RU2795103C2 - Combination therapy for cancer treatment using recombinant mva and antibodies introduced intravenously - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to oncology, and is intended to reduce the size of a tumor and/or increase the survival rate of an oncological patient. The pharmaceutical combination contains a recombinant modified vaccinia virus Ankara (MVA) and an antibody. In another embodiment, a method for reducing tumor size and/or improving survival of a cancer patient is provided, comprising administering said pharmaceutical combination to the patient.

EFFECT: use of the group of inventions provides a reduction in tumor volume and/or an increase in the survival of a cancer patient due to the effective targeting and destruction of tumor cells.

32 cl, 33 dwg, 3 tbl, 34 ex

Description

FIELD OF TECHNOLOGY

[001] The present invention relates to a combination therapy for the treatment of cancer, the combination comprising an intravenously administered recombinant modified vaccinia virus Ankara (MVA) containing a nucleic acid encoding a heterologous tumor associated antigen (TAA) and an antibody.

BACKGROUND OF THE INVENTION

[002] Recombinant poxviruses have been used as immunotherapeutic vaccines against infectious organisms and, more recently, against tumors. Mastrangelo et al. J Clin Invest. 2000;105(8):1031-1034.

[003] One of the poxvirus strains that has proven useful as an immunotherapeutic vaccine against infectious diseases and cancer is modified vaccinia virus Ankara (MVA). MVA was generated by 516 serial passages in chicken embryonic fibroblasts of the Ankara strain of vaccinia virus (CVA) (for a review, see Mayr, A., et al. Infection 3, 6-14 (1975)). Due to these long passages, the genome of the resulting MVA virus has had about 31 kb deleted in its genomic sequence and is therefore highly replication-restricted towards avian host cells (Meyer, H. et al., J. Gen. Virol 72, 1031-1038 (1991)). In a number of animal models, the resulting MVA has been shown to be essentially avirulent (Mayr, A. & Danner, K., Dev. Biol. Stand. 41: 225-34 (1978)). MVA strains having improved safety profiles have been described for the development of safer products such as vaccines or pharmaceuticals. See PCT International Publication WO2002042480 (also see US Patent Nos. 6,761,893 and 6,913,752), which are all incorporated herein by reference. Such variants are capable of replicating reproductively in non-human cells and cell lines, especially chicken embryonic fibroblasts (CEF), but are not capable of replicating in human cell lines, in particular, including the HeLa, HaCat and 143B cell lines. Such strains are also not capable of reproductive replication in vivo, for example in certain mouse strains such as the AGR 129 transgenic mouse model, which is very immunocompromised and highly susceptible to replicating viruses. See US Pat. No. 6,761,893. Such variants of MVA and its derivatives have been described, including recombinant variants referred to as "MVA-BN". See PCT International Publication WO2002042480 (also see US Patent Nos. 6761893 and 6913752).

[004] The use of poxvirus vectors that encode tumor-associated antigens (TAA) has been shown to successfully reduce tumor size as well as improve the overall survival of cancer patients. See for example WO 2014/062778 . It has been demonstrated that when a poxvirus vector encoding TAA such as HER2, CEA, MUC1 and/or Brachyury is administered to a cancer patient, a strong and specific T cell response is generated in the patient to fight the cancer. Id. , also see Guardino et al. (2009), Heery et al., (2015)

[005] HER2 is one such TAA that has been shown to be effective when encoded as part of a poxvirus vector. Id. HER2 is a tumor-associated antigen that is overexpressed in certain types of tumor cells in some patients with various types of cancer such as breast, rectal, colon, lung, ovarian, cervix, bladder, stomach, and urothelial cancers. Immunization with various HER2 polypeptides has been used to generate an immune response against tumor cells expressing this antigen, as has vaccination with recombinant modified vaccinia Ankara ("MVA") vectors expressing a modified form of the HER2 protein (i.e., MVA-BN-HER2) . See, for example, Renard et al., J. Immunol. 171:1588-1595 (2003); Mittendorf et al., Cancer 106:2309-2317 (2006); Mandl et al., Cancer Immunol. Immunother. 61(1):19-29 (2012).

[006] Previous work on MVA-BN-HER2 has been shown to induce a TH1-biased immune response with both antibody and cellular components. See, for example, Mandl et al., 2012. The same researchers have shown that a balanced immune response, including both humoral and cell-mediated components, is important for protection against and clearance of various pathogens in the context of an infectious disease. See, for example, Hutchings et al., J. Immunol. 175:599-606 (2005).

[007] In addition to their efficacy in combination with TAA, poxviruses such as MVA have been shown to have increased efficacy in combination with a CD40 agonist such as CD40 ligand (CD40L). See WO 2014/037124. CD40/CD40L is a member of the tumor necrosis factor/tumor necrosis factor receptor ("TNFR/TNF") superfamily. Although CD40 is constitutively expressed on many cell types, including B cells, macrophages, DCs (dendritic cells), its CD40L ligand is predominantly expressed on activated CD4+ T cells [Lee et al., "CD40, but not CD154, expression on B -cells is necessary for optimal primary B-cell responses," J. Immunol. 171(11):5707-5717 (2002); D. Y. Ma and E. A. Clark, "The role of CD40 and CD154/CD40L in dendritic cells," Semin. Immunol. 21(5):265-272 (2009)]. Cognate interaction between DC and CD4+ T cells early after infection or immunization licenses DC to prime CD8+ T cell responses [J. P. Ridge et al., "A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell," Nature 393(6684):474-478 (1998)]. DC licensing leads to increased expression of co-stimulatory molecules, improved survival, and better ability to cross-present DCs. This process is mediated primarily through the CD40/CD40L interaction [S. R. Bennet et al., "Help for cytotoxic T-cell responses is mediated by CD40 signaling," Nature 393(6684):478-480 (1998); S. P. Schoenberger et al., "T-cell help for cytotoxic T-cell help is mediated by CD40-CD40L interactions," Nature 393(6684):480-483 (1998)], but CD40/CD40L-independent mechanisms also exist ( CD70, LT.beta.R). Interestingly, a direct interaction between DC-expressed CD40L and CD40 expressed on CD8+ T cells has also been suggested, providing a possible explanation for the generation of helper-independent CTL responses [S. Johnson et al., "Selected Toll-like receptor ligands and viruses promote helper-independent cytotoxic T-cell priming by upregulating CD40L on dendritic cells," Immunity 30(2):218-227 (2009)].

[008] Several studies have shown that anti-CD40 agonist antibodies can be used as an adjuvant in a vaccine. In addition, recombinant AdVs have been created [K. Kato et al., "Gene transfer of CD40-ligand induces autologous immune recognition of chronic lymphocytic leukemia B-cells," J. Clin. Invest. 101(5):1133-1141 (1998)] and VV [Bereta et al., "Immune properties of recombinant vaccinia virus encoding CD154 (CD40L) are determined by expression of virally encoded CD40L and the presence of CD40L protein in viral particles, Cancer Gen. Ther. 11(12):808-818 (2004)], encoding CD40L, which showed superior immunogenicity in vitro and in vivo compared to non-adjuvanted viruses.

[009] It has been shown that CD40L, encoded as part of MVA, is able to induce and enhance the overall T-cell response against a disease-associated antigen. WO 2014/037124. WO 2014/037124 showed that recombinant MVA encoding CD40L and a heterologous antigen was able to enhance DC activation in vivo , increase heterologous antigen-specific T cell responses, and increase the quality and quantity of CD8 T cells. Id.

[010] Over the past decade, the use of antibodies for cancer therapy has also achieved significant success. See, for example, Scott et al., Nature Reviews Cancer 12, 278-287 (2012). There are several antibody-based therapies that have been approved by the FDA and kill tumor cells in several ways. For example, antibody-based therapeutics can kill tumor cells through the direct action of the antibody, such as binding the antibody to a tumor antigen on the cell surface. Id . See, for example, Brodowicz et al., (2001). This can lead to apoptosis and death of tumor cells, as well as inhibition of the activity of tumor receptors. Prevention of receptor activity may include: prevention of tumor receptor dimerization, prevention of kinase activation, blocking of extracellular cleavage of receptors, induction of receptor internalization and subsequent signaling. Inhibition of tumor receptor activity by antibody-based therapeutic agents can prevent tumor proliferation. Id .

[011] Antibody-based therapeutics can further kill tumor cells by enhancing the cancer patient's own immune system to attack the tumor cells, which is referred to as immune-mediated tumor cell killing. Id. Immune-mediated killing of tumor cells may include phagocytosis, activation of the complement system, antibody-dependent cellular cytotoxicity (ADCC), antibody targeting of genetically modified T cells to the tumor, and inhibition of T cell inhibitor receptors such as CTLA-4. Id.

[012] ADCC is one of the most important pathways by which antibody therapeutics attack and destroy tumor cells. ADCC is triggered by activating the interaction between the target-bound antibody on the tumor cell membrane and the effector cells of the patient's immune system. Wang et al., NK Cell mediated Antibody Dependent Cellular Cytotoxicity in Cancer Immunotherapy Front Immunol vol. 6 (2015). The antitumor efficacy of many antibody-based therapeutics has been shown to be dependent on natural killer (NK) cells. Id. Human NK cells can express proteins that bind to the Fc portion of antibodies. Once bound and activated, NK cells mediate tumor killing in several ways, including exocytosis of cytotoxic granules, TNF family receptor signaling, and release of pro-inflammatory cytokines such as IFNγ. Id.

[013] Although there are successful options for cancer treatment based on poxviruses, chemotherapy and radiation therapy, and antibody-based therapeutics for cancer patients, there are many mechanisms that are used by tumor cells to evade and/or reduce the effect of these options. treatment. For example, to evade a particular patient's immune system, many tumor cells use immune checkpoint molecules and/or downregulate the specific expression of major histocompatibility complex (MHC) molecules so as to suppress and/or avoid detection by specific CD8 T cells of the immune system. Scott et al. (2012). Additionally, tumors have been shown to evade the patient's innate immune response by modifying or reducing the expression of tumor antigens on the surface of tumor cells, which can reduce both antibody binding and tumor killing by NK cells. Id.

[014] More recently, it has been found that tumor cells can evade the immune system and cancer therapy by entering an equilibrium phase with the immune system of the cancer patient. See Bhatia et al., Cancer Microenvironment vol. 4:209-217 (2011). In at least one aspect of the equilibrium phase, tumor cells may remain in the body below the limit of conventional morphological recognition or cytogenetic recognition.Id.For example, expression of tumor antigen receptors on tumor cells will fluctuate and, in many cases, drop to a point below the limit at which the immune system can recognize the tumor cells.Id.

[015] Given the ability of cancer and tumor cells to actively evade anti-cancer therapy and the patient's immune system, there is a significant need to develop anti-cancer treatments that effectively target and destroy tumor cells that actively evade the immune system. In addition, there is a need for anti-cancer treatments that can attack and destroy tumors and tumor cells that use the equilibrium phase to evade therapy and the immune system. In at least one aspect, various embodiments of the present invention successfully overcome the difficulties associated with treating tumor cells that actively evade the immune system.

BRIEF DESCRIPTION OF THE INVENTION

[016] In various embodiments of the present invention, it has been determined that recombinant MVA, intravenously administered to a patient in combination with an antibody against a tumor surface antigen, enhances the treatment of a cancer patient, in particular, increases the reduction in tumor volume and / or increases the survival of the cancer patient.

[017] Accordingly, in one embodiment, the present invention includes a pharmaceutical combination for use in reducing tumor size and/or improving survival of a cancer patient, wherein the pharmaceutical combination comprises: a) a recombinant modified vaccinia virus Ankara (MVA) comprising a first nucleic acid encoding the first heterologous tumor-associated antigen (TAA) that, when administered intravenously, induces both an enhanced natural killer (NK) cell response and an enhanced T cell response in a cancer patient compared to the NK cell and T cell response induced by non-intravenous administration of a recombinant MVA containing a nucleic acid encoding a heterologous tumor-associated antigen; and b) an antibody containing an Fc domain and being specific for an antigen that is expressed on the cell membrane of the tumor cell; wherein administration of a) and b) to a cancer patient reduces tumor size and/or improves survival of the cancer patient compared to non-intravenous administration of any of a) or b) alone. In additional embodiments, the recombinant MVA further comprises a second nucleic acid encoding a second heterologous TAA.

[018] In one or more preferred embodiments, the pharmaceutical combination further comprises CD40L. In the most preferred embodiment, CD40L is encoded by recombinant MVA.

[019] In various embodiments, the antibody is approved for the treatment of a cancer patient. In one more specific embodiment, the antibody is selected from the group consisting of: anti-CD20 (eg, rituximab; ofatumumab; tositumumab), anti-CD52 (eg, alemtuzumab Campat®), anti-EGFR (eg, cetuximab Erbitux®, panitumumab ), anti-CD2 (eg, ciplizumab), anti-CD37 (eg, BI836826), anti-CD123 (eg, JNJ-56022473), anti-CD30 (eg, XmAb2513), anti-CD38 (eg, daratumumab Darzalex®) , anti-PDL1 (eg, avelumab, atezolizumab, durvalumab), CTLA-4 (eg, ipilimumab), anti-GD2 (eg, 3F8, ch14.18, KW-2871, dinutuximab), anti-CEA, anti-MUC1, anti-FLT3, anti-CD19, anti-CD40, anti-SLAMF7, anti-CCR4, anti-B7-H3, anti-ICAM1, anti-CSF1R, anti-CA125 (eg oregovomab), anti-FRα (eg MOv18-IgG1 , mirvetuximab soravtansine (IMGN853), MORAb-202), anti-mesothelin (eg MORAb-009) and anti-HER2. In one more preferred embodiment, the antibody is an anti-HER2 antibody. In the most preferred embodiment, the antibody is an anti-HER2 antibody selected from pertuzumab, trastuzumab, Gerzuma, ABP 980, and ado-trastuzumab emtansine.

[020] In various additional embodiments, the first and/or second TAA contains one or more mutations to prevent the first and/or second TAA from binding and/or interacting with the combination therapy antibody. In one or more preferred embodiments, the first TAA is a HER2 antigen. In a more preferred embodiment, the HER2 antigen contains one or more mutations to prevent the first TAA from binding to the anti-HER2 antibody. In further preferred embodiments, the second TAA is a Brachyury antigen (brachyuria). In a more preferred embodiment, the Brachyury antigen contains one or more mutations in the nuclear localization signaling domain (SLD).

[021] In one or more preferred embodiments, the recombinant MVA is MVA-BN or a derivative thereof.

[022] In various embodiments, the present invention relates to one or more methods for reducing tumor size and/or improving the survival of a cancer patient. In one embodiment, a method is provided, comprising: a) intravenously administering to a cancer patient a recombinant MVA comprising a first nucleic acid encoding a first heterologous tumor-associated antigen (TAA) that, when administered intravenously, induces an enhanced natural killer (NK) cell response in a cancer patient. and enhanced T cell response compared to NK cell response and T cell response induced by non-intravenous administration of a recombinant MVA virus containing a nucleic acid encoding a heterologous tumor-associated antigen; and b) administering to the cancer patient an antibody comprising an Fc domain and which is specific for an antigen that is expressed on the cell membrane of the tumor cell; wherein administration of a) and b) to a cancer patient reduces tumor size in the cancer patient and/or improves survival of the cancer patient, as compared to non-intravenous administration of any of a) or b) alone.

[023] In one or more preferred embodiments, said method comprises administering CD40L intravenously to a cancer patient. In a more preferred embodiment, CD40L is encoded by recombinant MVA.

[024] In another embodiment, the implementation of the recombinant MVA of the present invention is administered simultaneously with or after the introduction of antibodies. In a more preferred embodiment, the recombinant MVA is administered after the antibody.

[025] In another embodiment, the present invention includes a method of enhancing antibody therapy in a cancer patient, comprising administering to the cancer patient a pharmaceutical combination of the present invention, wherein administering the pharmaceutical combination enhances antibody-dependent cell-mediated cytotoxicity (ADCC) induced by antibody-based therapy compared to using only antibody based therapy.

[026] In another embodiment, a method for inducing both an enhanced innate and an enhanced adaptive immune response in a cancer patient is provided, comprising administering a pharmaceutical combination of the present invention, wherein administering the pharmaceutical combination enhances both the innate and adaptive immune responses of the cancer patient compared to non-intravenous administration of the pharmaceutical combination or elements of the combination separately.

[027] In various additional embodiments, the present invention relates to one or more synthetic peptides and to nucleic acids encoding synthetic peptides. In more specific embodiments, a synthetic HER2 peptide and nucleic acid is provided. In more preferred embodiments, the synthetic HER2 peptide contains one or more mutations that prevent the HER2 peptide from binding to an anti-HER2 antibody, the antibody being preferably selected from pertuzumab, trastuzumab, Gerzuma, ABP 980, and ado-trastuzumab emtansine. In further preferred embodiments, the synthetic HER2 peptide contains one or more mutations that prevent extracellular dimerization, tyrosine kinase activity, and/or phosphorylation of the HER2 antigen.

[028] In another more specific embodiment, one or more synthetic peptides and one or more Brachyury nucleic acids are provided. In more preferred embodiments, the Brachyury synthetic peptides and nucleic acids contain one or more mutations in the nuclear localization signaling domain (SLD).

[029] Additional objects and advantages of this invention will be set forth in part in the description which follows, and in part will become apparent from the description, or may be learned by practice of this invention. The objects and advantages of this invention will be realized and achieved by means of the elements and combinations specifically set forth in the appended claims.

[030] The accompanying drawings, which are included and form part of this specification, illustrate one or more embodiments of this invention and, together with the description, serve to explain the principles of this invention.

BRIEF DESCRIPTION OF GRAPHICS

[031] In FIG. 1A-1G show that intravenous (IV) administration of MVA-OVA (rMVA) results in greater systemic activation of NK cells compared to subcutaneous (SC) administration. NK cell activation is further enhanced when MVA encodes CD40L (rMVA-CD40L). Illustrated are the results of Example 1, in which NK cell frequency staining and expression (given as geometric mean fluorescence intensity (GSIF)) of these protein markers in NKp46 ⁺ CD3 ^- cells were assessed in the spleen. A) NKp46 ⁺ CD3 ^- cells; B) CD69; C) NKG2D; D) FaSL; E); Bcl-X _L ; F), CD70; and G) IFN-γ.

[032] In FIG. 2A-2G show that iv administration of MVA-OVA (rMVA) results in greater systemic activation of NK cells compared to s.c. administration. NK cell activation is further enhanced when MVA encodes CD40L (rMVA-CD40L). Illustrated are the results of Example 1, in which NK cell frequency staining and expression (given as geometric mean fluorescence intensity (GSIF)) of these protein markers in NKp46 ⁺ CD3 ^- cells were assessed in the liver. A) NKp46 ⁺ CD3 ^- cells; B) CD69; C) NKG2D; D) FaSL; E); Bcl-X _L ; F), CD70; and G) IFN-γ.

[033] In FIG. 3A-3G show that iv administration of MVA-OVA (rMVA) results in greater systemic NK cell activation compared to s.c. administration. NK cell activation is further enhanced when MVA encodes CD40L (rMVA-CD40L). Illustrated are the results of Example 1, in which NK cell frequency staining and expression (given as geometric mean fluorescence intensity (GSIF)) of these protein markers in NKp46 ⁺ CD3 ^- cells were assessed in the lung. A) NKp46 ⁺ CD3 ^- cells; B) CD69; C) NKG2D; D) FaSL; E); Bcl-X _L ; F), CD70; and G) IFN-γ.

[034] In FIG. 4A-4F show that intravenous (IV) administration of MVA-HER2-Twist-CD40L results in greater systemic activation of NK cells compared to subcutaneous (SC) administration. Illustrated are the results of Example 1, in which NK cell frequency staining and expression (given as geometric mean fluorescence intensity (GSIF)) of these protein markers in NKp46 ⁺ CD3 ^- cells were assessed in the spleen. A) NKp46 ⁺ CD3 ^- cells; B) CD69; C) FaSL; D); Bcl-X _L ; E), CD70; and F) IFN-γ.

[035] In FIG. 5A-5F show that IV administration of MVA-HER2-Twist-CD40L results in greater systemic activation of NK cells compared to SC administration. Illustrated are the results of Example 1, in which NK cell frequency staining and expression (given as geometric mean fluorescence intensity (GSIF)) of these protein markers in NKp46 ⁺ CD3 ^- cells were assessed in the liver. A) NKp46 ⁺ CD3 ^- cells; B) CD69; C) FaSL; D); Bcl-X _L ; E), CD70; and F) IFN-γ.

[036] In FIG. 6A-6F show that iv administration of MVA-HER2-Twist-CD40L results in greater systemic NK cell activation compared to s.c. administration. Illustrated are the results of Example 1, in which NK cell frequency staining and expression (given as geometric mean fluorescence intensity (GSIF)) of these protein markers in NKp46 ⁺ CD3 ^- cells were assessed in the lung. A) NKp46 ⁺ CD3 ^- cells; B) CD69; C) FaSL; D); Bcl-X _L ; E), CD70; and F) IFN-γ.

[037] In FIG. 7A-7F show that iv administration of MVA-OVA-CD40L (rMVA-CD40L) results in increased levels of IL-12p70 and IFN-γ. The results of Example 2 are illustrated. A) IFN-γ concentration was higher after rMVA-CD40L compared to MVA-OVA (rMVA) immunization. B) The NK cell activating cytokine IL-12p70 was detectable only after immunization with MVA-CD40L. High serum levels of IFN-γ are consistent with a higher frequency of IFN-γ ⁺ NK cells (see Fig. 1G) and CD69 ⁺ granzyme B ⁺ NK cells in spleen C) after immunization with rMVA-CD40L. Similar responses have been observed in OCP (non-human primates) ( Macaca fascicularis ) following iv injection of MVA-MARV-GP-huCD40L (rMVA-CD40L), namely higher serum concentrations of IFN-γ (D) and IL-12p40 /70 (E), as well as more proliferating (Ki67 ⁺ ) NK cells (F) compared to MVA-MARV-GP (rMVA).

[038] In FIG. 8A-8C illustrate the time course of NK cell activation and proliferation. Illustrated are the results of Example 3, in which NK cell frequency staining and expression of these protein markers in NKp46 ⁺ CD3 ^- cells were assessed in spleen, liver, lung and blood. A) CD3 ^- CD19 ^- NKp46 ⁺ ) B) NK cell proliferation marker Ki67; and C) CD69 expression (given as geometric mean fluorescence intensity (GSIF)).

[039] In FIG. 9 illustrates ex vivo enhanced NK cell mediated toxicity after systemic immunization with MVA-OVA (rMVA) and MVA-CD40L (rMVA-CD40L). Spleen NK cells were purified and used as effectors in a kill assay as described in Example 4. NK cells were cultured with CFSE-labeled MHC class I deficient YAC-1 cells at the indicated ratios overnight. Specific killing was assessed by quantifying non-viable CFSE ⁺ YAC-1 cells by flow cytometry.

[040] In FIG. 10A-10E illustrate in vivo and ex vivo enhanced antibody-dependent cellular cytotoxicity (ADCC) following systemic immunization with MVA-OVA (rMVA), MVA-CD40L (rMVA-CD40L), and MVA-HER2-Twist-CD40L as described in Example 5. A ) C57BL/6 mice were treated i.v. with 25 µg anti-CD4, rMVA + 5 µg rat IgG2b, 1 µg anti-CD4 or MVA-OVA (rMVA) + 1 µg anti-CD4. Analyzed the depletion of CD4 T-cells (CD3 ⁺ CD4 ⁺ ) in the liver and gave it as a percentage of specific destruction. To assess ADCC activity of NK cells ex vivo , B) C57BL/6 or C) Balb/c mice were immunized iv with PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L). E) Balb/c mice were immunized iv with PBS or MVA-HER2-Twist-CD40L. Spleen NK cells were purified and used as effectors in an antibody-dependent killing assay. B) B16.F10 cells were coated with mouse anti-human/mouse Trp1 mAb (clone TA99) and C) CT26-HER2 cells were coated with mouse anti-human HER2 mAb (clone 7.16.4). Purified NK cells were added to antibody-coated target cells at a ratio of 5:1 and 4:1, respectively. D) CT26-HER2 cells that were incubated with different concentrations of anti-human HER2 antibody were also more efficiently killed by rMVA-CD40L activated NK cells compared to rMVA activated NK cells. E) CT26-HER2 cells were coated with various concentrations of mouse anti-human HER2 mAb (clone 7.16.4). Purified NK cells were added to antibody-coated target cells in a 5:1 ratio.

[041] In FIG. 11 shows that iv immunization induces stronger CD8 T cell responses than s/c immunization. As described in Example 6, C57BL/6 mice were immunized SC or IV with MVA-OVA on days 0 and 15. Responses of OVA-specific CD8 T cells in blood were assessed after staining with H-2K ^b /OVA _257-264 dextramers.

[042] In FIG. 12 shows that CD8 T cell responses can be further enhanced with MVA-CD40L. As described in Example 7, C57BL/6 mice were immunized iv with MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L) on days 0 and 35. Responses of OVA-specific CD8 T cells in blood were assessed after staining with H-2K ^b /OVA _257-264 dextramers.

[043] In FIG. 13A-13B illustrate NK cell reactivation and proliferation after primary/booster immunization. As described in Example 8, C57BL/6 mice were immunized iv with PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L) as shown in Table 1. NK cells (NKp46 ⁺ CD3 ^- ) were analyzed in blood by flow cytometry one and four days after the second and third immunizations. A) Illustrates the GSIF (Geometric Mean Fluorescence Intensity) of CD69, and B) Illustrates the frequency of Ki67 ⁺ NK cells.

[044] In FIG. 14A-14M illustrate systemic cytokine responses after primary/booster immunization. As described in Example 9, C57BL/6 mice were immunized iv with PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L) as shown in Table 1. Serum cytokine levels were measured 6 hours after immunization . Illustrated results for A) IL-6; B) CXCL10; C) IFN-α; D) IL-22; E) IFN-γ; F) CXCL1; G) CCL4; H) CCL7); I) CCL2; J) CCL5; K) TNF-α; L) IL-12p70; and M) IL-18.

[045] In FIG. 15A-15D illustrate strong antigen-specific CD8 T cell responses after primary/booster immunizations with MVA and MVA-CD40L. As described in Example 10, C57BL/6 mice were immunized iv with PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L) as shown in Table 1. The induction of antigen-specific CD8 T- cells after booster immunization. A) assessed the frequency of CD8 T cells; B) assessed B8-specific responses of CD8 T cells, C) assessed transgene-specific (OVA) responses; and D) ratios of OVA/B8-specific CD8 T cells were assessed.

[046] In FIG. 16A-16B illustrate the induction of CD8 and CD4 effector T cells after primary/booster immunization with MVA and MVA-CD40L. As described in Example 11, C57BL/6 mice were immunized iv with PBS, MVA-OVA (rMVA), or MVA-OVA-CD40L (rMVA-CD40L) as shown in Table 1. Phenotypically, effector T cells were identified by expression CD44 and the absence of superficial CD62L. Tracked A) CD44 ⁺ CD62L ^- CD8 T cells and B) CD4 T cells in the blood.

[047] In FIG. 17A-17B illustrate the superior antitumor effect of iv immunization with rMVA-CD40L in a heterologous prime-boost regimen in a melanoma model. C57BL/6 mice bearing palpable B16.OVA tumors were primed (dotted line) with PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L) s.c. or i.v. as described in Example 12 Mice received subsequent booster injections of FPV-OVA 7 and 14 days after priming (dash-dot line). Tumor growth was measured at regular intervals. Illustrated are A) mean tumor volume and B) survival of tumor-bearing mice at day 45 after tumor inoculation.

[048] In FIG. 18 illustrates effective tumor suppression after a single IV immunization with MVA-OVA-CD40L (rMVA-CD40L). C57BL/6 mice bearing palpable B16.OVA tumors were primed i.v. or received i.v. primary and booster injections as described in Example 13. Tumor growth was measured at regular intervals. The average volume of the tumor is given.

[049] In FIG. 19A-19C show that CD8 T cells play an essential role in rMVA-CD40L mediated tumor suppression. C57BL/6 mice bearing palpable B16.OVA tumors were immunized iv with PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L) as described in Example 14. Where indicated, mice received 200 μg anti-CD8 antibodies intraperitoneally (ip). A) CD8 T cell responses were measured. B) responses of OVA-specific CD8 T cells were measured. C) represents overall survival.

[050] In FIG. 20A-20C show that simultaneously targeting two TAAs is more effective than targeting just one. C57BL/6 mice bearing palpable B16.OVA tumors were immunized iv with PBS, MVA-OVA-CD40L, MVA-OVA-TRP2, or MVA-OVA-TRP2-CD40L as described in Example 15. Tumor growth was measured using equal intervals and were presented as A) average diameter for an individual mouse; and B) medium volume. C) Illustrated overall survival.

[051] In FIG. 21A-21G illustrate increased T cell infiltration into the tumor microenvironment (TMO) following rMVA-CD40L immunization. C57BL/6 mice bearing palpable B16.OVA tumors were immunized iv with PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L) as described in Example 16. The mice were sacrificed seven days later. A) the frequency of CD8 ⁺ T cells among CD45 ⁺ leukocytes in the spleen, tumor-draining lymph nodes (DONA) and tumor tissues; B) distribution of OVA _257-264 -specific CD8 ⁺ T cells in different organs after immunization; C) GSIF for PD-1 and Lag3 on tumor-infiltrating OVA _257-264 -specific CD8 ⁺ T cells; D) Representative dot plots of tumor-infiltrating CD8 ⁺ T cells showing Ki67 and PD-1 expression; E) frequency of tumor-infiltrating Ki67 ⁺ CD8 ⁺ T cells and GSIF for PD-1; F) the frequency of tumor-infiltrating regulatory T cells (Treg) among CD45 ⁺ leukocytes; and G) PD-1 ^{high frequency.} - and PD-1 ^neg. -tumor-infiltrating Treg.

