and GM-CSF (FIG. 27). The P2A sequence used in VB4196 to isolate the nucleic acid sequences encoding the FLT3L and GM-CSF proteins appeared glycosylated, such that changes in protein size were observed in Western blots. The deglycosylation scheme using PNGase F reduces this size variation. Furthermore, the P2A peptide left a 21 amino acid tail attached to the FLT3LC terminus, which can be observed by producing a size change of about 2.2kDa in western blot. Importantly, no additional bands were observed in the anti-FLT 3L and anti-GM-CSF probed membranes, indicating that ribosome skipping of the P2A and T2A sequences was successful, resulting in expression of multiple individual proteins from a single DNA plasmid.

In summary, ELISA and western blot data indicate that by using different 2A peptides as co-expression elements, the complete dimeric protein, including targeting units, dimeric units and antigenic units, can be co-expressed from DNA plasmids together with one or more other proteins (immunostimulatory compounds).

Example 5:

A DNA plasmid VB4204 is designed and produced comprising a nucleic acid sequence encoding the elements/units listed in table 4 and further comprising a nucleic acid sequence encoding the elements/units listed in table 11 below:

TABLE 11

Furthermore, the DNA plasmid pGM-CSF was designed and produced by cloning the sequences of the native leader sequence of mouse GM-CSF (SEQ ID NO: 12) and mouse GM-CSF (SEQ ID NO: 13) into the expression vector pUMVC4 a.

Assessment of expression and secretion of proteins encoded by VB4204

HEK293 cells were obtained from ATCC and transiently transfected with VB4204 or VB1020 (comparative) as described in example 1.

Sandwich ELISA was performed on supernatants (1:10 dilutions) using mouse anti-human IgG CH3 domain antibodies (capture antibody, 100 μl/well, 1 μg/ml, MCA878G, bio-Rad) and goat anti-human MIP-1α antibodies (biotinylated detection antibody, 100 μl/well, 0.2 μg/ml, R & D Systems, BAF 270) to characterize secreted proteins encoded by VB4204 or VB 1020.

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

The results presented in fig. 28 demonstrate that the first polypeptide/dimer protein encoded in VB4204 comprising targeting units, dimerization units and antigenic units is well expressed and secreted in transfected HEK293 cells. In addition, GM-CSF encoded in VB4204 as the second protein alone was also expressed and secreted at high levels as shown in FIG. 29.

Evaluation of immunogenicity of VB4204 (1)

Immunogenicity of VB4204, VB1020 (comparative) and VB1026 (negative control) was determined in C57BL/6 mice as described in example 2, however, no data was obtained for splenocytes depleted of cd4+ T cells. The T cell response in spleen cells was then tested in FluoroSpot assay for production of INF-gamma. In addition, the immunogenicity of coinjected DNA plasmids (6. Mu.g of total DNA) VB1020 (encoding the same polypeptide as VB4204 but not GM-CSF) and pGM-CSF (encoding GM-CSF but not VB 4204) were determined as described in this paragraph.

Peptides corresponding to the E6 and E7 antigens described in table 12 below were used to re-stimulate spleen cells harvested from mice administered VB1020, VB4204, VB1026 and (VB 1020 plus pGM-CSF).

Table 12

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

As shown in fig. 30, no IFN- γ production was detected in response to administration of VB 1026.

In addition, VB1020 induced a strong T cell response against the peptides in table 12, and VB4202 induced an even stronger T cell response compared to VB 1020. In addition, VB4202 induced a stronger T cell response than the co-injection of VB1020 and pGM-CSF (FIG. 30).

Evaluation of immunogenicity of VB4204 (2)

Immunogenicity of VB4204, VB1020 (comparative) and VB1026 (negative control) was determined by flow cytometry. C57BL/6 mice were treated as described in example 2, splenocytes were combined and re-stimulated with a single peptide corresponding to HPV 16E 7 (49-57) antigen described in table 12 at RT for 1 hour, followed by the addition of monensin (monensin) and brefeldin (brefeldin) to each well to inhibit endocytosis. The cells were further incubated at 37℃for 15 hours. After re-stimulation, the cells were harvested for flow cytometry analysis. Briefly, single cell suspensions were first incubated with a vital dye (eFluor 780, invitrogen) for 10 minutes at RT. The vital dye was rinsed off with PBS (centrifuged twice at 400Xg for 6 min each at 4 ℃). The cells were then incubated with the Fc blocker for 10 minutes at RT to block non-specific binding of the fluorescent antibody. Following the blocking step, the cells were stained with a pool of surface marker specific antibodies (table 9) for 30 minutes on ice. The antibody was washed off with PBS (centrifuged at 400xg twice for 6 min each at 4 ℃) and the cells incubated with the fixation/permeabilization solution (60 min at 4 ℃). Cells were centrifuged, washed and resuspended in permeabilization buffer containing 100. Mu.l of the antibody mixture and incubated at 4℃for 30 min. Stained cells were run on BD FACSymphony A flow cytometer. Flow cytometry data were analyzed using FlowJo software.

The ability of DNA plasmid VB4202 to elicit T cell immune responses against the single peptide HPV 16E 7 (49-57) in table 12 was compared. VB1026 was included as a negative control.

As shown in fig. 31, no IFN- γ or TNF- α production was detected in response to administration of VB 1026.

The number of IFN-gamma secreting CD8+ T cells (CD4+ T cell depleted samples), TNF-alpha secreting CD8+ T cells (CD4+ T cell depleted samples) and INF-gamma+ TNF-alpha co-secreting cells were all increased from VB1020 to VB4202 (FIG. 31).

These results indicate that the DNA plasmids of the invention encoding the first polypeptide and the immunostimulatory compound co-expressed as separate molecules from the DNA plasmids of the invention can enhance antigen-specific T cell responses against the antigen comprised in the first polypeptide compared to DNA plasmids encoding only the first polypeptide and compared to co-injecting a DNA plasmid encoding the same first polypeptide and a plasmid encoding the same immunostimulatory compound.

Example 6:

a DNA plasmid VB4205 was designed and produced comprising a nucleic acid sequence encoding the elements/units listed in table 4 and further comprising a nucleic acid sequence encoding the elements/units listed in table 13 below:

TABLE 13

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Assessment of expression and secretion of proteins encoded by VB4205

HEK293 cells were obtained from ATCC and transiently transfected with VB4205 or VB1020 (comparative) as described in example 1. Sandwich ELISA was performed on supernatants (1:10 dilutions) using mouse anti-human IgG CH3 domain antibodies (capture antibody, 100 μl/well, 1 μg/ml, MCA878G, bio-Rad) and goat anti-human MIP-1α antibodies (biotinylated detection antibody, 100 μl/well, 0.2 μg/ml, R & D systems, BAF 270) to characterize secreted proteins encoded by VB4205 or VB 1020.

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

The results presented in fig. 32 demonstrate that the first polypeptide/dimer protein encoded in VB4205 comprising targeting units, dimerization units and antigenic units is well expressed and secreted in transfected HEK293 cells. In addition, CCL5 encoded as the second protein in VB4205 is expressed and secreted at high levels as shown in fig. 33.

Evaluation of immunogenicity of VB4205

The immunogenicity of VB4205 was determined in C57BL/6 mice and compared to the immunogenicity of VB1020 (comparison) and VB1026 (negative control) as described in example 5 (1).

The ability of VB1020 (first polypeptide only) and VB4205 (first polypeptide and CCL 5) to elicit T cell immune responses against the peptides in table 12 was compared.

As shown in fig. 34, no IFN- γ production was detected in response to administration of VB 1026. VB1020 induced a strong T cell response against the peptides in Table 12, while VB4205 induced an even stronger T cell response compared to VB 1020.

These results also indicate that the DNA plasmids of the invention encoding the first polypeptide and the immunostimulatory compound co-expressed as separate molecules from the DNA plasmids of the invention can enhance antigen-specific T cell responses against the antigen contained in the first polypeptide compared to DNA plasmids encoding only the first polypeptide.

Example 7:

DNA plasmids VB1026, VB4208, VB4194, VB4202 and pGM-CSF, comprising the nucleic acid sequences encoding the elements/units listed in table 14, were designed and produced as described herein:

TABLE 14

The DNA plasmid encodes the following proteins:

VB4202 encodes and allows co-expression of the first polypeptide and the immunostimulatory compound mGM-CSF as described above as separate molecules

VB4194: encoding only the first polypeptide comprising an antigenic unit comprising a CT26 epitope, and not encoding an immunostimulatory compound, for comparison

VB1026: encoding a polypeptide having the sequence of SEQ ID NO:1, which is identical to the first polypeptide encoded by VB4194, but does not comprise a linker unit nor an antigenic unit. It served as a negative control

VB4208 (SEQ ID NO: 24): encodes a first polypeptide in the form of a single molecule that does not comprise an antigenic unit (i.e., does not encode any CT26 epitope) and mGM-CSF. It served as a negative control

PGM-CSF: coding mGM-CSF for comparison

Treatment of CT26 tumor challenged mice

The antitumor efficacy of VB4202 was evaluated in CT26 tumor challenge. VB4202 is compared to VB4194 encoding a first polypeptide identical to VB 4202. In addition, the antitumor efficacy of VB4202 was compared with that of co-injected VB4194 and pGM-CSF. VB1026 and VB4208 were included as negative controls.

Each of the A-F groups contained 8 BALB/c mice, and CT26 tumor cells were inoculated on day 0 (D) by injecting 1X10 ⁵ tumor cells in the left leg. On days 4 and 11, the DNA plasmids described in table 15 were administered intramuscularly to the right leg of the mice in their respective amounts. Due to the plasmid size differences between VB4194 and pGM-CSF plasmids, a second co-injection group (group F) was included, where the amount of each plasmid was adjusted to match the plasmid copy number of a single plasmid injection in group D (table 16) to ensure equivalent levels of expressed protein.

TABLE 15

Table 16

Tumor size was measured using calipers. Two-dimensional measurements, i.e. length and width, are made on the tumor and the height is set equal to the width. Tumor volume was calculated by the following formula: tumor volume = length (mm) x width (mm) x height (mm)/2000. The treatment ended on day 32.

Tumor growth was slower in the groups treated with VB4194 (group C), VB4202 (group D) and VB4194 and pGM-CSF coinjection (group E and group F) compared to the VB1026 and VB4208 negative control groups (group A and group B, respectively).

Administration of VB4202 (group D) co-expressing the same first polypeptide as VB4194 and GM-CSF resulted in a reduced tumor growth rate compared to VB4194 alone (group C). Furthermore, administration of VB4202 resulted in a reduced tumor growth rate compared to the two co-injected groups, wherein VB4194 and pGM-CSF were administered either together at 10 μg (E) or adjusted to comparable copy number (F). The tumor growth rate resulting from such coinjection was similar to that of administration of VB4194 alone.

These results (shown in fig. 35) demonstrate that VB4194 provides a further increase in tumor growth inhibition efficacy by co-expression of GM-CSF from VB4202, VB4202 encoding the same first polypeptide as VB 4194. Tumor growth inhibition in animals treated with VB4202 was accompanied by an increase in survival compared to the other groups, as shown in fig. 36. Antitumor efficacy was driven by antigen-specific immune responses as shown by comparing VB4202 with negative controls VB1026 and VB 4208. Furthermore, VB4202 provided a stronger antitumor efficacy than that observed when VB4194 was co-injected with pGM-CSF as two separate plasmids.

Example 8:

DNA plasmids TECH001-CV021, TECH001-CV022 and TECH001-CV023 were designed and produced, comprising a nucleic acid sequence encoding the elements/units listed in table 4, and further comprising a nucleic acid sequence encoding the elements/units listed in table 17 below:

TABLE 17

The DNA plasmids TECH001-CV021, TECH001-CV022 and TECH001-CV023 comprise a nucleic acid sequence encoding a first polypeptide comprising an antigenic unit containing a SARS-CoV-2 Receptor Binding Domain (RBD) antigen.

Each of these DNA plasmids is a model of the DNA plasmid of the invention encoding the first polypeptide, for use in the treatment of infectious diseases, i.e. a plasmid comprising antigenic units comprising an antigen derived from a pathogen (here an antigen derived from SARS-CoV-2).

DNA plasmids TECH001-CV021, TECH001-CV022 and TECH001-CV023 allow co-expression of the first polypeptide as described above and the following immunostimulatory compounds as separate molecules:

VB2060: encoding only the first polypeptide, not the immunostimulatory compound, for comparison

·TECH001-CV021：mGM-CSF

·TECH001-CV022：mIL-12

·TECH001-CV023：mIL-21

IL12 is a heterodimeric cytokine encoded by two separate genes, IL-12A (p 35) and IL-12B (p 40). Active heterodimers (called p 70) and p40 homodimers are formed after protein synthesis.

Assessment of expression and secretion of proteins encoded by DNA plasmids

Briefly, the Expi293F cells (2×10 ⁶ cells/ml, 1 ml) were seeded in 96-well plates. Cells were transfected with 0.64 μg/ml plasmid DNA using ExpiFectamine reagents (Thermo Fisher sci.) and plates were incubated on an orbital shaker (diameter 3mm,900 rpm) in a humidified CO ₂ cell incubator (8% CO ₂, 37 ℃). Plates were incubated for 72 hours and then supernatants were harvested.

Secreted first polypeptide/dimer protein in the supernatant (dilution 1:1500) was characterized by sandwich ELISA using mouse anti-human IgG CH3 domain antibodies (capture antibody, 100 μl/well, 1 μg/ml, MCA878G, bio-Rad) and goat anti-human MIP-1α antibodies (biotinylated detection antibody, 100 μl/well, 0.2 μg/ml, BAF270, R & D systems) (fig. 37).

Anti-mouse GM-CSF Ab (rat anti-mouse GM-CSF capture antibody, 100. Mu.l/well, 1.0. Mu.g/ml mouse GM-CSF antibody, MAB415, R & D Systems, respectively; goat anti-mouse GM-CSF biotinylated detection antibody, 100. Mu.l/well, 0.2. Mu.g/ml, BAM215, R & D Systems) (FIG. 38 a), anti-mouse IL-12Ab (rat anti-mouse IL-12 capture antibody, 100. Mu.l/well, 1.0. Mu.g/ml, MAB419R & D Systems; goat anti-mouse IL-12 biotinylated detection antibody, 100. Mu.l/well, 0.4. Mu.g/ml, BAF419, R & D Systems) (FIG. 38 b) and anti-mouse IL-21Ab (goat anti-mouse IL-21 capture antibody, 100. Mu.l/well, 0.1. Mu.g/ml, AF594, R & D Systems; goat anti-mouse IL-21 biotinylated detection antibody, 100. Mu.l/well, 0.4. Mu.g/ml, BAF594, R & D Systems) (FIG. 38 c), sandwich GM-12 and IL-21 expression and IL-21 secretion of ELISA in supernatants were measured as shown in FIGS. 38b and 38c, respectively.

The results presented in fig. 37 demonstrate that the first polypeptide/dimer protein encoded in TECH001-CV021, TECH001-CV022 and TECH001-CV023 comprising a targeting unit, a dimerization unit and an antigenic unit is expressed and secreted from transfected Expi293F cells. GM-CSF, IL-12 and IL-21 encoded as the second proteins in TECH001-CV021, TECH001-CV022 and TECH001-CV023, respectively, were also expressed and secreted at high levels, as shown in FIGS. 38a-38 c.

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 further characterize the proteins encoded by TECH001-CV021, TECH001-CV022 and TECH001-CV 023. VB2060 encoding the same first polypeptide as the aforementioned DNA plasmid was included as a comparison.

Samples were prepared by mixing 70 μl of supernatant of transfected Expi293F cells with 25 μl of 4x Laemmli sample buffer (Bio-Rad) and 5 μl of DTT (Thermo Fisher sci.) or 5 μl of ultrapure water (for reducing and non-reducing conditions, respectively). The sample (reduced or non-reduced) was heated at 70℃for 10 minutes and then added to 4% -20% Criterion TGX Stain-Free pre-gel (Bio-Rad) at 20. Mu.L per lane. SDS-PAGE was performed in 1 XTris/glycine/SDS running buffer (Bio-Rad) using Precision Plus Protein All Blue pre-stained and unstained protein standards (Bio-Rad). Proteins were transferred from the gel onto an ethanol activated Low Fluorescence (LF) 0.45 μm PVDF membrane (Bio-Rad) using a Tran-Blot Turbo semi-dry transfer system (Bio-Rad). PVDF membrane was blocked in EveryBlot buffer (Bio-Rad) for 5min and probed with goat anti-human MIP-1α (AF 270, R & D Systems), goat anti-mouse GM-CSF (BAF 415, R & D Systems), goat anti-mouse IL-12 (BAF 419, R & D Systems) or goat anti-mouse IL-21 (BAF 594, R & D Systems) to detect the first polypeptide, GM-CSF, IL-12 and IL-21, respectively. The membrane was washed, and the anti-goat secondary conjugated with fluorescent dye was incubated for 1 hour at RT, then washed and dried (rinsed in ethanol). Images were acquired using a ChemiDoc ^TM MP imaging system (set up dlight 650 and 800, auto-optimization). Cells treated with Expifectamine (transfection control) were included as negative controls on each gel.