[052] In FIG. 22A-22F illustrate the persistence of OAA-specific CD8 T cells with a less depleted phenotype in OMO following immunization with rMVA-CD40L. Purified OVA-specific TCR transgenic CD8 T cells (OT-I) were transferred iv to B16.OVA tumor-bearing mice when tumors became palpable as described in Example 17. When tumors were at least 60 mm ³ in volume, animals were iv immunized with MVA-BN®, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L). After 17 days, mice were sacrificed and analyzed for: A) frequency of CD8 ⁺ T cells among leukocytes in tumor tissues; B) frequencies of Lag3 ⁺ PD1 ⁺ among CD8+ T cells; C) frequencies of Eomes ⁺ PD1 ⁺ T cells among CD8+ T cells; D) the presence of OT-I transgenic CD8 ⁺ T cells in OMO after immunization; E) frequency of Lag3 ⁺ PD1 ⁺ depleted T cells among OT-I ⁺ CD8 ⁺ T cells; and F) frequency of Eomes ⁺ PD1 ⁺ T cells depleted among OT-I ⁺ CD8 ⁺ T cells.

[053] In FIG. 23A-23B illustrate the long-term decline in regulatory T cells (Treg) in OMO after immunization with rMVA-CD40L. Purified OVA-specific TCR transgenic CD8 T cells (OT-I) were transferred intravenously to tumor-bearing B16.OVA mice when tumors became palpable as described in Example 18. When tumors were at least 60 mm ³ in volume, animals were iv immunized with MVA-BN®, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L). After 17 days, mice were sacrificed for additional analysis: A) the frequency of Foxp3 ⁺ CD4 ⁺ Treg among CD4 ⁺ T cells in tumor tissues; B) ratio of CD8 ⁺ CD44 ⁺ effector T cells (Teff) to Foxp3 ⁺ CD4 ⁺ Tregs.

[054] In FIG. 24 illustrates lower serum levels of alanine aminotransferase (ALT) after rMVA and rMVA-CD40L immunization compared to anti-CD40 immunoglobulin injection. C57BL/6 mice were injected intravenously with MVA-OVA (rMVA), MVA-OVA-CD40L (rMVA-CD40L) or anti-CD40 (FGK4.5) as in Example 19. Serum ALT concentration was analyzed one day after each immunization by ELISA.

[055] In FIG. 25A-25B illustrate extended survival in a colon cancer model following iv immunization with rMVA-CD40L. Balb/c mice bearing palpable CT26.HER2 tumors were immunized iv with MVA-AH1A5-p15-Trp2-CD40L (rMVA-CD40L) or received PBS as described in Example 20. Tumor growth was measured at regular intervals . A) mean tumor volume and B) survival are shown.

[056] In FIG. 26A-26B illustrate the enhanced antitumor effect of rMVA-CD40L in combination with anti-Trp1. C57BL/6 mice bearing palpable B16.OVA tumors were immunized iv with MVA-OVA-CD40L (rMVA-CD40L) on day 8 as described in Example 21. Where indicated, starting on day 5 twice a week iv 200 μg of anti-Trp1 (clone TA99) was injected. A) mean tumor volume and B) overall survival are shown.

[057] In FIG. 27A-27D illustrate transgenic expression of MVA-HER2-Brachyury-CD40L. HeLa cells were left untreated (control; solid gray line) or infected with MVA-BN (solid black line) or MVA-HER2-Brachyury-CD40L (open black line) as described in Example 23. Protein expression was then determined A) MVA, B ) HER2, C) Brachyury and D) CD40L by flow cytometry (see histograms).

[058] In FIG. 28 shows that the anti-HER2 antibodies Herceptin® (also known as trastuzumab) and Perjeta® (also known as pertuzumab) do not bind to the modified HER2 sequence described in Example 24. CT26 cells were infected with MVA-HER2-Brachyury-CD40L at multiplicity of infection (MOH) 1. After 24 hours, cells were incubated with 5 μg/ml of anti-HER2 antibody Herceptin®, Perjeta® or 24D2 and analyzed by flow cytometry.

[059] In FIG. 29A-29D illustrate dose-dependent and enhanced human DC activation by MVA-HER2-brachyury-CD40L versus MVA-HER2-brachyury. Monocytic DCs were generated after enrichment of CD14 ⁺ monocytes from human PBMCs and cultured for 7 days in the presence of GM-CSF (GM-CSF) and IL-4 as described in Example 25. DCs were stimulated with MVA-HER2-brachyury or MVA-HER2-brachyury- CD40L. Expression A) CD40L; B) CD86; and C) MHC class II was measured by flow cytometry. D) IL-12p70 concentration was quantified.

[060] In FIG. 30A and 30B illustrate the increased antitumor effect of IV immunization with MVA-HER2-Twist-CD40L compared to IV immunization with MVA-CD40L in a HER2-positive colon carcinoma model. C57BL/6 mice bearing palpable MC38.HER2 tumors were iv immunized (dashed line) with PBS, MVA-CD40L, or MVA-HER2-Twist-CD40L as described in Example 26. Tumor growth was measured at regular intervals. A) mean tumor volume and B) overall survival are shown.

[061] In FIG. 31A and 31B illustrate the increased antitumor effect of IV immunization with MVA-HER2-Twist-CD40L compared with IV immunization with MVA-CD40L in a HER2-positive colon carcinoma model. Balb/c mice bearing palpable CT26.HER2 tumors were iv immunized (dashed line) with PBS, MVA-CD40L, or MVA-HER2-Twist-CD40L as described in Example 26. Tumor growth was measured at regular intervals. A) mean tumor volume and B) overall survival are shown.

[062] In FIG. 32 illustrates increased infiltration of HER2-specific CD8 ⁺ T cells producing IFN-γ in the tumor microenvironment after iv immunization with MVA-HER2-Twist-CD40L. Balb/c mice bearing palpable CT26.HER2 tumors were intravenously immunized with PBS or MVA-HER2-Twist-CD40L as described in Example 27. Seven days later, splenic and tumor-infiltrating CD8 ⁺ T cells were isolated by magnetic cell sorting. and cultured in the presence of HER2 peptide-loaded dendritic cells for 5 hours. The graphs show the percentage of CD44 ⁺ IFN-γ ⁺ cells among CD8 ⁺ T cells.

[063] In FIG. 33A and 33B illustrate the enhanced antitumor effect of MVA-HER2-Twist-CD40L and trastuzumab (anti-HER2). Balb/c mice carrying mature 17-day CT26.HER2 tumors were iv immunized with MVA-HER2-Twist-CD40L (dashed line) as described in Example 28. Where indicated, injected ip (dash-dotted line) 5 mcg anti-HER2 or huIgG1. A) mean tumor volume and B) overall survival are shown.

DETAILED DESCRIPTION OF THE INVENTION

[064] It should be understood that both the above summary of the invention and the following detailed description are merely illustrative and explanatory and do not limit the claimed invention.

[065] Antibody-dependent cellular cytotoxicity (ADCC) is a cell-mediated immune defense mechanism that allows the immune system to actively lyse and destroy target cells that express an antigen or receptor that has been bound by specific antibodies. See Hashimoto et al., Journal of Infectious Disease, 148: 785-794 (1983). ADCC uses natural killer (NK) cells that interact with antibodies to lyse and kill target cells. Id. With this in mind, over the past decade, therapeutic monoclonal antibodies have been developed that target tumor-associated antigens and enhance ADCC and thus the ability to kill tumor cells. See, for example, Scott et al. (2012) and Kohrt et al. Although antibody-based therapy has been shown to be effective in increasing tumor cell killing by ADCC, it has been shown that tumor cells can evade ADCC and the immune system through a number of mechanisms, such as heterogeneity in tumor antigen expression (e.g., decreased expression of tumor antigens on the surface of tumor cells). ), antibody stability, low concentration of antibodies relative to receptors, saturation of receptors, immune suppression, for example, by regulatory T cells, immune surveillance evasion, and NK cell dysfunction. Scott et al. (2012). Some aspects of tumor cell evasion have been described as the entry of tumor cells into an "equilibrium phase" with the patient's immune system, whereby tumor cells can remain in the body below the threshold of traditional morphological, cytological, or immune cell recognition. Bhatia et al., 2011.

[066] To induce synergistic antitumor responses, various pharmaceutical combinations of the present invention have been developed. In several aspects, various pharmaceutical combinations induce both highly potent tumor-specific killer T cells and natural killer (NK) cells that are capable of killing tumor cells coated with antibodies against tumor-expressed receptors via ADCC. In preferred aspects, as described herein, intravenous injection of recombinant MVA encoding CD40L induces enhanced NK cell activation and dramatically increases the killing of antibody-coated tumor cells. This enhanced NK cell activation, in combination with the enhanced killer T cell response also induced by MVA, has been shown to act synergistically in therapeutic antitumor vaccination.

[067] In various additional aspects, embodiments of the present invention induce an immune response that is characterized by enhanced and/or increased ability to target and kill those tumor and cancer cells that evade the patient's immune system. In additional aspects, embodiments of the present invention induce an immune response that enhances and/or enhances the effectiveness of both the patient's innate and adaptive immune responses. The ability to enhance and/or increase both innate and adaptive immune responses provides an exceptional advantage, as the present invention allows for the targeting and killing of those tumor cells that evade and/or suppress the patient's adaptive immune response, as well as those tumor cells that which evade and/or suppress the patient's innate immune response.

[068] In one embodiment, the present invention is a combination therapy comprising: a) intravenous (IV) administration of a recombinant MVA that contains a nucleic acid encoding one or more heterologous tumor-associated antigens (TAAs), and b) an antibody that contains an Fc domain and is specific for the antigen expressed on the tumor cell. In another embodiment, the combination therapy further comprises iv administration of CD40L. In a preferred embodiment, CD40L is encoded by recombinant MVA.

[069] The pharmaceutical combination and/or combination therapy of the present invention described and illustrated herein enhances a number of aspects of the cancer patient's immune response. In at least one aspect, the pharmaceutical combination enhances both innate and adaptive immune responses in a synergistic manner to reduce tumor volume and improve cancer patient survival. One or more enhanced effects of the pharmaceutical combination and/or therapy are summarized below.

[070] In/in the introduction of recombinant MVA enhances the response of NK cells. In one aspect, the present invention includes intravenous administration of recombinant MVA to a subject, wherein the intravenous administration induces an enhanced innate immune response, in particular, an enhanced NK cell response in the subject compared to the NK cell response induced by non-intravenous administration of recombinant MVA to the subject. As illustrated in FIG. 1-9 and 13, i.v. administration of recombinant MVA induced a strong systemic NK cell response in several compartments, both when administered intravenously and intravenously as a homologous prime-boost injection, compared with non-injection. / in the introduction.

[071] As illustrated in FIG. 1-6, the quality of the NK cell response was improved compared to non-i.v. administration. The CD69 activation marker is elevated in all organs (spleen, liver and lungs). Levels of a member of the anti-apoptotic Bcl family, Bcl-X _L , which enhances NK cell survival, co-stimulatory CD70, and effector cytokine IFN-γ, were elevated in both the spleen and lungs. Expression of the natural killer cell-activating group 2D receptor (NKG2D) was particularly elevated in the liver and lungs after IV compared with SC injection. NKG2D binds to ligands on tumor cells, promoting their elimination (Garcia-Cuesta et al., 2015, reviewed in Spear et al., 2013).

[072] IV administration of recombinant MVA encoding CD40L further enhances the response of NK cells. In another aspect of the present invention, it has been determined that i.v. administration of the CD40L antigen in addition to recombinant MVA further enhances the NK cell response compared to i.v. administration of recombinant MVA alone. As illustrated in FIG. 1-9 and 13, recombinant MVA encoding the CD40L antigen induced a stronger NK cell response compared to recombinant MVA without CD40L both when administered alone and as a homologous prime boost injection. In addition, the quality of the NK cell response was improved compared to i.v. administration of recombinant MVA alone. Increased expression by NK cells of the effector cytokine IFN-γ was observed in all analyzed organs (Fig. 1-6, spleen, liver, lungs), as was the expression of CD69 by NK cells in all analyzed organs (Fig. 5C). In addition, in FIG. 7 illustrates elevated serum levels of IFN-γ 6 hours after iv immunization with rMVA-CD40L compared to recombinant MVA and, more importantly, NK-activating cytokine IL-12p70, in both mice and OCP. Additionally, enhanced NK cell proliferation exhibited by Ki67 expression was systematically observed not only in the peripheral blood of mice (FIG. 7B), but also in OCP (FIG. 6F). These results indicate that iv immunization with rMVA-CD40L compared with rMVA improves NK cell quality in several animal research models.

[073] Although recombinant MVA viruses have previously been administered intravenously, see e.g. WO2002/42480, WO2014/037124, it was previously thought that administration of and treatment with recombinant MVA was associated with increased adaptive immune responses such as CD8 T cell responses. . For example, in WO2002/42480, CTL responses were measured following MVA immunizations by iv administration of 10 ⁷ PFU of MVA-BN per mouse or subcutaneous administration of 10 ⁷ PFU or 10 ⁸ PFU of MVA-BN per mouse. In WO2014/037124, mice were intravenously inoculated with recombinant MVA and recombinant MVA encoding CD40L. See WO2014/037124. CTL responses were enhanced and it was determined that the increased immunogenicity of recombinant MVA-CD40L was independent of CD4 ⁺ T cells, but dependent on CD40 in the host.

[074] In at least one aspect, the enhanced NK cell response observed in the present invention is unexpected, as it has been shown in the art that MVA-induced NK cell activation is dependent on intrinsic lymph node CD169-positive subcapsular macrophages. sinus (SCS) after subcutaneous immunization (Garcia et al. Blood 2012).

[075] IV administration of recombinant MVA enhances ADCC of tumor cells. In a further aspect, the present invention includes a recombinant MVA comprising a nucleic acid encoding one or more heterologous antigens, wherein i.v. administration to a subject enhances the ADCC response in the subject compared to the ADCC response when non-i.v. administered the recombinant MVA to the subject. In a more specific aspect, enhancement of the ADCC response resulting from intravenous administration of recombinant MVA is characterized by an increase in the ability of NK cells to target and kill antibody-coated tumor cells. As illustrated in FIG. 10A-10D, tumor cell lysis representing mouse anti-human HER2 mAb or mouse anti-human/mouse Trp1 mAb was increased for NK cells activated by iv administration of rMVA. Cell lysis of tumor cells was further enhanced for NK cells activated with recombinant MVA together with CD40L compared to rMVA without CD40L.

[076] As illustrated in FIG. 10D and 10E, enhancement of the ADCC response in a subject is further characterized as an increase in the efficiency of the NK cell response such that the NK cells are able to target and kill tumor cells for which the concentration of antibody associated with the tumor antigen (antibody-tumor antigen) is lower. The ability to target and kill tumor cells at reduced concentrations of antibody-coated tumor antigen provides a unique advantage, as one of the mechanisms by which tumor cells have been shown to evade the immune system is to reduce the expression of tumor antigens (see Scott et al. . (2012)); which reduces the concentration of antibodies - tumor antigens on tumor cells.

[077] IV administration of recombinant MVA enhances NK cell killing of tumor cells having low levels of MHC. In a further aspect illustrated in FIG. 9, enhanced NK cell activation and NK cell response lead to enhanced killing of tumor cells having low levels of MHC. This aspect provides an exceptional advantage, since the destruction of tumor cells with low levels of MHC by NK cells occurs independently of ADCC, thus providing an additional route for tumor cells to attack. This provides an additional benefit as many tumors and/or cancers reduce MHC expression levels in an attempt to evade the patient's immune responses.

[078] In/in the introduction of recombinant MVA increases the activation and proliferation of NK cells. In another aspect of the present invention, recombinant MVA is administered via a number of booster injections, resulting in increased activation and proliferation of NK cells, as shown in FIG. 10A through increased expression of CD69. On FIG. 13B illustrates increased proliferative capacity of blood NK cells by Ki67 expression 24 hours after booster iv immunizations compared to non-iv immunizations. This effect was observed when recombinant MVA priming was followed by recombinant MVA booster, when recombinant MVA-CD40L injection was followed by rMVA-CD40L booster, or when rMVA priming was followed by rMVA-CD40L booster.

[079] In at least one aspect of the present invention, enhanced NK cell responses following repeated iv administration of recombinant MVA and recombinant MVA-CD40L were unexpected. It was unexpected to observe increased activation and proliferation of NK cells 24 hours after booster IV immunizations in the absence of an increase in IFN-α levels. Indeed, NK cell activation and priming in secondary infections and cancers has been known to be heavily controlled by IFN-α. See, for example, Stackaruk et al., Expert Rev Vaccines. 2013 Aug;12(8):875-84; and Muller et al., Front Immunol (2017). Surprisingly, no increase in serum IFN-α levels was observed 6 hours after iv booster immunizations with rMVA hom, rMVA-CD40L hom, or rMVA-CD40L het (FIG. 14C). Together, repeated homologous or heterologous IV immunizations with rMVA containing a nucleic acid encoding one or more heterologous antigens resulted in unexpected activation and proliferation of IFN-α-independent NK cells.

[080] IV administration of recombinant MVA encoding a heterologous antigen enhances the adaptive immune responses of a cancer patient. In another aspect of the present invention, there is provided a recombinant MVA comprising a nucleic acid encoding one or more heterologous antigens, wherein intravenous administration to a subject enhances the subject's adaptive immune response to the one or more heterologous antigens. In a preferred aspect, the recombinant MVA further encodes for CD40L. As illustrated in FIG. 11-12, IV administration of recombinant MVA expressing a heterologous antigen elicited a stronger CTL response compared to subcutaneous (SC) administration. Also, as illustrated in the figures, when CD40L is included as part of recombinant MVA, i.v. administration of recombinant MVA and antigen elicited a stronger CTL response compared to i.v. administration of recombinant MVA without CD40L.

[081] Additionally, as illustrated in FIG. 14-16, when recombinant MVA encoding a heterologous antigen and CD40L was administered intravenously, the quality of the T cell response was improved after booster IV immunizations. On FIG. 15 illustrates increased specific for MVA antigen, B8 (FIG. 15B) and heterologous antigen, OVA (FIG. 15C), CD8 T cell proliferation enhanced by booster IV immunizations. This effect is related to the expression profile of cytokines and chemokines observed in FIG. 14, which quantifies the T-cell activating cytokines CXCL10, IFN-γ, CXCL1, CCL4, CCL7, CCL2, CCL5, TNF-α, IL-12p70, and IL-18 in serum after IV booster injections. Accordingly, in FIG. 16A and 16B illustrate the expansion of memory T cells, a key characteristic of vaccines, following IV booster immunizations. The ratio of CD8 T cells specific for the heterologous antigen to MVA specific CD8 T cells increased with each immunization (FIG. 15D).

[082] Before the present invention, it was known that CD40L encoded by recombinant MVA can replace helper CD4 T cells (Lauterbach et al., 2013). No further effect of recombinant MVA-encoded CD40L on CD4 T cells was known. Surprisingly, we observed the proliferation of CD4 ⁺ T memory cells 25 days after primary immunization (Fig. 16B), corresponding to 4 days after booster IV immunization with rMVA-CD40L (rMVA-CD40L hom and rMVA-CD40L het) (day 21 , see table 1). This fact is confirmed by increased production of IL-22, an important cytokine indicative of helper T cell responses, which was quantified 6 hours after the IV booster in the MVA-CD40L hom and MVA-CD40L het groups (Fig. 14D). This unexpected observation is relevant for maintaining rMVA-CD40L memory responses. In addition, CD4 T cells can maintain tumor-specific CD8 T cells at the tumor site, avoid activation-induced cell death, and can also become cytotoxic themselves (reviewed in Kennedy and Celis, 2008; Knutson and Disis, 2005). These results are unexpected because other viral vectors, such as those based on adenovirus and herpes simplex virus, induce vector-specific immunity that reduces the induction of immune responses to vaccine-encoded antigens after booster immunization (Lauterbach et al., 2005; Pine et al. ., 2011).

[083] In / in the introduction of recombinant MVA simultaneously or after the introduction of antibodies increases the efficiency of the destruction of tumor cells. In additional aspects, the present invention provides one or more regimens for administering to a subject a pharmaceutical combination of the present invention. In at least one aspect, the regimens of the present invention enhance the efficacy of the pharmaceutical combination and/or therapy to enhance ADCC-mediated tumor cell killing. In one embodiment, the administration schedule for the pharmaceutical combination comprises a) administering an antibody as described herein, and b) simultaneously or after administration of the antibody, administering recombinant MVA of the present invention intravenously to a subject.

[084] In one advantageous aspect of the present invention, the administration of the recombinant MVA simultaneously with or after the antibody allows the administered antibody to bind tumor cells simultaneously or prior to the enhancement of the NK cell response resulting from the administration of the recombinant MVA. Accordingly, during a typical first step, an antibody is administered, which results in the binding of the antibody to the tumor or infected cells. During a typical second step, recombinant MVA is administered intravenously, which, as described herein, enhances and enhances the subject's NK cell response. The enhanced NK cell response then leads to aggressive targeting and killing of tumor cells having the bound antibody.

[085] In other aspects, the pharmaceutical combination of the present invention is administered as part of a homologous and/or heterologous prime-boost regimen. As illustrated in FIG. 11-16, a homologous and/or heterologous prime-boost regimen prolongs and re-activates enhanced NK cell responses as well as increases specific CD8 and CD4 T cell responses of the subject.

[086] In/in the introduction of recombinant MVA enhances antitumor effects. In another aspect of the present invention, there is provided a recombinant MVA comprising a nucleic acid encoding one or more heterologous antigens, wherein i.v. administration to a subject results in an increase in the survival rate of the subject, as well as a decrease in overall tumor volume compared to non-i.v. administration. In a preferred aspect, the recombinant MVA further encodes for CD40L. As illustrated in FIG. 17 and 18, i.v. administration of recombinant MVA resulted in a greater overall survival rate and greater reduction in tumor volume or longer suppression of tumor growth compared to sc administration. It is also illustrated that CD40L included as part of recombinant MVA resulted in improved overall survival and reduced tumor volume compared to iv administration of recombinant MVA without CD40L. As illustrated in FIG. 19, CD40L included as part of recombinant MVA increased the total accumulation of CD8 and antigen (OVA)-specific T cells in the peripheral blood of tumor-bearing animals compared to iv administration of recombinant MVA without CD40L. In addition, CD8 T cells are important mediators of the antitumor effect of rMVA-CD40L, as antibody-mediated depletion of CD8 cells results in a lack of prolongation of overall survival.

[087] In / in the introduction of MVA, encoding two heterologous antigens, increases antitumor efficacy. In another aspect of the present invention, illustrated in FIG. 291, IV immunization with MVA encoding two heterologous antigens (OVA and TRP2) induces prolongation of tumor growth suppression and improves overall survival. CD40L included as part of recombinant MVA encoding two heterologous antigens enhances the antitumor response compared to iv administration of recombinant MVA with CD40L and only one heterologous antigen. Since malignancies use a variety of mechanisms to evade immunological surveillance, such as a decrease in the expression of tumor antigens (Jensen et al., 2012), encoding two or more tumor antigens in a vaccine limits the development of mechanisms to evade the antitumor immune response.

[088]In/in the introduction of MVA reduces the immunosuppressive effects of the tumor. As illustrated in FIG. 29 and 32, intravenously administered recombinant MVA encoding a heterologous antigen and optionally CD40L induced CD8 infiltration.⁺ T cells into the tumor and reduced some of the immunosuppressive effects typically used by tumors to evade the immune system. In addition to increasing the number of endogenous CD8 cells⁺ in tumors after stimulation with recombinant MVA with or without CD40L, there was an increased number of antigen (OVA)-specific T cells in the spleen and tumors after i.v. administration of recombinant MVA with CD40L compared with MVA without CD40L. In addition, tumor-infiltrating T lymphocytes expressed less immunosuppressive PD-1 and Lag3 surface molecules when tested in endogenous T cells (Fig. 22C-E, 23B) or antigen-transferred (OVA)-specific T cells (Fig. 21D). ) and proliferated in the tumor microenvironment. Decreased expression of PD-1 on tumor-infiltrating T cells after immunization with rMVA and rMVA-CD40L was unexpected since type I and II interferons, which are induced by both vectors (Fig. 14C and E), are known inducers of PD-1 and PD expression. -L1 (reviewed in Dong et al., 2017). Unexpectedly, the number of immunosuppressive regulatory T cells (Treg) in the tumor microenvironment was reduced (22F, 23A) by administration of recombinant MVA encoding a heterologous antigen and optionally CD40L, resulting in an increase in the ratio of effector T cells to Tregs in the tumor microenvironment (Fig. .23B). Decreased expression of immune-attenuating molecules such as PD-1 and Lag3, as well as reduced Tregs in tumor tissue correlate with enhanced antitumor effects observed after immunization with rMVA and rMVA-CD40L.

[089] The pharmaceutical combination of the present invention reduces tumor burden and improves survival in cancer patients. In various embodiments, the pharmaceutical combination comprises a) IV administration of a recombinant MVA encoding a heterologous TAA and optionally CD40L, and b) administration of an antibody. As illustrated in FIG. 26 and 33, the pharmaceutical combination results in a reduction in tumor volume and an increase in overall survival.

[090] In at least one aspect, the enhanced antitumor effects of the pharmaceutical combination (e.g., reduced tumor volume and/or increased survival rate) are provided by a synergistic combination of the individual effects of enhancing innate and adaptive T cell responses described herein. In one exemplary embodiment, these individual enhancing effects include one or more of the above, e.g., enhanced innate response (e.g., NK cells), enhanced ADCC-mediated tumor cell killing, enhanced NK cell killing of tumor cells having lower levels of the MHC class I, and enhanced adaptive T cell response. In addition, one or more dosing regimens of the present invention further improve and enable the destruction of tumor cells by the patient's immune system over a period of time.

[091] IV administration of MVA-HER2-Brachyury plus anti-HER2 antibodies. In one preferred embodiment, the pharmaceutical combination comprises a) intravenous administration of recombinant MVA encoding HER2 and Brachyury antigens and optionally CD40L, and b) administration of an anti-HER2 antibody. This embodiment induces the enhanced immune response described herein and further focuses on killing tumor cells expressing HER2 and Brachyury. In additional advantageous aspects, the HER2 antigen contains one or more modifications that further enhance the effectiveness of the combination therapy of the present invention. As illustrated in FIG. 29, rMVA-HER2-Brachyury and rMVA-HER2-Brachyury-CD40L demonstrated dendritic cell (DC) activation. DC activation was enhanced by rMVA-HER2-Brachyury-CD40L compared to rMVA-HER2-Brachyury. As illustrated in FIG. 33, administration of the pharmaceutical combination of the present invention resulted in a significant increase in tumor volume reduction and overall survival of subjects.

[092] In one specific embodiment, the present invention relates to and is adapted to the treatment of cancer patients with HER2-expressing malignancies, such as HER2-positive breast or gastric cancers, who are treated with HER2-binding antibodies. The recombinant MVA encoding HER2 induces highly effective killer T cells against HER2 expressing tumor cells, while the Brachyury transgene encoded by recombinant MVA induces highly effective killer T cells against Brachyury expressing tumor cells that have the potential to be metastatic.

[093] In the context of this document, singular forms include references to plural forms, unless the context clearly implies otherwise. Thus, for example, reference to "nucleic acid" includes one or more nucleic acids, and reference to "method" includes reference to equivalent steps and methods known to those skilled in the art that may modify or replace the methods described herein. .

[094] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element of the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, the existence of numerous equivalents to the particular embodiments of the invention described herein. It is implied that such equivalents are covered by the scope of the present invention.

[095] In the text of this description and the following claims, unless the context otherwise requires, the word "comprise" and its variations, such as "comprises" and "comprising", should be understood as the inclusion of the specified integer or step or group integers or steps, but not to the exclusion of any other integer or step or group of integers or steps. In the context of this document, the term "comprising" may be replaced by the term "including" or sometimes, in the context of this document, the term "having". Any of the above terms (comprising, including, having), although less preferred, in each case when used herein in the context of an aspect of an embodiment of the present invention, may be replaced by the term "consisting of". In the context of this document, the term "consisting of" excludes any element, step or ingredient not listed in the claim element. In the context of this document, the term "consisting predominantly of" does not exclude materials or steps that do not significantly affect the essential and new characteristics of the claim.

[096] In the context of this document, the connecting term "and/or" between a plurality of enumerated elements should be understood as including both separate and combined options. For example, if two elements are connected "and/or", the first option refers to the use of the first element without the second. The second option refers to the use of the second element without the first. The third option refers to the use of the first and second elements together. It is understood that any of these options corresponds to the meaning and, accordingly, satisfies the requirement of the term "and/or" in the context of this document. It is also understood that the simultaneous use of more than one of these options corresponds to the meaning and, accordingly, satisfies the requirement of the term "and/or".

[097] A "mutation" as defined herein is any modification in a nucleic acid or amino acid, such as deletions, additions, insertions, and/or substitutions.

[098] As used herein, a "host cell" is a cell into which a foreign molecule, virus, or microorganism has been introduced. In one non-limiting example described herein, a cell from a suitable cell culture, such as, for example, CEF cells, can be infected with a poxvirus or, in other alternative embodiments, with a plasmid vector containing a foreign or heterologous gene. Thus, suitable host cells and cell cultures serve as a host for the poxvirus and/or the foreign or heterologous gene.

[099] "Percentage (%) sequence homology or identity" with respect to the nucleic acid sequences described herein is defined as the percentage of nucleotides in a candidate sequence that are identical to nucleotides in a reference sequence (i.e., a nucleic acid sequence from which it is derived), after aligning the sequences and introducing gaps where necessary to achieve the maximum percentage of sequence identity and not counting any conservative substitutions as part of the sequence identity. Alignment to determine percent identity or homology of a nucleotide sequence can be performed in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR). Those skilled in the art can determine the appropriate parameters for determining the alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the compared sequences.

[0100] For example, Smith and Waterman's local homology algorithm provides appropriate alignment for nucleic acid sequences (Smith and Waterman, (1981), Advances in Applied Mathematics 2:482-489). This algorithm can be applied to amino acid sequences using the weight matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov (1986), Nucl. Acids Res. 14(6):6745-6763. An example implementation of this algorithm for determining percent sequence identity is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). The preferred method for determining percent identity in the context of the present invention is to use the MPSRCH software package, copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok and distributed by IntelliGenetics, Inc. (Mountain View, Calif). From this set of packages, the Smith-Waterman algorithm can be used, which uses the default parameters for the weight table (for example, a gap opening penalty of 12, a gap extension penalty of one, and a gap penalty of six). From the generated data, the "Match" value reflects "sequence identity". Other suitable programs for calculating percent identity or similarity between sequences are generally known in the art, for example another alignment program is BLAST used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code=standard; filter=none; chain=both; cutoff=60; wait=10; matrix=BLOSUM62; descriptions=50 sequences; sort by=HIGHEST RATE; databases=non-duplicate, GenBank+EMBL+DDBJ+PDB+GenBank CDS+Swiss protein+Spupdate+PIR transfers. Detailed information on these programs can be found at the following Internet address: blast.ncbi.nlm.nih.gov/.

[0101] The term “prime boost vaccination” or “prime boost schedule” refers to a vaccination strategy that uses a primary (priming) injection of a vaccine targeted to a particular antigen followed, at intervals, by one or more booster injections of the same vaccines. Prime boost vaccination can be homologous or heterologous. Homologous prime boost vaccination uses a vaccine containing the same antigen and vector for both the primary injection and one or more booster injections. A heterologous prime boost vaccine uses a vaccine containing the same antigen for both the primary injection and one or more booster injections, but different vectors for the primary injection and one or more booster injections. For example, a homologous prime boost vaccination may use a recombinant poxvirus containing nucleic acids expressing one or more antigens for a primary injection and the same recombinant poxvirus expressing one or more antigens for one or more booster injections. In contrast, a heterologous prime boost vaccination may use a recombinant poxvirus containing nucleic acids expressing one or more antigens for the primary injection and another recombinant poxvirus expressing one or more antigens for one or more booster injections.

[0102] The term "recombinant" means a polynucleotide, virus, or vector of semi-synthetic or synthetic origin, which either does not exist in nature or is linked to another polynucleotide in an order not found in nature.

[0103] In the context of this document, the reduction or reduction in tumor volume can be characterized as a decrease in tumor volume and/or size, but can also be characterized in terms of the end results of a clinical study known in the art. Some typical clinical trial endpoints associated with reduction in tumor volume and/or size may include, but are not limited to, response rate (RR), objective response rate (ORR), etc.

[0104] In the context of this document, the improvement in survival rate can be characterized as an increase in the survival of a cancer patient, but can also be characterized in terms of the end results of a clinical study known in the art. Some typical clinical trial outcomes associated with improved survival include, but are not limited to, overall survival (OS), progression-free survival (PFS), etc.

[0105] As used herein, a "transgene" or "heterologous" gene is to be understood as a nucleotide or amino acid sequence that is not present in the genome of a wild-type poxvirus (eg, vaccinia virus, fowl pox virus, or MVA). One skilled in the art will understand that a "transgene" or "heterologous gene", if present in a poxvirus such as vaccinia, must be incorporated into the genome of the poxvirus such that, upon introduction of the recombinant poxvirus into a host cell, it is expressed as the corresponding heterologous gene product, i.e. as a "heterologous antigen" and/or "heterologous protein". Expression is usually achieved by operably linking the heterologous gene to regulatory elements that allow expression in a poxvirus-infected cell. Preferably, the regulatory elements include natural and synthetic poxvirus promoter.

[0106] "Vector" refers to a recombinant DNA or RNA plasmid or virus that may contain a heterologous polynucleotide. The heterologous polynucleotide may contain a sequence of interest for prevention or therapy purposes, and may optionally be in the form of an expression cassette. In the context of this document, the vector does not necessarily have to be capable of replication in the final target cell or body of the subject. This term includes cloning vectors and viral vectors.

Pharmaceutical combinations and methods

[0107] In various embodiments, the present invention includes a pharmaceutical combination for the treatment of a cancer patient by reducing tumor volume and/or improving the survival of a cancer patient. The pharmaceutical combination contains a recombinant MVA containing the first nucleic acid encoding the first heterologous tumor-associated antigen (TAA) and, when administered intravenously, induces both an enhanced natural killer (NK) cell response and an enhanced T cell response compared to the NK cell response. and a T cell response induced by non-intravenous administration of a recombinant MVA virus containing a nucleic acid encoding a heterologous tumor-associated antigen.

Enhanced NK cell response

[0108] In one aspect, an enhanced NK cell response as described herein is characterized by one or more of 1) an increase in NK cell frequency, 2) an increase in NK cell activation, and/or 3) an increase in NK cell proliferation. Thus, whether an enhanced NK cell response as described herein can be determined by measuring the expression of one or more cytokines indicative of an increase in NK cell frequency, an increase in NK cell activation, and/or an increase in NK cell proliferation. Exemplary markers useful in determining the frequency and/or activity of NK cells include one or more of: NKp46, IFN-γ, CD69, CD70, NKG2D, FasL, granzyme B, CD56 and/or Bcl-X _L . Exemplary markers useful in determining NK cell activation include one or more of IFN-γ, CD69, CD70, NKG2D, FasL, granzyme B, and/or Bcl-X _L . Exemplary markers useful in determining NK cell proliferation include Ki67. These molecules and their measurements are validated methods of analysis known in the art, which can be carried out in accordance with known techniques. See, for example, Borrego et al., Immunology 1999; Fogeln et al., 2013. In addition, analysis methods for measuring molecules can be found in examples 1-3 and 9 of this description. In at least one aspect, 1) an increase in the frequency of NK cells can be defined as at least a 2-fold increase in the number of CD3 ^- NKp46 ⁺ cells compared with the number before treatment/baseline; 2) increased NK cell activation can be defined as at least a 2-fold increase in expression of IFN-γ, CD69, CD70, NKG2D, FasL, granzyme B and/or Bcl-X _L compared to pre-treatment/baseline expression; and/or 3) increased NK cell proliferation can be defined as at least a 1.5-fold increase in Ki67 expression over pre-treatment/baseline expression.

[0109] In a more preferred aspect, as used herein, "enhanced NK cell response" is characterized by increased NK cell-mediated killing of tumor cells. NK cell-mediated killing of tumor cells can be analyzed by measuring the release of lactate dehydrogenase (LDH) into the cell culture medium as shown in Example 5. Thus, in the context of the present invention, whether or not a pharmaceutical combination enhances the "NK cell response" can be determine by the presence of an increase in NK cell-mediated killing of tumor cells. NK-mediated killing of tumor cells can be analyzed by measuring the release of lactate dehydrogenase, as shown in example 5. Assay methods related to LDH and its measurements are validated and known in the art. In at least one aspect, the increase in NK cell-mediated tumor cell killing can be defined as at least a 2-fold increase in tumor cell killing.

Enhanced ADCC response

[0110] In at least one aspect, the enhanced NK cell response induced by the present invention results in an enhanced ADCC response. The "enhanced ADCC response" according to the present invention is characterized by an increase in the ability of innate immune effector cells to target and kill antibody-coated tumor cells. As used herein, innate immune effectors may include, but are not limited to, NK cells, macrophages, neutrophils, basophils, eosinophils, mast cells, dendritic cells, etc. In a more preferred aspect, the innate immune effector is an NK cell. Assay methods applicable to determine the ability of effector cells to kill tumor cells includeex vivo effector:target kill assay, where "effector" is isolated effector cells and "target" is tumor cells in the presence of antibodies. See Yamashita et al., (2016) and Broussas et al. (2013). The ex vivo effector:target kill assay has been validated and is known in the art. In addition, assay methods for determining the ability of effector cells to kill tumor cells can be found in Example 5. Thus, in the context of the present application, whether or not enhanced innate immune response, including enhanced NK cell response, ADCC, can be determined by measuring levels killing tumor cells by one or more of: 1) applying different ratios of effector cells to coated target cells (tumor cells coated with antibodies); and 2) using a constant ratio of effector cells and one or more antibody concentrations, eg, lower concentrations of antibodies bound to a tumor antigen are compared to tumor cell killing rates at higher concentrations.

[0111] In a preferred aspect, an "enhanced ADCC response" as used herein is characterized by an increase in the ability of NK cells to target and kill antibody-coated tumor cells. Assay methods useful for determining the ability of NK cells to kill tumor cells include ex vivo effector:target kill assays, where "effector" is isolated NK cells and "target" is tumor cells in the presence of antibodies. In addition, assay methods for determining the ability of NK cells to kill tumor cells can be found in Example 5. Thus, in one aspect, in the context of the present application, whether or not an ADCC NK cell response is enhanced can be determined by measuring levels of tumor cell killing: 1) using different ratios of NK cells to coated target cells (tumor cells coated with antibodies); and 2) using a constant ratio of NK cells and one or more antibody concentrations, eg, lower concentrations of antibodies bound to a tumor antigen are compared to tumor cell killing rates at higher concentrations.

Stronger NK cell-mediated toxicity

[0112] In additional aspects, an enhanced NK cell response results in an increased ability of innate immune effectors such as NK cells to attack and kill tumor cells having low levels of MHC expression. In one aspect, killing of tumor cells having low levels of MHC can be characterized by innate immune effectors, such as NK cells having stronger NK cell-mediated toxicity, meaning that NK cells are more effective at killing tumor cells. NK cells with stronger NK cell-mediated toxicity can be determined by the number of tumor cells destroyed by the NK cells. This can be determined using an effector:target kill analysis. Such assays have been validated and are known in the art and are also demonstrated in this application at least in Example 4 and in FIG. 9.

Effector cells of enhanced innate immunity

[0113] In additional aspects, IV administration of the recombinant poxviruses of the present application not only enhances the response of NK cells, but also enhances all aspects of the innate immune response. An increase in the innate immune response can be characterized as an increase in 1) frequency, 2) activation and/or 3) proliferation of innate immune effector cells. Innate immune effector cells may include NK cells, innate immune lymphoid cells (ILCs), macrophages, neutrophils, basophils, eosinophils, mast cells, monocytes, and/or dendritic cells. In various aspects, whether the innate immune response is enhanced according to the present application can be determined by measuring one or more of the expression of cytokines and/or activation markers associated with the described innate response effector cells. These cytokines and activation markers and their measurements are validated and known in the art and can be performed according to known techniques. See, for example, Borrego et al., Immunology 1999; Fogeln et al., 2013.

Enhanced T cell response

[0114] In accordance with the present application, "enhanced T cell response" is characterized by one or more of the following: 1) an increase in the frequency of CD8 T cells; 2) increased activation of CD8 T cells; and/or 3) increased proliferation of CD8 T cells. Thus, whether a T cell response is enhanced according to the present application can be determined by measuring the expression of one or more cytokines indicative of 1) an increase in CD8 T cell frequency, 2) an increase in CD8 T cell activation, and/or 3 ) increase the proliferation of CD8 T cells. Exemplary markers useful for determining the frequency, activation and proliferation of CD8 T cells include CD3, CD8, IFN-γ, TNF-α, IL-2, CD69 and/or CD44 and Ki67, respectively. Measurement of the frequency of antigen-specific T cells can also be performed using ELIspot or MHC multimers such as pentamers or dextramers, as illustrated in FIG. 11, 12, 15, 19 and 21. Such measurements and methods of analysis are validated and known in the art.

[0115] In one aspect, the increase in the frequency of CD8 T cells is characterized by at least a 2-fold increase in the number of IFN-γ and/or dextramer ⁺ CD8 T cells compared to the number before treatment/baseline. The increase in CD8 T cell activation is characterized as at least a 2-fold increase in CD69 and/or CD44 expression compared to pre-treatment/baseline expression. The increase in CD8 T cell proliferation is characterized as at least a 2-fold increase in Ki67 expression compared to pre-treatment/baseline expression.

[0116] In an alternative aspect, an enhanced T cell response is characterized by an increase in CD8 T cell expression of effector cytokines and/or an increase in cytotoxic effector functions. An increase in the expression of effector cytokines can be determined by the expression of one or more of IFN-γ, TNF-α and/or IL-2 compared to pre-treatment/baseline expression. An increase in cytotoxic effector functions can be determined by the expression of one or more of CD107a, granzyme B and/or perforin and/or antigen-specific killing of target cells.

[0117] The assays, cytokines, markers, and molecules described herein, as well as their measurements, are validated and known in the art, and can be performed according to known techniques. In addition, assay methods for determining T cell responses can be found in Examples 6 and 7, to which T cell responses were analyzed.

[0118] The enhanced T cell response implemented by the present invention provides an exceptional advantage in combination with the enhanced NK cell response, as the enhanced T cell response effectively targets and kills those tumor cells that have evaded and/or survived the initial responses. innate immunity in a cancer patient. In addition, antibody treatment can enhance the presentation of MHC class I TAA, leading to greater susceptibility of TAA-expressing tumors to lysis by TAA-specific T cells (Kono et al., Clin Cancer Res, 2004).

[0119] In additional embodiments, the pharmaceutical combination further comprises an antibody specific for an antigen that is expressed or overexpressed on the cell membrane, preferably the outer cell membrane of the tumor cell. It is understood that the antibody may be any antibody described in the present invention. In preferred embodiments, the antibody contains an Fc domain.

[0120] In additional embodiments, a method for reducing tumor volume and/or improving survival in a cancer patient is provided. Said method comprises a) intravenously administering to a cancer patient a recombinant MVA containing a first nucleic acid encoding a first heterologous tumor-associated antigen (TAA) that, when administered intravenously, induces both an enhanced innate immune response and an enhanced T cell response compared to the innate immune response and a T cell response induced by non-intravenous administration of a recombinant MVA virus containing a nucleic acid encoding a heterologous tumor-associated antigen; and b) administering to the cancer patient an antibody comprising an Fc domain and being specific for an antigen that is expressed on the cell membrane of the tumor cell, wherein administration of a) and b) to the cancer patient reduces tumor size in the cancer patient and/or increases the cancer patient's survival rate by compared with non-intravenous administration a) or administration b) alone. In a more preferred embodiment, the enhanced innate immune response includes the enhanced NK cell response described herein.

[0121] In another embodiment, a method for enhancing antibody-based therapy in a patient is provided, comprising a) administering to an oncology patient an antibody comprising an Fc domain and being specific for an antigen that is expressed on the cell membrane of a tumor cell, wherein administration of the antibody induces ADCC ; and b) administering intravenously to a cancer patient a recombinant MVA comprising a first nucleic acid encoding a first heterologous tumor associated antigen (TAA) that induces both an enhanced innate immune response and an enhanced T cell response in the patient, wherein the enhanced innate immune response enhances ADCC in patient compared with non-intravenous administration of a combination of a) and b) or a) and b) alone. In a more preferred embodiment, the enhanced innate immune response includes the enhanced NK cell response described herein.

[0122] In another embodiment, a method for enhancing the efficacy of antibody-based therapy in a cancer patient is provided, comprising administering a pharmaceutical combination of the present invention, wherein administering the combination to the patient reduces the antibody concentration required for NK cell-mediated toxicity in tumor cells. In at least one advantageous aspect, reducing the concentration of antibodies required for NK cell-mediated toxicity allows for an enhanced NK cell response to kill tumor cells that have reduced extracellular antigen expression to evade bound antibody from being targeted. In addition, lowering the concentration of antibodies may allow treatment with smaller amounts of antibodies.

[0123] In the context of the present application, the decrease in the concentration of antibodies required for NK-mediated toxicity can be characterized as the decrease in the concentration of antibodies necessary to kill tumor cells. The reduction in antibody concentration required to kill tumor cells can be determined using an ex vivo effector:target kill assay, where "effector" is isolated NK cells and "target" is tumor cells in the presence of antibodies. In addition, assay methods for detection include measuring levels of tumor cell killing, for example, by 1) applying different ratios of NK cells to coated target cells (antibody coated tumor cells); and 2) using a constant ratio of NK cells and one or more antibody concentrations, eg, lower concentrations of antibodies bound to a tumor antigen are compared to tumor cell killing rates at higher concentrations. Such analysis methods are known in the art and are also described in Examples 5 and 31 of this application.

[0124] In additional embodiments, the pharmaceutical combination and methods described herein are for the treatment of a human cancer patient. In preferred embodiments, the cancer patient has and/or has been diagnosed with cancer selected from the group consisting of: breast cancer, lung cancer, head and neck cancer, thyroid cancer, melanoma, stomach cancer, bladder cancer, kidney cancer, liver cancer, melanoma, pancreatic cancer, prostate cancer, ovarian cancer, urothelial cancer, cervical cancer or colorectal cancer.

[0125] In additional preferred embodiments, pharmaceutical combinations and methods of the present invention contain CD40L. Preferably CD40L is encoded by recombinant MVA.

Typical tumor-associated antigens

[0126] In certain preferred embodiments, the first and/or second tumor associated antigen (TAA) includes, but is not limited to, HER2, PSA, PAP, CEA, MUC-1, survivin, TYRP1, TYRP2, or Brachyury (brachiuria) alone, or in combinations. Such exemplary combinations may contain HER2 and Brachyury, CEA and MUC-1, or PAP and PSA.

[0127] Numerous TAAs are known in the art. Typical TAAs include, but are not limited to, 5-alpha-reductase, alpha-fetoprotein, AM-1, APC, April, BAGE, beta-catenin, Bcl12, bcr-abl, Brachyury (brachyuria), CA-125, CASP- 8/FLICE, cathepsins, CD19, CD20, CD21, CD23, CD22, CD33 CD35, CD44, CD45, CD46, CD5, CD52, CD55, CD59, CDC27, CDK4, CEA, c-myc, Cox-2, DCC, DcR3 , E6/E7, CGFR, EMBP, Dna78, farnesyl transferase, FGF8b, FGF8a, FLK-1/KDR, folic acid receptor, G250, GAGE family, gastrin 17, gastrin-releasing hormone, GD2/GD3/GM2, GnRH, GnTV , GP1, gp100/Pmel17, gp-100-in4, gp15, gp75/TRP-1, hCG, heparanase, HER2/neu, HMTV, Hsp70, hTERT, IGFR1, IL-13R, iNOS, Ki67, KIAA0205, K-ras , H-ras, N-ras, KSA, LKLR-FUT, MAGE family, mammaglobin, MAP17, melan-A/MART-1, mesothelin, MIC A/B, MT-MMPs, mucin, NY-ESO-1, osteonectin, p15, P170/MDR1, p53, p97/melanotransferrin, PAI-1, PDGF, uPA, PRAME, probasin, progenipointin, PSA, PSM, RAGE-1, Rb, RCAS1, SART-1, SSX-family, STAT3, STn, TAG-72, TGF-alpha, TGF-beta, thymosin-beta-15, TNF-alpha, TYRP-, TYRP-2, tyrosinase, VEGF, ZAG, p16INK4 and glutathione S-transferase.

[0128] A preferred PSA antigen contains the amino acid substitution of isoleucine for leucine at U.S. position 155, as described in US Pat. No. 7,247,615, which is incorporated herein by reference.

[0129] In more preferred embodiments of the present invention, the first and/or second heterologous TAAs are selected from HER2 and Brachyury. In even more preferred embodiments, the first and/or second heterologous TAAs are selected from the HER2 and Brachyury synthetic proteins described herein.

[0130] In further preferred embodiments, the first and/or second heterologous TAAs contain one or more mutations. In at least one embodiment, the one or more mutations include mutations that prevent the antibodies of the present disclosure from binding to the first and/or second heterologous TAAs. In additional embodiments, the one or more mutations include mutations that prevent the first and/or second heterologous TAA from performing one or more of the normal cellular functions of the TAA. Exemplary embodiments of one or more mutations are described by the synthetic HER2 proteins and the synthetic Brachyury proteins of the present invention.

Synthetic HER2 proteins

[0131] In the course of working on the present invention, the inventors have determined that one or more modifications of the HER2 antigens and/or nucleic acids encoding HER2 antigens described herein increase the effectiveness of combination therapy.

[0132] Accordingly, in various embodiments, the present invention includes a nucleic acid encoding a HER2 antigen selected from HER2v1 and HER2v2. HER2v1 and HER2v2 contain SEQ ID NO: 1 and SEQ ID NO: 13, respectively.

[0133] In at least one aspect, the nucleic acid encoded by SEQ ID NO: 1 and/or 13 provides an exceptional advantage, since SEQ ID NO: 1 and/or 13 are designed to function synergistically with antibodies to the HER2 antigen when administration as part of combination therapy.

[0134] In one exemplary embodiment, anti-HER2 antibodies include those approved for the treatment of HER2-expressing cancer, or, in a more specific embodiment, HER2-expressing breast cancer. Two humanized monoclonal antibodies targeting HER2 have been developed and approved for the treatment of HER2-expressing breast cancer. Both antibodies bind to different regions of the extracellular domain of HER2. Binding of trastuzumab (sold under the brand name Herceptin® and having biosimilars of Gerzum and ABP 980) results in blocking signal transduction and preventing HER2 cleavage. In contrast, pertuzumab (Perjeta®) sterically blocks HER2 dimerization with other EGF receptors and blocks ligand-activated signaling. Both trastuzumab and pertuzumab provide dual blocking of HER2-gated signaling pathways and are additionally able to mediate ADCC against breast cancer. Thus, in various specific embodiments of the present invention, the antibodies of the present invention include trastuzumab, Gerzuma, ABP 980 and/or pertuzumab.

Synthetic HER2 v1

[0135] In one or more embodiments, the synthetic HER2 protein is HER2v1 (SEQ ID NO: 1). As previously described, to enhance the efficacy of combination therapy, recombinant MVA-encoded transgenes are mutated to minimize any potential interaction and/or binding between the transgene and the administered antibodies. Accordingly, one or more mutations in the HER2 antigen have been created to increase the efficacy of therapy involving the co-administration of an anti-HER2 antibody such as trastuzumab and pertuzumab. In particular, the corresponding antibody binding sites in HER2 were mutated. Thus, in one or more embodiments, the synthetic HER2 protein contains one or more mutations in the following amino acid domains: 579-625 (trastuzumab binding domain), 267-337 (pertuzumab binding domain), 274-288 (HER2 dimerization domain) , 721-987 (kinase domain) and 1139-1248 (phosphorylation domain). In one or more exemplary embodiments of the present invention, the HER2 antigen contains one or more of the following mutations: E580A, F595A, K615A, L317A, H318A, D277R, E280K, K753M, Y1023A. Mutations in the HER2v1 antigen (based on HER2 (NP_004439.2) are illustrated below.

Amino acid sequence of HER2 v1. The above synthetic HER is based on HER2 NP_004439.2. Mutated amino acids are underlined and crossed out. Amino acids 579-625 represent the Herceptin binding site. Amino acids 267-337 represent the Perjeta binding site. Amino acids 274-288 represent residues of the dimerization domain. Amino acids 721-987 represent residues of the kinase domain. Amino acids 1139-1248 represent residues in the phosphorylation domain. [SEQ ID NO: 1]

Synthetic HER2 v2

[0136] In one or more embodiments, the synthetic HER2 protein is HER2v2 (SEQ ID NO: 13). As previously described, to enhance the effectiveness of combination therapy, the transgenes encoded by the recombinant MVA are mutated to minimize any potential interaction and/or binding between the transgene and the administered antibodies. Accordingly, one or more mutations in the HER2 antigen have been created to increase the efficacy of therapy involving the co-administration of an anti-HER2 antibody such as trastuzumab and pertuzumab. In particular, the corresponding antibody binding sites in HER2 were mutated. Thus, in one or more embodiments, the synthetic HER2 protein contains one or more mutations in the following amino acid domains: 579-625 (trastuzumab binding domain), 267-337 (pertuzumab binding domain), 274-288 (HER2 dimerization domain) , 721-987 (kinase domain) and 1139-1248 (phosphorylation domain). In one or more exemplary embodiments of the present invention, the HER2 antigen contains one or more of the following mutations: E580A, F595A, K615A, L317A, H318A, D277R, E280K, K753M, Y1023A.

[0137] In at least one aspect, the above mutations enhance combination therapy since their function is to minimize any interaction and/or unwanted binding of the MVA-HER2 antigen with the anti-HER2 antibodies trastuzumab and pertuzumab. The interaction of trastuzumab with HER2 involves 3 loops in the near-membrane region of HER2, formed by amino acids 579-583 (loop 1), 592-595 (loop 2), and 615-625 (loop 3) [6]. See, for example, H. S. Cho, “Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab,” Nature, 2003. These loops contain several key residues for HER2-trastuzumab binding, including E580, D582, P594 , F595, K615, Q624S. Satyanarayanajois, “Design, Synthesis, and Docking Studies of Peptidomimetics based on HER2-Herceptin Binding Site with Potential Antiproliferative Activity Against Breast Cancer Cell lines,” Chem Biol Drug Des, 2009.

[0138] Accordingly, in one embodiment, the HER2 antigen of the present invention contains one or more mutations in residues that affect the binding of the HER2 antigen to trastuzumab. Some type residues include, but are not limited to, E580, D582, P594, F595, K615, and Q624. Thus, in a more specific embodiment, the HER2 antigen of the present invention contains one or more mutations in residues selected from the group consisting of E580, D582, P594, F595, K615, Q624, and combinations thereof. In an even more specific embodiment, the HER2 antigen of the present invention contains one or more mutations in residues selected from E580, F595 and K615. In another specific embodiment, one or more mutations in the HER2 antigen include Ala substitutions in E580, F595 and K615 (eg, E580A, F595A and K615A).

[0139] In another exemplary embodiment, the HER2 antigen contains one or more mutations in the pertuzumab binding domain. Pertuzumab binds near a loop in the dimerization domain (domain II) of HER2, including key residues H267, Y274, S310, L317, H318 and K333. Mutations on the binding surface greatly reduce Perjeta's binding to HER2. M. C. Franklin, “Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complex,” Cancer Cell, 2004. Additional amino acid residues in this binding domain are potentially involved in the pertuzumab-HER2 interaction, including F279, V308, and P337.

[0140] Accordingly, in one embodiment, the HER2 antigen of the present invention contains one or more mutations in residues that affect the binding of the HER2 antigen to pertuzumab. In a more specific embodiment, the HER2 antigen of the present invention contains one or more mutations in residues selected from the group consisting of H267, F279, V308, S310, L317, H318, K333 and P337 and combinations thereof. In another specific embodiment, one or more mutations in the HER2 antigen include Ala substitutions in H267, F279, V308, S310, L317, H318, K333, and P337 (e.g., H267A, F279A, V308A, S310A, L317A, H318A, K333A, and P337A) .

[0141] In at least one aspect of the present invention, it has been suggested that if HER2 dimerization is minimized, this minimizes interaction and binding between the MVA-encoded HER2 and other members of the HER family, such as EGFR, HER3, or HER4, expressed by the transduced MVA cell. Minimization of HER dimerization would provide an exceptional advantage, since HER2 realizes its oncogenic potential through intracellular signaling initiated by dimerization. Accordingly, in one embodiment, the HER2 antigen of the present invention contains one or more mutations in residues that affect HER2 dimerization. Some type residues include, but are not limited to, L317, H318, D277, and E280. Thus, in a more specific embodiment, the HER2 antigen of the present invention contains one or more mutations in residues selected from the group consisting of L317, H318, D277, E280, and combinations thereof. In a more specific embodiment, the HER2 antigen of the present invention contains one or more mutations in residues selected from D277, E280. In another specific embodiment, one or more mutations in the HER2 antigen include an Arg substitution at D277 (D277R) and a Lys substitution at E280 (E280K).

[0142] In additional aspects, it has been suggested that if tyrosine kinase activity is minimized, this minimizes the subsequent activation of cellular signals that can induce cell proliferation and angiogenesis. Accordingly, in one embodiment, the HER2 antigen of the present invention contains one or more mutations in residues that affect the activity of HER2 tyrosine kinase. A typical residue includes, but is not limited to, K753. Thus, in a more specific embodiment, the HER2 antigen of the present invention contains a mutation at residue K753. In a more specific embodiment, the HER2 antigen of the present invention contains a Met substitution at residue K753 (K753M).

[0143] In additional aspects, it has been suggested that if potential phosphorylation sites are removed, this minimizes the subsequent activation of cell signals that can induce cell proliferation and angiogenesis. Accordingly, in one embodiment, the HER2 antigen of the present invention contains one or more mutations in residues that affect potential phosphorylation sites in HER2. Some exemplary residues include, but are not limited to, amino acids 1139 to 1248 and Y1023. Thus, in a more specific embodiment, the HER2 antigen of the present invention contains one or more mutations at residues selected from amino acids 1139 to 1248, Y1023, and combinations thereof. In a more specific embodiment, the HER2 antigen of the present invention comprises a deletion of amino acids 1139 to 1248 and/or a Y1023 to Ala (Y1023A) substitution.

[0144] In one preferred embodiment, the HER2 antigen contains one or more mutations in the pertuzumab binding domain, trastuzumab binding domain, dimerization domain, kinase domain, and/or phosphorylation domain found in HER2. Typical mutations of the HER2 antigen (based on NP_004439.2) in the previously described domains are illustrated below as synthetic HER2v2.

Amino acid sequence of HER2_v2. (SEQ ID: NO 13) The above synthetic HER_version2 is based on HER2 NP_004439.2. Mutated amino acids are underlined and crossed out. Amino acids 579-625 represent the trastuzumab binding site. Amino acids 267-337 represent the binding site for pertuzumab. Amino acids 274-288 represent residues of the dimerization domain. Amino acids 721-987 represent residues of the kinase domain. Amino acids 1139-1248 represent residues in the phosphorylation domain.

[0145] In accordance with the advantages defined herein, in various embodiments, the present invention includes a synthetic HER2 antigen in which one or more amino acids of HER2 are mutated to prevent binding of the HER2 antigen to an anti-HER2 antibody. In a more preferred embodiment, the synthetic HER2 antigen is mutated to prevent binding of an antibody selected from pertuzumab, trastuzumab, and ado-trastuzumab emtansine.