WB analysis confirmed ELISA results, indicating that the complete first polypeptide was expressed from all four DNA plasmids TECH001-CV021, TECH001-CV022, TECH001-CV023 and VB2060 (fig. 39). TECH001-CV021 further expressed (heterologous glycosylation) GM-CSF (FIG. 40). FIG. 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 also expressed glycosylated IL-12B (p 40) and IL-12A (p 35) (FIG. 41, left panel). Previous studies reported that cells secreting bioactive IL-12 (p 70 heterodimer) also secrete p40 (monomer) in free form (Jalah et al, J Biol Chem Vol 288, no.9, 6763-6776, 2013). In fact, bands were detected for both IL-12p70 heterodimer and p40 monomer under non-reducing conditions (FIG. 41, right panel). In addition to the first polypeptide, TECH001-CV023 also expressed IL-21 (FIG. 42). Importantly, no additional bands were observed on the anti-GM-CSF, anti-IL-12 and anti-IL-21 probed membranes, indicating that the ribosome was successfully hopped at the T2A sequence, resulting in the expression of multiple individual proteins from a single DNA plasmid.

In summary, ELISA and western blot data demonstrate that by using the same co-expression element 2A peptide, the complete first polypeptide comprising the targeting unit, dimerization unit and antigenic unit can be co-expressed from a DNA plasmid together with one or more immunostimulatory compounds.

Immunogenicity of TECH001-CV021, TECH001-CV022 and TECH001-CV023 were evaluated

Immunogenicity of TECH001-CV021, TECH001-CV022 and TECH001-CV023 were determined and compared to immunogenicity of VB2060 and VB1026 (negative control).

Female BALB/c mice of 6 weeks of age were obtained from Janvier Labs (France). All animals were kept in animal facilities at the university of oslo (norwegian oslo). All animal protocols were approved by the norwegian food safety agency (norwegian oslo). 5 mice/group were used for the test of TECH001-CV021, TECH001-CV022, TECH001-CV023 and VB2060, while 3 mice/group were used for the negative control.

A final dose of 1 μg DNA plasmid was injected into each tibialis anterior (2 x25 μl,20 μg/ml) via intramuscular needle injection, and then electroporated using AgilePulse in vivo electroporation system (BTX, USA).

Humoral immune responses induced in mice against SARS-CoV-2RBD were assessed.

Serum from mice administered with these DNA plasmids was collected 13 days after administration of the DNA plasmids, and binding of anti-RBD IgG antibodies (of the martial arts) to RBD proteins was detected.

Briefly, blood was collected from saphenous veins of vaccinated mice. The coagulated blood was centrifuged twice (1000 g,15 min), serum was collected and transferred to a clean tube. The humoral immune response was assessed by ELISA to detect binding of total IgG in serum to SARS-CoV2 (Wuhan variant) RBD (aa 319-542). ELISA plates (MaxiSorp Nunc-Immuno plates) were coated overnight at 4℃with 1. Mu.g/ml recombinant RBD-His protein antigen in PBS. Plates were blocked with 4% BSA in PBS for 1 hour at RT. Plates were then incubated with serial dilutions of mouse serum (diluted in PBS with 0.1% BSA) and incubated for 2 hours at 37 ℃. Plates were washed 3 times and incubated with anti-mouse total IgG-HRP antibody (Southern Biotech) diluted 1:50 000 in PBS containing 0.1% BSA and incubated for 1 hour at 37 ℃. After the final wash, the plates were developed using TMB substrate (Merck, cat. Number CL 07-1000). UsingThe plates were read in 30 minutes with a multimode microplate reader (Tecan) at a wavelength of 450 nm. The bound antibody endpoint titer was calculated as the reciprocal of the highest dilution (resulting in a signal above the cutoff). The binding antigens tested included SARS-CoV-2 antigen: RBD (Sino Biological40592-V08H, (SEQ ID NO: 30)). /(I)

The results shown in FIG. 43 demonstrate that TECH001-CV021 and TECH001-CV023, which encode a first polypeptide comprising RBD (aa 319-542) from SARS-CoV-2 (Wuhan variant) in the antigenic unit and GM-CSF and IL-21, respectively, as a second protein, induced a stronger total IgG response against RBD than comparative VB2060, which encoded only the same first polypeptide (Mann-Whitney test, TECH001-CV021: P=0.008, TECH001-CV023: P=0.047). Furthermore, TECH001-CV022, which encodes the above-described first polypeptide and two subdomains of IL-12 as second and third proteins, also induced a stronger IgG response against RBD compared to comparison VB2060 (Mann-Whitney test, p=0.047).

Assessment of T cell response induced against SARS-CoV-2RBD

Spleens of mice administered with DNA plasmids were collected 14 days after administration and triturated in a cell filter to obtain a single cell suspension. Erythrocytes were lysed using potassium Ammonium Chloride (ACK) lysis buffer. Spleen cells were counted using a NucleoCounter NC-202 (ChemoMetec, denmark) and resuspended to a final concentration of 6x10 ⁶ cells/ml. For each plasmid tested, a portion of the single cell suspension was used to deplete cd4+ T cells using Dynabeads ^TM anti-CD 4 beads. The total splenocytes and splenocytes depleted of CD4+ T cells were then tested for INF-gamma production in a FluoroSpot assay by seeding 6X10 ⁵ cells/well and re-stimulating with a 2. Mu.g/ml RBD peptide pool (Table 18) for 22.5 hours. The RBD peptide library contains 15-mer peptides that overlap with 12 amino acids across the RBD region.

TABLE 18

Pool ID	Composition of the composition
		Pool 1	RBD-1,2,3,4,5,6,7,8,9,10
Pool 2	RBD-11,12,13,14,15,16,17,18,19,24
		Pool 3	RBD-20,21,22,23,25,26,27,28,29,30
Pool 4	RBD-31,32,33,34,36,37,38,39,40
		Pool 5	RBD-41,42,43,44,45,46,47,48,49,50,51
Pool 6	RBD-52,53,54,55,56,57,58,59,60,61

The results shown in FIG. 44 demonstrate that the encoding of RBDs (aa 319-542) from SARS-CoV-2 (Wuhan variant) and TECH001-CV021 and TECH001-CV023 of the GM-CSF and IL-21 first polypeptides, respectively, as second proteins, induced a much stronger total T cell response against the RBDs in the antigenic unit than the comparative VB2060 encoding only the same first polypeptide (FIG. 44A). In addition, TECH001-CV021 and TECH001-CV023 induced a stronger CD8 ⁺ T cell response (cd4+ depleted splenocyte fraction) compared to VB2060 (fig. 44B). The TECH001-CV022, encoding the aforementioned first polypeptide and the two subdomains of IL-12 as the second and third proteins, also induced a much stronger total T cell response against RBD than VB2060 (FIG. 44A). The increase in T cell response induced by the addition of IL-12 cytokine appears to be due mainly to the increase in IFN- γ secretion by cd4+ T cells, as a substantial decrease in response was observed in samples depleted of TECH001-CV022 CD4 ⁺ T cells compared to the TECH001-CV021 and TECH001-CV023 treated groups (fig. 44B).

Taken together, the results presented demonstrate that the humoral and cellular immune responses elicited against SARS-CoV-2RBD in mice are enhanced by coexpression of a first polypeptide/dimer protein comprising a targeting unit, a dimerization unit and an antigenic unit comprising an infectious antigen (RBD derived from the pathogen SARS-CoV-2) and an immunostimulatory compound (GM-CSF, IL-12 or IL-21) compared to expression of only the first polypeptide/dimer protein described above.

Sequence overview

SEQ ID NO:1

M¹QVSTAALAVLLCTMALCNQVLS²³A²⁴PLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSA⁹³E⁹⁴LKTPLGDTTHT¹⁰⁵E¹⁰⁶PKSCDTPPPCPRCP¹²⁰G¹²¹GGSSGGGSG¹ ³⁰G¹³¹QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK²³⁷

SEQ ID NO:2

Signal peptides

MNFGLRLIFLVLTLKGVQC

SEQ ID NO:3

Signal peptides

MDAMKRGLCCVLLLCGAVFVSP

SEQ ID NO:4

Signal peptide of human FLT3L

MTVLAPAWSPTTYLLLLLLLSSGLSG

SEQ ID NO:5

VB4194

MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLKSWIHCWKYLSVQSQLFRGSSLLFRRVGGGGSGGGGSNNLQKYIEIYVQKINPSRLPVVIGGLLGGGGSGGGGSEVIQTSKYYMRDVIAIESAWLLELAPHGGGGSGGGGSVILPQAPSGPSYATYLQPAQAQMLTPPGGGGSGGGGSFVSPMAHYVPGIMAIESVVARFQFIVPGGGGSGGGGSGDVKIHAHKVVLANISPYFKAMFTGNLGGGGSGGGGSTPLRKHTVHAIRKFYLEFKGSSPPPRLGGGGSGGGGSKIYEFDYHLYGQNITMIMTSVSGHLLA

SEQ ID NO:6

VB4168

MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLKSWIHCWKYLSVQSQLFRGSSLLFRRVGGGGSGGGGSNNLQKYIEIYVQKINPSRLPVVIGGLLGGGGSGGGGSEVIQTSKYYMRDVIAIESAWLLELAPHGGGGSGGGGSVILPQAPSGPSYATYLQPAQAQMLTPPGGGGSGGGGSFVSPMAHYVPGIMAIESVVARFQFIVPGGGGSGGGGSGDVKIHAHKVVLANISPYFKAMFTGNLGGGGSGGGGSTPLRKHTVHAIRKFYLEFKGSSPPPRLGGGGSGGGGSKIYEFDYHLYGQNITMIMTSVSGHLLAGSGEGRGSLLTCGDVEENPGPMTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQP

SEQ ID NO:7

VB4169

MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLKSWIHCWKYLSVQSQLFRGSSLLFRRVGGGGSGGGGSNNLQKYIEIYVQKINPSRLPVVIGGLLGGGGSGGGGSEVIQTSKYYMRDVIAIESAWLLELAPHGGGGSGGGGSVILPQAPSGPSYATYLQPAQAQMLTPPGGGGSGGGGSFVSPMAHYVPGIMAIESVVARFQFIVPGGGGSGGGGSGDVKIHAHKVVLANISPYFKAMFTGNLGGGGSGGGGSTPLRKHTVHAIRKFYLEFKGSSPPPRLGGGGSGGGGSKIYEFDYHLYGQNITMIMTSVSGHLLAGSGEGRGSLLTCGDVEENPGPMTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPGSGATNFSLLKQAGDVEENPGPMWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQK

SEQ ID NO:8

VB4170

MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLKSWIHCWKYLSVQSQLFRGSSLLFRRVGGGGSGGGGSNNLQKYIEIYVQKINPSRLPVVIGGLLGGGGSGGGGSEVIQTSKYYMRDVIAIESAWLLELAPHGGGGSGGGGSVILPQAPSGPSYATYLQPAQAQMLTPPGGGGSGGGGSFVSPMAHYVPGIMAIESVVARFQFIVPGGGGSGGGGSGDVKIHAHKVVLANISPYFKAMFTGNLGGGGSGGGGSTPLRKHTVHAIRKFYLEFKGSSPPPRLGGGGSGGGGSKIYEFDYHLYGQNITMIMTSVSGHLLAGSGEGRGSLLTCGDVEENPGPMTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPGSGATNFSLLKQAGDVEENPGPMWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQKGSGQCTNYALLKLAGDVESNPGPMKISAAALTIILTAAALCTPAPASPYGSDTTPCCFAYLSLALPRAHVKEYFYTSSKCSNLAVVFVTRRNRQVCANPEKKWVQEYINYLEMS

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

Joint

GGGGSGGGGS

SEQ ID NO:18

VB4202

MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLKSWIHCWKYLSVQSQLFRGSSLLFRRVGGGGSGGGGSNNLQKYIEIYVQKINPSRLPVVIGGLLGGGGSGGGGSEVIQTSKYYMRDVIAIESAWLLELAPHGGGGSGGGGSVILPQAPSGPSYATYLQPAQAQMLTPPGGGGSGGGGSFVSPMAHYVPGIMAIESVVARFQFIVPGGGGSGGGGSGDVKIHAHKVVLANISPYFKAMFTGNLGGGGSGGGGSTPLRKHTVHAIRKFYLEFKGSSPPPRLGGGGSGGGGSKIYEFDYHLYGQNITMIMTSVSGHLLAGSGEGRGSLLTCGDVEENPGPMWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQK

SEQ ID NO:19

VB1020

MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLMHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGSSGGGSGMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFARRDLCIVYRDGNPYAVRDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINRQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL

SEQ ID NO:20

VB4195

MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLMHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGSSGGGSGMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFARRDLCIVYRDGNPYAVRDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINRQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLGSGEGRGSLLTCGDVEENPGPMTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQP

SEQ ID NO:21

VB4196

MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLMHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGSSGGGSGMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFARRDLCIVYRDGNPYAVRDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINRQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLGSGEGRGSLLTCGDVEENPGPMTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPGSGATNFSLLKQAGDVEENPGPMWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQK

SEQ ID NO:22

VB4204

MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLMHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGSSGGGSGMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFARRDLCIVYRDGNPYAVRDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINRQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLGSGEGRGSLLTCGDVEENPGPMWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQK

SEQ ID NO:23

VB4205

MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLMHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGGGSSGGGSGMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFARRDLCIVYRDGNPYAVRDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINRQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLGSGEGRGSLLTCGDVEENPGPMKISAAALTIILTAAALCTPAPASPYGSDTTPCCFAYLSLALPRAHVKEYFYTSSKCSNLAVVFVTRRNRQVCANPEKKWVQEYINYLEMS

SEQ ID NO:24

VB4208

MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLGSGEGRGSLLTCGDVEENPGPMWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQK

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

SEQ ID NO:28:

Nucleotide sequence encoding amino acids 94-237 of SEQ ID NO. 1 GAGCTCAAAACCCCACTTGGTGACACAACTCACACAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGGCGGTGGAAGCAGCGGAGGTGGAAGTGGAGGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

SEQ ID NO:29

Nucleotide sequence encoding amino acids 1-23 of SEQ ID NO. 1

ATGCAGGTCTCCACTGCTGCCCTTGCCGTCCTCCTCTGCACCATGGCTCTCTGCAACCAGGTCCTCTCT

SEQ ID NO:30

SARS-CoV-2RBD (amino acids 319-542)

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

SEQ ID NO:31

TECH001-CV021

MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFGSGEGRGSLLTCGDVEENPGPMWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQK

SEQ ID NO:32

TECH001-CV022

MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFGSGEGRGSLLTCGDVEENPGPMCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAEGRGSLLTCGDVEENPGPMCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS

SEQ ID NO:33

TECH001-CV023

MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFGSGEGRGSLLTCGDVEENPGPMERTLVCLVVIFLGTVAHKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLDPELLSAPQDVKGHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSCDSYEKRTPKEFLERLKWLLQKMIHQHLS

SEQ ID NO:34

Mouse IL-12A signal peptide

MCQSRYLLFLATLALLNHLSLA

SEQ ID NO:35

Mouse IL-12A

RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA

SEQ ID NO:36

Mouse IL-12B signal peptide

MCPQKLTISWFAIVLLVSPLMA

SEQ ID NO:37

Mouse IL-12B

MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS

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

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

SEQ ID NO:48

Human IL-21 signal peptide

MRSSPGNMERIVICLMVIFLGTLV

SEQ ID NO:49

Human IL-21

HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

Description of the embodiments

1. A carrier, comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerization unit, e.g., a dimerization unit, and an antigenic unit comprising one or more antigens or portions thereof, e.g., one or more disease-associated antigens or portions thereof; and

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 wherein 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).

3. The vector according to embodiment 1 or 2, 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.

4. The vector according to any one of embodiments 1 to 3, wherein the one or more immunostimulatory compounds promote the attraction of antigen presenting cells (attraction).

5. The vector according to embodiment 4, wherein the one or more immunostimulatory compounds are chemokines, preferably human chemokines.

6. The vector according to embodiment 5, wherein the one or more immunostimulatory compounds may interact with a surface molecule selected from CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 and XCR1 on antigen presenting cells, preferably wherein the one or more immunostimulatory compounds may interact with a surface molecule selected from hCCR1, hCCR3, hCCR4, hCCR5, hCCR6, hCCR7, hCCR8 and hXCR1 on human antigen presenting cells.

7. The vector according to any one of embodiments 5 to 6, wherein the one or more immunostimulatory compounds are selected from macrophage inflammatory protein a, including isoforms thereof, such as mouse CCL3, human CCL3L1, human CCL3L2, and human CCL3L3, CCL4, preferably hCCL4, CCL5, preferably hCCL, CCL19, preferably hCCL, CCL20, preferably hCCL, CCL21, preferably hCCL21, XCL1, preferably hXCL1, and XCL2, preferably hXCL2.