[0146] In other embodiments, the synthetic HER2 antigen contains one or more mutations to prevent extracellular dimerization, tyrosine kinase activity, and/or phosphorylation of the HER2 antigen (after rMVA expression). In further specific embodiments, the synthetic HER2 antigen contains one or more mutations in at least one of the 3 loops of the peri-membrane region of HER2.

[0147] In various additional embodiments, the present invention includes a nucleic acid encoding a HER2 antigen comprising SEQ ID NO: 1. In additional embodiments, the present invention includes a nucleic acid encoding a HER2 antigen having at least 90%, 95%, 97 %, 98% or 99% identity with SEQ ID NO:1.

[0148] In one embodiment, the invention includes a nucleic acid comprising SEQ ID NO:2 that encodes a HER2 antigen of SEQ ID NO:1. In further embodiments, the invention includes a nucleic acid encoding a HER2 antigen, wherein the nucleic acid shares at least 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:2.

[0149] In various additional embodiments, the present invention includes a nucleic acid encoding a HER2 antigen comprising SEQ ID NO: 13. In additional embodiments, the present invention includes a nucleic acid encoding a HER2 antigen having at least 90%, 95%, 97 %, 98% or 99% identity with SEQ ID NO:13.

[0150] In one embodiment, the invention includes a nucleic acid comprising SEQ ID NO:14 that encodes a HER2 antigen of SEQ ID NO:13. In further embodiments, the present invention includes a nucleic acid encoding a HER2 antigen, wherein the nucleic acid shares at least 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:14.

Synthetic Brachyury Proteins

[0151] As previously illustrated, the addition of a second disease/tumor-associated antigen encoded by MVA enhanced the antitumor response/activity of the combination therapy. Accordingly, in various embodiments of the invention, the recombinant MVA further encodes a Brachyury antigen (brachiuria).

[0152] The inventors have determined that one or more modifications of the Brachyury antigens and/or nucleic acids encoding the Brachyury antigens described herein enhance the efficacy of the combination therapy. In particular, one or more mutations have been introduced into the nuclear localization signal (NLS) domain to minimize and/or prevent any nuclear localization of the Brachyury antigen by the host cell.

[0153] Accordingly, in various embodiments, the synthetic Brachyury polypeptide contains one or more mutations to prevent nuclear localization of Brachyury. "Nuclear localization" can be defined as localization and/or transfer to the nucleus. Conducting an assay to determine whether one or more mutations prevent the nuclear localization of the Brachyury antigen is within the skill of the art. Such assay methods may include immunofluorescence assay and immunofluorescence microscopy.

[0154] In more specific embodiments, the synthetic Brachyury polypeptide contains one or more mutations in the SNL domain. In a more specific and preferred embodiment, amino acids 286-293 of the Brachyury antigen (GenBank reference NP_003172.1) are removed from the synthetic Brachyury polypeptide as shown below.

Amino acid sequence of a typical modified Brachyury. Residues of the conserved core motif RSSPYPSP in the potential NLS of Brachyury have been removed (shown with a strikethrough) [SEQ ID NO:3]

[0155] In more specific embodiments, the present invention includes a nucleic acid encoding a Brachyury antigen selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7. In a preferred aspect, various embodiments of the present invention include a nucleic acid encoding SEQ ID NO:3.

[0156] In at least one specific aspect, the nucleic acid encoded by SEQ ID NO: 3 provides an exceptional advantage, since SEQ ID NO: 3 is designed to prevent or minimize the localization and/or transfer of the MVA-encoded Brachyury antigen to a core in which the possibility of unwanted transcriptional activity may exist.

[0157] In accordance with certain advantages of combination therapy, in various aspects, embodiments of the present invention include a nucleic acid encoding a Brachyury antigen containing SEQ ID NO: 3. In additional aspects, embodiments of the present invention include a nucleic acid encoding a Brachyury antigen having at least 90%, 95%, 97%, 98% or 99% identity with SEQ ID NO:3.

[0158] In additional aspects, embodiments of the present invention include a nucleic acid comprising SEQ ID NO:4 that encodes a Brachyury antigen of SEQ ID NO:3. In additional aspects, embodiments of the present invention include a nucleic acid encoding a Brachyury antigen, wherein the nucleic acid has at least 90%, 95%, 97%, 98% or 99% identity with SEQ ID NO:4.

CD40L

[0159] As illustrated herein, the inclusion of CD40L as part of a pharmaceutical combination and the associated method further enhance tumor volume reduction, prolong progression-free survival, and increase survival rates realized by the present invention. Thus, in various embodiments, the pharmaceutical combination further comprises administering CD40L to a cancer patient.

[0160] Although CD40 is constitutively expressed in many cell types, including B cells, macrophages, and dendritic cells, its CD40L ligand is predominantly expressed on activated helper T cells. The cognate interaction between dendritic cells and helper T cells at early time points after infection or immunization licenses dendritic cells to prime CTL responses. Dendritic cell licensing results in increased expression of co-stimulatory molecules, improved survival, and better cross-presentation ability. This process is mediated mainly by the CD40/CD40L interaction. This describes various configurations of CD40L, from membrane-bound to soluble (monomeric or trimeric), which induce a variety of stimuli that induce or suppress the activation, proliferation and differentiation of APC.

[0161] As illustrated by the results of Example 19, MVA-encoded CD40L is a much safer alternative for the cancer patient because MVA-encoded CD40L has reduced toxicity compared to a soluble CD40 agonist.

[0162] In one or more preferred embodiments, CD40L is encoded by the MVA of the present invention. In more preferred embodiments, CD40L contains a nucleic acid encoding SEQ ID NO: 11. In more preferred embodiments, CD40L contains a nucleic acid having at least 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO: :12. In the most preferred embodiment, CD40L contains SEQ ID NO:12.

Generic antibodies

[0163] In various embodiments, the pharmaceutical combination and related methods comprise an antibody specific for an antigen that is expressed on the cell membrane of a tumor cell. It is known in the art that in many types of cancer, one or more antigens are expressed or overexpressed on the tumor cell membrane. See for example Durig et al., 2002; Mocellin et al., 2013; Arteaga 2017; Fin 2017; Ginaldi et al., 1998. Assay methods for determining whether an antigen is expressed or overexpressed on tumor cells are well known in the art (ID), as well as methods for obtaining antibodies to a specific antigen.

[0164] In more specific embodiments, the pharmaceutical combination and related methods comprise an antibody, wherein the antibody is a) specific for an antigen that is expressed on the cell membrane of the tumor cell, and b) contains an Fc domain. In at least one aspect, the characteristics of the antibody (e.g., a) and b)) allow the antibody to bind and interact with an effector cell, such as an NK cell, a macrophage, a basophil, a neutrophil, an eosinophil, monocytes, mast cells, and/or dendritic cells. and allow the antibody to bind to a tumor antigen that is expressed on the tumor cell. In a preferred embodiment, the antibody contains an Fc domain. In a further preferred embodiment, the antibody is capable of binding to and interacting with an NK cell.

[0165] Some exemplary antibodies to antigens expressed on tumor cells provided by the present invention include, but are not limited to, anti-CD20 (e.g., rituximab; ofatumumab; tositumomab), anti-CD52 (e.g., alemtuzumab Campat®), anti- -EGFR (eg cetuximab Erbitux®, panitumumab), anti-CD2 (eg ciplizumab), anti-CD37 (eg BI836826), anti-CD123 (eg JNJ-56022473), anti-CD30 (eg XmAb2513), anti-CD38 (eg, daratumumab Darzalex®), anti-PDL1 (eg, avelumab, atezolizumab, durvalumab), anti-GD2 (eg, 3F8, ch14.18, KW-2871, dinutuximab), anti-CEA, anti-MUC1 , anti-FLT3, anti-CD19, anti-CD40, anti-SLAMF7, anti-CCR4, anti-B7-H3, anti-ICAM1, anti-CSF1R, anti-CA125 (eg oregovomab), anti-FRα (eg MOv18- IgG1, mirvetuximab soravtansine (IMGN853), MORAb-202), anti-mesothelin (eg MORAb-009) and anti-HER2 (eg trastuzumab, Gerzuma, ABP 980 and/or pertuzumab).

[0166] In a more preferred embodiment, an antibody included as part of the present invention includes an antibody that, when administered to a patient, binds to an appropriate antigen on a tumor cell and induces antibody-dependent cell-mediated cytotoxicity (ADCC). In an even more preferred embodiment, the antibody comprises an antibody that is approved or is under pre-approval for the treatment of cancer.

[0167] In even more preferred embodiments, the antibody is an anti-HER2 antibody. In the most preferred embodiment, the antibody is selected from pertuzumab, trastuzumab, Gerzuma, ABP 980, and ado-trastuzumab emtansine.

[0168] In additional embodiments, the antibody comprises a fusion of one or more antibodies and/or antibody fragments. Exemplary fusion antibodies and/or antibody fragments include, but are not limited to, bispecific killer cell attracting antibodies (BiKE) and trispecific killer cell attracting antibodies (TriKE). Demonstrated by Tay et al., (2016), BiKE and TriKE are known to effectively control the antitumor activity of NK cells and enable NK cell-mediated ADCC. See Tay et al., (2016). It is contemplated that 161533 TriKE and/or 1633 BiKE can be used as an antibody in the present invention. It is further contemplated that the antibodies of the present invention may be implemented as bispecific, killer cell-attracting antibodies or BiTE. See, for example, Huehls et al. Immunol Cell Biol. 2015 March, 93: 290-296.

Recombinant poxviruses

[0169] In one or more aspects of the present invention, the nucleotide and protein sequences of the present invention may be incorporated into recombinant poxviruses.

[0170] In various embodiments of the present invention, the recombinant poxvirus is preferably an orthopoxvirus, such as, without limitation, vaccinia virus, modified vaccinia virus Ankara (MVA), MVA-BN, or a derivative of MVA-BN.

[0171] Examples of vaccinia strains include Temple of Heaven, Copenhagen, Paris, Budapest, Dairen, Gam, MRIVP, Per, Tashkent, TBK, Tom, Bern, Patwadangar, BIEM, B-15, Lister, EM-63, New York City Board of Health, Elstree, Ikeda and WR. The preferred strain of vaccinia virus (VV - from the English. "vaccinia virus") is a strain of Wyeth (DRYVAX) (US patent 7410644).

Recombinant MVA

[0172] In more preferred embodiments of the present invention, one or more of the proteins or nucleotides described herein are included in a recombinant MVA. As described and illustrated herein, intravenous administration of the recombinant MVAs of the present invention induces, in various aspects, an enhanced immune response in cancer patients. Thus, in one or more preferred embodiments, the invention includes a recombinant MVA comprising one or more nucleic acids encoding HER2, Brachyury and/or CD40L described herein. In more preferred embodiments, the recombinant MVA comprises one or more nucleic acids encoding a synthetic HER2 antigen and a synthetic Brachyury antigen described herein. In another more preferred embodiment, the recombinant MVA contains one or more nucleic acids encoding the HER2 synthetic antigen and the Brachyury synthetic antigen described herein, as well as CD40L. In another more preferred embodiment, the recombinant MVA contains one or more nucleic acids encoding SEQ ID NO: 1 or SEQ ID NO: 13 (Her2) and SEQ ID NO: 3 (Brachyury). In another preferred embodiment, the recombinant MVA contains one or more nucleic acids encoding SEQ ID NO: 1 or SEQ ID NO: 13, SEQ ID NO: 3, and CD40L. In a more preferred embodiment, the recombinant MVA contains one or more nucleic acids encoding SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11, and/or SEQ ID NO: 13. In the most preferred embodiment, the recombinant MVA contains one or more nucleic acids encoding SEQ ID NO:13, SEQ ID NO:3 and SEQ ID NO:11.

[0173] In additional embodiments, the recombinant MVA comprises one or more nucleic acids selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and/or SEQ ID NO: 14. In another more preferred embodiment, the recombinant MVA contains one or more nucleic acids selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and CD40L. In another preferred embodiment, the recombinant MVA contains one or more nucleic acids selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 12. In a more preferred embodiment, the recombinant MVA contains SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 12. In the most preferred embodiment, the implementation of the recombinant MVA contains SEQ ID NO: 14, SEQ ID NO: 4 and SEQ ID NO: 12.

[0174] Examples of strains of the MVA virus that can be used in the practice of the present invention and which have been deposited in accordance with the requirements of the Budapest Treaty are strains MVA 572 deposited with the European Collection of Animal Cell Cultures (ECACC), Vaccine Research and Production Laboratory, Public Health Laboratory Service, Center for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom, deposit number ECACC 94012707 January 27, 1994, and MVA 575 deposited with ECACC 00120707 December 7, 2000, MVA- BN, deposited on August 30, 2000 in the European Cell Culture Collection (ECACC) under the number V00083008, and their derivatives, as well as additional type strains.

[0175] "Derivatives" of MVA-BN refer to viruses showing essentially the same replication characteristics as MVA-BN described herein, but showing differences in one or more parts of their genomes. MVA-BN, as well as its derivatives, are replication incompetent, meaning they are unable to replicate reproductively in vivo and in vitro . In particular, in vitro , MVA-BN or its derivatives have been described as capable of reproductive replication in chicken embryonic fibroblasts (CEF), but not reproductively replicating in the human keratinocyte cell line HaCat (Boukamp et al (1988), J. Cell Biol. 106:761-771), Human Bone Osteosarcoma Cell Line 143B (ECACC, Deposit No. 91112502), Human Embryonic Kidney Cell Line 293 (ECACC, Deposit No. 85120602), and Human Cervical Adenocarcinoma Cell Line HeLa (ATCC, Deposit No. CCL -2). In addition, MVA-BN or derivatives thereof have a viral amplification rate at least two times lower, more preferably three times lower than MVA-575 in Hela cells and HaCaT cell lines. Studies and analysis of these properties of MVA-BN and its derivatives are described in WO 02/42480 (US Patent Application No. 2003/0206926) and WO 03/048184 (US Patent Application No. 2006/0159699).

[0176] The term "incapable of reproductive replication" or "lack of reproductive replication capability" in human cell lines in vitro, as described in the previous paragraphs, is described, for example, in WO 02/42480, which also describes how to obtain MVA A that has the required properties as above. This term refers to a virus that has an in vitro viral amplification index 4 days post-infection of less than 1 as defined in the assay described in WO 02/42480 or US Pat. No. 6,761,893.

[0177] The term "failure to replicate" refers to a virus that has an amplification rate of the virus in human cell lines in vitro, as described in the previous paragraphs, 4 days after infection less than 1. Assay methods described in WO 02/42480 or in US Pat. No. 6,761,893 are applicable to determine the virus amplification rate.

[0178] Viral amplification or replication in in vitro human cell lines, as described in the previous paragraphs, is usually expressed as the ratio of virus obtained from an infected cell (yield) to the amount originally used to infect the cell in the first place (input), and called "amplification index". An amplification score of "1" defines an amplification status in which the amount of virus obtained from the infected cells is the same as the amount originally used to infect the cells, which means that the infected cells are susceptible to viral infection and reproduction. In contrast, a score less than 1, i.e. a decrease in the output compared to the input level, indicates a lack of reproductive replication and therefore a weakening of the virus.

[0179] In another embodiment, a recombinant poxvirus, including the synthetic nucleotides and proteins of the present invention, can be implemented in an avipoxvirus, such as, but not limited to, chicken poxvirus.

[0180] The term "avipoxvirus" refers to any avipoxvirus, such as chicken poxvirus, canary poxvirus, junco poxvirus, lane poxvirus, pigeon poxvirus, parrot poxvirus, quail poxvirus, peacock poxvirus, penguin poxvirus, sparrow poxvirus, starling poxvirus, and turkey poxvirus. Preferred avipoxviruses include canary poxvirus and chicken poxvirus.

[0181] An example of a canary poxvirus is the Rentschler strain. A plaque-purified canary poxvirus strain named ALVAC (US Patent No. 5,766,598) was deposited under the Budapest Treaty with the American Type Culture Collection (ATCC) under accession number VR-2547. Another strain of canary poxvirus is a commercial canary poxvirus vaccine strain designated LF2 CEP 524 24 10 75 available from Institute Merieux, Inc.

[0182] Examples of chicken poxvirus are strains FP-1, FP-5, TROVAC (US patent No. 5766598), POXVAC-TC (US patent 7410644), TBC-FPV (Therion Biologics-FPV), FP-1 is a Duvette strain modified for use as a vaccine for one-day-old chicks. This strain is a commercial chicken poxvirus vaccine strain designated O DCEP 25/CEP67/2309, October 1980, available from Institute Merieux, Inc. FP-5 is a commercial chicken poxvirus vaccine strain available from American Scientific Laboratories (Division of Schering Corp.) Madison, Wis., United States Veterinary License No. 165, serial no. 30321.

Expression cassettes/regulatory sequences

[0183] In various aspects, one or more of the nucleic acids described herein are implemented in one or more expression cassettes in which the one or more nucleic acids are operably linked to expression control sequences. "Operably linked" means that the described components are in a relationship that allows them to function in the intended way, for example, a promoter for transcription of the nucleic acid to be expressed. An expression control sequence operably linked to a coding sequence is linked to allow expression of the coding sequence under conditions compatible with the expression control sequence. Expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon at the beginning of the protein-coding open reading frame, splicing signals for introns, and in-frame stop codons. Suitable promoters include, but are not limited to, the SV40 early promoter, RSV promoter, retroviral PrEP, adenoviral major late promoter, human CMV immediate early promoter I, and various poxvirus promoters including, but not limited to, the following vaccinia virus promoters or derived from MVA and derived from FPV: 30K promoter, I3 promoter, PrS promoter, PrS5E promoter, Pr7.5K promoter, PrHyb promoter, Pr13.5 long promoter, 40K promoter, MVA-40K promoter, FPV 40K promoter, 30k promoter, PrSynIIm promoter, Promoter PrLE1 and PR1238 promoter. Additional promoters are described in WO 2010/060632, WO 2010/102822, WO 2013/189611, WO 2014/063832 and WO 2017/021776, which are incorporated herein by reference in their entirety.

[0184] Additional expression control sequences include, but are not limited to, leader sequences, termination codons, polyadenylation signals, and any other sequences necessary for proper transcription and subsequent translation of the nucleic acid sequence encoding the desired recombinant protein (e.g., HER2, Brachyury and/ or CD40L) in the required host system. The poxvirus vector may also contain additional elements necessary for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the desired host system. One of skill in the art will further appreciate that such vectors are not difficult to construct using conventional methods (Ausubel et al., (1987) in "Current Protocols in Molecular Biology," John Wiley and Sons, New York, N.Y.), or that they are commercially available.

[0185] In certain embodiments, one or more recombinant MVAs of the present invention comprise one or more cytokines such as IL-2, IL-6, IL-12, IL-15, IL-7, IL-21 RANTES, GM- CSF, TNF-α or IFN-γ, one or more growth factors such as GM-CSF or G-CSF, one or more costimulatory molecules such as ICAM-1, LFA-3, CD72, B7-1, B7- 2 or other related B7 molecules; one or more molecules such as CD70, OX-40L or 4-1 BBL, or combinations of these molecules, which can be used as biological adjuvants (see, for example, Salgaller et al., 1998, J. Surg. Oncol. 68( 2):122-38;Lotze et al., 2000, Cancer J. Sci.Am.6(Suppl 1):S61-6;Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv Exp Med Biol 465:381-90). These molecules can be administered systemically (or topically) to the host. In a few examples, IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1 B7-2, CD70, OX -40L, 4-1 BBL and ICAM-1.

Methods and dosing regimens for the administration of a pharmaceutical combination

[0186] In additional aspects, the present invention provides one or more schemes for administering a pharmaceutical combination and/or applying the methods of the present invention. In at least one aspect, the regimens of the present invention increase the efficacy of a pharmaceutical combination and/or therapy to reduce tumor volume and improve the survival rate of a cancer patient.

[0187] Thus, in one embodiment, a pharmaceutical combination and/or method for reducing tumor size and/or improving survival of a cancer patient is provided, comprising administering to the cancer patient a pharmaceutical combination of the present invention, wherein the recombinant MVA is administered simultaneously or after administration of the antibody.

[0188] In one particular exemplary embodiment, a pharmaceutical combination and/or method for reducing tumor size and/or improving survival of a cancer patient is provided, comprising a) administering to the cancer patient an antibody comprising an Fc domain and specific for an antigen that is expressed on a cell membrane tumor cell; and b) administering intravenously to a cancer patient a modified Ankara vaccinia virus (MVA) containing a first nucleic acid encoding a first heterologous tumor-associated antigen (TAA) that, when administered intravenously, induces in the cancer patient both an enhanced natural killer (NK) cell response and enhanced response of cytotoxic T cells; wherein b) is administered simultaneously or after a).

[0189] In several specific embodiments, the recombinant MVA can be administered simultaneously with the antibody. In other specific embodiments, the recombinant MVA is administered after or following administration of the antibody. In various embodiments, the recombinant MVA is administered within 1 to 6 days after the antibody. In more preferred embodiments, the recombinant MVA is administered within 0 to 5 days, 0 to 4 days, 0 to 3 days, or 0-2 days after administration of the antibody. In the most preferred embodiment, the recombinant MVA is administered 0-3 days after antibody administration.

[0190] In various additional embodiments, the recombinant MVA is administered within 1 to 6 days after the antibody. In more preferred embodiments, the recombinant MVA is administered within 1 to 5 days, 1 to 4 days, 1 to 3 days, or 1 to 2 days after administration of the antibody. In the most preferred embodiment, the recombinant MVA is administered 1-3 days after antibody administration.

[0191] In one advantageous aspect of the present invention, the administration of the recombinant MVA simultaneously with or after the antibody allows the administered antibody to bind tumor cells simultaneously or prior to the enhancement of the NK cell response resulting from the administration of the recombinant MVA. Accordingly, during a typical first step, an antibody is administered, which results in the binding of the antibody to an antigen on the surface of one or more tumor cells. During a typical second step, recombinant MVA is administered intravenously, which, as described herein, enhances and/or enhances both the subject's innate immune response (eg, NK cell response) and T cell response. The enhanced innate immune response leads to aggressive targeting and killing of tumor cells having the bound antibody.

[0192] In another advantageous aspect, the regimens of the present invention are designed to effectively attack a tumor from both innate and adaptive immunity of a subject. In one exemplary aspect, the pharmaceutical combination is designed to administer recombinant MVA intravenously simultaneously with or after administration of an antibody, wherein the recombinant MVA induces an enhanced NK cell response and an innate immune response that attack and destroy tumor cells having the bound antibody. During the period when the innate immune response destroys antibody-coated tumor cells, recombinant MVA also induces a specific T cell response (including both CD8 and CD4 T cells) in the patient. The scheme described is designed so that when the innate immune response, such as NK cells, is naturally reduced, the enhanced response of specific CD8/CD4 T cells will kill the tumor cells. The developed scheme provides an exceptional advantage, since an enhanced response of specific CD8/CD4 T cells can destroy those tumor cells that could potentially evade the patient's innate and/or non-specific immune response.

[0193] In other aspects, the pharmaceutical combination of the present invention is administered as part of a homologous and/or heterologous prime-boost regimen. As illustrated in FIG. 8-13, a homologous and/or heterologous prime-boost regimen prolongs and re-activates enhanced NK cell responses as well as increases specific CD8 and CD4 T cell responses of the subject. Thus, in one or more embodiments, a pharmaceutical combination and/or method for reducing tumor size and/or improving survival of a cancer patient is provided, comprising administering to the cancer patient a pharmaceutical combination of the present invention, wherein the pharmaceutical combination is administered as part of a homologous or heterologous prime boost. scheme. In a more preferred aspect of the prime-boost regimen, the recombinant MVA is administered intravenously at the same time as or after administration of the antibody.

Creation of recombinant MVA viruses containing transgenes

[0194] The recombinant MVA viruses provided herein can be generated by conventional methods known in the art. Methods for producing recombinant poxviruses or inserting exogenous coding sequences into the poxvirus genome are well known to those skilled in the art. For example, methods for performing standard molecular biology techniques such as DNA cloning, DNA and RNA isolation, Western blot analysis, RT-PCR and PCR amplification techniques are described in Molecular Cloning, A laboratory Manual ( ^2nd Ed.) [J . Samb rook et al., Cold Spring Harbor Laboratory Press (1989)], and methods for handling and manipulating viruses are described in the Virology Methods Manual [BWJ Mahy et al. (eds.), Academic Press (1996)]. Likewise, techniques and best practices for operating, manipulating and genetically engineering MVA are described in Molecular Virology: A Practical Approach [AJ Davison & RM Elliott (Eds.), The Practical Approach Series, IRL Press at Oxford University Press, Oxford, UK (1993 ) (see, for example, Chapter 9: Expression of genes by Vaccinia virus vectors)] and Current Protocols in Molecular Biology [John Wiley & Son, Inc. (1998) (see, for example, Chapter 16, Section IV: Expression of proteins in mammalian cells using vaccinia viral vector)].

[0195] Various methods can be used to generate the various recombinant MVA viruses described herein. The DNA sequence to be inserted into the virus can be placed in an E. coli plasmid construct into which DNA homologous to the DNA region of the poxvirus has been inserted. Otherwise, the DNA sequence to be inserted can be ligated to the promoter. The promoter-gene linkage can be placed in a plasmid construct such that the promoter-gene linkage is flanked at both ends of a DNA homologous DNA sequence flanking a region of poxvirus DNA containing a non-essential locus. The resulting plasmid construct can be amplified by propagation in E. coli bacteria and isolated. The isolated plasmid containing the DNA gene sequence to be inserted can be transferred into a cell culture, such as chicken embryonic fibroblasts (CEF), and at the same time the culture is infected with the MVA virus. Recombination between the homologous MVA viral DNA in the plasmid and the viral genome, respectively, can lead to the creation of a poxvirus modified by the presence of foreign DNA sequences.

[0196] According to a preferred embodiment, cells from a suitable cell culture, such as CEF cells, can be infected with the MVA virus. The infected cells can then be transfected with a first plasmid vector containing a foreign or heterologous gene or genes, such as one or more of the nucleic acids of the present invention; preferably under the transcriptional control of a poxvirus expression control element. As explained above, the plasmid vector also contains sequences capable of directing the insertion of an exogenous sequence into a selected region of the MVA viral genome. Optionally, the plasmid vector also contains a cassette containing a marker and/or selection gene operably linked to a poxvirus promoter. Suitable marker or selection genes are, for example, genes encoding green fluorescent protein, β-galactosidase, neomycin phosphoribosyltransferase, or other markers. The use of selectable marker cassettes simplifies the identification and isolation of the generated recombinant poxvirus. In this case, the recombinant poxvirus can also be identified using PCR technology. Subsequently, additional cells can be infected with the recombinant poxvirus prepared as described above and transfected with a second vector containing a second foreign or heterologous gene or genes. In the event that this gene needs to be introduced into a different insertion site of the poxvirus genome, the second vector also differs in sequences homologous to the poxvirus that direct the integration of the second foreign gene or genes into the poxvirus genome. Once homologous recombination has occurred, a recombinant virus containing two or more foreign or heterologous genes can be isolated. To introduce additional foreign genes into the recombinant virus, the infection and transfection steps can be repeated using the recombinant virus isolated in the previous steps for infection and using an additional vector containing the additional foreign gene or genes for transfection.

[0197] Alternatively, the steps of infection and transfection described above are interchangeable, i.e., a suitable cell can first be transfected with a plasmid vector containing a foreign gene and then infected with a poxvirus. As a further alternative, it is also possible to introduce each foreign gene into different viruses, co-infect a cell with all of the resulting recombinant viruses, and screen for a recombinant containing all of the foreign genes. A third alternative is to ligate the DNA genome and foreign sequences in vitro and restore the recombined vaccinia DNA genome with a helper virus. A fourth alternative is homologous recombination in E. coli or another bacterial species between the MVA virus genome cloned as a bacterial artificial chromosome (BIC) and a linear foreign sequence flanked by DNA sequences homologous to sequences flanking the desired integration site into the MVA virus genome.

[0198] One or more nucleic acids of the present invention can be inserted into a suitable region of an MVA virus or an MVA viral vector. Suitable regions of the MVA virus are non-essential regions of the MVA genome. Non-essential regions of the MVA genome may be intergenic regions or known deletion sites 1-6 of the MVA genome. Alternatively or additionally, non-essential regions of the recombinant MVA may be a coding region of the MVA genome that is not essential for viral growth. However, insertion sites are not limited to these preferred insertion sites in the MVA genome, as it is within the scope of the present invention that the nucleic acids of the present invention (e.g., HER2, Brachyury, and CD40L) and their associated promoters described herein can be inserted anywhere in the viral genome, provided that recombinants can be obtained that can be amplified and propagated in at least one cell culture system, such as chicken embryonic fibroblasts (CEF cells).

[0199] Preferably, the nucleic acids of the present invention can be inserted into one or more intergenic regions (IGOs) of the MVA virus. The term "intergene region" refers primarily to regions of the viral genome located between two adjacent open reading frames (ORFs) of the MVA virus genome, preferably between two significant ORFs of the MVA virus genome. In the case of MVA, in certain embodiments, OPC is selected from OPC 07/08, OPC 44/45, OPC 64/65, OPC 88/89, OPC 136/137, and OPC 148/149.

[0200] In the case of an MVA virus, the nucleotide sequences may additionally or alternatively be inserted into one or more known deletion sites, i.e., deletion sites I, II, III, IV, V or VI of the MVA genome. The term "known deletion site" refers to those regions of the MVA genome that have been deleted by continuous passaging on CEF cells characterized at passage 20 out of 516, relative to the genome of the parent virus from which the MVA is derived, in particular the parent vaccinia virus Ankara, cultivated on chorioallantois (CVA), for example, as described in Meisinger-Henschel et al. (2007), Journal of General Virology 88:3249-3259.

Vaccines

[0201] In certain embodiments, the recombinant MVA of the present invention can be obtained as part of a vaccine. For the preparation of vaccines, the MVA virus can be converted into a physiologically acceptable form. In certain embodiments, such preparation is based on experience in the preparation of poxvirus vaccines used for vaccination against smallpox, as described, for example, in Stickl, H. et al., Dtsch. med. Wschr. 99, 2386-2392 (1974).

[0202] A typical preparation method is shown below. The purified virus is stored at -80°C with a titer of 5×10 ⁸ TCID50/ml in a ready mix of 10 mM Tris, 140 mM NaCl, pH 7.4. To prepare vaccine doses, for example, 1x10 ⁸ -1x10 ⁹ virus particles can be lyophilized in phosphate buffered saline (PBS) in the presence of 2% peptone and 1% human albumin in an ampoule, preferably a glass ampoule. Alternatively, doses of the vaccine can be prepared by stepwise lyophilization of the virus into a dosage form. In certain embodiments, the dosage form contains additional additives such as mannitol, dextran, sugar, glycine, lactose, polyvinylpyrrolidone, or other additives such as, but not limited to, antioxidants or inert gas, stabilizers, or recombinant proteins (e.g., human serum albumin) suitable for in vivo administration. The ampoule is then sealed and can be stored at a suitable temperature, eg 4° C. to room temperature, for several months. While there is no need, the ampoule is stored preferably at temperatures below -20°C, most preferably at about -80°C.

[0203] In various embodiments involving vaccination or therapy, the lyophilisate is dissolved in 0.1-0.5 ml of an aqueous solution, preferably saline or Tris buffer, for example, 10 mM Tris, 140 mM NaCl, pH 7.7. It is contemplated that the recombinant MVA, vaccine or pharmaceutical composition of the present disclosure may be prepared in solution in a concentration range of 10 ⁴ to 10 ¹⁰ SCID ₅₀ /ml, 10 ⁵ to 5×10 ⁸ SCID ₅₀ /ml, 10 ⁶ to 10 ⁸ SCID ₅₀ /ml or 10 ⁷ to 10 ⁸ SCID ₅₀ /ml. The preferred dose for humans is 10 ⁶ to 10 ¹⁰ SCID ₅₀ , including a dose of 10 ⁶ SCID ₅₀ , 10 ⁷ SCID ₅₀ , 10 ⁸ SCID ₅₀ , 5 x 10 ⁸ SCID ₅₀ , 10 ⁹ SCID ₅₀ , 5 x 10 ⁹ SCID ₅₀ or 10 ¹⁰ TCID ₅₀ . Optimization of the dose and number of injections is within the skill and ability of a person skilled in the art.

[0204] In one or more preferred embodiments provided herein, recombinant MVA is intravenously administered to a cancer patient.

[0205] In additional embodiments, the antibody can be administered systemically or topically, i. via intraperitoneal, parenteral, subcutaneous, intravenous, intramuscular, intranasal, intradermal or any other route of administration known to those skilled in the art.

Kits, compositions and methods of use

[0206] In various embodiments, this invention includes kits, pharmaceutical combinations, pharmaceutical compositions, and/or immunogenic combinations comprising a) a recombinant MVA that contains the nucleic acids described herein, and b) one or more of the antibodies described herein. document.

[0207] It is contemplated that a kit and/or composition may contain one or more containers or vials of a recombinant poxvirus of the present invention, one or more containers or vials of an antibody of the present invention, along with instructions for administering the recombinant MVA and the antibody. It is contemplated that, in a more specific embodiment, the kit may contain instructions for administering the recombinant MVA and the antibody as a first priming, followed by one or more booster administrations of the recombinant MVA and the antibody.

[0208] The kits and/or compositions provided herein may generally contain one or more pharmaceutically acceptable and/or approved carriers, additives, antibiotics, preservatives, adjuvants, diluents, and/or stabilizers. Such excipients may be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffers, and the like. Suitable carriers are generally large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and the like.

CERTAIN TYPICAL IMPLEMENTATIONS

[0209] Embodiment 1 is a method for reducing tumor size and/or improving survival in a cancer patient, comprising a) administering intravenously to a cancer patient a recombinant modified vaccinia virus Ankara (MVA) containing a first nucleic acid encoding a first heterologous tumor associated antigen (TAA) , which, when administered intravenously, induces both an enhanced natural killer (NK) cell response and an enhanced cytotoxic T cell response compared to both NK cell and T cell responses induced by non-intravenous administration of recombinant MVA containing the first nucleic acid encoding the first heterologous tumor-associated antigen; and b) administering to the cancer patient an antibody comprising an Fc domain and being specific for an antigen that is expressed or overexpressed on the cell membrane of the tumor cell; wherein administration of a) and b) to a cancer patient reduces tumor size in the cancer patient and/or improves survival of the cancer patient compared to non-intravenous administration of a) or administration of b) alone.

[0210] Embodiment 2 is a method of reducing tumor size and/or improving survival in a cancer patient, comprising a) administering to the cancer patient an antibody comprising an Fc domain that is specific for an antigen that is expressed on the cell membrane of the tumor cell; and b) administering intravenously to a cancer patient a recombinant modified vaccinia virus Ankara (MVA) containing a first nucleic acid encoding a first heterologous tumor-associated antigen (TAA) that, when administered intravenously, induces both an enhanced natural killer (NK) cell response and an enhanced response cytotoxic T cells compared to the response of NK cells and the response of T cells induced by non-intravenous administration of recombinant MVA containing the first nucleic acid encoding the first heterologous tumor-associated antigen; wherein administration of a) and b) to a cancer patient reduces tumor size in the cancer patient and/or improves survival of the cancer patient compared to non-intravenous administration of a) or administration of b) alone.

[0211] Embodiment 3 is a method for reducing tumor size and/or improving survival in a cancer patient, comprising a) administering to the cancer patient an antibody comprising an Fc domain that is specific for an antigen that is expressed on the outer surface of the tumor cell's cell membrane, which when administered, it induces ADCC in a cancer patient; and b) administering intravenously to a cancer patient a recombinant modified vaccinia virus Ankara (MVA) containing a first nucleic acid encoding a first heterologous tumor-associated antigen (TAA) that, when administered intravenously, induces both an enhanced natural killer (NK) cell response and an enhanced response T cells versus NK cell response and T cell response induced by non-intravenous administration of recombinant MVA containing a first nucleic acid encoding a first heterologous tumor-associated antigen; wherein administration of a) and b) to a cancer patient reduces tumor size in the cancer patient and/or improves survival of the cancer patient compared to non-intravenous administration of a) or administration of b) alone.

[0212] Embodiment 4 is a method according to any one of embodiments 1-3, wherein the antibody is specific for an antigen that is overexpressed on the cell membrane of the tumor cell.

[0213] Embodiment 5 is the method of any one of Embodiments 1-4, further comprising administering CD40L intravenously to a cancer patient.

[0214] Embodiment 6 is the method of any one of Embodiments 1-5, wherein CD40L is encoded by MVA.

[0215] Embodiment 7 is a method according to any one of embodiments 1-6, wherein the induction of an enhanced NK cell response comprises at least one of a) induction of an enhanced ADCC response and b) induction of NK cells to target, and destruction of tumor cells with low MHC expression.

[0216] Embodiment 8 is the method of any one of Embodiments 1-6, wherein the induction and enhanced response of NK cells comprise the induction of an enhanced ADCC response.

[0217] Embodiment 9 is a method according to any one of embodiments 1-8, wherein the recombinant MVA is administered simultaneously or after the antibody.

[0218] Embodiment 10 is a method according to any one of embodiments 1-8, wherein the recombinant MVA is administered after the antibody.

[0219] Embodiment 11 is a method according to any one of embodiments 1-10, wherein the MVA further comprises a second nucleic acid encoding a second heterologous TAA.

[0220] Embodiment 12 is a method according to any one of embodiments 1-11, wherein the first and/or second TAA are selected from the group consisting of: 5-α-reductase, α-fetoprotein (“AFP”), AM-1, APC, April, melanoma B antigen gene ("BAGE"), β-catenin, Bcl12, bcr-abl, Brachyury, CA-125, caspase-8 ("CASP-8", also known as "FLICE" ), cathepsins, CD19, CD20, CD21/complement receptor 2 ("CR2"), CD22/BL-CAM, CD23/F _with εRII, CD33, CD35/complement receptor 1 ("CR1"), CD44/PGP-1, CD45/leukocyte common antigen ("LCA"), CD46/membrane cofactor protein ("MCP"), CD52/CAMPATH-1, CD55/degradation accelerator factor ("DAF"), CD59/protectin, CDC27, CDK4, carcinoembryonic antigen ( "CEA"), c-myc, cyclooxygenase-2 ("cox-2"), colorectal cancer deleted gene ("DCC"), DcR3, E6/E7, CGFR, EMBP, Dna78, farnesyl transferase, fibroblast growth factor-8a ("FGF8a"), fibroblast growth factor-8b ("FGF8b"), FLK-1/KDR, folate receptor, G250, melanoma antigen G gene family ("GAGE family"), gastrin 17, gastrin-releasing hormone, ganglioside 2 ("GD2")/ganglioside 3 ("GD3")/ganglioside-monosialic acid-2 ("GM2"), gonadotropin-releasing hormone ("GnRH"), UDP-GlcNAc:R ₁ Man(α1-6) R ₂ [GlcNAc to Man(α1-6)] β1,6-N-acetylglucosaminyltransferase V ("GnT V"), GP1, gp100/Pme117, gp-100-in4, gp15, gp75/tyrosine-related protein-1 ( "gp75/TRP-1"), human chorionic gonadotropin ("hCG"), heparanase, HER2, human breast tumor virus ("HMTV"), 70 kDa heat shock protein ("HSP70"), human telomerase reverse transcriptase ("hTERT"), insulin-like growth factor receptor-1 ("IGFR-1"), interleukin-13 receptor ("IL-13R"), inducible nitric oxide synthase ("iNOS"), Ki67, KIAA0205, K-ras, H-ras, N-ras, KSA, LKLR-FUT, gene encoding melanoma antigen 1 (“MAGE-1”), gene encoding melanoma antigen 2 (“MAGE-2”), gene encoding melanoma antigen 3 (“ MAGE-3"), melanoma antigen 4 ("MAGE-4"), mammaglobin, MAP17, melanoma-A/melanoma antigen T-cell-recognized 1 ("MART-1"), mesothelin, MIC A/B , MT-MMP, mucin, testis-specific antigen NY-ESO-1, osteonectin, p15, P170/MDR1, p53, p97/melanotransferrin, PAI-1, platelet-derived growth factor (“PDGF”), µPA, PRAME, probasin, progenipoietin, prostate specific antigen ("PSA"), prostate specific membrane antigen ("PSMA"), RAGE-1, Rb, RCAS1, SART-1, SSX family, STAT3, STn, TAG-72, transforming growth factor alpha ("TGF-α"), transforming growth factor beta ("TGF-β"), thymosin-beta-15, tumor necrosis factor alpha ("TNF-α"), TP1, TRP-2, tyrosinase, vascular endothelial growth factor ("VEGF"), ZAG, p16INK4 and glutathione-S-transferase ("GST").

[0221] Embodiment 13 is the method of any one of Embodiments 1-12, wherein the antibody is an antibody approved for the treatment of a cancer patient.

[0222] Embodiment 14 is a method according to any one of embodiments 1-13, wherein the antibody is selected from the group consisting of: anti-CD20 (e.g., rituximab; ofatumumab; tositumomab), anti-CD52 (e.g., alemtuzumab Campat®), anti-EGFR (eg, cetuximab Erbitux®, panitumumab), anti-CD2 (eg, ciplizumab), anti-CD37 (eg, BI836826), anti-CD123 (eg, JNJ-56022473), anti-CD30 (eg, e.g. XmAb2513), anti-CD38 (e.g. daratumumab Darzalex®), anti-PDL1 (e.g. avelumab, atezolizumab, durvalumab), CTLA-4 (e.g. ipilimumab), anti-GD2 (e.g. 3F8, ch14.18, KW-2871, dinutuximab), anti-CEA, anti-MUC1, anti-FLT3, anti-CD19, anti-CD40, anti-SLAMF7, anti-CCR4, anti-B7-H3, anti-ICAM1, anti-CSF1R, anti -CA125 (eg oregovomab), anti-FRα (eg MOv18-IgG1, mirvetuximab soravtansine (IMGN853), MORAb-202), anti-mesothelin (eg MORAb-009), and anti-HER2.

[0223] Embodiment 15 is the method of any of embodiments 1-14, wherein the antibody is specific for the HER2 antigen.

[0224] Embodiment 16 is a method according to any one of embodiments 1-15, wherein the antibody is selected from pertuzumab, trastuzumab, Gerzuma, ABP 980, and ado-trastuzumab emtansine.

[0225] Embodiment 17 is a method according to any one of embodiments 1-16, wherein the first and/or second TAA have been modified to prevent the antibody from binding to the first and/or second TAA.

[0226] Embodiment 18 is the method of any one of Embodiments 1-17, wherein the first TAA is HER2.

[0227] Embodiment 19 is the method of Embodiment 18 wherein one or more HER2 amino acids are mutated to prevent anti-HER2 antibody from binding to HER2 antigen.

[0228] Embodiment 20 is a method according to any one of embodiments 18-19, wherein one or more HER2 amino acids are mutated to prevent extracellular dimerization, tyrosine kinase activity, and/or phosphorylation of a HER2 polypeptide and/or antigen.

[0229] Embodiment 21 is the method of any one of embodiments 18-20, wherein one or more mutations are introduced in at least one of the 3 loops of the peri-membrane region of HER2.

[0230] Embodiment 22 is the method of any one of embodiments 18-21, wherein the 3 loops of the HER2 peri-membrane region are selected from amino acids 579-583 (loop 1), 592-595 (loop 2) and 615-625 ( loop 3).

[0231] Embodiment 23 is a method according to any one of embodiments 18-22, wherein the HER2 antigen contains at least one mutation in at least one of the amino acids H267, Y274, S310, L317, H318, K333, E580 , D582, P594, F595, K615 and Q624.

[0232] Embodiment 24 is a method according to any one of embodiments 18-23, wherein the HER2 antigen contains at least one mutation selected from the group consisting of: E580A, F595A, K615A, S310A, H318A, L317A, D277R, E280K, K573M and Y1023A.

[0233] Embodiment 25 is the method of any one of embodiments 18-24, wherein the HER2 antigen contains at least one mutation at amino acids 1139-1248.

[0234] Embodiment 26 is the method of any one of embodiments 18-25, wherein the HER2 antigen contains at least one deletion at amino acids 1139-1248.

[0235] Embodiment 27 is the method of any of embodiments 18-26, wherein the HER2 antigen contains a deletion of amino acids 1139-1248.

[0236] Embodiment 28 is a method according to any one of embodiments 18-27, wherein the first nucleic acid encodes a HER2 antigen having at least 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:1.

[0237] Embodiment 29 is the method of any one of embodiments 18-28, wherein the first nucleic acid encodes a HER2 antigen comprising SEQ ID NO:1.

[0238] Embodiment 30 is the method of any one of embodiments 18-29, wherein the first nucleic acid encodes a HER2 antigen, wherein the first nucleic acid has at least 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:2.

[0239] Embodiment 31 is the method of any one of embodiments 18-30, wherein the first nucleic acid comprises SEQ ID NO:2.

[0240] Embodiment 32 is the method of any one of Embodiments 1-31, wherein the MVA contains a second nucleic acid encoding a second heterologous TAA other than the first TAA.

[0241] Embodiment 33 is the method of Embodiment 32, wherein the second TAA is Brachyury.

[0242] Embodiment 34 is the method of Embodiment 33, wherein the Brachyury antigen contains one or more mutations to prevent nuclear localization of the Brachyury antigen.

[0243] Embodiment 35 is the method of any of embodiments 33-34, wherein the Brachyury antigen contains one or more mutations in the nuclear localization signal (NLS) domain.

[0244] Embodiment 36 is a method according to any one of embodiments 33-35, wherein the SNL domain is deleted from the Brachyury antigen.

[0245] Embodiment 37 is the method of any of embodiments 33-36, wherein the second nucleic acid comprises a Brachyury antigen having at least 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:3.

[0246] Embodiment 38 is the method of any one of embodiments 33-37, wherein the second nucleic acid comprises SEQ ID NO:3.

[0247] Embodiment 39 is the method of any of embodiments 33-38, wherein the second nucleic acid encodes a Brachyury antigen, wherein the second nucleic acid is at least 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:4.

[0248] Embodiment 40 is the method of any one of embodiments 33-39, wherein the second nucleic acid comprises SEQ ID NO:4.

[0249] Embodiment 41 is a method according to any one of embodiments 1-40, wherein the antibody is selected from the group consisting of: anti-CD20 (rituximab; ofatumumab; tositumomab), anti-CD52 (alemtuzumab Campat®), anti-EGFR (cetuximab Erbitux®, panitumumab), anti-CD38 (daratumumab Darzalex®), anti-PDL1 (avelumab), anti-CTLA-4 (ipilimumab), anti-GD2 (3F8, ch14.18, KW-2871, dinutuximab), anti-CEA, anti-Muc1, anti-FLT3L, anti-CD19, anti-CD40, anti-SLAMF7, anti-CCR4, anti-B7-H3, anti-ICAM1, anti-CSF1R, and anti-HER2.

[0250] Embodiment 42 is the method of any one of embodiments 1-41, wherein the antibody is specific for the HER2 antigen.

[0251] Embodiment 43 is the method of Embodiment 42 wherein the anti-HER2 antibody is selected from pertuzumab, trastuzumab ado-trastuzumab emtansine.

[0252] Embodiment 44 is a method according to any one of embodiments 1-43, wherein the antibody and MVA are administered in a homologous prime-boost regimen.

[0253] Embodiment 45 is the method of any one of Embodiments 1-44, wherein MVA is MVA-BN or a derivative of MVA-BN.

[0254] Embodiment 46 is a method according to any one of embodiments 1-45, wherein the cancer patient has and/or has been diagnosed as having cancer selected from the group consisting of breast cancer, lung cancer, head cancer, and neck, thyroid cancer, melanoma, stomach cancer, bladder cancer, kidney cancer, liver cancer, melanoma, pancreatic cancer, prostate cancer, ovarian cancer or colorectal cancer.

[0255] Embodiment 47 is a pharmaceutical combination for use in reducing tumor size and/or improving survival in a cancer patient, comprising a) a recombinant modified vaccinia virus Ankara (MVA) containing a first nucleic acid encoding a first heterologous tumor associated antigen (TAA) , which, when administered intravenously, induces both an enhanced natural killer (NK) cell response and an enhanced T cell response compared to both NK cell and T cell responses induced by non-intravenous administration of a recombinant MVA virus containing a nucleic acid encoding a heterologous OAA; and b) an antibody containing an Fc domain and being specific for an antigen that is expressed on the cell membrane of the tumor cell; wherein administration of a) and b) to a cancer patient reduces tumor size and/or improves survival of the cancer patient compared to non-IV administration of a) or administration of b) alone.

[0256] Embodiment 48 is a pharmaceutical combination for use in reducing tumor size and/or improving survival in a cancer patient, comprising a) a recombinant modified vaccinia virus Ankara (MVA) containing a first nucleic acid encoding a first heterologous tumor associated antigen (TAA) , which, when administered intravenously, induces both an enhanced natural killer (NK) cell response and an enhanced T cell response compared to both NK and T cell responses induced by non-intravenous administration of recombinant MVA containing a nucleic acid encoding a heterologous TAA ; and b) an Fc-domain-containing antibody that is specific for an antigen that is expressed on the cell membrane of a tumor cell, wherein when administered, the antibody binds to an antigen on a tumor cell in a human cancer patient and induces antibody-dependent cell-mediated cytotoxicity (ADCC); wherein administration of a) and b) to a cancer patient reduces tumor size and/or improves survival of the cancer patient compared to non-IV administration of a) or administration of b) alone.

[0257] Embodiment 49 is a pharmaceutical combination according to any one of embodiments 47-48, wherein CD40L is encoded by recombinant MVA.

[0258] Embodiment 50 is a pharmaceutical combination according to any one of embodiments 47-49, wherein the recombinant MVA further comprises a second nucleic acid encoding a second heterologous OAA.

[0259] Embodiment 51 is a pharmaceutical combination according to any of embodiments 47-50, wherein the first and/or second TAA are selected from the group consisting of: 5-α-reductase, α-fetoprotein ("AFP") , AM-1, APC, April, melanoma B antigen gene ("BAGE"), β-catenin, Bcl12, bcr-abl, Brachyury, CA-125, caspase-8 ("CASP-8", also known as "FLICE ”), cathepsins, CD19, CD20, CD21/complement receptor 2 (“CR2”), CD22/BL-CAM, CD23/F _with εRII, CD33, CD35/complement receptor 1 (“CR1”), CD44/PGP-1 , CD45/leukocyte common antigen ("LCA"), CD46/membrane cofactor protein ("MCP"), CD52/CAMPATH-1, CD55/degradation accelerator factor ("DAF"), CD59/protectin, CDC27, CDK4, carcinoembryonic antigen ("CEA"), c-myc, cyclooxygenase-2 ("cox-2"), colorectal cancer deleted gene ("DCC"), DcR3, E6/E7, CGFR, EMBP, Dna78, farnesyl transferase, fibroblast growth factor- 8a ("FGF8a"), fibroblast growth factor-8b ("FGF8b"), FLK-1/KDR, folic acid receptor, G250, melanoma antigen G gene family ("GAGE family"), gastrin 17, gastrin-releasing hormone , ganglioside 2 ("GD2")/ganglioside 3 ("GD3")/ganglioside-monosialic acid-2 ("GM2"), gonadotropin-releasing hormone ("GnRH"), UDP-GlcNAc:R ₁ Man(α1-6 )R ₂ [GlcNAc to Man(α1-6)] β1,6-N-acetylglucosaminyltransferase V ("GnT V"), GP1, gp100/Pme117, gp-100-in4, gp15, gp75/tyrosine-related protein-1 ("gp75/TRP-1"), human chorionic gonadotropin ("hCG"), heparanase, HER2, human breast tumor virus ("HMTV"), 70 kDa heat shock protein ("HSP70"), human telomerase reverse transcriptase ( "hTERT"), insulin-like growth factor receptor-1 ("IGFR-1"), interleukin-13 receptor ("IL-13R"), inducible nitric oxide synthase ("iNOS"), Ki67, KIAA0205, K-ras , H-ras, N-ras, KSA, LKLR-FUT, gene encoding melanoma antigen 1 (“MAGE-1”), gene encoding melanoma antigen 2 (“MAGE-2”), gene encoding melanoma antigen 3 ( "MAGE-3"), gene encoding melanoma antigen 4 ("MAGE-4"), mammaglobin, MAP17, melanoma-A/melanoma antigen recognized by T-cells 1 ("MART-1"), mesothelin, MIC A/ B, MT-MMP, mucin, testis-specific antigen NY-ESO-1, osteonectin, p15, P170/MDR1, p53, p97/melanotransferrin, PAI-1, platelet-derived growth factor (“PDGF”), µPA, PRAME, probasin , progenipoietin, prostate specific antigen ("PSA"), prostate specific membrane antigen ("PSMA"), RAGE-1, Rb, RCAS1, SART-1, SSX family, STAT3, STn, TAG-72, transforming growth factor alpha ("TGF-α"), transforming growth factor beta ("TGF-β"), thymosin-beta-15, tumor necrosis factor alpha ("TNF-α"), TP1, TRP-2, tyrosinase, endothelial growth factor vessels ("VEGF"), ZAG, p16INK4 and glutathione-S-transferase ("GST").

[0260] Embodiment 52 is a pharmaceutical combination according to any one of embodiments 47-51, wherein the first TAA is HER2.

[0261] Embodiment 53 is a pharmaceutical combination according to embodiment 52, wherein the HER2 antigen contains at least one mutation selected from the group consisting of: E580A, F595A, K615A, S310A, H318A, L317A, D277R, E280K , K573M and Y1023A.

[0262] Embodiment 54 is a pharmaceutical combination according to any one of embodiments 52-53, wherein the first nucleic acid contains a HER2 antigen having at least 90%, 95%, 97%, 98%, or 99% identity with SEQID NO:1.

[0263] Embodiment 55 is a pharmaceutical combination according to any one of embodiments 52-54, wherein the first nucleic acid comprises SEQ ID NO:1.

[0264] Embodiment 56 is a pharmaceutical combination according to any one of embodiments 52-55, wherein the first nucleic acid encodes a HER2 antigen, wherein the first nucleic acid has at least 90%, 95%, 97%, 98% or 99% identity with SEQ ID NO:2.

[0265] Embodiment 57 is a pharmaceutical combination according to any one of embodiments 52-56, wherein the first nucleic acid comprises SEQ ID NO:2.

[0266] Embodiment 58 is a pharmaceutical combination according to any one of embodiments 47-57, wherein the MVA contains a second nucleic acid encoding a second heterologous TAA different from the first TAA.

[0267] Embodiment 59 is the pharmaceutical combination of Embodiment 58, wherein the second TAA is Brachyury.

[0268] Embodiment 60 is a pharmaceutical combination according to embodiment 59, wherein the second nucleic acid contains a Brachyury antigen having at least 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO: 3.

[0269] Embodiment 61 is a pharmaceutical combination according to any one of embodiments 59-60, wherein the second nucleic acid encodes an antigen comprising SEQ ID NO:3.

[0270] Embodiment 62 is a pharmaceutical combination according to any one of embodiments 59-61, wherein the second nucleic acid encodes a Brachyury antigen, wherein the second nucleic acid has at least 90%, 95%, 97%, 98% or 99% identity with SEQ ID NO:4.

[0271] Embodiment 63 is a pharmaceutical combination according to any one of embodiments 59-62, wherein the second nucleic acid comprises SEQ ID NO:4.

[0272] Embodiment 64 is a pharmaceutical combination according to any of embodiments 47-63, wherein the antibody is selected from the group consisting of: anti-CD20 (e.g., rituximab; ofatumumab; tositumomab), anti-CD52 (e.g., alemtuzumab Campat®), anti-EGFR (eg, cetuximab Erbitux®, panitumumab), anti-CD2 (eg, ciplizumab), anti-CD37 (eg, BI836826), anti-CD123 (eg, JNJ-56022473), anti-CD30 (eg, XmAb2513), anti-CD38 (eg, daratumumab Darzalex®), anti-PDL1 (eg, avelumab, atezolizumab, durvalumab), CTLA-4 (eg, ipilimumab), anti-GD2 (eg, 3F8, ch14.18 , KW-2871, dinutuximab), anti-CEA, anti-MUC1, anti-FLT3, anti-CD19, anti-CD40, anti-SLAMF7, anti-CCR4, anti-B7-H3, anti-ICAM1, anti-CSF1R, anti-CA125 (eg oregovomab), anti-FRα (eg MOv18-IgG1, mirvetuximab soravtansine (IMGN853), MORAb-202), anti-mesothelin (eg MORAb-009), and anti-HER2.

[0273] Embodiment 65 is a pharmaceutical combination according to any one of embodiments 47-64, wherein the antibody is specific for the HER2 antigen.

[0274] Embodiment 66 is the pharmaceutical combination of Embodiment 65 wherein the antibody is selected from pertuzumab, trastuzumab, Gerzuma, ABP 980, and ado-trastuzumab emtansine.

[0275] Embodiment 67 is a pharmaceutical combination according to any one of Embodiments 47-66, wherein MVA is MVA-BN or a derivative of MVA-BN.

[0276] Embodiment 68 is a method of inducing an enhanced natural killer (NK) cell response in a cancer patient, comprising administering the pharmaceutical combination of any one of Embodiments 47-66, wherein administering the combination enhances the NK cell response in the cancer patient compared to non-intravenous administration of combination a) or administration of b) alone.

[0277] Embodiment 69 is a method of inducing both an enhanced innate and an enhanced adaptive immune response in a cancer patient, comprising administering the pharmaceutical combination of any of Embodiments 47-67, wherein administering the combination enhances the NK cell response in the cancer patient compared to with non-intravenous administration of combination a) or administration of b) alone.

[0278] Embodiment 70 is the method of Embodiment 69 wherein the enhanced adaptive immune response includes an enhanced T cell response.

[0279] Embodiment 71 is the method of Embodiment 70, wherein the enhanced T cell response includes an enhanced CD8 T cell response and an enhanced CD4 T cell response.

[0280] Embodiment 72 is a method of enhancing antibody-based therapy in a cancer patient, comprising administering the pharmaceutical combination of any one of embodiments 47-67, wherein administering the pharmaceutical combination enhances antibody-dependent cell-mediated cytotoxicity (ADCC) induced by antibody-based therapy by compared to antibody-based therapy alone.

[0281] Embodiment 73 is a method of enhancing the killing of tumor cells having reduced levels of MHC in a cancer patient, comprising administering the pharmaceutical combination of any one of Embodiments 47-67, wherein administering the combination enhances the killing of tumor cells having reduced levels of MHC by compared with the introduction only b).

[0282] Embodiment 74 is a method of treating a human cancer patient, comprising administering to the cancer patient the pharmaceutical combination of any one of Embodiments 47-67.

[0283] Embodiment 75 is the method of any one of embodiments 68-74, wherein the recombinant MVA is administered simultaneously or after the antibody.

[0284] Embodiment 76 is the method of any one of embodiments 68-74, wherein the recombinant MVA is administered after the antibody.

[0285] Embodiment 77 is a method according to any one of embodiments 68-75, wherein the recombinant MVA is administered after 0-8 days, 0-7 days, 0-6 days, 0-5 days, 0-4 days , 0-3 days, 0-2 days, or 0-1 days after antibody.

[0286] Embodiment 78 is a method according to any one of embodiments 68-75, wherein recombinant MVA is administered after 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days , 1-3 days, 1-2 days or 1 day after the antibody.

[0287] Embodiment 79 is the method of any of embodiments 68-77, wherein recombinant MVA is administered 0-3 days, 0-2 days, 0-1 days after the antibody.

[0288] Embodiment 80 is the method of Embodiment 79, wherein the recombinant MVA is administered 0-3 days after the antibody.