8. The vector according to any one of embodiments 3 to 7, wherein the one or more immunostimulatory compounds promote activation and/or maturation of antigen presenting cells.

9. The vector according to any one of embodiments 3 to 8, wherein the one or more immunostimulatory compounds may interact with a surface molecule on an antigen presenting cell, the surface molecule being selected from receptors of the TNF receptor superfamily, including CD40 (cluster of differentiation 40), CD137 (4-1 BB), CD27, RANK, and ICOS (CD 278), preferably wherein the one or more immunostimulatory compounds may interact with a surface molecule on a human antigen presenting cell, the surface molecule being selected from receptors of the human TNF receptor superfamily, including human CD40, hCD137, hCD27, hRANK, and hcicos.

10. The vector according to embodiment 9, wherein the one or more immunostimulatory compounds are selected from the group consisting of CD40L, CD137L, CD, RANKL and ICOSL, preferably wherein the one or more immunostimulatory compounds are selected from the group consisting of hCD40L, hCD137L, hCD, hRANKL and hICOSL.

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, TNFa, IFNy and IL-1β, preferably wherein the one or more immunostimulatory compounds are human cytokines selected from the group consisting of hll-2, hll-10, hll-12, hll-21, htnfα, hlfγ and hll-1β.

12. The vector according to any of embodiments 3 to 8, wherein the one or more immunostimulatory compounds are viral infection sensors, such as MyD88 or TRIF, preferably human viral infection sensors, such as human MyD88 or human TRIF.

13. The vector according to any one of embodiments 3 to 8, wherein the one or more immunostimulatory compounds may interact with a pattern recognition receptor on antigen presenting cells, such as a human Toll-like receptor comprising hTLR2, hTLR4, TLR5 and TLR9, and/or with a receptor on antigen presenting cells selected from RAGE, TIM-3, FPR, SREC1, LOX1 and CD91, preferably wherein the one or more immunostimulatory compounds may interact with a pattern recognition receptor on human antigen presenting cells selected from hTLR2, hTLR4, hTLR5 and hTLR9, and/or with a receptor on human antigen presenting cells selected from htnge, htmr, hsec 1, hlop 1 and hCD91.

14. The vector according to embodiment 13, wherein the one or more immunostimulatory compounds are selected from pathogen-associated molecular patterns (PAMPs) such as flagellin, protein damage-associated molecular patterns (DAMP) such as HMGB1, heat Shock Proteins (HSP), calreticulin and annexin A1, preferably wherein the one or more immunostimulatory compounds are selected from human pathogen-associated molecular patterns (PAMPs), human protein damage-associated molecular patterns (DAMP) such as HMGB1, human Heat Shock Proteins (HSP), human calreticulin and human annexin A1.

15. The vector according to any one of embodiments 3 to 14, wherein the one or more immunostimulatory compounds promote growth and/or expansion of antigen presenting cells.

16. The vector according to any one of embodiments 3 to 15, wherein the one or more immunostimulatory compounds are growth factors, preferably human growth factors.

17. The vector according to any one of embodiments 3 to 15, wherein the one or more immunostimulatory compounds may interact with a surface molecule on an antigen presenting cell, the surface molecule being selected from the group consisting of GM-CSF receptor, FLT-3R, IL-15R and IL-4R, preferably wherein the one or more immunostimulatory compounds may interact with a surface molecule on a human antigen presenting cell, the surface molecule being selected from the group consisting of hGM-CSF receptor, hFLT-3R, hIL-15R and hIL-4R.

18. The vector according to any one of embodiments 16 to 17, wherein the one or more immunostimulatory compounds are selected from GM-CSF, FLT-3L, IL-15 and IL-4, preferably wherein the one or more immunostimulatory compounds are selected from hGM-CSF, hFLT-3L, hIL-15 and hll-4.

19. The vector according to any one of embodiments 1 to 17, wherein the one or more immunostimulatory compounds are selected from the group consisting of IL-4, IL-1 β, ifnγ, ifnα, IL-15, tnfα, IL-10, IL-12, IL-21, IL-2, myD88, tri, RIG-1, MDA-5, P28 region of C3d, IL-13, ifnε, ifnκ, ifnω, ifnβ, and IL-6, preferably wherein the one or more immunostimulatory compounds are selected from the group consisting of hll-4, hll-1 β, hifnγ, hlnfα, hll-15, htnfα, hll-10, hll-12, hll-21, hll-2, hMyD, hf, hlig-I, hMDA-5, P28 region of hC3d, hll-13, hε, hlfn, hlω, and hl-6.

20. The vector according to any one of embodiments 1 to 19, comprising a plurality of further nucleic acid sequences encoding a plurality of immunostimulatory compounds, e.g. 2, 3, 4,5, 6, 7 or 8 different immunostimulatory compounds.

21. The vector according to embodiment 20, wherein the plurality of immunostimulatory compounds are different immunostimulatory compounds that affect (e.g., stimulate) antigen presenting cells in different ways.

22. The vector according to any one of the preceding embodiments, wherein the vector comprises one or more co-expression elements.

23. The vector according to embodiment 22, wherein the one or more co-expression elements cause transcription of the first polypeptide and the one or more immunostimulatory compounds on a single transcript and independent translation into the first polypeptide alone and the one or more immunostimulatory compounds alone.

24. The vector according to any one of embodiments 22 to 23, wherein the one or more co-expression elements are IRES elements or nucleic acid sequences encoding a 2A self-cleaving peptide.

25. The vector according to any one of embodiments 22 to 23, wherein the vector comprises more than one co-expression element, which is an IRES element or a nucleic acid sequence encoding a 2A self-cleaving peptide, or an IRES element and a nucleic acid sequence encoding a 2A self-cleaving peptide.

26. The vector according to any one of embodiments 22 to 25, wherein the 2A self-cleaving peptide is selected from the group consisting of T2A peptide, P2A peptide, E2A peptide and F2A peptide.

27. The vector according to any one of embodiments 22 to 26, wherein the 2A self-cleaving peptide is selected from the group consisting of a polypeptide having a sequence identical to SEQ ID NO:9, a T2A peptide having an amino acid sequence with 80% to 100% sequence identity to the amino acid sequence of SEQ ID NO:11, a P2A peptide having an amino acid sequence with 80% to 100% sequence identity to the amino acid sequence of SEQ ID No. 14, an E2A peptide having an amino acid sequence with 80% to 100% sequence identity to the amino acid sequence of SEQ ID No. 51, and an F2A peptide having an amino acid sequence with 80% to 100% sequence identity to the amino acid sequence of SEQ ID No. 51.

28. The vector according to any one of embodiments 22 to 27, wherein the 2A self-cleaving peptide is selected from the group consisting of a T2A peptide having the amino acid sequence of SEQ ID NO. 9, a P2A peptide having the amino acid sequence of SEQ ID NO. 11, an E2A peptide having the amino acid sequence of SEQ ID NO. 14, and an F2A peptide having the amino acid sequence of SEQ ID NO. 51.

29. The vector according to embodiment 23, wherein the one or more co-expression elements cause transcription of the first polypeptide and the one or more immunostimulatory compounds as separate transcripts.

30. The vector according to embodiment 29, wherein the one or more co-expression elements is a bi-directional promoter.

31. The vector according to embodiment 29, wherein the one or more co-expression elements are promoters, and wherein the vector comprises a separate promoter for each of the nucleic acid sequences encoding the first polypeptide and the one or more immunostimulatory compounds.

32. The vector according to embodiment 29, wherein the one or more co-expression elements are a bi-directional promoter and a promoter.

33. The vector according to any one of embodiments 23 to 33, wherein the vector comprises one or more co-expression elements selected from the group consisting of an IRES element, a nucleic acid sequence encoding a 2A self-cleaving peptide, a bi-directional promoter, and a promoter.

34. The vector according to any one of embodiments 1 to 33, wherein the antigenic unit comprises one or more neoantigens or parts thereof.

35. The vector according to embodiment 34, wherein the antigenic units comprise one or more portions of one or more neoantigens.

36. The vector according to embodiment 35, wherein said moiety is a neoepitope.

37. The vector according to embodiment 36, wherein the antigenic unit comprises a plurality of neo-epitopes, e.g. a plurality of neo-epitopes separated from each other by a linker.

38. The vector according to any one of embodiments 36 to 37, wherein the antigenic unit comprises n-1 antigenic subunits and terminal neo-epitopes, each subunit comprising a neo-epitope and subunit linker, and wherein n is the number of neo-epitopes in the antigenic unit and n is an integer from 1 to 50.

39. The vector according to any one of embodiments 36 to 38, wherein the neoepitope has a length of 7 to 30 amino acids, e.g., 7 to 10 amino acids (e.g., 7, 8, 9, or 10 amino acids) or 13 to 30 amino acids (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids), e.g., 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

40. The vector according to any one of embodiments 34 to 35, wherein the antigenic unit further comprises a consensus cancer antigen or portion thereof present in one or more patients.

41. The vector according to embodiment 40, wherein the antigenic units further comprise one or more portions of a common cancer antigen present in one or more patients.

42. The vector according to embodiment 41, wherein said moiety is an epitope.

43. The vector according to any one of embodiments 40 to 41, wherein the antigenic unit further comprises a common cancer epitope present in a plurality of patients.

44. The vector according to any one of embodiments 40 to 43, wherein the patient has a consensus cancer antigen selected from the group consisting of an overexpressed or aberrantly expressed human cellular protein, a cancer testis antigen, a differentiation antigen, a viral antigen, a mutated oncogene, a mutated tumor suppressor gene, a carcinoembryonic antigen, a consensus intron retention antigen, a consensus antigen resulting from a frameshift mutation, a dark matter antigen, and a consensus antigen resulting from a splice mutation.

45. The vector according to any one of embodiments 1 to 33, wherein the antigenic units comprise one or more patient-present consensus cancer antigens or parts thereof.

46. The vector according to embodiment 45, wherein the antigenic units comprise one or more portions of a common cancer antigen present in one or more patients.

47. The vector according to embodiment 46, wherein said moiety is an epitope.

48. The vector according to embodiment 47, wherein said antigenic unit comprises a plurality of epitopes.

49. The vector of any one of embodiments 45-46, wherein the patient has a consensus cancer antigen selected from the group consisting of an overexpressed or aberrantly expressed human cellular protein, a cancer testis antigen, a differentiation antigen, a viral antigen, a mutated oncogene, a mutated tumor suppressor gene, a carcinoembryonic antigen, a consensus intron retention antigen, a consensus antigen resulting from a frameshift mutation, a dark matter antigen (DARK MATTER ANTIGEN), and a consensus antigen resulting from a splice mutation.

50. The vector according to any one of embodiments 1 to 33, wherein the antigenic units comprise one or more consensus cancer antigens or parts thereof.

51. The vector according to embodiment 50, wherein the antigenic units comprise one or more portions of one or more consensus cancer antigens.

52. The vector according to embodiment 51, wherein said moiety is an epitope.

53. The vector according to embodiment 52, wherein the antigenic unit comprises a plurality of epitopes.

54. The vector according to any one of embodiments 50 to 53, wherein the consensus cancer antigen is selected from the group consisting of an overexpressed or aberrantly expressed human cellular protein, a cancer testis antigen, a differentiation antigen, a viral antigen, a mutated oncogene, a mutated tumor suppressor gene, a carcinoembryonic antigen, a consensus intron retention antigen, a consensus antigen resulting from a frameshift mutation, a dark matter antigen, and a consensus antigen resulting from a splice mutation, a monoclonal Ig-derived scFv derived from a myeloma or lymphoma, a telomerase, an HIV antigen, a tyrosinase-related protein (TRP) -1, TRP-2, a melanoma antigen, a prostate-specific antigen, and an HPV antigen.

55. The vector according to any one of embodiments 1 to 33, wherein the antigenic unit comprises one or more infectious antigens or parts thereof.

56. The vector according to embodiment 55, wherein the antigenic units comprise one or more full-length infectious antigens or one or more portions of such full-length infectious antigens, or one or more full-length infectious antigens and one or more portions of such full-length infectious antigens.

57. The vector according to any one of embodiments 55 to 56, wherein said antigenic units comprise one or more portions of one or more infectious antigens.

58. The vector according to embodiment 57, wherein such moiety is a B cell epitope such that the antigenic unit comprises one or more B cell epitopes from one or more infectious antigens.

59. The vector according to embodiment 57, wherein such moiety is a T cell epitope such that the antigenic unit comprises one or more T cell epitopes from one or more infectious antigens.

60. The vector according to any one of embodiments 55 to 56, wherein the antigenic units comprise (i) one or more full length infectious antigens or one or more portions of such antigens, and (ii) one or more T cell epitopes from one or more infectious antigens.

61. The vector according to embodiment 60, wherein if more than one T cell epitope is contained in a subunit, the antigenic unit comprises a subunit containing one or more T cell epitopes separated from each other by a subunit linker; and wherein the subunits are linked to the multimerization unit by a first linker (e.g., a unit linker) and are separated from one or more full-length infectious antigens or portions of such antigens by a second linker.

62. The vector according to any one of embodiments 1 to 33, wherein the antigenic units comprise one or more antigens derived from one or more pathogens or parts of such antigens.

63. The vector according to embodiment 62, wherein the antigenic units comprise one or more full length antigens derived from one or more pathogens or one or more portions of such full length antigens or one or more full length antigens derived from one or more pathogens and one or more portions of such full length antigens.

64. The vector according to any one of embodiments 62 to 63, wherein said antigenic units comprise one or more portions of one or more antigens derived from one or more pathogens.

65. The vector according to embodiment 64, wherein such moiety is a B cell epitope such that the antigenic unit comprises one or more B cell epitopes derived from one or more pathogens.

66. The vector according to embodiment 64, wherein such moiety is a T cell epitope such that the antigenic unit comprises one or more T cell epitopes derived from one or more pathogens.

67. The vector according to any one of embodiments 62 to 63, wherein the antigenic units comprise (i) one or more full length antigens derived from one or more pathogens or one or more portions of such antigens and (ii) one or more T cell epitopes derived from one or more pathogens.

68. The vector according to embodiment 67, wherein if more than one T cell epitope is contained in a subunit, the antigenic unit comprises subunits containing one or more T cell epitopes separated from each other by subunit linkers; and wherein the subunits are linked to the multimerization unit by a first linker, e.g., a unit linker, and are separated from one or more full-length infectious antigens or portions of such antigens by a second linker.

69. The vector according to any one of embodiments 62 to 68, wherein said one or more pathogens are selected from the group consisting of viruses, bacteria, fungi and parasites.

70. The vector according to any one of embodiments 55 to 68, wherein the one or more antigens are selected from tuberculosis antigens, brucellosis antigens such as OMP31, HIV antigens such as gp120 derived sequences, HSV-2 antigens such as glycoprotein D, influenza virus antigens such as hemagglutinin, nucleoprotein and M2, HPV antigens such as E1, E2, E6, E7, L1 or L2, e.g. E6 and E7 of HPV16 or HPV18, CMV antigens, HBV antigens, coronavirus antigens such as SARS-CoV antigen, MERS-CoV antigen and SARS-CoV-2 antigen.

71. The vector according to any of the preceding embodiments, wherein the antigenic unit comprises up to 3500 amino acids, e.g. about 21 to about 2000 amino acids or about 60 to 3500 amino acids, e.g. about 80 or about 100 amino acids or about 150 amino acids to about 3000 amino acids, e.g. about 200 to about 2500 amino acids, e.g. about 300 to about 2000 amino acids or about 400 to about 1500 amino acids or about 500 to about 1000 amino acids.

72. The vector according to any of the preceding embodiments, wherein the targeting unit is or comprises a moiety that interacts with a surface molecule on an antigen presenting cell, preferably wherein the targeting unit is or comprises a moiety that interacts with a surface molecule on a human antigen presenting cell.

73. The vector according to embodiment 72, wherein the surface molecule is selected from the group consisting of MHC, CD14, CD40, CLEC9A, chemokine receptors such as CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 and XCR1 and Toll-like receptors such as TLR-2, TLR-4 or TLR-5, preferably wherein the surface molecule is selected from the group consisting of HLA, hCD14, hCD40, hCLEC a, human chemokine receptors such as hCCR1, hCCR3, hCCR4, hCCR5, hCCR6, hCCR7, hCCR8 and hXCR1 and Toll-like receptors such as hTLR-2, hTLR-4 or hTLR-5.

74. The vector according to any one of embodiments 72 and 73, wherein the targeting unit comprises or consists of: soluble CD40 ligand, preferably human soluble CD40 ligand, CCL4 and isoforms thereof, preferably human CCL4 and isoforms thereof, CCL5, preferably human CCL5, CCL19, preferably human CCL19, CCL20, preferably human CCL20, CCL21, preferably human CCL21, macrophage inflammatory protein α, including isoforms thereof, e.g., mouse CCL3, human CCL3L1, human CCL3L2 and human CCL3L3, XCL1, preferably human XCL1, XCL2, preferably human XCL2, flagellin, anti-HLA-DP, anti-HLA-DR, anti-pan-HLA class II, anti-CD 40, preferably anti-human CD40, anti-human CCL2, preferably anti-human CCL4, preferably anti-TLR 4, TLR-5, preferably anti-human-5, or anti-CLEC 9A, preferably anti-human CLEC9A.

75. The vector according to embodiment 74, wherein the targeting unit comprises or consists of human MIP-1α (ld78β, CCL3L 1).

76. The vector according to embodiment 75, wherein the targeting unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of 24-93 of SEQ ID NO.1, e.g. comprising the amino acid sequence of 26-93 of SEQ ID NO.1 or comprising the amino acid sequence of 28-93 of SEQ ID NO. 1.

77. The vector according to embodiment 76, wherein the targeting unit consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of 24-93 of SEQ ID NO. 1, e.g. consisting of the amino acid sequence of 26-93 of SEQ ID NO. 1 or consisting of the amino acid sequence of 28-93 of SEQ ID NO. 1.

78. The vector according to embodiment 77, wherein said targeting unit consists of the amino acid sequence of 24-93 of SEQ ID NO.1.

79. The vector according to any of the preceding embodiments, wherein the multimerization unit is selected from the group consisting of a dimerization unit, a trimerization unit, e.g. a collagen-derived trimerization unit, e.g. a human collagen-derived trimerization domain, e.g. a human collagen-derived XVIII trimerization domain or a human collagen XV trimerization domain or the C-terminal domain of T4 fibrin, and a tetramerization unit, e.g. a domain derived from p53, and wherein the multimerization unit optionally comprises a hinge region, e.g. hinge exon h1 and hinge exon h4.

80. The vector according to embodiment 79, wherein the vector comprises a hinge region capable of forming one or more covalent bonds.

81. The vector according to any one of embodiments 79 to 80, wherein said hinge region is Ig-derived.

82. The vector according to any one of embodiments 79 to 81, wherein the multimerization unit is a dimerization unit and the dimerization unit further comprises another domain that promotes dimerization.

83. The vector according to embodiment 82, wherein said another domain is an immunoglobulin domain, preferably an immunoglobulin constant domain.

84. The vector according to any one of embodiments 82 to 83, wherein the further domain is a carboxy-terminal C domain derived from IgG, preferably derived from IgG 3.

85. The vector according to any one of embodiments 82 to 84, wherein said dimerization unit further comprises a dimerization unit linker, such as a glycine-serine rich linker, e.g., GGGSSGGGSG (SEQ ID NO: 134).

86. The vector according to embodiment 85, wherein the dimerization unit linker connects the hinge region and another domain that promotes dimerization.

87. The vector according to any one of embodiments 82 to 86, wherein the dimerization unit comprises hinge exon h1 and hinge exon h4, a dimerization unit linker and a CH3 domain of human IgG 3.

88. The vector according to embodiment 87, wherein said dimerization unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of 94-237 of SEQ ID NO. 1.

89. The vector according to embodiment 88, wherein said dimerization unit consists of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of 94-237 of SEQ ID NO. 1.

90. The vector according to embodiment 89, wherein said dimerization unit consists of the amino acid sequence of 94-237 of SEQ ID NO. 1.

91. The vector according to any one of the preceding embodiments, wherein the first nucleic acid sequence encodes a first polypeptide further comprising a unit linker linking the antigenic unit to the multimerization unit, and wherein the unit linker is a non-immunogenic linker and/or a flexible or rigid linker.

92. The vector according to any one of the preceding embodiments, wherein the first nucleic acid sequence encodes a first polypeptide further comprising a signal peptide, preferably wherein the signal peptide is a native leader sequence of a protein as a targeting unit.

93. The vector according to embodiment 92, wherein the signal peptide is selected from the group consisting of an Ig VH signal peptide, a human TPA signal peptide, and a human MIP-1 a signal peptide.

94. The vector according to any one of embodiments 92 to 93, wherein the targeting unit is human MIP-1 a and the signal peptide comprises a sequence identical to SEQ ID NO: 1-23 having an amino acid sequence with at least 85% sequence identity.

95. The vector according to embodiment 94, wherein said signal peptide consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of 1-23 of SEQ ID NO. 1.

96. The vector according to embodiment 95, wherein said signal peptide consists of the amino acid sequence of 1-23 of SEQ ID NO. 1.

97. The vector according to any one of the preceding embodiments, wherein the one or more additional nucleic acid sequences further encode a signal peptide.

98. The vector according to embodiment 97, wherein said signal peptide is a natural leader sequence of an immunostimulatory compound.

99. The vector according to any one of the preceding embodiments, 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.

100. A method of producing a vector as defined in any one of embodiments 1 to 99, the method comprising:

a) Transfecting the cells in vitro with a vector;

b) Culturing the cells;

c) Optionally, lysing the cells to release the carrier from the cells; and

D) The vector is collected and optionally purified.

101. A host cell comprising a vector as defined in any one of embodiments 1 to 99, e.g. a host cell selected from a prokaryotic cell, a yeast cell, an insect cell, a higher eukaryotic cell, e.g. a cell from an animal or a human.

102. A carrier as defined in any one of embodiments 1 to 99 for use as a medicament.

103. A pharmaceutical composition comprising a carrier as defined in any one of embodiments 1 to 99 and a pharmaceutically acceptable carrier or diluent.

104. The pharmaceutical composition according to embodiment 103, wherein the pharmaceutically acceptable carrier or diluent is selected from the group consisting of saline, buffered saline such as PBS, dextrose, water, glycerol, ethanol, isotonic aqueous buffer, and Tyrode's buffer, and combinations thereof.

105. The pharmaceutical composition according to any one of embodiments 103 to 104, wherein the composition further comprises a transfection agent.

106. The composition according to any one of embodiments 103 to 105, wherein the composition further comprises a pharmaceutically acceptable amphiphilic block copolymer comprising poly (ethylene oxide) and poly (propylene oxide) blocks, e.g., further comprising a pharmaceutically acceptable amphiphilic block copolymer comprising poly (ethylene oxide) and poly (propylene oxide) blocks in an amount of 0.2% w/v to 20% w/v.

107. The pharmaceutical composition according to any one of embodiments 103 to 106, wherein the composition comprises 0.1 to 10mg of the vector, e.g., the DNA plasmid.

108. A method of treating a subject suffering from a disease or in need of prevention of the disease, the method comprising administering to the subject a vector as defined in any one of embodiments 1 to 99 or a pharmaceutical composition as defined in any one of embodiments 103 to 108.

109. The method according to embodiment 108, wherein the carrier or pharmaceutical composition is administered in a therapeutically or prophylactically effective amount.

110. The method of any one of embodiments 108 to 109, wherein the carrier or pharmaceutical composition is administered by intradermal, intramuscular or subcutaneous injection, or by mucosal or epithelial administration, e.g., intranasal or oral administration.

111. A method of treating a subject suffering from cancer, the method comprising administering to the subject a vector as defined in any one of embodiments 1 to 54 and 71 to 99 or a pharmaceutical composition comprising such a vector as defined in any one of embodiments 103 to 107.

112. The method according to embodiment 111, wherein the carrier or pharmaceutical composition is administered in a therapeutically effective amount.

113. The method according to any one of embodiments 111 to 112, wherein the carrier or pharmaceutical composition is administered by intradermal, intramuscular or subcutaneous injection, or by mucosal or epithelial administration, e.g. intranasal or oral administration.

114. The method according to any one of embodiments 111 to 113, wherein the cancer is a liquid cancer or a solid cancer, for example a cancer selected from 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, renal cancer, cholangiocarcinoma, brain cancer, cervical cancer, bladder cancer, esophageal cancer, hodgkin's disease, and adrenocortical cancer.

115. A method for treating a subject suffering from an infectious disease or in need of prevention of an infectious disease, the method comprising administering to the subject a vector as defined in any one of embodiments 1 to 33, 55 to 70 and 71 to 99 or a pharmaceutical composition comprising such a vector as defined in any one of embodiments 103 to 107.

116. The method according to embodiment 115, wherein the carrier or pharmaceutical composition is administered in a therapeutically or prophylactically effective amount.

117. The method according to any one of embodiments 116 to 116, wherein the carrier or pharmaceutical composition is administered by intradermal, intramuscular or subcutaneous injection, or by mucosal or epithelial administration, e.g. intranasal or oral administration.

Sequence listing

<110> Kaiko rule of virtue therapy Co., ltd (Nykode Therapeutics AS)

Co-expression of <120> constructs and immunostimulatory compounds

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Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

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Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

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Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

50 55 60

Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

65 70 75 80

Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

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Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr

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Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly

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Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

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Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

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Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

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Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

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Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

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Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

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Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

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Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

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Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

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Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

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Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

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Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr

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Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly

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Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

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Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

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Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

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Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

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Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

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Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

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Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly

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Gly Leu Lys Ser Trp Ile His Cys Trp Lys Tyr Leu Ser Val Gln Ser

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Gln Leu Phe Arg Gly Ser Ser Leu Leu Phe Arg Arg Val Gly Gly Gly

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Gly Ser Gly Gly Gly Gly Ser Asn Asn Leu Gln Lys Tyr Ile Glu Ile

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Tyr Val Gln Lys Ile Asn Pro Ser Arg Leu Pro Val Val Ile Gly Gly

290 295 300

Leu Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Ile Gln

305 310 315 320

Thr Ser Lys Tyr Tyr Met Arg Asp Val Ile Ala Ile Glu Ser Ala Trp

325 330 335

Leu Leu Glu Leu Ala Pro His Gly Gly Gly Gly Ser Gly Gly Gly Gly

340 345 350

Ser Val Ile Leu Pro Gln Ala Pro Ser Gly Pro Ser Tyr Ala Thr Tyr

355 360 365

Leu Gln Pro Ala Gln Ala Gln Met Leu Thr Pro Pro Gly Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Phe Val Ser Pro Met Ala His Tyr Val Pro

385 390 395 400

Gly Ile Met Ala Ile Glu Ser Val Val Ala Arg Phe Gln Phe Ile Val

405 410 415

Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asp Val Lys Ile

420 425 430

His Ala His Lys Val Val Leu Ala Asn Ile Ser Pro Tyr Phe Lys Ala

435 440 445

Met Phe Thr Gly Asn Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Thr Pro Leu Arg Lys His Thr Val His Ala Ile Arg Lys Phe Tyr Leu

465 470 475 480

Glu Phe Lys Gly Ser Ser Pro Pro Pro Arg Leu Gly Gly Gly Gly Ser

485 490 495

Gly Gly Gly Gly Ser Lys Ile Tyr Glu Phe Asp Tyr His Leu Tyr Gly

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Gln Asn Ile Thr Met Ile Met Thr Ser Val Ser Gly His Leu Leu Ala

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Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

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Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

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Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

35 40 45

Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

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Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

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Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

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Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr

100 105 110

Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

130 135 140

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

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Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

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Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly

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Gly Leu Lys Ser Trp Ile His Cys Trp Lys Tyr Leu Ser Val Gln Ser

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Gln Leu Phe Arg Gly Ser Ser Leu Leu Phe Arg Arg Val Gly Gly Gly

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Gly Ser Gly Gly Gly Gly Ser Asn Asn Leu Gln Lys Tyr Ile Glu Ile

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Tyr Val Gln Lys Ile Asn Pro Ser Arg Leu Pro Val Val Ile Gly Gly

290 295 300

Leu Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Ile Gln

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Thr Ser Lys Tyr Tyr Met Arg Asp Val Ile Ala Ile Glu Ser Ala Trp

325 330 335

Leu Leu Glu Leu Ala Pro His Gly Gly Gly Gly Ser Gly Gly Gly Gly

340 345 350

Ser Val Ile Leu Pro Gln Ala Pro Ser Gly Pro Ser Tyr Ala Thr Tyr

355 360 365

Leu Gln Pro Ala Gln Ala Gln Met Leu Thr Pro Pro Gly Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Phe Val Ser Pro Met Ala His Tyr Val Pro

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Gly Ile Met Ala Ile Glu Ser Val Val Ala Arg Phe Gln Phe Ile Val

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Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asp Val Lys Ile

420 425 430

His Ala His Lys Val Val Leu Ala Asn Ile Ser Pro Tyr Phe Lys Ala

435 440 445

Met Phe Thr Gly Asn Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Thr Pro Leu Arg Lys His Thr Val His Ala Ile Arg Lys Phe Tyr Leu

465 470 475 480

Glu Phe Lys Gly Ser Ser Pro Pro Pro Arg Leu Gly Gly Gly Gly Ser

485 490 495

Gly Gly Gly Gly Ser Lys Ile Tyr Glu Phe Asp Tyr His Leu Tyr Gly

500 505 510

Gln Asn Ile Thr Met Ile Met Thr Ser Val Ser Gly His Leu Leu Ala

515 520 525

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

530 535 540

Glu Asn Pro Gly Pro Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr

545 550 555 560

Thr Tyr Leu Leu Leu Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr

565 570 575

Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val

580 585 590

Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr

595 600 605

Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg

610 615 620

Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly

625 630 635 640

Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe

645 650 655

Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val

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Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val

675 680 685

Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu

690 695 700

Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro

705 710 715 720

Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln Pro

725 730

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<213> Artificial sequence

<220>

<223> VB4169

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Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

1 5 10 15

Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

35 40 45

Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

50 55 60

Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

65 70 75 80

Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr

100 105 110

Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

130 135 140

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

210 215 220

Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly

225 230 235 240

Gly Leu Lys Ser Trp Ile His Cys Trp Lys Tyr Leu Ser Val Gln Ser

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Gln Leu Phe Arg Gly Ser Ser Leu Leu Phe Arg Arg Val Gly Gly Gly

260 265 270

Gly Ser Gly Gly Gly Gly Ser Asn Asn Leu Gln Lys Tyr Ile Glu Ile

275 280 285

Tyr Val Gln Lys Ile Asn Pro Ser Arg Leu Pro Val Val Ile Gly Gly

290 295 300

Leu Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Ile Gln

305 310 315 320

Thr Ser Lys Tyr Tyr Met Arg Asp Val Ile Ala Ile Glu Ser Ala Trp

325 330 335

Leu Leu Glu Leu Ala Pro His Gly Gly Gly Gly Ser Gly Gly Gly Gly

340 345 350

Ser Val Ile Leu Pro Gln Ala Pro Ser Gly Pro Ser Tyr Ala Thr Tyr

355 360 365

Leu Gln Pro Ala Gln Ala Gln Met Leu Thr Pro Pro Gly Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Phe Val Ser Pro Met Ala His Tyr Val Pro

385 390 395 400

Gly Ile Met Ala Ile Glu Ser Val Val Ala Arg Phe Gln Phe Ile Val

405 410 415

Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asp Val Lys Ile

420 425 430

His Ala His Lys Val Val Leu Ala Asn Ile Ser Pro Tyr Phe Lys Ala

435 440 445

Met Phe Thr Gly Asn Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Thr Pro Leu Arg Lys His Thr Val His Ala Ile Arg Lys Phe Tyr Leu

465 470 475 480

Glu Phe Lys Gly Ser Ser Pro Pro Pro Arg Leu Gly Gly Gly Gly Ser

485 490 495

Gly Gly Gly Gly Ser Lys Ile Tyr Glu Phe Asp Tyr His Leu Tyr Gly

500 505 510

Gln Asn Ile Thr Met Ile Met Thr Ser Val Ser Gly His Leu Leu Ala

515 520 525

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

530 535 540

Glu Asn Pro Gly Pro Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr

545 550 555 560

Thr Tyr Leu Leu Leu Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr

565 570 575

Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val

580 585 590

Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr

595 600 605

Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg

610 615 620

Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly

625 630 635 640

Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe

645 650 655

Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val

660 665 670

Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val

675 680 685

Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu

690 695 700

Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro

705 710 715 720

Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln Pro Gly Ser Gly

725 730 735

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

740 745 750

Pro Gly Pro Met Trp Leu Gln Asn Leu Leu Phe Leu Gly Ile Val Val

755 760 765

Tyr Ser Leu Ser Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro

770 775 780

Trp Lys His Val Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp

785 790 795 800

Met Pro Val Thr Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe

805 810 815

Ser Phe Lys Lys Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu

820 825 830

Gln Gly Leu Arg Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met

835 840 845

Thr Ala Ser Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp

850 855 860

Cys Glu Thr Gln Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys

865 870 875 880

Thr Phe Leu Thr Asp Ile Pro Phe Glu Cys Lys Lys Pro Val Gln Lys

885 890 895

<210> 8

<211> 1010

<212> PRT

<213> Artificial sequence

<220>

<223> VB4170

<400> 8

Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

1 5 10 15

Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

35 40 45

Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

50 55 60

Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

65 70 75 80

Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr

100 105 110

Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

130 135 140

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

210 215 220

Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly

225 230 235 240

Gly Leu Lys Ser Trp Ile His Cys Trp Lys Tyr Leu Ser Val Gln Ser

245 250 255

Gln Leu Phe Arg Gly Ser Ser Leu Leu Phe Arg Arg Val Gly Gly Gly

260 265 270

Gly Ser Gly Gly Gly Gly Ser Asn Asn Leu Gln Lys Tyr Ile Glu Ile

275 280 285

Tyr Val Gln Lys Ile Asn Pro Ser Arg Leu Pro Val Val Ile Gly Gly

290 295 300

Leu Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Ile Gln

305 310 315 320

Thr Ser Lys Tyr Tyr Met Arg Asp Val Ile Ala Ile Glu Ser Ala Trp

325 330 335

Leu Leu Glu Leu Ala Pro His Gly Gly Gly Gly Ser Gly Gly Gly Gly

340 345 350

Ser Val Ile Leu Pro Gln Ala Pro Ser Gly Pro Ser Tyr Ala Thr Tyr

355 360 365

Leu Gln Pro Ala Gln Ala Gln Met Leu Thr Pro Pro Gly Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Phe Val Ser Pro Met Ala His Tyr Val Pro