[0289] Embodiment 81 is a method according to any one of embodiments 68-80, wherein the antibody and MVA are administered as part of a homologous or heterologous prime-boost regimen.

[0290] Embodiment 82 is a method according to any one of embodiments 68-80, wherein the antibody and MVA are administered as part of a homologous prime boost regimen.

[0291] Embodiment 83 is the method of any one of Embodiments 1-45 and 68-82, wherein the cancer patient has and/or has been diagnosed with a cancer selected from the group consisting of: breast cancer, lung cancer , head and neck cancer, thyroid cancer, melanoma, stomach cancer, bladder cancer, kidney cancer, liver cancer, melanoma, pancreatic cancer, prostate cancer, ovarian cancer or colorectal cancer.

[0292] Embodiment 84 is a pharmaceutical combination for use in reducing tumor volume and/or improving survival of a cancer patient, comprising the pharmaceutical combination of any one of Embodiments 47-67, wherein administering the combination reduces tumor volume and/or improves patient survival compared to with non-intravenous administration of combination a) or administration of b) alone.

[0293] Embodiment 85 is the use of the pharmaceutical combination of any one of Embodiments 47-67 in a method for reducing tumor volume and/or improving cancer patient survival.

[0294] Embodiment 86 is the use of the pharmaceutical combination of any one of Embodiments 47-67 in the preparation of a pharmaceutical or medicament to reduce tumor volume and/or improve survival of a cancer patient.

[0295] Embodiment 87 is a method of reducing tumor size and/or improving survival in a cancer patient, comprising a) administering intravenously to a cancer patient a recombinant modified vaccinia virus Ankara (MVA) containing a first nucleic acid encoding a first heterologous tumor associated antigen (TAA) , which, when administered intravenously, induces both an enhanced innate immune response and an enhanced T cell response compared to the innate immune response and T cell response induced by non-intravenous administration of a recombinant MVA containing a first nucleic acid encoding a first heterologous tumor-associated antigen; and b) administering to the cancer patient an antibody containing an Fc domain and being specific for an antigen that is expressed on the cell membrane of the tumor cell; wherein administration of a) and b) to a cancer patient reduces tumor size in the cancer patient and/or improves survival of the cancer patient compared to non-intravenous administration of a) or administration of b) alone.

[0296] Embodiment 88 is the method of Embodiment 87, wherein the enhanced innate immune response comprises at least one of enhanced NK cell response, enhanced macrophage response, enhanced monocyte response, enhanced neutrophil response, enhanced basophil response, enhanced eosinophil response, enhanced mast cell response, and enhanced dendritic cell response.

[0297] Embodiment 89 is the method of any one of embodiments 87-88, wherein the recombinant MVA further encodes CD40L.

[0298] Embodiment 90 is the method of any one of embodiments 88-89, wherein inducing an enhanced NK cell response comprises at least one of a) inducing an enhanced ADCC response and b) inducing NK cells to target, and destruction of tumor cells with low MHC expression.

[0299] Embodiment 91 is the method of any one of embodiments 87-90, wherein the recombinant MVA is administered simultaneously or after the antibody.

[0300] Embodiment 92 is the method of any one of embodiments 87-91, wherein the recombinant MVA is administered after the antibody.

[0301] Embodiment 93 is the method of any of embodiments 87-92, wherein the MVA comprises a first and/or second nucleic acid encoding a first and/or second heterologous TAA of any of embodiments 51-63.

[0302] Embodiment 94 is a method according to any one of embodiments 87-93, wherein the antibody comprises an antibody according to any one of embodiments 64-66.

[0303] Embodiment 95 is a pharmaceutical combination for reducing tumor size and/or improving survival in a cancer patient, comprising a) a recombinant modified vaccinia virus Ankara (MVA) containing a first nucleic acid encoding a first heterologous tumor associated antigen (TAA) that when administered intravenously, it induces both an enhanced innate immune response and an enhanced T cell response compared to the innate immune response and T cell response induced by non-intravenous administration of a recombinant MVA virus containing a nucleic acid encoding a heterologous TAA; and b) an antibody containing an Fc domain and being specific for an antigen that is expressed on the cell membrane of the tumor cell; wherein administration of a) and b) to a cancer patient reduces tumor size and/or improves survival of the cancer patient compared to non-IV administration of a) or administration of b) alone.

[0304] Embodiment 96 is the pharmaceutical combination of Embodiment 95, wherein the recombinant MVA further encodes CD40L.

[0305] Embodiment 97 is a pharmaceutical combination according to any one of embodiments 95-96, wherein the enhanced innate immune response comprises at least one of enhanced NK cell response, enhanced macrophage response, enhanced neutrophil response, enhanced basophil response , enhanced eosinophil response, enhanced mast cell response, and enhanced dendritic cell response.

[0306] Embodiment 98 is a pharmaceutical combination according to any one of embodiments 95-97, wherein the induction of an enhanced NK cell response comprises at least one of a) induction of an enhanced ADCC response and b) induction of NK cells to target and destruction of tumor cells with low MHC expression.

[0307] Embodiment 99 is a pharmaceutical combination according to any one of embodiments 95-98, wherein the MVA contains a first and/or second nucleic acid encoding a first and/or second heterologous TAA according to any one of embodiments 51-63.

[0308] Embodiment 100 is a pharmaceutical combination according to any one of embodiments 95-99, wherein the antibody comprises an antibody according to any one of embodiments 64-66.

[0309] Embodiment 101 is a pharmaceutical combination for reducing tumor size and/or improving survival in a cancer patient, comprising a) a recombinant poxvirus containing a first nucleic acid encoding a first heterologous tumor-associated antigen (TAA) that, when administered intravenously, induces both enhanced natural killer (NK) cell response and enhanced T cell response compared to NK cell response and T cell response induced by non-intravenous administration of a recombinant poxvirus containing a nucleic acid encoding a heterologous TAA; and b) an antibody containing an Fc domain and being specific for an antigen that is expressed on the cell membrane of the tumor cell; wherein administration of a) and b) to a cancer patient reduces tumor size and/or improves survival of the cancer patient compared to non-IV administration of a) or administration of b) alone.

[0310] Embodiment 102 is the pharmaceutical combination of Embodiment 101, wherein the recombinant poxvirus further encodes CD40L.

[0311] Embodiment 103 is a pharmaceutical combination according to any one of embodiments 101-102, wherein the MVA contains a first and/or second nucleic acid encoding a first and/or second heterologous TAA according to any one of embodiments 51-63.

[0312] Embodiment 104 is a pharmaceutical combination according to any one of embodiments 101-103, wherein the antibody comprises an antibody according to any one of embodiments 64-66.

[0313] Embodiment 105 is a method of reducing tumor size and/or improving survival in a cancer patient, comprising a) administering intravenously to a cancer patient a recombinant modified vaccinia virus Ankara (MVA) containing a first nucleic acid encoding a first heterologous tumor-associated antigen (TAA) , which, when administered intravenously, induces both an enhanced natural killer (NK) cell response and an enhanced T cell response compared to both NK cell and T cell responses induced by non-intravenous administration of recombinant MVA containing a nucleic acid encoding a heterologous tumor-associated antigen; and b) administering to the cancer patient an antibody that is specific for an antigen that is expressed on the cell membrane of the tumor cell; wherein administration of a) and b) to a cancer patient reduces tumor size in the cancer patient and/or improves survival of the cancer patient compared to non-intravenous administration of a) or administration of b) alone.

[0314] Embodiment 106 is a pharmaceutical combination for reducing tumor size and/or improving survival in a cancer patient, comprising a) an Fc-domain-containing antibody that is specific for an antigen that is expressed on the cell membrane of a tumor cell, which, when administered to the patient induces ADCC in a patient; and b) a recombinant modified vaccinia virus Ankara (MVA) containing a first nucleic acid encoding a first heterologous tumor-associated antigen (TAA), wherein MVA, when administered intravenously to a patient, induces an enhanced natural killer (NK) cell response that enhances ADCC in the patient and enhances NK cell-mediated toxicity, wherein MVA induces an enhanced T cell response compared to NK cell and T cell responses induced by non-intravenous administration of a recombinant MVA virus containing a nucleic acid encoding a heterologous tumor-associated antigen; and wherein administration of a) and b) to a cancer patient reduces tumor size and/or improves survival of the cancer patient compared to non-IV administration of a) or administration of b) alone.

[0315] Embodiment 107 is a pharmaceutical combination according to embodiment 106, wherein the recombinant MVA further comprises a nucleic acid encoding CD40L.

[0316] Embodiment 108 is a pharmaceutical combination according to any of embodiments 106-107, wherein the MVA contains a first and/or second nucleic acid encoding a first and/or second heterologous TAA according to any of embodiments 51-63.

[0317] Embodiment 109 is a method for reducing tumor volume and/or improving survival in a cancer patient, comprising administering the pharmaceutical combination of any of embodiments 106-108.

[0318] Embodiment 110 is a pharmaceutical combination for use in reducing tumor volume and/or improving survival of a cancer patient, comprising the pharmaceutical combination of any one of embodiments 106-108, wherein administration of the combination reduces tumor volume and/or improves patient survival compared to with the introduction of a) or non-intravenous administration b) separately.

[0319] Embodiment 111 is the use of the pharmaceutical combination of any of embodiments 106-108 in a method for reducing tumor volume and/or improving survival of a cancer patient.

[0320] Embodiment 112 is the use of a pharmaceutical combination according to any one of embodiments 106-108 in the preparation of a pharmaceutical or medicament to reduce tumor volume and/or improve survival of a cancer patient.

[0321] Embodiment 113 is a method of enhancing the efficacy of antibody-based therapy in a cancer patient, comprising administering the pharmaceutical combination of any of embodiments 47-67 and 106-108, wherein administering the combination to the patient reduces the antibody concentration required for NK cell-mediated toxicity in tumor cells.

[0322] Embodiment 114 is a pharmaceutical combination according to any of embodiments 47-67 and 106-108, wherein the recombinant MVA contains a nucleic acid encoding CD40L and CD40L contains SEQ ID NO:11.

[0323] Embodiment 115 is a pharmaceutical combination according to embodiment 114, wherein the nucleic acid encoding CD40L is a nucleic acid having at least 95%, 98%, 99%, or 100% identity with SEQ ID NO :12.

[0324] Embodiment 116 is a method according to any one of embodiments 1-46, wherein the recombinant MVA contains a nucleic acid encoding CD40L and CD40L contains SEQ ID NO:11.

[0325] Embodiment 117 is the method of Embodiment 116, wherein the nucleic acid encoding CD40L is a nucleic acid having at least 95%, 98%, 99%, or 100% identity with SEQ ID NO: 12.

[0326] Embodiment 118 is the method of Embodiment 83 wherein the breast cancer is a HER2 overexpressing breast cancer.

[0327] Embodiment 119 is a method according to any one of embodiments 18-19, wherein one or more HER2 amino acids are mutated to prevent extracellular dimerization, tyrosine kinase activity, and/or phosphorylation of a HER2 polypeptide and/or antigen upon expression by recombinant MVA.

[0328] Embodiment 120 is the method of any of embodiments 18-19, wherein the HER antigen contains one or more mutations to prevent the anti-HER2 antibody from binding to the HER2 antigen expressed by the recombinant MVA.

[0329] Embodiment 121 is a pharmaceutical combination according to any one of embodiments 50-63, wherein the first and/or second TAA contain one or more mutations to prevent the antibody from binding to the first and/or second TAA.

[0330] Embodiment 122 is the method of any one of embodiments 18-20, wherein one or more mutations are introduced in at least one of the 3 loops of the peri-membrane region of HER2.

[0331] Embodiment 123 is the method of any of embodiments 18-21, wherein the 3 loops of the HER2 peri-membrane region are selected from amino acids 579-583 (loop 1), 592-595 (loop 2), and 615-625 ( loop 3).

[0332] Embodiment 124 is a method according to any one of embodiments 18-22, wherein the HER2 antigen contains at least one mutation in at least one of the amino acids E580A, F595A, K615A, H267A, F279A, V308A, S310A , L317A, H318A, K333A, P337A.

[0333] Embodiment 125 is a method according to any one of embodiments 18-23, wherein the HER2 antigen contains at least one mutation selected from the group consisting of: E580A, F595A, K615A, H267A, F279A, V308A, S310A, L317A, H318A, K333A, P337A, D277R, E280K, K753M and Y1023A.

[0334] Embodiment 126 is a pharmaceutical combination for use in therapy by reducing tumor volume and/or improving survival of a cancer patient (preferably wherein the cancer patient has and/or is diagnosed as having a cancer selected from the group consisting of: breast cancer , lung cancer, head and neck cancer, thyroid cancer, melanoma, stomach cancer, bladder cancer, kidney cancer, liver cancer, melanoma, pancreatic cancer, prostate cancer, ovarian cancer or colorectal cancer), containing a pharmaceutical combination according to any of embodiments 47-67, wherein administering the combination reduces tumor volume and/or improves patient survival compared to non-intravenous administration of combination a) or administration of b) alone.

[0335] Embodiment 127 is the use of the pharmaceutical combination of any of Embodiments 47-67 in the preparation of a pharmaceutical or medicament to reduce tumor volume and/or improve survival of a cancer patient, preferably wherein the cancer patient has and/or is diagnosed as having cancer selected from the group consisting of: breast cancer, lung cancer, head and neck cancer, thyroid cancer, melanoma, stomach cancer, bladder cancer, kidney cancer, liver cancer, melanoma, pancreatic cancer, prostate cancer, cancer ovarian or colorectal cancer.

[0336] Embodiment 128 is the method of any of embodiments 18-27, wherein the first nucleic acid encodes a HER2 antigen having at least 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO: 13.

[0337] Embodiment 129 is the method of any of embodiments 18-28, wherein the first nucleic acid encodes a HER2 antigen comprising SEQ ID NO: 13.

[0338] Embodiment 130 is the method of any one of embodiments 18-29, wherein the first nucleic acid encodes a HER2 antigen, wherein the first nucleic acid has at least 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO: 14.

[0339] Embodiment 131 is the method of any of embodiments 18-30, wherein the first nucleic acid comprises SEQ ID NO: 14.

[0340] Embodiment 132 is the method of any one of embodiments 18-27, wherein the first nucleic acid encodes a HER2 antigen having at least 90%, 95%, 97%, 98%, or 99% identity with the full length SEQ ID NO: 13.

[0341] Embodiment 133 is a method according to any one of embodiments 18-29, wherein the first nucleic acid encodes a HER2 antigen, wherein the first nucleic acid has at least 90%, 95%, 97%, 98%, or 99% identity with full size SEQ ID NO: 14.

[0342] Embodiment 134 is a method according to any one of embodiments 18-27, wherein the first nucleic acid encodes a HER2 antigen having at least 90%, 95%, 97%, 98%, or 99% identity with the full length SEQ ID NO: 1.

[0343] Embodiment 135 is a method according to any one of embodiments 18-29, wherein the first nucleic acid encodes a HER2 antigen, wherein the first nucleic acid has at least 90%, 95%, 97%, 98%, or 99% identity with full size SEQ ID NO: 2.

[0344] Embodiment 136 is a method according to any one of embodiments 34-36, wherein the first nucleic acid encodes a HER2 antigen having at least 90%, 95%, 97%, 98%, or 99% identity with the full length SEQ ID NO: 3.

[0345] Embodiment 137 is a method according to any one of embodiments 18-29, wherein the first nucleic acid encodes a HER2 antigen, wherein the first nucleic acid has at least 90%, 95%, 97%, 98%, or 99% identity with full length SEQ ID NO: 4.

[0346]

EXAMPLES

[0347] The following examples illustrate the invention, but should not be taken as limiting the scope of the claims in any way.

Example 1 Intravenous Administration of Recombinant MVA Leads to Stronger NK Cell Activation

[0348] C57BL/6 mice were immunized subcutaneously (SC) or intravenously (IV) with 5×10 ⁷ SCID ₅₀ MVA-OVA (shown as rMVA) or MVA-OVA-CD40L (shown as rMVA-CD40L). The FSB was administered s / c. One day later, using the method of flow cytometry, the frequency of NK cells and protein expression (given as geometric mean fluorescence intensity (GSIF)) in the spleen (illustrated in Fig. 1A-1G), in the liver (illustrated in Fig. 2A- 2G) and in the lungs (illustrated in Figs. 3A-3G) by staining for A) NKp46 ⁺ CD3 ^- cells; B) CD69; C) NKG2D; D) FaSL; E); Bcl-X _L ; F), CD70; and G) IFN-γ.

[0349] Additionally, C57BL/6 mice were subcutaneously (SC) or intravenously (IV) immunized with 5 x 10 ⁷ SCID ₅₀ of recombinant MVA encoding HER2, TWIST and CD40L antigens (shown as MVA-HER2-TWIST-CD40L ). The FSB was administered s / c. One day later, using the method of flow cytometry, the frequency of NK cells and protein expression (given as geometric mean fluorescence intensity (GSIF)) in the spleen (illustrated in Fig. 4A-4F), in the liver (illustrated in Fig. 5A- 5F) and in the lungs (illustrated in Figs. 6A-6F) by staining for A) NKp46 ⁺ CD3 ^- cells; B) CD69; C) FaSL; D); Bcl-X _L ; E), CD70; and F) IFN-γ.

[0350] As illustrated in the figures, the frequency of NK cells fell or remained after the injection of rMVA, rMVA-CD40L and MVA-HER2-TWIST-CD40L, regardless of the route of administration. In A), iv administration of rMVA increased the incidence of NK cells in the liver and spleen compared with s.c. administration. In B), CD69 is a stimulatory receptor for NK cells (Borrego et al., Immunology 1999) and is highly overexpressed after i.v. but not s.c. injection of rMVA, rMVA-CD40L and MVA-HER2-TWIST-CD40L . The highest expression of CD69 was induced by i.v. administration of rMVA-CD40L. On FIG. 1-3C) there is increased expression of the activating lectin-like C-type receptor NKG2D on NK cells after immunization with rMVA and rMVA-CD40L compared to PBS treatment. On FIG. 1-3(D) and FIG. 4-6(C) there is increased expression of the apoptosis inducing factor FasL (CD95L) on NK cells after immunization with rMVA and rMVA-CD40L compared to PBS treatment. On FIG. 1-3(D) and FIG. 4-6(C) FasL expression in the spleen and lungs was greatest after iv injection of rMVA-CD40L and MVA-HER2-TWIST-CD40L. On FIG. 1-3(E) and 4-6(D) i.v. immunization with rMVA-CD40L and MVA-HER2-TWIST-CD40L also resulted in higher expression of a member of the anti-apoptotic Bcl family, Bcl-x _L , compared to p / to immunization. On FIG. 1-3(F) and 4-6(E) increased expression of the costimulatory molecule CD70, a member of the tumor necrosis factors (TNF) superfamily, is induced by intravenous injection of rMVA, rMVA-CD40L and MVA-HER2-TWIST-CD40L, especially on NK cells of the spleen. On FIG. 1-3(G) and 4-6(F), importantly, the effector cytokine IFN-γ is most strongly expressed after iv immunization with rMVA-CD40L or iv immunization with MVA-HER2-TWIST-CD40L in the spleen, lungs, and liver. These data show that iv immunization with either rMVA-CD40L or MVA-HER2-TWIST-CD40L, but not SC immunization, results in strong, systemic NK cell activation.

Example 2: Intravenous Administration of Recombinant MVA-CD40L Leads to Stronger Systemic NK Cell Activation

[0351] C57BL/6 mice were iv immunized with 5×10 ⁷ SCID ₅₀ MVA-OVA (rMVA), MVA-OVA-CD40L (rMVA-CD40L) or PBS. Six hours post-injection, serum levels of cytokines (A) IFN-γ, (B) IL-12p70, and (C) CD69 ⁺ granzyme B ⁺ were quantified by bead assay (Luminex) (A and B) and flow cytometry (C ), as illustrated in FIG. 7A-7F. The NK cell activating cytokine IL-12p70 was only detectable after immunization with rMVA-CD40L. IFN-γ concentration was higher after rMVA-CD40L compared to rMVA immunization. Elevated serum levels of IFN-γ are consistent with greater GSIF IFN-γ NK cells (compared to Fig. 1G) and a higher frequency of CD69 ⁺ granzyme B ⁺ NK cells in the spleen 48 hours after immunization with rMVA-CD40L.

[0352] Similar responses were observed in OCP ( Macaca fascicularis ) after IV injection of MVA-MARV-GP-huCD40L, namely higher serum concentrations of IFN-γ (D) and IL-12p40/70 (E), as well as more proliferating (Ki67 ⁺ ) NK cells (F) compared to MVA-MARV-GP. These data demonstrate that CD40L-encoding MVA-based vaccines have comparable immunological properties in mice and PCVs.

Example 3: Intravenous Administration Leads to Stronger NK Cell Activation and Proliferation Over Time

[0353] C57BL/6 mice were iv immunized with 5×10 ⁷ SCID ₅₀ MVA-OVA (rMVA), MVA-OVA-CD40L (rMVA-CD40L) or PBS. Activation and proliferation of NK cells was determined for (A) CD3 ^- CD19 ^- NKp46 ⁺ , (B), Ki67 and (C) CD69 in the spleen, liver and lungs after 1 day and 4 days after immunization. The results are illustrated in FIG. 8.

[0354] As illustrated in FIG. 8A), the frequency of NK cells in the spleen (defined as CD3 ^- CD19 ^- NKp46 ⁺ ) fell temporarily on day 1 after immunization with rMVA and rMVA-CD40L, as seen in example 1, but increased on day 4 compared with PBS. In the liver and lungs, both vectors induced a higher frequency of NK cells as early as 1 day. Surprisingly, on day 4, the frequency of NK cells in the liver was 2.4 times higher after immunization with rMVA and 4 times higher after immunization with rMVA-CD40L compared to PBS. Likewise, the frequency of NK cells in the lungs and blood was dramatically increased on the 4th day after immunization, as illustrated in 8A. Consistent with the systemic increase in NK cells, more than 80% of all NK cells were proliferating on day 4 post-immunization with rMVA and rMVA-CD40L, as indicated by expression of the Ki67 proliferation marker on 8B. Peak expression of CD69 (given as geometric mean fluorescence intensity (GSIF)) in all analyzed organs occurred on day 1. On day 4 post-immunization, CD69 expression returned to baseline (FIG. 8C).

Example 4 Intravenous Administration of Recombinant MVA Leads to Stronger Activation of NK Cell Mediated Toxicity

[0355] C57BL/6 mice were iv immunized with 5×10 ⁷ SCID ₅₀ MVA-OVA (rMVA), MVA-OVA-CD40L (rMVA-CD40L) or PBS. Mice were sacrificed 24 hours later and spleen NK cells were purified by magnetic cell sorting and used as effectors in an effector:target kill assay. Briefly, NK cells were cultured with CFSE-labeled MHC class I deficient YAC-1 cells at the ratios indicated in FIG. 9. Specific killing was assessed by quantifying non-viable CFSE ⁺ YAC-1 cells by flow cytometry. As a result, activated rMVA and rMVA-CD40L NK cells after iv immunization effectively destroy target cells deficient in MHC class I.

Example 5 Intravenous Administration of Recombinant MVA Leads to Increased ADCC

[0356] As illustrated in FIG. 10A, C57BL/6 mice were treated iv with 25 µg anti-CD4 (clone GK1.5), MVA-OVA (rMVA) + 5 µg rat IgG2b or 1 µg anti-CD4 or MVA-OVA (rMVA) + 1 µg anti-CD4. Mice were sacrificed 24 hours later and the depletion of CD4 T cells (CD3 ⁺ CD4 ⁺ ) in the liver was analyzed by flow cytometry. By definition, 25 µg of anti-CD4 corresponded to 100% specific killing. Compared to rMVA+IgG2b (as defined by 0% specific killing), 1 μg of anti-CD4 depleted 61% of all liver CD4 T cells. The combination of rMVA+1 µg anti-CD4 resulted in 93% CD4 T cell depletion, which is almost as effective as 25 µg anti-CD4. Thus, the combination of rMVA and depleting antibodies results in synergistic killing of target cells in vivo.

[0357] As illustrated in FIG. 10B and 10C, to evaluate ADCC activity of ex vivo NK cells, C57BL/6 (B) or Balb/c (C) mice were immunized with iv PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA -CD40L). Mice were sacrificed 24 hours later; Spleen NK cells were purified by magnetic cell sorting and used as effectors in an effector:target antibody dependent kill assay. (B) B16.F10 target cells were coated with mouse anti-human/mouse Trp1 mAb (clone TA99) and (C) CT26-HER2 target cells were coated with mouse anti-human HER2 mAb (clone 7.16.4). Purified NK cells were added to antibody-coated target cells at a ratio of 5:1 and 4:1, respectively. Cell death was determined by measuring the release of lactate dehydrogenase (LDH) into the cell culture medium. In both assays, target cell lysis was greater when NK cells were activated in vivo with rMVA or rMVA-CD40L compared to PBS. Activation of rMVA-CD40L resulted in the highest lytic activity. CT26-HER2 cells that were incubated with different concentrations of anti-human HER2 antibody (from 0.1 to 10 μg/ml anti-HER2) were also killed more efficiently by rMVA-CD40L-activated NK cells compared to rMVA-activated NK cells (illustrated to D).

[0358] As illustrated in FIG. 10E, ex vivo ADCC activity of NK cells was analyzed using MVA-HER2-Twist-CD40L. Balb/c mice were immunized iv with PBS or MVA-HER2-Twist-CD40L. CT26-HER2 cells were coated with 5, 0.5 and 0.05 μg/ml mouse anti-human HER2 mAb (clone 7.16.4). Purified NK cells were added to antibody-coated target cells in a 5:1 ratio. Cell death was determined by measuring the release of lactate dehydrogenase (LDH) into the cell culture medium. Target cell lysis was stronger when NK cells were activated in vivo with MVA-HER2-Twist-CD40L compared to PBS.

[0359] Thus, ADCC-mediated killing of tumor cells expressing appropriate tumor antigens can be enhanced by rMVA and, in particular, rMVA-CD40L and MVA-HER2-Twist-CD40L mediated NK cell activation. In addition, enhanced rMVA, and especially rMVA-CD40L and MVA-HER2-Twist-CD40L, ADCC activity was observed over a wide range of antibody doses.

Example 6 Intravenous Administration of Recombinant MVA Induces Strong CD8 T Cell Responses

[0360] C57BL/6 mice were immunized intravenously or sc 5×10 ⁷ SCID ₅₀ MVA-OVA on days 0 and 16. On days 7 and 22, the responses of OVA-specific CD8 T cells in the blood were evaluated by flow cytometry after staining with H-2K ^b /OVA _257-264 dextramers. As illustrated in FIG. 11, on day 7, the frequency of OVA-specific CD8 T cells was 9-fold higher compared with sc injections. On day 22, the number of OVA-specific T cells was 4 times higher than after s/c injections.

Example 7 Intravenous Administration of Recombinant MVA-CD40L Induces Strong CD8 T Cell Responses

[0361] As illustrated in FIG. 12, C57BL/6 mice were immunized intravenously with 5×10 ⁷ SCID ₅₀ MVA-OVA or MVA-OVA-CD40L on days 0 and 35. Flow cytometry was used to evaluate the responses of OVA-specific CD8 T cells in blood after staining with H-2Kb/OVA _257-264 dextramers. At the peak of the primary (day 7) and secondary (day 39) responses, the frequency of OVA-specific CD8 T cells was increased 4-fold and 2-fold, respectively, after immunization with MVA-OVA-CD40L compared with MVA-OVA (Lauterbach et al. ., 2013).

Example 8 Prime Boost Immunization Demonstrates Reactivation and Proliferation of NK Cells

Table 1 IV Immunization Schedule for Examples 8-10

[0362] C57BL/6 mice were iv immunized as shown in Table 1 (recombinant MVA doses were 5×10 ⁷ SCID ₅₀ ). NK cells (CD3 ^- NKp46 ⁺ ) were analyzed in blood by flow cytometry one and four days after the second and third immunizations. On FIG. 13A and 13B illustrate CD69 GSIF (A) and Ki67 ⁺ NK cell frequency (B). On FIG. 13A and 13B illustrate that NK cell activation occurs after each immunization despite the presence of immunity against the vector. This surprising finding favors combination therapy based on antibodies with booster immunizations that activate NK cells. Thus, when cancer patients are treated a number of times with recombinant MVA and develop responses against the vector, NK cell activation remains intact. On the contrary, each stage of treatment leads to de novo activation of NK cells.

Example 9 Prime Boost Immunization Demonstrates Stronger Induction of CD4 Helper T Cells

[0363] C57BL/6 mice were iv immunized as shown in Table 1 (recombinant MVA doses were 5×10 ⁷ SCID ₅₀ ). Serum levels of cytokines were quantified 6 hours after immunization by a multichannel bead assay (Luminex). The results on the expression of these cytokines are presented. 14A) IL-6; 14B) CXCL10; 14C) IFN-α; 14D) IL-22; 14E) IFN-γ; 14F) CXCL1; 14G) CCL4; 14H) CCL7; 14I) CCL2; 14J) CCL5; 14K) TNF-α; 14L) IL-12p70; and 14M) IL-18.

[0364] As illustrated in FIG. 14A-14M, mice treated with rMVA-CD40L in the homologous regimen had a similar cytokine profile to mice treated with rMVA primer and rMVA-CD40L booster (rMVA-CD40L het). Homologous rMVA-treated mice exhibited lower levels of IL-6, IL12p70, IL-22, IFN-α, TNF-α, CCL2, CCL5 and CXCL1 after the first and second immunizations compared to mice primed with rMVA-CD40L. The cytokine that was absent after the primary but was produced at high levels after the second and third immunizations was IL-22. IL-22 is produced in large quantities by effector helper T cells and lymphocyte subpopulations of the innate immune system. Higher expression of IL-22 in rMVA-CD40L het or rMVA-CD40L hom treated mice thus indicates a stronger induction of CD4 T helper responses when immunized with rMVA-CD40L. Overall, iv immunization with rMVA and rMVA-CD40L induced high systemic cytokine responses, which were highest in mice primed with rMVA-CD40L.