385 390 395 400

Gly Ile Met Ala Ile Glu Ser Val Val Ala Arg Phe Gln Phe Ile Val

405 410 415

Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asp Val Lys Ile

420 425 430

His Ala His Lys Val Val Leu Ala Asn Ile Ser Pro Tyr Phe Lys Ala

435 440 445

Met Phe Thr Gly Asn Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Thr Pro Leu Arg Lys His Thr Val His Ala Ile Arg Lys Phe Tyr Leu

465 470 475 480

Glu Phe Lys Gly Ser Ser Pro Pro Pro Arg Leu Gly Gly Gly Gly Ser

485 490 495

Gly Gly Gly Gly Ser Lys Ile Tyr Glu Phe Asp Tyr His Leu Tyr Gly

500 505 510

Gln Asn Ile Thr Met Ile Met Thr Ser Val Ser Gly His Leu Leu Ala

515 520 525

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

530 535 540

Glu Asn Pro Gly Pro Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr

545 550 555 560

Thr Tyr Leu Leu Leu Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr

565 570 575

Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val

580 585 590

Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr

595 600 605

Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg

610 615 620

Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly

625 630 635 640

Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe

645 650 655

Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val

660 665 670

Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val

675 680 685

Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu

690 695 700

Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro

705 710 715 720

Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln Pro Gly Ser Gly

725 730 735

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

740 745 750

Pro Gly Pro Met Trp Leu Gln Asn Leu Leu Phe Leu Gly Ile Val Val

755 760 765

Tyr Ser Leu Ser Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro

770 775 780

Trp Lys His Val Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp

785 790 795 800

Met Pro Val Thr Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe

805 810 815

Ser Phe Lys Lys Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu

820 825 830

Gln Gly Leu Arg Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met

835 840 845

Thr Ala Ser Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp

850 855 860

Cys Glu Thr Gln Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys

865 870 875 880

Thr Phe Leu Thr Asp Ile Pro Phe Glu Cys Lys Lys Pro Val Gln Lys

885 890 895

Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp

900 905 910

Val Glu Ser Asn Pro Gly Pro Met Lys Ile Ser Ala Ala Ala Leu Thr

915 920 925

Ile Ile Leu Thr Ala Ala Ala Leu Cys Thr Pro Ala Pro Ala Ser Pro

930 935 940

Tyr Gly Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Leu Ser Leu Ala

945 950 955 960

Leu Pro Arg Ala His Val Lys Glu Tyr Phe Tyr Thr Ser Ser Lys Cys

965 970 975

Ser Asn Leu Ala Val Val Phe Val Thr Arg Arg Asn Arg Gln Val Cys

980 985 990

Ala Asn Pro Glu Lys Lys Trp Val Gln Glu Tyr Ile Asn Tyr Leu Glu

995 1000 1005

Met Ser

1010

<210> 9

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Self-cleaving peptides

<400> 9

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro

<210> 10

<211> 158

<212> PRT

<213> Person (Homo sapiens)

<400> 10

Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala

1 5 10 15

Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val

20 25 30

Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp

35 40 45

Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala

50 55 60

Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His

65 70 75 80

Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe

85 90 95

Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu

100 105 110

Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu

115 120 125

Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser

130 135 140

Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln Pro

145 150 155

<210> 11

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Self-cleaving peptides

<400> 11

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210> 12

<211> 17

<212> PRT

<213> Mouse (Mus Musculus)

<400> 12

Met Trp Leu Gln Asn Leu Leu Phe Leu Gly Ile Val Val Tyr Ser Leu

1 5 10 15

Ser

<210> 13

<211> 124

<212> PRT

<213> Mouse (Mus Musculus)

<400> 13

Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp Lys His Val

1 5 10 15

Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp Met Pro Val Thr

20 25 30

Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe Ser Phe Lys Lys

35 40 45

Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu Gln Gly Leu Arg

50 55 60

Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met Thr Ala Ser Tyr

65 70 75 80

Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp Cys Glu Thr Gln

85 90 95

Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys Thr Phe Leu Thr

100 105 110

Asp Ile Pro Phe Glu Cys Lys Lys Pro Val Gln Lys

115 120

<210> 14

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Self-cleaving peptides

<400> 14

Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

1 5 10 15

Asn Pro Gly Pro

20

<210> 15

<211> 23

<212> PRT

<213> Mouse (Mus Musculus)

<400> 15

Met Lys Ile Ser Ala Ala Ala Leu Thr Ile Ile Leu Thr Ala Ala Ala

1 5 10 15

Leu Cys Thr Pro Ala Pro Ala

20

<210> 16

<211> 68

<212> PRT

<213> Mouse (Mus Musculus)

<400> 16

Ser Pro Tyr Gly Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Leu Ser

1 5 10 15

Leu Ala Leu Pro Arg Ala His Val Lys Glu Tyr Phe Tyr Thr Ser Ser

20 25 30

Lys Cys Ser Asn Leu Ala Val Val Phe Val Thr Arg Arg Asn Arg Gln

35 40 45

Val Cys Ala Asn Pro Glu Lys Lys Trp Val Gln Glu Tyr Ile Asn Tyr

50 55 60

Leu Glu Met Ser

65

<210> 17

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 17

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 18

<211> 690

<212> PRT

<213> Artificial sequence

<220>

<223> VB4202

<400> 18

Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

1 5 10 15

Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

35 40 45

Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

50 55 60

Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

65 70 75 80

Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr

100 105 110

Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

130 135 140

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

210 215 220

Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly

225 230 235 240

Gly Leu Lys Ser Trp Ile His Cys Trp Lys Tyr Leu Ser Val Gln Ser

245 250 255

Gln Leu Phe Arg Gly Ser Ser Leu Leu Phe Arg Arg Val Gly Gly Gly

260 265 270

Gly Ser Gly Gly Gly Gly Ser Asn Asn Leu Gln Lys Tyr Ile Glu Ile

275 280 285

Tyr Val Gln Lys Ile Asn Pro Ser Arg Leu Pro Val Val Ile Gly Gly

290 295 300

Leu Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Ile Gln

305 310 315 320

Thr Ser Lys Tyr Tyr Met Arg Asp Val Ile Ala Ile Glu Ser Ala Trp

325 330 335

Leu Leu Glu Leu Ala Pro His Gly Gly Gly Gly Ser Gly Gly Gly Gly

340 345 350

Ser Val Ile Leu Pro Gln Ala Pro Ser Gly Pro Ser Tyr Ala Thr Tyr

355 360 365

Leu Gln Pro Ala Gln Ala Gln Met Leu Thr Pro Pro Gly Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Phe Val Ser Pro Met Ala His Tyr Val Pro

385 390 395 400

Gly Ile Met Ala Ile Glu Ser Val Val Ala Arg Phe Gln Phe Ile Val

405 410 415

Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asp Val Lys Ile

420 425 430

His Ala His Lys Val Val Leu Ala Asn Ile Ser Pro Tyr Phe Lys Ala

435 440 445

Met Phe Thr Gly Asn Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Thr Pro Leu Arg Lys His Thr Val His Ala Ile Arg Lys Phe Tyr Leu

465 470 475 480

Glu Phe Lys Gly Ser Ser Pro Pro Pro Arg Leu Gly Gly Gly Gly Ser

485 490 495

Gly Gly Gly Gly Ser Lys Ile Tyr Glu Phe Asp Tyr His Leu Tyr Gly

500 505 510

Gln Asn Ile Thr Met Ile Met Thr Ser Val Ser Gly His Leu Leu Ala

515 520 525

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

530 535 540

Glu Asn Pro Gly Pro Met Trp Leu Gln Asn Leu Leu Phe Leu Gly Ile

545 550 555 560

Val Val Tyr Ser Leu Ser Ala Pro Thr Arg Ser Pro Ile Thr Val Thr

565 570 575

Arg Pro Trp Lys His Val Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu

580 585 590

Asp Asp Met Pro Val Thr Leu Asn Glu Glu Val Glu Val Val Ser Asn

595 600 605

Glu Phe Ser Phe Lys Lys Leu Thr Cys Val Gln Thr Arg Leu Lys Ile

610 615 620

Phe Glu Gln Gly Leu Arg Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu

625 630 635 640

Asn Met Thr Ala Ser Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu

645 650 655

Thr Asp Cys Glu Thr Gln Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser

660 665 670

Leu Lys Thr Phe Leu Thr Asp Ile Pro Phe Glu Cys Lys Lys Pro Val

675 680 685

Gln Lys

690

<210> 19

<211> 501

<212> PRT

<213> Artificial sequence

<220>

<223> VB1020

<400> 19

Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

1 5 10 15

Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

35 40 45

Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

50 55 60

Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

65 70 75 80

Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr

100 105 110

Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

130 135 140

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

210 215 220

Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly

225 230 235 240

Gly Leu Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp

245 250 255

Leu Gln Pro Glu Thr Thr Asp Leu Tyr Gly Tyr Gly Gln Leu Asn Asp

260 265 270

Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu

275 280 285

Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp

290 295 300

Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr

305 310 315 320

Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys

325 330 335

Ser Gln Lys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Met Phe

340 345 350

Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Leu Cys Thr Glu

355 360 365

Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val Tyr Cys Lys

370 375 380

Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Arg Arg Asp Leu

385 390 395 400

Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Arg Asp Lys Cys

405 410 415

Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser

420 425 430

Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp

435 440 445

Leu Leu Ile Arg Cys Ile Asn Arg Gln Lys Pro Leu Cys Pro Glu Glu

450 455 460

Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gly

465 470 475 480

Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser Arg Thr Arg

485 490 495

Arg Glu Thr Gln Leu

500

<210> 20

<211> 706

<212> PRT

<213> Artificial sequence

<220>

<223> VB4195

<400> 20

Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

1 5 10 15

Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

35 40 45

Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

50 55 60

Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

65 70 75 80

Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr

100 105 110

Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

130 135 140

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

210 215 220

Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly

225 230 235 240

Gly Leu Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp

245 250 255

Leu Gln Pro Glu Thr Thr Asp Leu Tyr Gly Tyr Gly Gln Leu Asn Asp

260 265 270

Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu

275 280 285

Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp

290 295 300

Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr

305 310 315 320

Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys

325 330 335

Ser Gln Lys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Met Phe

340 345 350

Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Leu Cys Thr Glu

355 360 365

Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val Tyr Cys Lys

370 375 380

Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Arg Arg Asp Leu

385 390 395 400

Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Arg Asp Lys Cys

405 410 415

Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser

420 425 430

Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp

435 440 445

Leu Leu Ile Arg Cys Ile Asn Arg Gln Lys Pro Leu Cys Pro Glu Glu

450 455 460

Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gly

465 470 475 480

Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser Arg Thr Arg

485 490 495

Arg Glu Thr Gln Leu Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr

500 505 510

Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Thr Val Leu Ala Pro

515 520 525

Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu Leu Leu Leu Leu Ser Ser

530 535 540

Gly Leu Ser Gly Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser

545 550 555 560

Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln

565 570 575

Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys

580 585 590

Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu

595 600 605

Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn

610 615 620

Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser

625 630 635 640

Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr

645 650 655

Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe

660 665 670

Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro

675 680 685

Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro

690 695 700

Gln Pro

705

<210> 21

<211> 869

<212> PRT

<213> Artificial sequence

<220>

<223> VB4196

<400> 21

Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

1 5 10 15

Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

35 40 45

Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

50 55 60

Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

65 70 75 80

Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr

100 105 110

Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

130 135 140

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

210 215 220

Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly

225 230 235 240

Gly Leu Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp

245 250 255

Leu Gln Pro Glu Thr Thr Asp Leu Tyr Gly Tyr Gly Gln Leu Asn Asp

260 265 270

Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu

275 280 285

Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp

290 295 300

Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr

305 310 315 320

Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys

325 330 335

Ser Gln Lys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Met Phe

340 345 350

Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Leu Cys Thr Glu

355 360 365

Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val Tyr Cys Lys

370 375 380

Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Arg Arg Asp Leu

385 390 395 400

Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Arg Asp Lys Cys

405 410 415

Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser

420 425 430

Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp

435 440 445

Leu Leu Ile Arg Cys Ile Asn Arg Gln Lys Pro Leu Cys Pro Glu Glu

450 455 460

Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gly

465 470 475 480

Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser Arg Thr Arg

485 490 495

Arg Glu Thr Gln Leu Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr

500 505 510

Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Thr Val Leu Ala Pro

515 520 525

Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu Leu Leu Leu Leu Ser Ser

530 535 540

Gly Leu Ser Gly Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser

545 550 555 560

Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln

565 570 575

Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys

580 585 590

Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu

595 600 605

Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn

610 615 620

Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser

625 630 635 640

Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr

645 650 655

Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe

660 665 670

Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro

675 680 685

Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro

690 695 700

Gln Pro Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly

705 710 715 720

Asp Val Glu Glu Asn Pro Gly Pro Met Trp Leu Gln Asn Leu Leu Phe

725 730 735

Leu Gly Ile Val Val Tyr Ser Leu Ser Ala Pro Thr Arg Ser Pro Ile

740 745 750

Thr Val Thr Arg Pro Trp Lys His Val Glu Ala Ile Lys Glu Ala Leu

755 760 765

Asn Leu Leu Asp Asp Met Pro Val Thr Leu Asn Glu Glu Val Glu Val

770 775 780

Val Ser Asn Glu Phe Ser Phe Lys Lys Leu Thr Cys Val Gln Thr Arg

785 790 795 800

Leu Lys Ile Phe Glu Gln Gly Leu Arg Gly Asn Phe Thr Lys Leu Lys

805 810 815

Gly Ala Leu Asn Met Thr Ala Ser Tyr Tyr Gln Thr Tyr Cys Pro Pro

820 825 830

Thr Pro Glu Thr Asp Cys Glu Thr Gln Val Thr Thr Tyr Ala Asp Phe

835 840 845

Ile Asp Ser Leu Lys Thr Phe Leu Thr Asp Ile Pro Phe Glu Cys Lys

850 855 860

Lys Pro Val Gln Lys

865

<210> 22

<211> 663

<212> PRT

<213> Artificial sequence

<220>

<223> VB4204

<400> 22

Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

1 5 10 15

Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

35 40 45

Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

50 55 60

Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

65 70 75 80

Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr

100 105 110

Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

130 135 140

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

210 215 220

Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly

225 230 235 240

Gly Leu Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp

245 250 255

Leu Gln Pro Glu Thr Thr Asp Leu Tyr Gly Tyr Gly Gln Leu Asn Asp

260 265 270

Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu

275 280 285

Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp

290 295 300

Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr

305 310 315 320

Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys

325 330 335

Ser Gln Lys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Met Phe

340 345 350

Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Leu Cys Thr Glu

355 360 365

Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val Tyr Cys Lys

370 375 380

Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Arg Arg Asp Leu

385 390 395 400

Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Arg Asp Lys Cys

405 410 415

Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser

420 425 430

Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp

435 440 445

Leu Leu Ile Arg Cys Ile Asn Arg Gln Lys Pro Leu Cys Pro Glu Glu

450 455 460

Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gly

465 470 475 480

Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser Arg Thr Arg

485 490 495

Arg Glu Thr Gln Leu Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr

500 505 510

Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Trp Leu Gln Asn Leu

515 520 525

Leu Phe Leu Gly Ile Val Val Tyr Ser Leu Ser Ala Pro Thr Arg Ser

530 535 540

Pro Ile Thr Val Thr Arg Pro Trp Lys His Val Glu Ala Ile Lys Glu

545 550 555 560

Ala Leu Asn Leu Leu Asp Asp Met Pro Val Thr Leu Asn Glu Glu Val

565 570 575

Glu Val Val Ser Asn Glu Phe Ser Phe Lys Lys Leu Thr Cys Val Gln

580 585 590

Thr Arg Leu Lys Ile Phe Glu Gln Gly Leu Arg Gly Asn Phe Thr Lys

595 600 605

Leu Lys Gly Ala Leu Asn Met Thr Ala Ser Tyr Tyr Gln Thr Tyr Cys

610 615 620

Pro Pro Thr Pro Glu Thr Asp Cys Glu Thr Gln Val Thr Thr Tyr Ala

625 630 635 640

Asp Phe Ile Asp Ser Leu Lys Thr Phe Leu Thr Asp Ile Pro Phe Glu

645 650 655

Cys Lys Lys Pro Val Gln Lys

660

<210> 23

<211> 613

<212> PRT

<213> Artificial sequence

<220>

<223> VB4205

<400> 23

Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

1 5 10 15

Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

35 40 45

Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

50 55 60

Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

65 70 75 80

Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr

100 105 110

Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

130 135 140

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

210 215 220

Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly

225 230 235 240

Gly Leu Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp

245 250 255

Leu Gln Pro Glu Thr Thr Asp Leu Tyr Gly Tyr Gly Gln Leu Asn Asp

260 265 270

Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu

275 280 285

Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp

290 295 300

Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr

305 310 315 320

Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys

325 330 335

Ser Gln Lys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Met Phe

340 345 350

Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Leu Cys Thr Glu

355 360 365

Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val Tyr Cys Lys

370 375 380

Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Arg Arg Asp Leu

385 390 395 400

Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Arg Asp Lys Cys

405 410 415

Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser

420 425 430

Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp

435 440 445

Leu Leu Ile Arg Cys Ile Asn Arg Gln Lys Pro Leu Cys Pro Glu Glu

450 455 460

Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gly

465 470 475 480

Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser Arg Thr Arg

485 490 495

Arg Glu Thr Gln Leu Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr

500 505 510

Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Lys Ile Ser Ala Ala

515 520 525

Ala Leu Thr Ile Ile Leu Thr Ala Ala Ala Leu Cys Thr Pro Ala Pro

530 535 540

Ala Ser Pro Tyr Gly Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Leu

545 550 555 560

Ser Leu Ala Leu Pro Arg Ala His Val Lys Glu Tyr Phe Tyr Thr Ser

565 570 575

Ser Lys Cys Ser Asn Leu Ala Val Val Phe Val Thr Arg Arg Asn Arg

580 585 590

Gln Val Cys Ala Asn Pro Glu Lys Lys Trp Val Gln Glu Tyr Ile Asn

595 600 605

Tyr Leu Glu Met Ser

610

<210> 24

<211> 404

<212> PRT

<213> Artificial sequence

<220>

<223> VB4208

<400> 24

Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

1 5 10 15

Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

35 40 45

Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

50 55 60

Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

65 70 75 80

Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr

100 105 110

Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

130 135 140

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

210 215 220

Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly

225 230 235 240

Gly Leu Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

245 250 255

Val Glu Glu Asn Pro Gly Pro Met Trp Leu Gln Asn Leu Leu Phe Leu

260 265 270

Gly Ile Val Val Tyr Ser Leu Ser Ala Pro Thr Arg Ser Pro Ile Thr

275 280 285

Val Thr Arg Pro Trp Lys His Val Glu Ala Ile Lys Glu Ala Leu Asn

290 295 300

Leu Leu Asp Asp Met Pro Val Thr Leu Asn Glu Glu Val Glu Val Val

305 310 315 320

Ser Asn Glu Phe Ser Phe Lys Lys Leu Thr Cys Val Gln Thr Arg Leu

325 330 335

Lys Ile Phe Glu Gln Gly Leu Arg Gly Asn Phe Thr Lys Leu Lys Gly

340 345 350

Ala Leu Asn Met Thr Ala Ser Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr

355 360 365

Pro Glu Thr Asp Cys Glu Thr Gln Val Thr Thr Tyr Ala Asp Phe Ile

370 375 380

Asp Ser Leu Lys Thr Phe Leu Thr Asp Ile Pro Phe Glu Cys Lys Lys

385 390 395 400

Pro Val Gln Lys

<210> 25

<211> 210

<212> DNA

<213> Artificial sequence

<220>

<223> Coding part of SEQ ID NO 1

<400> 25

gcaccacttg ctgctgacac gccgaccgcc tgctgcttca gctacacctc ccgacagatt 60

ccacagaatt tcatagctga ctactttgag acgagcagcc agtgctccaa gcccagtgtc 120

atcttcctaa ccaagagagg ccggcaggtc tgtgctgacc ccagtgagga gtgggtccag 180

aaatacgtca gtgacctgga gctgagtgcc 210

<210> 26

<211> 81

<212> DNA

<213> Artificial sequence

<220>

<223> Coding part of SEQ ID NO 1

<400> 26

gagctcaaaa ccccacttgg tgacacaact cacacagagc ccaaatcttg tgacacacct 60

cccccgtgcc caaggtgccc a 81

<210> 27

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Coding part of SEQ ID NO 1

<400> 27

ggacagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 60

aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 120

tgggagagca gcgggcagcc ggagaacaac tacaacacca cgcctcccat gctggactcc 180

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 240

aacatcttct catgctccgt gatgcatgag gctctgcaca accgcttcac gcagaagagc 300

ctctccctgt ctccgggtaa a 321

<210> 28

<211> 432

<212> DNA

<213> Artificial sequence

<220>

<223> Coding part of SEQ ID NO 1

<400> 28

gagctcaaaa ccccacttgg tgacacaact cacacagagc ccaaatcttg tgacacacct 60

cccccgtgcc caaggtgccc aggcggtgga agcagcggag gtggaagtgg aggacagccc 120

cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc 180

agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 240

agcgggcagc cggagaacaa ctacaacacc acgcctccca tgctggactc cgacggctcc 300

ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacatcttc 360

tcatgctccg tgatgcatga ggctctgcac aaccgcttca cgcagaagag cctctccctg 420

tctccgggta aa 432

<210> 29

<211> 69

<212> DNA

<213> Artificial sequence

<220>

<223> Coding part of SEQ ID NO 1

<400> 29

atgcaggtct ccactgctgc ccttgccgtc ctcctctgca ccatggctct ctgcaaccag 60

gtcctctct 69

<210> 30

<211> 223

<212> PRT

<213> Artificial sequence

<220>

<223> SARS-COV-2 RBD (amino acids 319-542)

<400> 30

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe

210 215 220

<210> 31

<211> 627

<212> PRT

<213> Artificial sequence

<220>

<223> TECH001-CV021

<400> 31

Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

1 5 10 15

Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

35 40 45

Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

50 55 60

Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

65 70 75 80

Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr

100 105 110

Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

130 135 140

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

210 215 220

Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly

225 230 235 240

Gly Leu Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile

245 250 255

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala

260 265 270

Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp

275 280 285

Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr

290 295 300

Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr

305 310 315 320

Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro

325 330 335

Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

340 345 350

Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys

355 360 365

Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn

370 375 380

Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly

385 390 395 400

Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu

405 410 415

Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr

420 425 430

Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val

435 440 445

Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn

450 455 460

Phe Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val

465 470 475 480

Glu Glu Asn Pro Gly Pro Met Trp Leu Gln Asn Leu Leu Phe Leu Gly

485 490 495

Ile Val Val Tyr Ser Leu Ser Ala Pro Thr Arg Ser Pro Ile Thr Val

500 505 510

Thr Arg Pro Trp Lys His Val Glu Ala Ile Lys Glu Ala Leu Asn Leu

515 520 525

Leu Asp Asp Met Pro Val Thr Leu Asn Glu Glu Val Glu Val Val Ser

530 535 540

Asn Glu Phe Ser Phe Lys Lys Leu Thr Cys Val Gln Thr Arg Leu Lys

545 550 555 560

Ile Phe Glu Gln Gly Leu Arg Gly Asn Phe Thr Lys Leu Lys Gly Ala

565 570 575

Leu Asn Met Thr Ala Ser Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr Pro

580 585 590

Glu Thr Asp Cys Glu Thr Gln Val Thr Thr Tyr Ala Asp Phe Ile Asp

595 600 605

Ser Leu Lys Thr Phe Leu Thr Asp Ile Pro Phe Glu Cys Lys Lys Pro

610 615 620

Val Gln Lys

625

<210> 32

<211> 1054

<212> PRT

<213> Artificial sequence

<220>

<223> TECH001-CV022

<400> 32

Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

1 5 10 15

Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

35 40 45

Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

50 55 60

Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

65 70 75 80

Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr

100 105 110

Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

130 135 140

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

210 215 220

Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly

225 230 235 240

Gly Leu Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile

245 250 255

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala

260 265 270

Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp

275 280 285

Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr

290 295 300

Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr

305 310 315 320

Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro

325 330 335

Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

340 345 350

Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys

355 360 365

Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn

370 375 380

Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly

385 390 395 400

Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu

405 410 415

Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr

420 425 430

Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val

435 440 445

Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn

450 455 460

Phe Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val

465 470 475 480

Glu Glu Asn Pro Gly Pro Met Cys Gln Ser Arg Tyr Leu Leu Phe Leu

485 490 495

Ala Thr Leu Ala Leu Leu Asn His Leu Ser Leu Ala Arg Val Ile Pro

500 505 510

Val Ser Gly Pro Ala Arg Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys

515 520 525

Thr Thr Asp Asp Met Val Lys Thr Ala Arg Glu Lys Leu Lys His Tyr

530 535 540

Ser Cys Thr Ala Glu Asp Ile Asp His Glu Asp Ile Thr Arg Asp Gln

545 550 555 560

Thr Ser Thr Leu Lys Thr Cys Leu Pro Leu Glu Leu His Lys Asn Glu

565 570 575

Ser Cys Leu Ala Thr Arg Glu Thr Ser Ser Thr Thr Arg Gly Ser Cys

580 585 590

Leu Pro Pro Gln Lys Thr Ser Leu Met Met Thr Leu Cys Leu Gly Ser

595 600 605

Ile Tyr Glu Asp Leu Lys Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn

610 615 620

Ala Ala Leu Gln Asn His Asn His Gln Gln Ile Ile Leu Asp Lys Gly

625 630 635 640

Met Leu Val Ala Ile Asp Glu Leu Met Gln Ser Leu Asn His Asn Gly

645 650 655

Glu Thr Leu Arg Gln Lys Pro Pro Val Gly Glu Ala Asp Pro Tyr Arg

660 665 670

Val Lys Met Lys Leu Cys Ile Leu Leu His Ala Phe Ser Thr Arg Val

675 680 685

Val Thr Ile Asn Arg Val Met Gly Tyr Leu Ser Ser Ala Glu Gly Arg

690 695 700

Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met

705 710 715 720

Cys Pro Gln Lys Leu Thr Ile Ser Trp Phe Ala Ile Val Leu Leu Val

725 730 735

Ser Pro Leu Met Ala Met Trp Glu Leu Glu Lys Asp Val Tyr Val Val

740 745 750

Glu Val Asp Trp Thr Pro Asp Ala Pro Gly Glu Thr Val Asn Leu Thr

755 760 765

Cys Asp Thr Pro Glu Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln Arg

770 775 780

His Gly Val Ile Gly Ser Gly Lys Thr Leu Thr Ile Thr Val Lys Glu

785 790 795 800

Phe Leu Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Thr Leu

805 810 815

Ser His Ser His Leu Leu Leu His Lys Lys Glu Asn Gly Ile Trp Ser

820 825 830

Thr Glu Ile Leu Lys Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys Glu

835 840 845

Ala Pro Asn Tyr Ser Gly Arg Phe Thr Cys Ser Trp Leu Val Gln Arg

850 855 860

Asn Met Asp Leu Lys Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro Asp

865 870 875 880

Ser Arg Ala Val Thr Cys Gly Met Ala Ser Leu Ser Ala Glu Lys Val

885 890 895

Thr Leu Asp Gln Arg Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln Glu

900 905 910

Asp Val Thr Cys Pro Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu Ala

915 920 925

Leu Glu Ala Arg Gln Gln Asn Lys Tyr Glu Asn Tyr Ser Thr Ser Phe

930 935 940

Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Met

945 950 955 960

Lys Pro Leu Lys Asn Ser Gln Val Glu Val Ser Trp Glu Tyr Pro Asp

965 970 975

Ser Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Lys Phe Phe Val Arg

980 985 990

Ile Gln Arg Lys Lys Glu Lys Met Lys Glu Thr Glu Glu Gly Cys Asn

995 1000 1005

Gln Lys Gly Ala Phe Leu Val Glu Lys Thr Ser Thr Glu Val Gln

1010 1015 1020

Cys Lys Gly Gly Asn Val Cys Val Gln Ala Gln Asp Arg Tyr Tyr

1025 1030 1035

Asn Ser Ser Cys Ser Lys Trp Ala Cys Val Pro Cys Arg Val Arg

1040 1045 1050

Ser

<210> 33

<211> 632

<212> PRT

<213> Artificial sequence

<220>

<223> TECH001-CV023

<400> 33

Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

1 5 10 15

Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

35 40 45

Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

50 55 60

Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

65 70 75 80

Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr

100 105 110

Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

130 135 140

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

210 215 220

Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly

225 230 235 240

Gly Leu Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile

245 250 255

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala

260 265 270

Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp

275 280 285

Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr

290 295 300

Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr

305 310 315 320

Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro

325 330 335

Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

340 345 350

Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys

355 360 365

Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn

370 375 380

Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly

385 390 395 400

Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu

405 410 415

Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr

420 425 430

Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val

435 440 445

Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn

450 455 460

Phe Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val

465 470 475 480

Glu Glu Asn Pro Gly Pro Met Glu Arg Thr Leu Val Cys Leu Val Val

485 490 495

Ile Phe Leu Gly Thr Val Ala His Lys Ser Ser Pro Gln Gly Pro Asp

500 505 510

Arg Leu Leu Ile Arg Leu Arg His Leu Ile Asp Ile Val Glu Gln Leu

515 520 525

Lys Ile Tyr Glu Asn Asp Leu Asp Pro Glu Leu Leu Ser Ala Pro Gln

530 535 540

Asp Val Lys Gly His Cys Glu His Ala Ala Phe Ala Cys Phe Gln Lys

545 550 555 560

Ala Lys Leu Lys Pro Ser Asn Pro Gly Asn Asn Lys Thr Phe Ile Ile

565 570 575

Asp Leu Val Ala Gln Leu Arg Arg Arg Leu Pro Ala Arg Arg Gly Gly

580 585 590

Lys Lys Gln Lys His Ile Ala Lys Cys Pro Ser Cys Asp Ser Tyr Glu

595 600 605

Lys Arg Thr Pro Lys Glu Phe Leu Glu Arg Leu Lys Trp Leu Leu Gln

610 615 620

Lys Met Ile His Gln His Leu Ser

625 630

<210> 34

<211> 22

<212> PRT

<213> Mouse (Mus Musculus)

<400> 34

Met Cys Gln Ser Arg Tyr Leu Leu Phe Leu Ala Thr Leu Ala Leu Leu

1 5 10 15

Asn His Leu Ser Leu Ala

20

<210> 35

<211> 193

<212> PRT

<213> Mouse (Mus Musculus)

<400> 35

Arg Val Ile Pro Val Ser Gly Pro Ala Arg Cys Leu Ser Gln Ser Arg

1 5 10 15

Asn Leu Leu Lys Thr Thr Asp Asp Met Val Lys Thr Ala Arg Glu Lys

20 25 30

Leu Lys His Tyr Ser Cys Thr Ala Glu Asp Ile Asp His Glu Asp Ile

35 40 45

Thr Arg Asp Gln Thr Ser Thr Leu Lys Thr Cys Leu Pro Leu Glu Leu

50 55 60

His Lys Asn Glu Ser Cys Leu Ala Thr Arg Glu Thr Ser Ser Thr Thr

65 70 75 80

Arg Gly Ser Cys Leu Pro Pro Gln Lys Thr Ser Leu Met Met Thr Leu

85 90 95

Cys Leu Gly Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Thr Glu Phe

100 105 110

Gln Ala Ile Asn Ala Ala Leu Gln Asn His Asn His Gln Gln Ile Ile

115 120 125

Leu Asp Lys Gly Met Leu Val Ala Ile Asp Glu Leu Met Gln Ser Leu

130 135 140

Asn His Asn Gly Glu Thr Leu Arg Gln Lys Pro Pro Val Gly Glu Ala

145 150 155 160

Asp Pro Tyr Arg Val Lys Met Lys Leu Cys Ile Leu Leu His Ala Phe

165 170 175

Ser Thr Arg Val Val Thr Ile Asn Arg Val Met Gly Tyr Leu Ser Ser

180 185 190

Ala

<210> 36

<211> 22

<212> PRT

<213> Mouse (Mus Musculus)

<400> 36

Met Cys Pro Gln Lys Leu Thr Ile Ser Trp Phe Ala Ile Val Leu Leu

1 5 10 15

Val Ser Pro Leu Met Ala

20

<210> 37

<211> 313

<212> PRT

<213> Mouse (Mus Musculus)

<400> 37

Met Trp Glu Leu Glu Lys Asp Val Tyr Val Val Glu Val Asp Trp Thr

1 5 10 15

Pro Asp Ala Pro Gly Glu Thr Val Asn Leu Thr Cys Asp Thr Pro Glu

20 25 30

Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln Arg His Gly Val Ile Gly

35 40 45

Ser Gly Lys Thr Leu Thr Ile Thr Val Lys Glu Phe Leu Asp Ala Gly

50 55 60

Gln Tyr Thr Cys His Lys Gly Gly Glu Thr Leu Ser His Ser His Leu

65 70 75 80

Leu Leu His Lys Lys Glu Asn Gly Ile Trp Ser Thr Glu Ile Leu Lys

85 90 95

Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys Glu Ala Pro Asn Tyr Ser

100 105 110

Gly Arg Phe Thr Cys Ser Trp Leu Val Gln Arg Asn Met Asp Leu Lys

115 120 125

Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro Asp Ser Arg Ala Val Thr

130 135 140

Cys Gly Met Ala Ser Leu Ser Ala Glu Lys Val Thr Leu Asp Gln Arg

145 150 155 160

Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln Glu Asp Val Thr Cys Pro

165 170 175

Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu Ala Leu Glu Ala Arg Gln

180 185 190

Gln Asn Lys Tyr Glu Asn Tyr Ser Thr Ser Phe Phe Ile Arg Asp Ile

195 200 205

Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Met Lys Pro Leu Lys Asn

210 215 220

Ser Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Ser Trp Ser Thr Pro

225 230 235 240

His Ser Tyr Phe Ser Leu Lys Phe Phe Val Arg Ile Gln Arg Lys Lys

245 250 255

Glu Lys Met Lys Glu Thr Glu Glu Gly Cys Asn Gln Lys Gly Ala Phe

260 265 270

Leu Val Glu Lys Thr Ser Thr Glu Val Gln Cys Lys Gly Gly Asn Val

275 280 285

Cys Val Gln Ala Gln Asp Arg Tyr Tyr Asn Ser Ser Cys Ser Lys Trp

290 295 300

Ala Cys Val Pro Cys Arg Val Arg Ser

305 310

<210> 38

<211> 17

<212> PRT

<213> Mouse (Mus Musculus)