Example 10 Prime Boost Immunization Demonstrates Stronger Antigen-Specific CD8 T Cell Responses

[0365] C57BL/6 mice were iv immunized as shown in Table 1 (recombinant MVA doses were 5×10 ⁷ SCID ₅₀ ). Similar to NK cells, the induction of antigen-specific responses of CD8 T cells was assessed after booster immunization. As illustrated in FIG. 15, the frequency of CD8 T cells increased dramatically after the second and third immunizations with rMVA and rMVA-CD40L. It should be noted that after the second immunization, about 80% of all PBLs were CD8 T cells (A). Although the vector-specific response (B8) of CD8 T cells peaked after the second immunization (B), transgene-specific (OVA) responses can be further enhanced by the third immunization (C). This resulted in an increase in the ratio of OVA/B8-specific CD8 T cells (D), indicating an immune focus on the transgene. Importantly, these results demonstrate that antigen-specific T cell responses can be enhanced by iv immunization in the presence of immunity to the vector. Thus, in cancer patients, a number of treatment steps may result in stronger antigen-specific CD8 T cell responses.

Example 11 Prime Boost Immunization Demonstrates Stronger CD8 and CD4 Memory T Cell Responses

[0366] IV C57BL/6 mice were immunized as shown in Table 1. The results are illustrated in FIG. 16. Phenotypically, effector T cells and memory effector T cells can be identified by CD44 expression and the absence of surface CD62L. Determination of CD44 ⁺ CD62L ^- CD8 (A) and CD4 (B) T cells in the blood demonstrated that repeated IV immunization induces the expansion of effector T cells and memory effector T cells. Interestingly, mice treated with rMVA-CD40L hom or rMVA-CD40L het had about 2.5 times more effector CD4 T cells in circulation than mice primed with rMVA (B, day 25). This indicates that systemic priming of rMVA-CD40L induces stronger CD4 T cell responses than rMVA.

Example 12: Intravenous administration of recombinant MVA results in a strong antitumor effect in the treatment of melanoma

[0367] C57BL/6 mice bearing palpable B16.OVA tumors were primed (dotted line) with PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L) s.c. or i.v. MVA was 5×10 ⁷ TCID ₅₀ ). On days 7 and 14 after the primary immunization, mice received subsequent booster injections of FPV-OVA at 5×10 ⁷ SCID ₅₀ (dashed-dotted lines). Tumor growth was measured at regular intervals. On FIG. 17 illustrates mean tumor volume (A) and survival of tumor-bearing mice at day 45 after tumor inoculation (B). Thus, iv priming of B16.OVA tumor-bearing mice with rMVA-CD40L provides a stronger antitumor effect compared to s/c rMVA-CD40L or s/c or iv rMVA.

Example 13: One Intravenous Administration of Recombinant MVA Leads to Strong Antitumor Effect

[0368] C57BL/6 mice bearing palpable B16.OVA tumors were iv vaccinated as shown in Table 2. Tumor growth was measured at regular intervals. On FIG. 18 illustrates the average tumor volume. The results indicate that a single rMVA-CD40L therapeutic immunization is as effective as homologous or heterologous prime/boost immunizations. Importantly, these data highlight the strong antitumor activity of rMVA-CD40L.

Table 2. The schedule of vaccination corresponds to example 13

Example 14: CD8 T cells are important for rMVA-CD40L mediated tumor suppression

[0369] C57BL/6 mice bearing palpable B16.OVA tumors were immunized by intravenous administration of PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L) (recombinant MVA doses were 510 ⁷ SCID ₅₀ ). Mice received intraperitoneal injections of 200 μg of anti-CD8 antibody, if indicated. As illustrated in FIG. 19, immunization with rMVA-CD40L induced a stronger overall CD8 T cell response (A) as well as stronger neoantigen-specific (OVA) CD8 T cell responses (B) compared to rMVA. C) represents overall survival. These data indicate that rMVA-CD40L induces superior CD8 T cell responses under tumor conditions and that the induced CD8 T cell responses are important for the antitumor effect observed after therapeutic immunization.

Example 15: IV administration of recombinant MVA encoding at least two TAAs is more efficient

[0370] C57BL/6 mice bearing palpable B16.OVA tumors were immunized iv with PBS, MVA-OVA-TRP2, MVA-OVA-TRP2-CD40L, or MVA-OVA-CD40L. As illustrated in FIG. 20, tumor growth was measured at regular intervals and presented as mean single mouse diameter (A) and mean volume (B). Survival is illustrated in (C). These data indicate that a single therapeutic immunization with rMVA-CD40L encoding two TAAs is more effective than immunization with rMVA-CD40L encoding only one TAA.

Example 16 Intravenous administration of recombinant MVA-CD40L increased T cell infiltration into the tumor microenvironment

[0371] C57BL/6 mice bearing palpable B16.OVA tumors were intravenously immunized with PBS, rMVA (MVA-OVA) or rMVA-CD40L (MVA-OVA-CD40L) (recombinant MVA doses were 5×10 ⁷ SCID ₅₀ ). Mice were sacrificed after 7 days. As illustrated in FIG. 21A and 21B, the frequency and distribution of CD8 ⁺ T cells and OVA _257-264 -specific CD8 + T cells were analyzed among leukocytes in the spleen, tumor-draining lymph nodes (DONA) and tumor tissues. As illustrated in FIG. 21C analyzed the geometric mean fluorescence intensity (GSIF) of PD-1 and Lag3 on tumor-infiltrating OVA _257-264 -specific CD8 ⁺ T cells; in FIG. 21D shows representative dotted plots of Ki67 and PD-1 expression of tumor-infiltrating CD8 ⁺ T cells. On FIG. 21E illustrates the frequencies of tumor-infiltrating Ki67 ⁺ CD8 ⁺ T cells and GSIF PD-1; in FIG. 21F illustrates the frequency of tumor infiltrating T cells (Treg) among leukocytes. On FIG. 21G illustrates the frequency of tumor-infiltrating PD-1 ^high. and PD-1 ^neg. Treg. Together, these data show that immunization with rMVA-CD40L leads to greater infiltration of TAA-specific CD8 T cells into the tumor microenvironment (TMO) and that these CD8 T cells express lower amounts of markers associated with "immune depletion" compared to immunization with PBS and rMVA. In addition, immunization with rMVA-CD40L leads to a decrease in the number of highly suppressive Tregs in OMO compared to PBS.

Example 17 Intravenous administration of recombinant MVA-CD40L increased the duration of T cell infiltration into the tumor microenvironment

[0372] TKR transgenic OVA-specific CD8 T cells (OT-I) were intravenously transferred to tumor-bearing B16.OVA mice when tumors became palpable. When tumors reached at least 60 mm ³ in volume, animals were immunized with MVA-BN, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L) (recombinant MVA doses were 510 ⁷ SCID ₅₀ ). After 17 days, mice were sacrificed and analyzed for A) the frequency of CD8 ⁺ T cells among leukocytes in tumor tissues; B) Lag3 ⁺ PD1 ⁺ frequencies among CD8 ⁺ T cells; C) frequencies of Eomes ⁺ PD1 ⁺ T cells among CD8 ⁺ T cells; D) the presence of OT-I transgenic CD8 ⁺ T cells in OMO after immunization; and E) frequency of Lag3 ⁺ PD1 ⁺ depleted T cells among OT-I ⁺ CD8 ⁺ T cells; and F) frequency of Eomes ⁺ PD1 ⁺ T cells depleted among OT-I ⁺ CD8 ⁺ T cells. The results are illustrated in FIG. 22. These data show that OAA-specific CD8 T cells recruited to OMO after immunization with rMVA-CD40L show fewer signs of immune depletion than after control treatment (MVA-BN without encoded OAA) or immunization with rMVA even after prolonged exposure to OMO. .

Example 18: Intravenous administration of recombinant MVA-CD40L reduced Treg levels in the tumor microenvironment

[0373] Purified OVA-specific TCR transgenic CD8 T cells (OT-I) were intravenously transferred to tumor-bearing B16.OVA mice when tumors became palpable. When tumors reached at least 60 mm ³ in volume, animals were immunized with MVA-BN, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L) (recombinant MVA doses were 5×10 ⁷ SCID ₅₀ ). After 17 days, mice were sacrificed and analyzed for: A) frequency of Foxp3 ⁺ CD4 ⁺ Treg among CD4 ⁺ T cells in tumor tissues; and B) ratio of CD8 ⁺ CD44 ⁺ effector T cells to Foxp3 ⁺ CD4 ⁺ Tregs. The results are illustrated in FIG. 23. Together, these data show that even after prolonged exposure to OMO, rMVA-CD40L immunization alone results in an increase in the Teff/Treg ratio compared to control (MVA-BN without encoded TAA) or rMVA immunization.

Example 19: MVA-OVA-CD40L is safer in humans compared to CD40 agonist mAb

Table 3 Immunization Schedule for Example 19

[0374] C57BL/6 mice were intravenously injected with PBS, MVA-OVA (rMVA), MVA-OVA-CD40L (rMVA-CD40L) or anti-CD40 (FGK4.5) as shown in Table 3 (doses of recombinant MVA were 5×10 ⁷ SCID ₅₀ dose of anti-CD40 was 100 μg). Serum concentration of alanine aminotransferase (ALT) was determined one day after each immunization by ELISA. As illustrated in FIG. 24, ALT levels were highest after the first anti-CD40 injection. The ALT concentration after the first immunization with rMVA-CD40L was 2 times lower than after anti-CD40 treatment. Importantly, serum ALT levels were not elevated after boosting with rMVA and rMVA-CD40L. Although the second injection of anti-CD40 also did not lead to an increase in ALT levels, 3 out of 4 mice in this group died (NM=found dead). Also, as illustrated in FIG. 21, lower serum levels of alanine aminotransferase (ALT) were observed after rMVA and rMVA-CD40L immunization compared to anti-CD40 immunoglobulin injection. Thus, in contrast to the described liver toxicity of anti-CD40 agonist (Medina-Echeverz et al., 2015), no such toxicity was observed after repeated IV injection of rMVA and rMVA-CD40L.

Example 20 Improved Overall Survival and Tumor Shrinkage After IV Administration of rMVA-CD40L

[0375] Balb/c mice carrying palpable CT26.HER2 tumors were immunized iv with MVA-AH1A5-p15-TRP2-CD40L (rMVA-CD40L) or received PBS. Tumor growth was measured at regular intervals. On FIG. 25 illustrates mean tumor volume (A) and survival (B). These data indicate that rMVA-CD40L therapeutic immunization alone effectively prolongs survival in a colon cancer model.

Example 21 Improved overall survival and tumor reduction with iv administration of rMVA-CD40L+anti-Trp1

[0376] C57BL/6 mice bearing palpable B16.OVA tumors (at least 60 mm ³ in volume) were intravenously immunized with MVA-OVA-CD40L (mBNbc115) at 5×10 ⁷ SCID ₅₀ on day 8. Mice were intraperitoneally injected with 200 μg of anti-Trp1 (clone TA99) twice a week starting on day 5. On FIG. 26A and 26B illustrate the results for mean tumor volume and overall survival, respectively. As illustrated in the figures, the combination of rMVA-CD40L and anti-Trp1 mAb demonstrated an overall reduction in tumor volume and an increase in overall survival compared to anti-Trp1 mAb and rMVA-CD40L alone. Thus, the intravenously administered combination of tumor-specific antibody and rMVA-CD40L had significantly greater antitumor activity compared to non-IV administration of rMVA-CD40L or administration of the antibody alone.

Example 22: Construction of recombinant MVA viruses MVA-mBN445, MVA-mBN451, MVA-mBNbc197, MVA-mBNbc195, MVA-mBNbc388, MVA-mBN bc389 and MVA-mBN484

[0377] The creation of recombinant MVA viruses that are part of combination therapy (eg MVA-mBN445, MVA-mBN451 and MVA-mBN484) was performed by inserting these transgenes with their promoters into the MVA-BN vector. The transgenes were inserted using recombination plasmids containing the transgenes and a selection cassette, as well as sequences homologous to the target loci in MVA-BN. Homologous recombination between the viral genome and the recombination plasmid was achieved by transfection of the recombination plasmid into MVA-BN infected CEF cells. Then, in a second step, the selection cassette was removed with a plasmid expressing a CRE recombinase that specifically targets the loxP sites flanking the selection cassette, thus excising an intermediate sequence.

[0378] Two or three transgene recombination plasmids for mBNbc197 (MVA-OVA-TRP2) and mBNbc195 (MVA-OVA-TRP2-CD40L), respectively, were used to construct MVA-BN mBNbc197 and MVA-BN mBNbc195, respectively. The included plasmids insert sequences that are also present in the MVA. Nucleotide sequences encoding OVA and TRP2 and/or CD40L antigens were interposed between the inserted MVA sequences to allow recombination into the MVA viral genome. Thus, for each construct, a plasmid was constructed that contained the coding sequences for OVA and TRP2 and/or CD40L, each downstream of a promoter.

[0379] For the construction of mBN451, the recombination plasmid contained two HER2v1 and Brachyury transgenes (SEQ ID NO: 1 and SEQ ID NO: 3, respectively), each preceded by a promoter sequence, as well as sequences identical to the target insertion site in MVA-BN, so that allow homologous recombination into the viral genome. The HER2 and Brachyury coding sequences (or nucleotide sequences) are SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0380] For the construction of mBN445, the recombination plasmid contained three HER2v1, Brachyury and CD40L transgenes (SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 11, respectively), each of which was preceded by a promoter sequence, as well as sequences identical to the target insertion site in MVA-BN to allow homologous recombination into the viral genome. The HER2, Brachyury and CD40L coding sequences (or nucleotide sequences) are SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 12, respectively.

[0381] For the construction of mBNbc388, the recombination plasmid contained three transgenes HER2v1, Twist and CD40L (SEQ ID NO: 1, SEQ ID NO: 15 and SEQ ID NO: 17, respectively), each of which was preceded by a promoter sequence, as well as sequences identical to the target insertion site in MVA-BN to allow homologous recombination into the viral genome. The HER2v1, Twist and CD40L coding sequences (or nucleotide sequences) are SEQ ID NO: 2, SEQ ID NO: 16 and SEQ ID NO: 18, respectively.

[0382] For the construction of mBNbc389, the recombination plasmid contained two transgenes, HER2v1 and Twist (SEQ ID NO: 1, SEQ ID NO: 15, respectively), each of which was preceded by a promoter sequence, as well as sequences identical to the target insertion site in MVA-BN, so that allow homologous recombination into the viral genome. The HER2, Twist and CD40L coding sequences (or nucleotide sequences) are SEQ ID NO: 2 and SEQ ID NO: 16, respectively.

[0383] For the construction of mBN484, the recombination plasmid contained three HER2 v.2 transgenes, Brachyury and CD40L (SEQ ID NO: 13, SEQ ID NO: 3 and SEQ ID NO: 11, respectively), each of which was preceded by a promoter sequence, as well as sequences , identical to the target insertion site in MVA-BN to allow homologous recombination into the viral genome. The coding sequences for HER2 v.2, Brachyury and CD40L (or nucleotide sequences) are SEQ ID NO: 14, SEQ ID NO: 4 and SEQ ID NO: 12, respectively.

[0384] To create all of the mBN MVAs described above (eg, mBN445, mBN451 and mBN484), CEF cell cultures were each inoculated with MVA-BN and transfected with the appropriate recombination plasmid. In turn, samples from these cell cultures were inoculated into CEF cultures in media containing drugs that induce selective pressure, and fluorescent viral clones were isolated by plaque-based purification. The disappearance of the selection cassette containing the fluorescent protein from these viral clones was mediated in a second step by CRE-mediated recombination involving two loxP sites flanking the selection cassette in each construct. After the second recombination step, only transgenic sequences (eg, HER2, Brachyury and/or CD40L) remained with promoters inserted at the target MVA-BN loci. A stock of plaque-purified virus without a selection cassette was prepared.

[0385] After that, the expression of the identified transgenes was demonstrated in cells inoculated with the described construct (see, for example, Fig. 27).

[0386] Creation of MVA-mBNbc197, mBNbc195, mBNbc388 and mBNbc389 was performed using a cloned version of MVA-BN in the bacterial artificial chromosome (BIC). Recombination plasmids containing different transgenes for mBNbc197 (MVA-Ova-Trp2), mBNbc195 (MVA-Ova-Trp2-CD40L), mBNbc388, and mBNbc389 were used. These plasmids contained sequences that are also present in MVA and therefore allow specific targeting to the integration site. Nucleotide sequences encoding the OVA, Her2 v1, Twist, Trp2 and/or CD40L antigens were placed between the MVA sequences to allow recombination into the MVA viral genome. Thus, for each construct, a plasmid was constructed that contained the coding sequences for OVA, Her2 v1, Twist, Trp2, and/or CD40L, each downstream of a promoter. Briefly, infectious viruses were recovered from CIR by transfecting CIR DNA into BHK-21 cells and superinfecting them with Shope's fibroma virus as a helper virus. After three additional passages on CEF cell cultures, helper virus-free MVA-mBNbc197, MVA-mBNbc195, MVA-mBNbc388 and MVA-mBNbc389 were obtained. An exemplary way to generate MVA can also be found in Baur et al., "Immediate-early expression of a recombinant antigen by modified vaccinia virus Ankara breaks the immunodominance of strong vector-specific B8R antigen in acute and memory CD8 T-cell responses,". Virol. 84(17):8743-52 (2010).

Example 23 Heterologous Expression of MVA-HER2-Brachyury-CD40L

[0387] HeLa cells were left untreated (control) or infected with MVA-BN or MVA-HER2-Brachyury-CD40L (MVA-mBN445). After overnight culture, cells were stained with anti-HER2-APC (clone 24D2), anti-Brachyury (rabbit polyclonal) + anti-rabbit IgG-PE and anti-CD40L-APC (clone TRAP1). As illustrated in FIG. 27A-27D, flow cytometry analysis revealed expression of all three transgenes.

Example 24 Synthetic HER2 Proteins Prevent Binding of Trastuzumab and Pertuzumab

[0388] Mouse CT26 colon carcinoma cells were infected with MVA-HER2-Brachyury-CD40L (MVA-mBN484) at MH 1. After 24 hours, cells were incubated with 5 μg/ml of anti-HER2 antibody trastuzumab, pertuzumab, or 24D2. The figures illustrate HER2 expression levels normalized to clone 24D2 HER2 staining. Data are given as mean±SPS and represent two independent experiments. The results are illustrated in FIG. 28.

Example 25 Enhanced Activation of Human DCs by MVA-HER2-Brachyury-CD40L

[0389] Monocytic dendritic cells (DCs) were generated after enrichment for CD14 ⁺ monocytes from human PBMCs and cultured for 7 days in the presence of GM-CSF and IL-4 according to the protocol (Miltenyi, MO-DC Construction Kit). DCs were stimulated with MVA-HER2-Brachyury or MVA-HER2-Brachyury-CD40L. As illustrated in FIG. 29, expression of A) CD40L; B) CD86 and C) MHC class II were analyzed by flow cytometry. As illustrated in D), the concentration of IL-12p70 in the supernatant was determined using luminex after culturing overnight.

[0390] This experiment demonstrates that rMVA-HER2-Brachyury-CD40L stimulates human DCs, inducing their activation and thus increasing their ability to present antigens. The production of the Th1 polarizing and NK cell activating cytokine IL-12p70 by stimulated human DCs indicates that MVA-HER2-Brachyury-CD40L activates human DCs towards a pro-inflammatory phenotype.

Example 26 Improved Overall Survival and Tumor Reduction with Intravenous Administration of mBNbc388 and mBNbc389

[0391] Mouse MC38 colon carcinoma cells expressing HER2 (MC38.HER2) were s.c. injected into C57BL/6 mice. Mouse CT26 colon carcinoma cells expressing HER2 (CT26.HER2) were s.c. injected into Balb/c mice. In both groups of mice, when tumors were larger than 50 mm ³ , mice bearing MC38.HER2 tumors (Fig. 30A and 30B) and CT26.HER2 tumors (Fig. 31A and Fig. 31B) were immunized with (dashed line) PBS, MVA-CD40L or MVA-HER2-Twist-CD40L IV. Tumor growth was measured at regular intervals. A) measured the mean tumor volume, and B) illustrates the overall survival of tumor-bearing mice at 60 days after tumor inoculation.

[0392] Since the murine homologue of Brachyury is not highly expressed in normal mouse tissues and is not predominantly expressed in mouse tumor tissues, the efficacy of Brachyury as a target for active immunotherapy cannot be effectively studied in a mouse model system. See WO 2014/043535, which is incorporated herein by reference. Twist, a mouse homologue of human Brachyury, is used in mouse models and is a predictive model of Brachyury function in humans. This has been demonstrated in WO 2014/043535. Like Brachyury, the murine homologue of the EMT regulator Twist stimulates EMT during development by reducing E-cadherin-mediated cell-cell adhesion and upregulating mesenchymal marker expression, and is predominantly expressed in mouse tumor tissue. See, for example, Fig. 5 and example 8 in WO 2014/043535. Therefore, a study of a Twist-specific cancer vaccine in mice is very likely to have a significant predictive value for the efficacy of a Brachyury-specific cancer vaccine in humans. Id.

Example 27 Intravenous administration of MVA-HER2-Twist-CD40L (mBNbc388) enhances infiltration of HER2-specific CD8+ T cells into a tumor.

[0393] Balb/c mice carrying CT26.HER2 tumors received PBS or 5×10 ⁷ SCID ₅₀ MVA-HER2-Twist-CD40L intravenously. Seven days later, mice were sacrificed, splenic and tumor-infiltrating CD8 ⁺ T cells were isolated by magnetic cell sorting, and cultured in the presence of HER2-loaded dendritic cells for 5 hours. The graphs show the percentage of CD44 ⁺ IFN-γ ⁺ cells among CD8 ⁺ T cells. Results are given as mean ± SPS. The results illustrated in FIG. 32 demonstrate that various embodiments of the present invention are tumor-specific.

Example 28 Improved Overall Survival and Tumor Reduction with Intravenous Administration of mBNbc388 and mBNbc389 in Combination with Trastuzumab

[0394] Balb/c mice carrying CT26.HER2 tumors with an average volume greater than 100 mm ³ received 5 μg of human IgG1 or trastuzumab intraperitoneally on day 15 after tumor inoculation. Two days after the first injection of human IgG1 or trastuzumab, mice received intravenous PBS or 5×10 ⁷ SCID ₅₀ MVA-HER2-Twist-CD40L as illustrated in FIG. 31. In this example, human IgG1 is used as an experimental control for trastuzumab because IgG1 does not bind HER2 like trastuzumab does. Tumor growth and overall survival time were measured at regular intervals. (A) Mean tumor volume and (B) mouse survival over time.

[0395] As illustrated in FIG. 33(A) and (B), the combination of MVA-HER2-Twist-CD40L with trastuzumab was exceptionally effective in shrinking larger tumors and increasing overall survival in mice with such large tumors, even at reduced antibody dosage compared to Example 21.

Example 29 Overall Survival and Tumor Reduction with IV MVA-HER2-Twist-CD40L in an Orthotopic Breast Cancer Model

[0396] Balb/c mice carrying 4T1.HER2 orthotopic tumors are primed iv 5×10 ⁷ SCID ₅₀ MVA-HER2-Twist (mBNbc389) and/or MVA-HER2-Twist-CD40L (mBNbc388). Tumor growth should be measured at regular intervals and overall survival recorded.

Example 30 Severe NK Cell Mediated Toxicity with IV MVA-HER2-Twist-CD40L+anti-HER2

[0397] C57BL/6 mice are iv immunized with 5×10 ⁷ SCID ₅₀ MVA-HER2-Twist, MVA-HER2-Twist plus anti-Her2 or PBS. Mice are sacrificed 24 hours later and spleen NK cells are purified by magnetic cell sorting and used as effectors in an effector:target kill assay. Briefly, NK cells are cultured with CFSE-labeled MHC class I deficient YAC-1 cells at the ratios shown in Example 4. Specific killing is assessed by quantifying non-viable CFSE+ YAC-1 cells by flow cytometry.

Example 31: IV administration of MVA-HER2-Twist-CD40L+anti-HER2 enhances ADCC

[0398] C57BL/6 mice are treated iv with 25 μg anti-CD4, MVA-HER2-TWIST-CD40L+5 μg rat IgG2b or 1 μg anti-CD4 or MVA-HER2-TWIST-CD40L+1 μg anti-CD4 . Mice are sacrificed 24 hours later and the depletion of CD4 T cells (CD3 ⁺ CD4 ⁺ ) in the liver is analyzed by flow cytometry. By definition, 25 µg of anti-CD4 corresponded to 100% specific killing.

[0399] To evaluate ADCC activity of NK cells ex vivo , C57BL/6 (B) or Balb/c (C) mice are immunized iv with PBS, MVA-HER2-TWIST, or MVA-HER2-TWIST-CD40L. Mice are sacrificed 24 hours later; Spleen NK cells are purified by magnetic cell sorting and used as effectors in an antibody dependent kill effector:target assay. (B) MC38-HER2 target cells are coated with mouse anti-human/mouse HER2 mAb. CT26-HER2 target cells are coated with mouse anti-human HER2 mAb. Purified NK cells are added to antibody-coated target cells in a ratio of 5:1 and 4:1, respectively. Cell death is determined by measuring the release of lactate dehydrogenase (LDH) into the cell culture medium.

Example 32 Improved Overall Survival and Tumor Reduction with IV rMVA-CD40+anti-HER2 with Decreasing Anti-HER2 Antibody Concentrations

[0400] Balb/c mice bearing palpable CT26-HER2 tumors are immunized iv with MVA-HER2-Twist-CD40L at 5×10 ⁷ SCID ₅₀ on day 8. Mice are intraperitoneally injected with decreasing doses of anti-HER2 (200 μg, 100 μg, 25 μg) twice a week starting on day 5. Mean tumor volume and overall survival were analyzed in comparison to mice injected with 200 μg anti-HER2 antibody alone and mice injected with MVA-HER2-TWIST-CD40L alone.

Example 33: Improved overall survival and tumor reduction with iv administration of rMVA-CD40L+anti-CD52 antibody.

[0401] Balb/c mice carrying CT26.CD52 tumors receive 5 μg of human IgG1 or anti-CD52 (alemtuzumab) intraperitoneally after tumor inoculation. After the first injection of human IgG1 or alemtuzumab, mice receive intravenous PBS or 5×10 ⁷ SCID ₅₀ MVA-CD52-CD40L. Tumor growth and overall survival time are measured at regular intervals. (A) Mean tumor volume and (B) mouse survival over time.

Example 34: Improved overall survival and tumor reduction with iv administration of rMVA-CD40L+anti-EGFR antibody.

[0402] Balb/c mice carrying CT26.EGFR tumors receive 5 μg of human IgG1 or anti-EGFR (cetuximab) intraperitoneally after tumor inoculation. After the first injection of human IgG1 or cetuximab, mice receive intravenous PBS or 5×10 ⁷ SCID ₅₀ MVA-EGFR-CD40L. Tumor growth and overall survival time are measured at regular intervals. (A) Mean tumor volume and (B) mouse survival over time.

[0403] It should be understood that certain details of the methods and compositions described herein may be varied or modified without departing from the spirit of the invention described. We declare that all such modifications and variations are within the spirit and scope of the following claims.

SEQUENCE LIST

Nucleotide and amino acid sequences listed in the appended sequence listing are given using the standard letter designations for nucleotide bases and the one letter or three letter code for amino acids as defined by 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is intended to be included in the given strand by any reference.