<400> 38

Met Glu Arg Thr Leu Val Cys Leu Val Val Ile Phe Leu Gly Thr Val

1 5 10 15

Ala

<210> 39

<211> 129

<212> PRT

<213> Mouse (Mus Musculus)

<400> 39

His Lys Ser Ser Pro Gln Gly Pro Asp Arg Leu Leu Ile Arg Leu Arg

1 5 10 15

His Leu Ile Asp Ile Val Glu Gln Leu Lys Ile Tyr Glu Asn Asp Leu

20 25 30

Asp Pro Glu Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys Glu

35 40 45

His Ala Ala Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys Pro Ser Asn

50 55 60

Pro Gly Asn Asn Lys Thr Phe Ile Ile Asp Leu Val Ala Gln Leu Arg

65 70 75 80

Arg Arg Leu Pro Ala Arg Arg Gly Gly Lys Lys Gln Lys His Ile Ala

85 90 95

Lys Cys Pro Ser Cys Asp Ser Tyr Glu Lys Arg Thr Pro Lys Glu Phe

100 105 110

Leu Glu Arg Leu Lys Trp Leu Leu Gln Lys Met Ile His Gln His Leu

115 120 125

Ser

<210> 40

<211> 17

<212> PRT

<213> Person (Homo sapiens)

<400> 40

Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile

1 5 10 15

Ser

<210> 41

<211> 127

<212> PRT

<213> Person (Homo sapiens)

<400> 41

Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His Val

1 5 10 15

Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp Thr

20 25 30

Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe Asp

35 40 45

Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln

50 55 60

Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met Met

65 70 75 80

Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser Cys

85 90 95

Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp

100 105 110

Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu

115 120 125

<210> 42

<211> 23

<212> PRT

<213> Person (Homo sapiens)

<400> 42

Met Lys Val Ser Ala Ala Ala Leu Ala Val Ile Leu Ile Ala Thr Ala

1 5 10 15

Leu Cys Ala Pro Ala Ser Ala

20

<210> 43

<211> 68

<212> PRT

<213> Person (Homo sapiens)

<400> 43

Ser Pro Tyr Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala

1 5 10 15

Arg Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly

20 25 30

Lys Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg Gln

35 40 45

Val Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser

50 55 60

Leu Glu Met Ser

65

<210> 44

<211> 22

<212> PRT

<213> Person (Homo sapiens)

<400> 44

Met Cys Pro Ala Arg Ser Leu Leu Leu Val Ala Thr Leu Val Leu Leu

1 5 10 15

Asp His Leu Ser Leu Ala

20

<210> 45

<211> 197

<212> PRT

<213> Person (Homo sapiens)

<400> 45

Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu

1 5 10 15

His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys

20 25 30

Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp

35 40 45

His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu

50 55 60

Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr

65 70 75 80

Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe

85 90 95

Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr

100 105 110

Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys

115 120 125

Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu

130 135 140

Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser

145 150 155 160

Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu

165 170 175

Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser

180 185 190

Tyr Leu Asn Ala Ser

195

<210> 46

<211> 22

<212> PRT

<213> Person (Homo sapiens)

<400> 46

Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu

1 5 10 15

Ala Ser Pro Leu Val Ala

20

<210> 47

<211> 306

<212> PRT

<213> Person (Homo sapiens)

<400> 47

Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr

1 5 10 15

Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu

20 25 30

Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly

35 40 45

Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly

50 55 60

Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu

65 70 75 80

Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys

85 90 95

Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys

100 105 110

Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr

115 120 125

Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln

130 135 140

Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly

145 150 155 160

Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala

165 170 175

Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala

180 185 190

Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg

195 200 205

Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu

210 215 220

Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp

225 230 235 240

Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln

245 250 255

Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr

260 265 270

Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala

275 280 285

Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro

290 295 300

Cys Ser

305

<210> 48

<211> 24

<212> PRT

<213> Person (Homo sapiens)

<400> 48

Met Arg Ser Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met

1 5 10 15

Val Ile Phe Leu Gly Thr Leu Val

20

<210> 49

<211> 138

<212> PRT

<213> Person (Homo sapiens)

<400> 49

His Lys Ser Ser Ser Gln Gly Gln Asp Arg His Met Ile Arg Met Arg

1 5 10 15

Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu

20 25 30

Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu

35 40 45

Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn

50 55 60

Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys

65 70 75 80

Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu

85 90 95

Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe

100 105 110

Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu

115 120 125

Ser Ser Arg Thr His Gly Ser Glu Asp Ser

130 135

<210> 50

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Self-cleaving peptides

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (4)..(4)

<223> Xaa can be any naturally occurring amino acid

<400> 50

Asp Xaa Glu Xaa Asn Pro Gly Pro

1 5

<210> 51

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> Self-cleaving peptides

<400> 51

Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val

1 5 10 15

Glu Ser Asn Pro Gly Pro

20

<210> 52

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Self-cleaving peptides

<400> 52

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 53

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> Self-cleaving peptides

<400> 53

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 54

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Self-cleaving peptides

<400> 54

Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp

1 5 10 15

Val Glu Ser Asn Pro Gly Pro

20

<210> 55

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Self-cleaving peptides

<400> 55

Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala

1 5 10 15

Gly Asp Val Glu Ser Asn Pro Gly Pro

20 25

<210> 56

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> Trimerization unit

<400> 56

Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys

1 5 10 15

Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

20 25

<210> 57

<211> 117

<212> DNA

<213> Artificial sequence

<220>

<223> Tetramerization Unit

<400> 57

aagcctctgg acggagagta tttcactctc cagatccggg gccccgaaag gttcgaaatg 60

ttccgggagc ttaacgaggc cttggagctg aaagacgcac aggccggaaa ggaaccg 117

<210> 58

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 58

Gly Gly Gly Gly Ser

1 5

<210> 59

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 59

Gly Gly Gly Ser Ser

1 5

<210> 60

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 60

Gly Gly Gly Ser Gly

1 5

<210> 61

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 61

Gly Gly Ser Gly Gly

1 5

<210> 62

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 62

Ser Gly Ser Ser Gly Ser

1 5

<210> 63

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<220>

<221> SITE

<222> (1)..(4)

<223> May be repeated m times, m being an integer of 1 to 5

<400> 63

Glu Ala Ala Lys Gly Ser

1 5

<210> 64

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<220>

<221> SITE

<222> (1)..(5)

<223> May be repeated m times, m being an integer of 1 to 5

<400> 64

Gly Gly Gly Gly Ser

1 5

<210> 65

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<220>

<221> SITE

<222> (1)..(5)

<223> May be repeated m times, m being an integer of 1 to 5

<400> 65

Gly Gly Gly Ser Ser

1 5

<210> 66

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<220>

<221> SITE

<222> (1)..(5)

<223> May be repeated m times, m being an integer of 1 to 5

<400> 66

Gly Gly Ser Gly Gly

1 5

<210> 67

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<220>

<221> SITE

<222> (1)..(5)

<223> May be repeated m times, m being an integer of 1 to 5

<400> 67

Gly Gly Gly Ser Gly

1 5

<210> 68

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 68

Ser Gly Ser Ser Gly Ser

1 5

<210> 69

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 69

Leu Gly Gly Gly Ser

1 5

<210> 70

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 70

Gly Leu Gly Gly Ser

1 5

<210> 71

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 71

Gly Gly Leu Gly Ser

1 5

<210> 72

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 72

Gly Gly Gly Leu Ser

1 5

<210> 73

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 73

Gly Gly Gly Gly Leu

1 5

<210> 74

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 74

Leu Gly Gly Ser Gly

1 5

<210> 75

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 75

Gly Leu Gly Ser Gly

1 5

<210> 76

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 76

Gly Gly Leu Ser Gly

1 5

<210> 77

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 77

Gly Gly Gly Leu Gly

1 5

<210> 78

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 78

Gly Gly Gly Ser Leu

1 5

<210> 79

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 79

Leu Gly Gly Ser Ser

1 5

<210> 80

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 80

Gly Leu Gly Ser Ser

1 5

<210> 81

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 81

Gly Gly Leu Ser Ser

1 5

<210> 82

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> T cell epitope

<400> 82

Cys Thr Glu Leu Lys Leu Ser Asp Tyr

1 5

<210> 83

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> T cell epitope

<400> 83

Asn Leu Val Pro Met Val Ala Thr Val

1 5

<210> 84

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> T cell epitope

<400> 84

Lys Leu Val Ala Asn Asn Thr Arg Leu

1 5

<210> 85

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 85

Leu Gly Leu Gly Ser

1 5

<210> 86

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 86

Gly Leu Gly Leu Ser

1 5

<210> 87

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 87

Gly Leu Leu Gly Ser

1 5

<210> 88

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 88

Leu Gly Gly Leu Ser

1 5

<210> 89

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 89

Gly Leu Gly Gly Leu

1 5

<210> 90

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 90

Leu Gly Leu Ser Gly

1 5

<210> 91

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 91

Gly Leu Leu Ser Gly

1 5

<210> 92

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 92

Gly Gly Leu Ser Leu

1 5

<210> 93

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 93

Gly Gly Leu Leu Gly

1 5

<210> 94

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 94

Gly Leu Gly Ser Leu

1 5

<210> 95

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 95

Leu Gly Leu Ser Ser

1 5

<210> 96

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 96

Gly Gly Leu Leu Ser

1 5

<210> 97

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 97

Leu Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 98

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 98

Gly Leu Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 99

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 99

Gly Gly Leu Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 100

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 100

Gly Gly Gly Leu Ser Gly Gly Gly Gly Ser

1 5 10

<210> 101

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 101

Gly Gly Gly Gly Leu Gly Gly Gly Gly Ser

1 5 10

<210> 102

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 102

Leu Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10

<210> 103

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 103

Gly Leu Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10

<210> 104

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 104

Gly Gly Leu Ser Gly Gly Gly Gly Ser Gly

1 5 10

<210> 105

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 105

Gly Gly Gly Leu Gly Gly Gly Gly Ser Gly

1 5 10

<210> 106

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 106

Gly Gly Gly Ser Leu Gly Gly Gly Ser Gly

1 5 10

<210> 107

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 107

Leu Gly Gly Ser Ser Gly Gly Gly Ser Ser

1 5 10

<210> 108

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 108

Gly Leu Gly Ser Ser Gly Gly Gly Ser Ser

1 5 10

<210> 109

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 109

Gly Gly Leu Ser Ser Gly Gly Gly Ser Ser

1 5 10

<210> 110

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 110

Gly Gly Gly Leu Ser Gly Gly Gly Ser Ser

1 5 10

<210> 111

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 111

Gly Gly Gly Ser Leu Gly Gly Gly Ser Ser

1 5 10

<210> 112

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 112

Leu Gly Gly Gly Ser Leu Gly Gly Gly Ser

1 5 10

<210> 113

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 113

Gly Leu Gly Gly Ser Gly Leu Gly Gly Ser

1 5 10

<210> 114

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 114

Gly Gly Leu Gly Ser Gly Gly Leu Gly Ser

1 5 10

<210> 115

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 115

Gly Gly Gly Leu Ser Gly Gly Gly Leu Ser

1 5 10

<210> 116

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 116

Gly Gly Gly Gly Leu Gly Gly Gly Gly Leu

1 5 10

<210> 117

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 117

Leu Gly Gly Ser Gly Leu Gly Gly Ser Gly

1 5 10

<210> 118

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 118

Gly Leu Gly Ser Gly Gly Leu Gly Ser Gly

1 5 10

<210> 119

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 119

Gly Gly Leu Ser Gly Gly Gly Leu Ser Gly

1 5 10

<210> 120

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 120

Gly Gly Gly Leu Gly Gly Gly Gly Leu Gly

1 5 10

<210> 121

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 121

Gly Gly Gly Ser Leu Gly Gly Gly Ser Leu

1 5 10

<210> 122

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 122

Leu Gly Gly Ser Ser Leu Gly Gly Ser Ser

1 5 10

<210> 123

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 123

Gly Leu Gly Ser Ser Gly Leu Gly Ser Ser

1 5 10

<210> 124

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 124

Gly Gly Leu Ser Ser Gly Gly Leu Ser Ser

1 5 10

<210> 125

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 125

Gly Ser Gly Gly Gly Ala

1 5

<210> 126

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 126

Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly Gly Ala

1 5 10

<210> 127

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 127

Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly

1 5 10 15

Gly Ala

<210> 128

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 128

Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly

1 5 10 15

Gly Ala Gly Ser Gly Gly Gly Ala

20

<210> 129

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 129

Gly Glu Asn Leu Tyr Phe Gln Ser Gly Gly

1 5 10

<210> 130

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 130

Ser Gly Gly Gly Ser Ser Gly Gly Gly Ser

1 5 10

<210> 131

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 131

Ser Ser Gly Gly Gly Ser Ser Gly Gly Gly

1 5 10

<210> 132

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 132

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

1 5 10

<210> 133

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 133

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

1 5 10

<210> 134

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 134

Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly

1 5 10

<210> 135

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 135

Gly Gly Gly Ser Ser Ser

1 5

<210> 136

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 136

Gly Gly Gly Ser Ser Gly Gly Gly Ser Ser Gly Gly Gly Ser Ser

1 5 10 15

<210> 137

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 137

Gly Leu Gly Gly Leu Ala Ala Ala

1 5

<210> 138

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 138

Lys Pro Glu Pro Lys Pro Ala Pro Ala Pro Lys Pro

1 5 10

<210> 139

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 139

Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala

1 5 10

<210> 140

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<220>

<221> SITE

<222> (1)..(5)

<223> May be repeated m times, m being an integer of 1 to 5

<400> 140

Glu Ala Ala Ala Lys

1 5

<210> 141

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 141

Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro

1 5 10 15

<210> 142

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 142

Ser Ala Cys Tyr Cys Glu Leu Ser

1 5

<210> 143

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 143

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu

1 5 10 15

<210> 144

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 144

Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu

1 5 10

<210> 145

<211> 36

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 145

Gly Gly Ser Ala Gly Gly Ser Gly Ser Gly Ser Ser Gly Gly Ser Ser

1 5 10 15

Gly Ala Ser Gly Thr Gly Thr Ala Gly Gly Thr Gly Ser Gly Ser Gly

20 25 30

Thr Gly Ser Gly

35

<210> 146

<211> 36

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 146

Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

1 5 10 15

Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser

20 25 30

Gly Gly Gly Ser

35

<210> 147

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 147

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr

1 5 10

<210> 148

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 148

Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

1 5 10 15

<210> 149

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 149

Gly Leu Ser Gly Leu

1 5

<210> 150

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 150

Glu Ala Ala Ala Lys

1 5

<210> 151

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<220>

<221> SITE

<222> (1)..(5)

<223> May be repeated m times, m being an integer of 1 to 5

<400> 151

Glu Ala Ala Ala Lys Gly Ser

1 5

<210> 152

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 152

Gly Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro

1 5 10 15

Gly

<210> 153

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Joint

<400> 153

His Glu Tyr Gly Ala Glu Ala Leu Glu Arg Ala Gly

1 5 10

<210> 154

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> CT26 epitope

<400> 154

Val Ile Leu Pro Gln Ala Pro Ser Gly Pro Ser Tyr Ala Thr Tyr Leu

1 5 10 15

Gln Pro Ala Gln Ala Gln Met Leu Thr Pro Pro

20 25

<210> 155

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> CT26 epitope

<400> 155

Glu Val Ile Gln Thr Ser Lys Tyr Tyr Met Arg Asp Val Ile Ala Ile

1 5 10 15

Glu Ser Ala Trp Leu Leu Glu Leu Ala Pro His

20 25

<210> 156

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> CT26 epitope

<400> 156

Lys Ser Trp Ile His Cys Trp Lys Tyr Leu Ser Val Gln Ser Gln Leu

1 5 10 15

Phe Arg Gly Ser Ser Leu Leu Phe Arg Arg Val

20 25

<210> 157

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> CT26 epitope

<400> 157

Gly Asp Val Lys Ile His Ala His Lys Val Val Leu Ala Asn Ile Ser

1 5 10 15

Pro Tyr Phe Lys Ala Met Phe Thr Gly Asn Leu

20 25

<210> 158

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> CT26 epitope

<400> 158

Phe Val Ser Pro Met Ala His Tyr Val Pro Gly Ile Met Ala Ile Glu

1 5 10 15

Ser Val Val Ala Arg Phe Gln Phe Ile Val Pro

20 25

<210> 159

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> CT26 epitope

<400> 159

Lys Ile Tyr Glu Phe Asp Tyr His Leu Tyr Gly Gln Asn Ile Thr Met

1 5 10 15

Ile Met Thr Ser Val Ser Gly His Leu Leu Ala

20 25

<210> 160

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> CT26 epitope

<400> 160

Asn Asn Leu Gln Lys Tyr Ile Glu Ile Tyr Val Gln Lys Ile Asn Pro

1 5 10 15

Ser Arg Leu Pro Val Val Ile Gly Gly Leu Leu

20 25

<210> 161

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> CT26 epitope

<400> 161

Thr Pro Leu Arg Lys His Thr Val His Ala Ile Arg Lys Phe Tyr Leu

1 5 10 15

Glu Phe Lys Gly Ser Ser Pro Pro Pro Arg Leu

20 25

<210> 162

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> HPV epitope

<400> 162

Arg Ala His Tyr Asn Ile Val Thr Phe

1 5

<210> 163

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> HPV epitope

<400> 163

Met Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Leu

1 5 10 15

<210> 164

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> HPV epitope

<400> 164

Arg Pro Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr

1 5 10 15

<210> 165

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> HPV epitope

<400> 165

Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu

1 5 10 15

<210> 166

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> HPV epitope

<400> 166

Thr Ile His Asp Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln

1 5 10 15

<210> 167

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> HPV epitope

<400> 167

Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr

1 5 10 15

<210> 168

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> HPV epitope

<400> 168

Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Arg Arg Asp Leu

1 5 10 15

<210> 169

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> HPV epitope

<400> 169

Tyr Asp Phe Ala Arg Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly

1 5 10 15

<210> 170

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> HPV epitope

<400> 170

Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Arg Asp

1 5 10 15

<210> 171

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> HPV epitope

<400> 171

Gly Asn Pro Tyr Ala Val Arg Asp Lys Cys Leu Lys Phe Tyr Ser

1 5 10 15

<210> 172

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> HPV epitope

<400> 172

Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His

1 5 10 15

<210> 173

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Self-cleaving peptides

<400> 173

Asp Val Glu Ser Asn Pro Gly Pro

1 5

Claims

1. A carrier, comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets an antigen presenting cell, a multimerization unit, e.g., a dimerization unit, and an antigenic unit comprising one or more antigens or portions thereof, e.g., one or more disease-associated antigens or portions thereof; and

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

3. The vector of any one of claims 1-2, wherein the one or more immunostimulatory compounds promote the attraction of antigen presenting cells.

4. A vector according to claim 3, wherein the one or more immunostimulatory compounds are chemokines, preferably human chemokines.

5. The vector according to claim 4, wherein the one or more immunostimulatory compounds may interact with a surface molecule on an antigen presenting cell selected from CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 and XCR1, preferably wherein the one or more immunostimulatory compounds may interact with a surface molecule on a human antigen presenting cell selected from hCCR1, hCCR3, hCCR4, hCCR5, hCCR6, hCCR7, hCCR8 and hXCR 1.

6. The vector according to any one of claims 4 to 5, wherein the one or more immunostimulatory compounds are selected from macrophage inflammatory protein a, including isoforms thereof, such as mouse 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.

7. The vector of any one of claims 2 to 6, wherein the one or more immunostimulatory compounds promote activation and/or maturation of antigen presenting cells.

8. The vector according to any one of claims 2 to 7, wherein the one or more immunostimulatory compounds may interact with a surface molecule on an antigen presenting cell, the surface molecule being selected from receptors of the TNF receptor superfamily, including CD40 (cluster of differentiation 40), CD137 (4-1 BB), CD27, RANK, and ICOS (CD 278), preferably wherein the one or more immunostimulatory compounds may interact with a surface molecule on a human antigen presenting cell, the surface molecule being selected from receptors of the human TNF receptor superfamily, including hCD40, hCD137, hCD27, hRANK, and hcicos.

9. The vector according to claim 8, wherein the one or more immunostimulatory compounds are selected from CD40L, CD137L, CD, RANKL and ICOSL, preferably wherein the one or more immunostimulatory compounds are selected from hCD40L, hCD137L, hCD, hRANKL and hICOSL.

10. The vector according to any one of claims 2 to 7, wherein the one or more immunostimulatory compounds are cytokines selected from the group consisting of IL-2, IL-10, IL-12, IL-21, tnfa, ifnγ and IL-1β, preferably wherein the one or more immunostimulatory compounds are human cytokines selected from the group consisting of hll-2, hll-10, hll-12, hll-21, htnfa, hlnγ and hll-1β.

11. The vector according to any one of claims 2 to 7, wherein the one or more immunostimulatory compounds are viral infection sensors, such as MyD88 or TRIF, preferably human viral infection sensors, such as human MyD88 or human TRIF.

12. The vector according to any one of claims 2 to 7, wherein the one or more immunostimulatory compounds may interact with a pattern recognition receptor on antigen presenting cells, such as Toll-like receptors, including TLR2, TLR4, TLR5 and TLR9, and/or with a receptor on antigen presenting cells selected from RAGE, TIM-3, FPR, SREC1, LOX1 and CD91, preferably wherein the one or more immunostimulatory compounds may interact with a pattern recognition receptor on human antigen presenting cells, such as a human Toll-like receptor, including hTLR2, hTLR4, hTLR5 and hTLR9, and/or with a receptor on human antigen presenting cells selected from htrage, hfpm-3, hFPR, hSREC1, hLOX1 and hCD 91.

13. The vector according to claim 12, wherein the one or more immunostimulatory compounds are selected from pathogen-associated molecular patterns (PAMPs) such as flagellin, protein damage-associated molecular patterns (DAMP) such as HMGB1, heat Shock Proteins (HSP), calreticulin and annexin A1, preferably wherein the one or more immunostimulatory compounds are selected from human pathogen-associated molecular patterns (PAMPs), human protein damage-associated molecular patterns (DAMP) such as hHMGB1, human Heat Shock Proteins (HSP), human calreticulin and human annexin A1.

14. The vector according to any one of claims 2 to 13, wherein the one or more immunostimulatory compounds promote growth and/or expansion of antigen presenting cells.

15. The vector according to any one of claims 2 to 14, wherein the one or more immunostimulatory compounds are growth factors, preferably human growth factors.

16. The vector according to any one of claims 2 to 14, wherein the one or more immunostimulatory compounds may interact with a surface molecule on antigen presenting cells selected from GM-CSF receptor, FLT-3R, IL-15R and IL-4R, preferably wherein the one or more immunostimulatory compounds may interact with a surface molecule on human antigen presenting cells selected from hGM-CSF receptor, hFLT-3R, hIL-15R and hIL-4R.

17. The vector according to any one of claims 15 to 16, wherein the one or more immunostimulatory compounds are selected from GM-CSF, FLT-3L, IL-15 and IL-4, preferably wherein the one or more immunostimulatory compounds are selected from hGM-CSF, hFLT-3L, hIL-15 and hll-4.

18. The vector according to any one of the preceding claims, wherein the one or more immunostimulatory compounds are selected from the group consisting of IL-4, IL-1 β, ifnγ, ifnα, IL-15, tnfα, IL-10, IL-12, IL-2, IL-21, myD88, tri, RIG-1, MDA-5, P28 region of C3d, IL-13, ifnε, ifnκ, ifnω, ifnβ and IL-6, preferably wherein the one or more immunostimulatory compounds are selected from the group consisting of hll-4, hll-1 β, hifnγ, hlnfα, hll-15, htnfα, hll-10, hll-12, hll-2, hll-21, hMyD, hlig-I, hMDA-5, P28 region of hC3d, hll-13, hκ, hln, hlω, hlnf, hlw and hll-6.

19. The vector according to any of the preceding claims, wherein the vector comprises a plurality of further nucleic acid sequences encoding more than one immunostimulatory compound, such as 2, 3, 4, 5, 6, 7 or 8 immunostimulatory compounds, such as 2, 3, 4, 5, 6, 7 or 8 different immunostimulatory compounds.

20. The vector of claim 19, wherein the plurality of immunostimulatory compounds are different immunostimulatory compounds that affect antigen presenting cells in different ways, such as stimulating antigen presenting cells.

21. The vector according to any one of the preceding claims, wherein the vector comprises one or more co-expression elements.

22. The vector of claim 21, wherein the one or more co-expression elements result in transcription of the first polypeptide and the one or more immunostimulatory compounds on a single transcript and independent translation into a separate first polypeptide and a separate one or more immunostimulatory compounds.

23. The vector of any one of claims 21 to 22, wherein the one or more co-expression elements are IRES elements or nucleic acid sequences encoding a 2A self-cleaving peptide.

24. The vector of claim 21, wherein the one or more co-expression elements result in transcription of the first polypeptide and the one or more immunostimulatory compounds as separate transcripts.

25. The vector of claim 24, wherein the one or more co-expression elements are a) a bi-directional promoter or b) a promoter, wherein the vector comprises a separate promoter for each of the nucleic acid sequences encoding the first polypeptide and the one or more immunostimulatory compounds.

26. The vector according to any one of the preceding claims, wherein the antigenic unit comprises one or more neoantigens or parts thereof, such as neoepitopes.

27. The vector according to claim 26, wherein the antigenic unit comprises a plurality of neo-epitopes, such as a plurality of neo-epitopes separated from each other by a linker.

28. The vector of any one of claims 26 to 27, wherein the antigenic units further comprise one or more patient-present consensus cancer antigens or portions thereof, such as patient-present consensus cancer epitopes.

29. The vector of any one of claims 1 to 25, wherein the antigenic units comprise one or more patient-present consensus cancer antigens or portions thereof, such as patient-present consensus cancer epitopes.

30. The vector according to any one of claims 1 to 25, wherein the antigenic units comprise one or more consensus cancer antigens or parts thereof, such as consensus cancer epitopes.

31. The vector of any one of claims 1 to 25, wherein the antigenic units comprise one or more antigens derived from one or more pathogens or portions of such antigens.

32. The vector of claim 31, wherein the antigenic units comprise one or more full length antigens derived from one or more pathogens or one or more portions of such full length antigens, or one or more full length antigens derived from one or more pathogens and one or more portions of such full length antigens.

33. The vector of any one of claims 31 to 32, wherein the antigenic units comprise one or more portions of one or more antigens derived from one or more pathogens.

34. The vector of claim 33, wherein the moiety is a B cell epitope such that the antigenic unit comprises one or more B cell epitopes derived from one or more pathogens.

35. The vector of claim 33, wherein the moiety is a T cell epitope such that the antigenic unit comprises one or more T cell epitopes derived from one or more pathogens.

36. The vector of any one of claims 31 to 32, wherein the antigenic units comprise (i) one or more full length antigens derived from one or more pathogens or one or more portions of such antigens and (ii) one or more T cell epitopes derived from one or more pathogens.

37. The vector of any one of claims 31 to 36, wherein the one or more pathogens are selected from the group consisting of viruses, bacteria, fungi, and parasites.

38. The vector according to any one of the preceding claims, wherein the targeting unit is or comprises a moiety that interacts with a surface molecule on the antigen presenting cell.

39. The vector according to claim 38, wherein the surface molecule is selected from MHC, HLA, CD, CD40, CLEC9A, chemokine receptors such as CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 or XCR1 and Toll-like receptors such as TLR-2, TLR-4 or TLR-5, preferably wherein the surface molecule is selected from HLA, hCD14, hCD40, hCLEC a, human chemokine receptors such as hCCR1, hCCR3, hCCR4, hCCR5, hCCR6, hCCR7, hCCR8 or hXCR1 and human Toll-like receptors such as hTLR-2, hTLR-4 or hTLR-5.

40. The vector of any one of claims 38 and 39, wherein the targeting unit comprises or consists of: soluble CD40 ligand, CCL4 and isoforms thereof, CCL5, CCL19, CCL20, CCL21, macrophage inflammatory protein a includes isoforms thereof such as mouse CCL3, human CCL3L1, human CCL3L2, and human CCL3L3, XCL1, XCL2, flagellin, anti-HLA-DP, anti-HLA-DR, anti-pan-HLA class II, anti-CD 40, anti-TLR-2, anti-TLR-4, anti-TLR-5, or anti-CLEC 9A, preferably wherein the targeting unit comprises or consists of: soluble hCD40 ligand, hCCL4 and isoforms thereof, hCCL5, hCCL19, hCCL20, hCCL, human macrophage inflammatory protein α include isoforms thereof 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-hCD 40, anti-hTLR-2, anti-hTLR-4, anti-hTLR-5, or anti-hCLEC 9.

41. The vector of claim 40, wherein the targeting unit comprises or consists of human MIP-1α (LD 78 β, CCL3L 1).

42. The vector according to any of the preceding claims, wherein the multimerization unit is selected from a dimerization unit, a trimerization unit, such as a collagen-derived trimerization unit, such as a human collagen-derived trimerization domain, such as a human collagen-derived XVIII trimerization domain or a human collagen XV trimerization domain or the C-terminal domain of T4 fibrin, and a tetramerization unit, such as a domain derived from p53, and wherein the multimerization unit optionally comprises a hinge region, such as hinge exon h1 and hinge exon h4.

43. The vector according to claim 42, wherein the vector comprises a hinge region having the ability to form one or more covalent bonds and preferably being of Ig origin.

44. The vector according to any one of claims 42 to 43, wherein the multimerization unit is a dimerization unit, and the dimerization unit further comprises a further domain which promotes dimerization, preferably wherein the further domain is an immunoglobulin domain, more preferably an immunoglobulin constant domain.

45. The vector according to claim 44, wherein said further domain is a carboxy terminal C domain derived from IgG, preferably derived from IgG 3.

46. The vector according to any one of claims 44 to 45, 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 wherein the dimerization unit linker connects the hinge region and another domain that facilitates dimerization.

47. The vector of any one of claims 44 to 46, wherein the dimerization unit comprises hinge exon h1 and hinge exon h4, a dimerization unit linker, and a CH3 domain of human IgG 3.

48. The vector according to any one of the preceding claims, wherein the first nucleic acid sequence encodes a first polypeptide further comprising a unit linker connecting the antigenic unit and the multimerization unit, and wherein the unit linker is a non-immunogenic linker and/or a flexible or rigid linker.

49. The vector according to any one of the preceding claims, wherein the first nucleic acid sequence encodes a first polypeptide further comprising a signal peptide, and preferably wherein the one or more further nucleic acid sequences further encode a signal peptide.

50. The vector according to any of the preceding claims, 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.

51. A method of producing a vector as defined in any one of the preceding claims, the method comprising:

a) Transfecting cells in vitro with the vector;

b) Culturing the cells;

c) Optionally, lysing the cells to release the carrier from the cells; and

D) The vector is collected and optionally purified.

52. A host cell comprising a vector as defined in any one of claims 1 to 50, for example a host cell selected from the group consisting of a prokaryotic cell, a yeast cell, an insect cell, a higher eukaryotic cell, for example a cell from an animal or a human.

53. A carrier as defined in any one of claims 1 to 50 for use as a medicament.

54. A pharmaceutical composition comprising a carrier as defined in any one of claims 1 to 50 and a pharmaceutically acceptable carrier or diluent.

55. The pharmaceutical composition of claim 54, wherein the composition further comprises a transfection agent.

56. The pharmaceutical composition of any one of claims 54-55, wherein the composition comprises in the range of 0.1-10 mg of the vector, e.g., the DNA plasmid.

57. A method of treating a subject suffering from a disease or in need of prevention of said disease, the method comprising administering to the subject a vector as defined in any one of claims 1 to 50 or a pharmaceutical composition as defined in any one of claims 54 to 56.

58. The method of claim 57, wherein the carrier or the pharmaceutical composition is administered in a therapeutically or prophylactically effective amount, such as by intradermal, intramuscular, or subcutaneous injection, or by mucosal or epithelial administration, such as intranasal or oral administration.

59. A method of treating a subject suffering from cancer, the method comprising administering to the subject a vector as defined in any one of claims 1 to 30 and 38 to 50 or a pharmaceutical composition as defined in any one of claims 54 to 56 comprising such a vector.

60. The method of claim 59, wherein the carrier or the pharmaceutical composition is administered in a therapeutically effective amount, such as by intradermal, intramuscular or subcutaneous injection, or by mucosal or epithelial administration, such as intranasal or oral administration.

61. A method of treating a subject suffering from an infectious disease or in need of prevention of an infectious disease, the method comprising administering to the subject a vector as defined in any one of claims 1 to 25 and 31 to 50 or a pharmaceutical composition as defined in any one of claims 54 to 56 comprising such a vector.

62. The method of claim 61, wherein the carrier or the pharmaceutical composition is administered in a therapeutically or prophylactically effective amount, such as by intradermal, intramuscular, or subcutaneous injection, or by mucosal or epithelial administration, such as intranasal or oral administration.