SEQID NO:1

Amino acid sequence of synthetic Her2 v1 (1145 amino acids):

MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMC KGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTRTFKSMPNPEGRYTFGASCVTACPYNYLSTDVGSCTTLVCPAANQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYISAWP DSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPAADQCVACAHYKDPPACVARCPSGVKPDLSYMPIWAFPDEEGAC QPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIMVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLL NWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEALVPQQGFFC PDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPELGLDVPV

SEQ ID NO:2

Nucleotide sequence of synthetic Her2 v1 (3441 nucleotides):

atggaactggctgctctgtgtagatggggactgctgcttgctctgttgcctcctggagctgcttctacccaagtgtgcacaggcaccgacatgaagctgagactgcctgcttctcctgagacacacctggacatgctgacacctgtaccagggatgtcaggtggtgcagggaaatctggaactgacctacctgcctaccaacgccagcct gagctttctgcaggacatccaagaggtgcagggatacgtgctgatcgctcacaatcaagtgagacaggtgccactgcagaggctgagaatcgttagaggcacccagctgttcgaggacaactatgctctggctgtgctggacaatggcgaccctctgaacaacaccacacctgtgacaggagcttcctggtggactgagagaactgcagctgaga agcctgaccgagatcctgaaaggaggagtgctgatccagcggaaccctcagctgtgctaccaggacacaccatcctgtggaaggacatcttccacaagaacaaccagctggctctgacactgatcgacaccaacagaagcagagcctgccatccttgctctcccatgtgcaagggctctagatgttggggagagagcagcgaggattgccagagcctga ccagaacagtgtgtgctggaggatgtgccagatgcaaaggacctctgcctaccgactgctgccacgagcaatgtgcagctggatgtacaggaccaaagcactctgattgcctggcctgcctgcacttcaaccactctggaatctgcgagctgcactgtcctgctctggtcacctacaacacacggaccttcaagagcatgcctaat cctgaaggcagatacacctttggagccagctgtgtgacagcctgtccttacaactacctgagcaccgacgtgggcagctgcacactcgtttgtcctgctgccaatcaagaagtgacagccgaggacggcacccagagatgcgagaagtgtagcaagccttgcgctagagtgtgttacggactcggcatggaacacctgagagaagtg agagccgtgaccagtgccaacatccaagagtttgctggctgcaagaagaatctttggcagcctcgccttcctgcctgagagcttcgatggcgatcctgccagcaatactgctcctctgcagcctgaacagctccaggtgttcgagacactggaagagatcacaggctacctgtacatcagcgcgcatggccagacagcctgcctgacctgtccg tgttccagaacctgcaagtgatcagaggcagaatcctgcacaacggagcctattctgaccctgcaaggcctgggaatcagctggctgggactgagatccctgagagagcttggatctggcctggctctgatccaccacaatacccacctgtgcttcgtgcacaccgtgccttgggaccagctgttcggaatcctcatcaggctctgctgcaca cagccaacagacctgaggatgagtgtgttggcgaaggcctggcttgtcaccagctctgtgctagaggacactgttggggacctggacctacacagtgtgtgaactgtagccagttcctgaggccaagaatgcgtggaagagtgtagagttctgcagggactgcctcgcgagtacgtgaacgctagacactgtctgccttgtcatccc gagtgccagcctcagaatggcagcgtgacatgttttggaccagctgccgatcagtgcgtggcctgtgctcactataaggaccctccagcctgcgtggccagatgtcctagcggagtgaagcctgacctgagctacatgcccatctgggcatttccagatgaggaaggagcttgccagccttgtcctatcaactgcacccacagctg cgtggacctggacgataagggatgtccagccgagcagagcctctccactgacctctatcatctgccgtcgtgggcatcctgctggtggtggttctgggagttgtgttcggcatcctgatcaagagacggcagcagaagatccggaagtacaccatgcggagactgctgcaagagactgagctggtggaacctctgacacccagcggagct atgcctaaccaggctcagatgcggattctgaaagaaaccgagctgcggaaagtgaaggtgctcggctctggagcctttggcacagtgtacaaaggcatctggatccctgacggagagaacgtgaagattcctgtggccatcatggtgctgagagaagaacacaagtcccaaggccaacaaagatcctggacgaggcctacgtgatggct ggtgttggcagcccttatgtgtctagactgctgggcatctgtctgaccagcaccgtgcagctggtcactcagctgatgccttacggctgcctgctggatcacgtgagagagaatagaggcagactgggctctcaggacctgctgaactggtgcatgcagatcgccaagggcatgagctacctcgaggatgtgagactggtccacagagatctgg ctgccagaaacgtgctcgtgaagtctcctaaccacgtgaagatcaccgacttcggactggctaggctgctggatatcgacgagacagagtaccacgctgatggaggcaaggtgcccatcaagtggatggctctggaatccatcctgagacggagattcacccaccagtccgatgtgtggtcttacggagtgacagtgtgggagctga tgaccttcggagccaagccttacgacggcatccctgccagagatcccagatctgctggaaaagggagagagactgcctcagcctcctatctgcaccatcgacgtgtacatgattatggtcaagtgttggatgatcgacagcgagtgcagacccagattcagagaactggtgtccgagttctctcggatggccagagatcctcagagattcg tggtcatccagaacgaggatctgggacctgccagccctctggacagcaccttctacagatccctgctggaagatgacgacatgggtgacctggtggacgctgaagaagctctctggttcctcagggcttcttctgccctgatcctgctccaggagcaggtggaatggtgcatcacagacacagaagctccagcaccagaagcggaggcggagatctgacactg ggactcgagccatctgaggaagaggctcctagatctcctctggctccttctgaaggagctggaagcgacgttttcgacggagatcttggaatgggagctgccaaaggactccagtctctgcccacacacgacccatctccactgcagagatacagcgaggaccctaccgtgcctctgccaagcgagacagatggatatgtggcacctctgacctgctct cctcagccagaactgggacttgatgtgcctgtttgatga

SEQ ID NO:3

Amino acid sequence of synthetic Brachyury (427 amino acids):

MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTA YQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWGWLLPGTSTLCPPANPHPQFGGALSLPSTHSCDRYPTLRSHYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGSPLYEGAAAATDDSQYDAAAQGR LIASWTPVSPPSM

SEQ ID NO:4

Nucleotide sequence of synthetic Brachyury (1287 nucleotides):

atgtctagccctggcacagagtctgctggcaagagcctccagtacagagtggaccatctgctgagcgctgtggagaatgaactgcaggctggaagcgagaagggagatcctacagaaagagagctgagagtcggactggaagagtccgagctgtggctgcggttcaaagaactgaccaacgagatgatcgtgaccaagaacggcagacggatgttccctgtgctgaaagtgaacgtgtccggactggaccctaacgccatgtacagctttctgctggatttcgtggcagctgacaaccacagatggaagtacgtgaacggagagtgggtgccaggaggaaaacctgaacctcaggctcctagctgcgtgtacattcaccctgacagccctaacttcggagcccactggatgaaggctcctgtgtccttcagcaaagtgaagctgaccaacaagctgaacggaggaggccagatcatgctgaacagcctgcacaagtatgagcctaggatccacatcgtcagagttggaggccctcagcggatgatcaccagccactgtttccctgagacacagttcatcgcagtgaccgcttaccagaacgaggaaatcacagccctgaagatcaagtacaatcccttcgccaaggccttcctggacgccaaagagcggagcgaccacaaagaaatgatggaagaacctggcgacagccagcagcctggctattctcaatggggatggctgctgccaggcacctccacattgtgccctccagccaatcctcatcctcagtttggcggagccctgagcctgcctagcacacacagctgcgacagataccctacactgagaagccactacgctcacagaaacaacagccctacctacagcgacaatagccctgcctgtctgagcatgctgcagtcccacgacaattggtccagcctgggaatgcctgctcacccttctatgctgcctgtctctcacaacgcctctccacctacaagcagctctcagtaccctagcctttggagcgtgtccaatggagctgtgacacctggatctcaggctgccgctgtgtctaatggactgggagcccagttcttcagaggcagccctgctcactacacacctctgacacatccagtgtctgctcctagcagcagcggaagccctctctatgaaggagccgctgcagccaccgacatcgtggattctcagtatgatgctgccgcacagggcagactgatcgcctcttggacacctgtgagcccaccttccatgtgatga

SEQ ID NO:5 (435 ac)

Isoform 1 of the Brachyury protein, GenBank Accession No. O15178.1

MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTA YQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWGWLLPGTSTLCPPANPHPQFGGALSLPSTHSCDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGSPLYEGAAAATDIVDSQ YDAAAQGRLIASWTPVSPPSM

SEQ ID NO:6 (1308 nt)

Coding sequence for isoform 1 of the Brachyury protein, GenBank Accession No. O15178.1

atgagcagccctggcacagagagcgccggcaagagcctgcagtaccgggtggaccatctgctgagcgccgtggagaatgagctgcaggccggctccgagaagggcgaccccaccgagagggaactgaggtgggcctggaagagtccgagctgtggctgcggttcaaagaactgaccaacgagatgatcgtgaccaagaacggcagacggatgtt ccccgtgctgaaagtgaacgtgtccggcctggaccccaacgccatgtacagctttctgctggacttcgtggccgccgacaaccaggtggaaatacgtgaacggcgagtgggtgccaggcggcaaacctgagcctcaggcccccagctgcgtgtacatccaccccgacagccccaatttcggcgcccactggatgaaggccccc gtgtccttcagcaaagtgaagctgaccaacaagctgaacggcggaggccagatcatgctgaacagcctgcacaagtacgagccccggatccacattgtgcgcgtgggcggaccccagagaatgatcaccagccactgcttccccgagacacagtttatcgccgtgaccgcctaccagaacgaggaaatcaccgccctgaagatcaagtacaac cccttcgccaaggccttcctggacgccaaagagcggagcgaccacaaagaaatgatggaagaacccggcgacagccagcagcctggctacagccagtggggctggctgctgccaggcacctccactctgtgcccccctgccaaccctcaccctcagttcggcggagccctgagcctgcctagcacacacagctgcgacagataccccaccctgcggag ccacagaagcagcccctaccccagcccatacgcccaccggaacaacagccccacctacagcgacaactcccccgcctgcctgagcatgctgcagagccacgacaactggtccagcctgggcatgcctgcccaccctagcatgctgcccgtgtcccacaatgccagcccccctaccagcagctcccagtaccctagcctgtggagcgtgtccaatgg cgccgtgacacctggatctcaggccgctgccgtgagcaatggcctgggagcccagttctttgaggcagccctgcccactacacccctgacccaccctgtgtccgcccctagctccagcggcagccctctgtatgaaggcgccgctgcagccaccgatatcgtggacagccagtacgatgccgccgctcagggcagactgatcg ccagctggacccccgtgtctccccccagcatgtga

SEQ ID NO:7 (435 ac)

Isoform 1 of the Brachyury protein (L254V).

MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTA YQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWGWLLPGTST V CPPANPHPQFGGALSLPSTHSCDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGSPLYEGAAAATDIVDS QYDAAAQGRLIASWTPVSPPSM

SEQ ID NO:8 (1308 nt)

Coding sequence encoding Brachyury protein isoform 1 with L254V

atgagctcccctggcaccgagagcgcgggaaagagcctgcagtaccgagtggaccacctgctgagcgccgtggagaatgagctgcaggcgggcagcgagaagggcgaccccacagagcgcgaactgcgcgtgggcctggaggagagcgagctgtggctgcgcttcaaggagctcaccaatgagatgatcgtgacca agaacggcaggaggatgtttccggtgctgaaggtgaacgtgtctggcctggaccccaacgccatgtactccttcctgctggacttcgtggcggcggacaaccaccgctggaagtacgtgaacgggggaatgggtgccggggggcaagccggagccgcaggcgcccagctgcgtctacatccaccccgactcgcccaacttcggggc ccactggatgaaggctcccgtctccttcagcaaagtcaagctcaccaacaagctcaacggagggggccagatcatgctgaactccttgcataagtatgagcctcgaatccacatagtgagttgggggtccacagcgcatgatcaccagccactgcttccctgagacccagttcatagcggtgactgcttatcagaacgaggagatcacagcttaaaattaag tacaatccatttgcaaaggctttccttgatgcaaaggaaagaagtgatcacaaagagatgatggaggaacccggggagacagccagcaacctgggtactcccaatgggggtggcttcttcctggaaccagcaccgttgtccacctgcaaatcctcatcctcagtttggaggtgccctctccctcccctccacgcacagctgtgacaggtac ccaaccctgaggagccaccggtcctcaccctaccccagcccctatgctcatcggaacaattctccaacctattctgacaactcacctgcatgtttatccatgctgcaatcccatgacaattggtccagccttggaatgcctgcccatcccagcatgctccccgtgagccacaatgccagcccacctaccagctccagtcagtaccccagcctgtggtctg tgagcaacggcgccgtcaccccgggctcccaggcagcagccgtgtccaacgggctgggggcccagttcttccggggctcccccgcgcactacacacccctcacccatccggtctcggcgccctcttcctcgggatccccactgtacgaaggggcggccgcggccacagacatcgtggacagccagtacgacgccgcagcccaaggcc gcctcatagcctcatggacacctgtgtcgccaccttccatgtga

SEQ ID NO:9 (449 ac)

Brachyury-I3 fusion protein

MKNNLYEEKMNMSKKSSPPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQ RMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWGWLLPGTST V CPPANPHPQFGGALSLPSTHSCDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPS SSGSPLYEGAAAATDIVDSQYDAAAQGRLIASWTPVSPPSM

SEQ ID NO:10 (1350 nt)

A coding sequence encoding an I3-Brachyury fusion protein of SEQ ID NO 9.

atgaaaaataacttgtatgaagaaaaaatgaacatgagtaagaaaagctcccctggcaccgagagcgcgggaaagagcctgcagtaccgagtggaccacctgctgagcgccgtggagaatgagctgcaggcgggcagcgagaagggcgaccccacagagcgcgaactgcgcgtgggcctggaggagagcgagctgt ggctgcgcttcaaggagctcaccaatgagatgatcgtgaccaagaacggcaggaggatgtttccggtgctgaaggtgaacgtgtctggcctggaccccaacgccatgtactccttcctgctggacttcgtggcggcggacaaccaccgctggaagtacgtgaacggggaatgggtgccggggggcaagccggagccgcaggcgc ccagctgcgtctacatccaccccgactcgcccaacttcggggcccactggatgaaggctcccgtctccttcagcaaagtcaagctcaccaacaagctcaacggagggggccagatcatgctgaactccttgcataagtatgagcctcgaatccacatagtgagagttgggggtccacagcgcatgatcaccagccactgcttccctgagacccagttcatagc ggtgactgcttatcagaacgaggagatcacagctcttaaaattaagtacaatccatttgcaaaggctttccttgatgcaaaggaaagaagtgatcacaaagagatgatggaggaacccggagacagccagcaacctgggtactcccaatgggggtggcttcttcctggaaccagcaccgtttgtccacctgcaaatcctcatcctcagtttgga ggtgccctctccctcccctccacgcacagctgtgacaggtacccaaccctgaggagccaccggtcctcaccctaccccagcccctatgctcatcggaacaattctccaacctattctgacaactcacctgcatgtttatccatgctgcaatcccatgacaattggtccagccttggaatgcctgcccatcccagcatgctccccgtgagccacaatgcca gcccacctaccagctccagtcagtaccccagcctgtggtctgtgagcaacggcgccgtcaccccgggctcccaggcagcagccgtgtccaacggggctgggggcccagttcttccggggctcccccgcgcactacacacccctcacccatccggtctcggcgccctcttcctcgggatccccactgtacgaaggggcggccgcggcca cagacatcgtggacagccagtacgacgccgcagcccaaggccgcctcatagcctcatggacacctgtgtcgccaccttccatgtga

SEQ ID NO:11

hCD40L , NCBI RefSeq NP_000065.1. (261 amino acids)

MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCL KSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

SEQ ID NO:12

hCD40L , NCBI RefSeq NP_000065.1. (789 nucleotides)

nt-sequence:

atgatcgagacatacaaccagacaagccctagaagcgccgccacaggactgcctatcagcatgaagatcttcatgtacctgctgaccgtgttcctgatcacccagatgatcggcagcgccctgtttgccgtgtacctgcacagacggctggacaagatcgaggacgagagaaacctgcacgaggacttcgtgttcatgaagaccatccag cggtgcaacaccggcgagagaagtctgagcctgctgaactgcgaggaaatcaagagccagttcgagggcttcgtgaaggacatcatgctgaacaaagaggaaacgaagaaagagaactccttcgagatgcagaagggcgaccagaatcctcagatcgccgctcacgtgatcagcgaggccagcagcaagacaacaagcgtgctgcag tgggccgagaagggctactacaccatgagcaacaacctggtcaccctggagaacggcaagcagctgacagtgaagcggcagggcctgtactacatctacgcccaagtgaccttctgcagcaacagagaggccagctctcaggctccttcatcgccagcctgtgcctgaagtctcctggcagattcgagcggattctgctgagagccgccaacacacac agcagcgccaaaccttgtggccagcagtctattcacctcggcggagtgtttgagctgcagcctggcgcaagcgtgttcgtgaatgtgacagaccctagccaggtgtcccacggcaccggctttacatctttcggactgctgaagctgtgatga

SEQ ID NO:13

Amino acid sequence of synthetic Her2 v2 (1145 amino acids):

MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMC KGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELACPALVTYNTRTAKSMPNPEGRYTFGASCVTACPYNYLSTDAGACTLVCPAANQEVTAEDGTQRCEACSKACARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLP DLSVFQNLQVIRGRILHAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPAADQCVACAHYKDPPACVARCPSGVKPDLSYMPIWAFPDEEGACQPC PINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIMVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNW CMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEALVPQQGFFCPDPA PGAGGMVHHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPELGLDVPV

SEQ ID NO:14

Nucleotide sequence of synthetic Her2 v2 (3441 nucleotides):

atggaactggctgctctgtgtagatggggactgctgcttgctctgttgcctcctggagctgcttctacccaagtgtgcacaggcaccgacatgaagctgagactgcctgcttctcctgagacacacctggacatgctgacacctgtaccagggatgtcaggtggtgcagggaaatctggaactgacctacctgcctaccaacgccagcct gagctttctgcaggacatccaagaggtgcagggatacgtgctgatcgctcacaatcaagtgagacaggtgccactgcagaggctgagaatcgttagaggcacccagctgttcgaggacaactatgctctggctgtgctggacaatggcgaccctctgaacaacaccacacctgtgacaggagcttcctggtggactgagagaactgcagctgaga agcctgaccgagatcctgaaaggaggagtgctgatccagcggaaccctcagctgtgctaccaggacacaccatcctgtggaaggacatcttccacaagaacaaccagctggctctgacactgatcgacaccaacagaagcagagcctgccatccttgctctcccatgtgcaagggctctagatgttggggagagagcagcgaggattgccagagcctga ccagaacagtgtgtgctggaggatgtgccagatgcaaaggacctctgcctaccgactgctgccacgagcaatgtgcagctggatgtacaggaccaaagcactctgattgcctggcctgcctgcacttcaaccactctggaatctgcgagctcgcctgtcctgctctggtcacctacaacacacggaccgccaagagcatgccta atcctgaaggcagatacacctttggagccagctgtgtgacagcctgtccttacaactacctgagcaccgacgctggagcctgcacactcgtttgtcctgctgccaatcaagaagtgacggccgaggacggcacccagagatgcgaggcctgtagcaaggcttgcgctagagtgtgttacggactcggcatggaacacctgagagaagt gagagccgtgaccagtgccaacatccaagagtttgctggctgcaagaagaagatctttggcagcctcgccttcctgcctgagagcttcgatggcgatcctgccagcaatactgctcctctgcagcctgaacagctccaggtgttcgagacactggaagagatcacaggctacctgtacatcagcgcgcatggccagacagcctgcctgacctgtcc gtgttccagaacctgcaagtgatcagaggcagaatcctgcacaacggagcctattctgaccctgcaaggcctgggaatcagctggctgggactgagatccctgagagagcttggatctggcctggctctgatccaccacaatacccacctgtgcttcgtgcacaccgtgccttgggaccagctgttcggaatcctcatcaggctctgctgca cacagccaacagacctgaggatgagtgtgttggcgaaggcctggcttgtcaccagctgtgctagaggacactgttggggacctggacctacacagtgtgtgaactgtagccagttcctgagaggccaagaatgcgtggaagagtgtagagttctgcagggactgcctcgcgagtacgtgaacgctagacactgtctgccttgtcatc ccgagtgccagcctcagaatggcagcgtgacatgttttggaccagctgccgatcagtgcgtggcctgtgctcactataaggaccctccagcctgcgtggccagatgtcctagcggagtgaagcctgacctgagctacatgcccatctgggcatttccagatgaggaaggagcttgccagccttgtcctatcaactgcacccacagct gcgtggacctggacgataagggatgtccagccgagcagagcctctccactgacctctatcatctctgccgtcgtgggcatcctgctggtggtggttctgggagttgtgttcggcatcctgatcaagagacggcagcagaagatccggaagtacaccatgcggagactgctgcaagagactgagctggtggaacctctgacacccagcggag ctatgcctaaccaggctcagatgcggattctgaaagaaaccgagctgcggaaagtgaaggtgctcggctctggagcctttggcacagtgtacaaaggcatctggatccctgacggagagaacgtgaagattcctgtggccatcatggtgctgagagagaacacaagtcccaaggccaacaaagagatcctggacgaggcctacgtgatgg ctggtgttggcagcccttatgtgtctagactgctgggcatctgtctgaccagcaccgtgcagctggtcactcagctgatgccttacggctgcctgctggatcacgtgagagagaatagaggcagactgggctctcaggacctgctgaactggtgcatgcagatcgccaagggcatgagctacctcgaggatgtgagactggtccacagagatct ggctgccagaaacgtgctcgtgaagtctcctaaccacgtgaagatcaccgacttcggactggctaggctgctggatatcgacgagacagagtaccacgctgatggaggcaaggtgcccatcaagtggatggctctggaatccatcctgagacggagattcacccaccagtccgatgtgtgtggtcttacggagtgacagtgtgggagct gatgaccttcggagccaagccttacgacggcatccctgccagagagatcccagatctgctggaaaagggagagagactgcctcagcctcctatctgcaccatcgacgtgtacatgattatggtcaagtgttggatgatcgacagcgagtgcagacccagattcagagaactggtgtccgagttctctcggatggccagagatcctcagagattc gtggtcatccagaacgaggatctgggacctgccagccctctggacagcaccttctacagatccctgctggaagatgacgacatgggtgacctggtggacgctgaagaagctctctggttcctcagcagggcttcttctgccctgatcctgctccaggagcaggtggaatggtgcatcacagacacagaagctccagcaccagaagcggaggcggagatctgacact gggactcgagccatctgaggaagaggctcctagatctcctctggctccttctgaaggagctggaagcgacgttttcgacggagatcttggaatgggagctgccaaaggactccagtctctgcccacacacgacccatctccactgcagagatacagcgaggaccctaccgtgcctctgccaagcgagacagatggatatgtggcacctctgacctgct ctcctcagccagaactgggacttgatgtgcctgtttgatga

SEQ ID NO:15

Amino acid sequence of synthetic Twist (205 amino acids):

MQDVSSSPVSPADDSLSNSEEEPDRQQPASGKRGARKRRSSRRSAGGSAGPGGATGGGIGGGDEPGSPAQGKRGKKSAGGGGGGGAGGGGGGGGGSSSGGGSPQSYEELQTQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDSKMASCSYVAHERLSYAFSVW RMEGAWSMSASH

SEQ ID NO:16

Nucleotide sequence of synthetic Twist (618 nucleotides):

Atgcaggacgtgtccagcagccctgtgtctcctgccgacgacagcctgagcaacagcgaggaagaacccgacagacagcagcccgcctctggcaagagaggcgccagaaagaagaagaagctccagaagaagcgctggcggctctgctggacctggcggagctacaggcggaggaattggaggcggagatgagcctggctctccagcccaggg caagaggggcaagaaatctgctggcggaggcggcggaggaggagctggaggcggaggaggaggcggcggaggatcaagttctggcggaggaagccctcagagctacgaggaactgcagacccagcgcgtgatggccaacgtgcgcgagagacagaacccagagcctgaacgaggccttcgccgccctgagaaagatcatccccaccctgcccag cgacaagctgagcaagatccagaccctgaagctggccgccagatatatcgacttcctgtatcaagtgctgcagagcgacgagctggacagcaagatggccagctgctcctacgtggcccacgagagactgagctacgccttcagcgtgtggcggatggaaggcgcctggtctatgagcgccagccactga

SEQ ID NO:17

Amino acid sequence of synthetic mouse CD40L (260 amino acids):

MIETYSQPSPRSVATGLPASMKIFMYLLTVFLITQMIGSVLFAVYLHRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENSFEMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGL WLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKL

SEQ ID NO:18

Nucleotide sequence of synthetic mouse CD40L (786 nucleotides):

atgatcgagacatacagccagcccagccccagaagcgtggccacaggactgcctgccagcatgaagatctttatgtacctgctgaccgtgttcctgatcacccagatgatcggcagcgtgctgttcgccgtgtacctgcacagacggctggacaaggtggaagaggaagtgaacctgcacgaggacttcgtgttcatcaagaaact gaagcggtgcaacaagggcgagggcagcctgagcctgctgaactgcgaggaaatgagaaggcagttcgaggacctcgtgaaggacatcaccctgaacaaagaggaaaagaaagaaaactccttcgagatgcagaggggcgacgaggaccctcagatcgctgctcacgtggtgtccgaggccaacagcaacgccgcttctgtg ctgcagtgggccaagaaaggctactacaccatgaagtccaacctcgtgatgctggaaaacggcaagcagctgacagtgaagcgcgagggcctgtactatgtgtacacccaagtgacattctgcagcaacagagagcccagcagccagaggcccttcatcgtgggactgtggctgaagcctagcagcggcagcgagagaatcctgctgaa ggccgccaacacccacagcagctctcagctgtgcgagcagcagagcgtgcacctgggcggagtgttcgagctgcaagctggcgcctccgtgttcgtgaacgtgacagaggccagccaagtgatccacagagtgggcttcagcagctttggactgctgaaactgtaatga

LITERATURE

Literature references included as part of this specification other than those listed below are incorporated herein by reference in their entirety.

[1] World Health Organization, World Health report, 2013.

[2] L. A. Torre, “Global Cancer Statistics,” CA: A Cancer Journal for Clinicians, 2012.

[3] J. S. Ross, “The Her-2/neu gene and protein in breast cancer 2003: biomarker and target of therapy”, Oncologist, 2003.

[4] C. Palena, “The human T-box mesodermal transcription factor Brachyury is a candidate target for T-cell-mediated cancer immunotherapy”, Clin Cancer Res, 2007.

[5] N. E. Hynes and H. A. Lane, “ERBB receptors and cancer: the complexity of targeted inhibitors”, Nat Rev Cancer, 2005.

[6] H. S. Cho, “Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab”, Nature, 2003.

[7] S. Satyanarayanajois, “Design, Synthesis, and Docking Studies of Peptidomimetics based on HER2-Herceptin Binding Site with Potential Antiproliferative Activity Against Breast Cancer Cell lines,” Chem Biol Drug Des, 2009.

[8] M. C. Franklin, “Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complex”, Cancer Cell, 2004.

[9] R. Yang, Targeting The Dimerization Of ERBB Receptor, All Theses and Dissertations (ETDs). Paper 391, 2009.

[10] M. Tan, “ErbB2 promotes Src synthesis and stability: novel mechanisms of Src activation that confer breast cancer metastasis”, Cancer Res, 2005.

[11] R. J. Roskoski, “ErbB/HER protein-tyrosine kinases: Structures and small molecule inhibitors”, Pharmacol Res, 2014.

[12] M. Roselli, “Brachyury, a driver of the epithelial-mesenchymal transition, is overexpressed in human lung tumors: an opportunity for novel interventions against lung cancer”, Clin Cancer Res, 2012.

[13] J. Z. Stoller und J. A. Epstein, “Identification of a novel nuclear localization signal in Tbx1 that is deleted in DiGeorge syndrome patients harboring the 1223delC mutation”, Hum Mol Genet, 2005.

[14] H. Lauterbach, “Genetic Adjuvantation of Recombinant MVA with CD40L Potentiates CD8 T Cell Mediated Immunity”, Front Immunol, 2013.

[15] A. Guardino et al., Results of Two Phase I Clinical Trials of MVA-BN®-HER2 in HER-2 Overexpressing Metastatic Breast Cancer Patients, Cancer Res, 69 (24 Supp Abstract nr 5089 (2009).

[16] C. Heery et al., Phase I, dose-escalation, clinical trial of MVA-Brachyury-TRICOM vaccine demonstrating safety and brachyury-specific T cell responses, J Immunother Cancer, 3: (Suppl 2) P132 (2015) .

[17] Brodowicz et al., Anti-Her-2/neu antibody induces apoptosis in Her-2/neu overexpressing breast cancer cells independently from p53 status, Br J Cancer (2001).

[18] Stackaruk et al., Type I interferon regulation of natural killer cell function in primary and secondary infections, Expert Rev Vaccines. 12(8):875-84 (2013 Aug).

[19] Muller et al., Type I Interferons and Natural Killer Cell Regulation in Cancer, Front Immunol. 8:304 (2017).

[20] Yamashita et al. A novel method for evaluating antibody dependent cell-mediated cytotoxicity by flow cytometry using human peripheral blood mononuclear cells, Scientific reports 6 (Article number 19772); (January 2016).

[21] Broussas et al., Evaluation of antibody-dependent cell cytotoxicity using lactate dehydrogenase (LDH) measurement. Methods Mol Biol, 988: 305-317 (2013).

[22] Tay et al., TriKEs and BiKEs join CARs on the cancer immunotherapy highway, Human Vaccines and Immunotherapeutics, vol 12: 2790-2796 (2016).

[23] Kono et al., Trastuzumab (Herceptin) Enhances Class I-Restricted Antigen Presentation Recognized by Her2/neu Specific T Cytotoxic Lymphocytes, Clinical Cancer Research 10: 2538-2544 (April 2004).

--->

SEQUENCE LIST

<110> Bavarian Nordic A/S

<120> COMBINATION THERAPY FOR CANCER TREATMENT BY INTRAVENOUS

INTRODUCTION OF RECOMBINANT MVA AND ANTIBODY

<130> BNIT0012PCT

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 1145

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221> synthetic

<222> (1)..(1145)

<223> Synthetic amino acid sequence v1 Her2

<400> 1

Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu

1 5 10 15

Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys

20 25 30

Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His

35 40 45

Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr

50 55 60

Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val

65 70 75 80

Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu

85 90 95

Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr

100 105 110

Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Pro

115 120 125

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

130 135 140

Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln

145 150 155 160

Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn

165 170 175

Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys

180 185 190

His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser

195 200 205

Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys

210 215 220

Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys

225 230 235 240

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

245 250 255

His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val

260 265 270

Thr Tyr Asn Thr Arg Thr Phe Lys Ser Met Pro Asn Pro Glu Gly Arg

275 280 285

Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu

290 295 300

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

305 310 315 320

Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys

325 330 335

Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu

340 345 350

Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys

355 360 365

Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp

370 375 380

Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe

385 390 395 400

Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro

405 410 415

Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg

420 425 430

Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu

435 440 445

Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly

450 455 460

Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val

465 470 475 480

Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr

485 490 495

Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His

500 505 510

Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys

515 520 525

Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys

530 535 540

Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys

545 550 555 560

Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys

565 570 575

Phe Gly Pro Ala Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp

580 585 590

Pro Pro Ala Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu

595 600 605

Ser Tyr Met Pro Ile Trp Ala Phe Pro Asp Glu Glu Gly Ala Cys Gln

610 615 620

Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys

625 630 635 640

Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser

645 650 655

Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly

660 665 670

Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg

675 680 685

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

690 695 700

Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu

705 710 715 720

Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys

725 730 735

Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile

740 745 750

Met Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu

755 760 765

Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg

770 775 780

Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu

785 790 795 800

Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg

805 810 815

Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly

820 825 830

Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala

835 840 845

Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe

850 855 860

Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp

865 870 875 880

Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg

885 890 895

Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val

900 905 910

Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala

915 920 925

Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro

930 935 940

Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met

945 950 955 960

Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe

965 970 975

Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu

980 985 990

Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu

995 1000 1005

Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Ala

1010 1015 1020

Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly

1025 1030 1035

Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg

1040 1045 1050

Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu

1055 1060 1065

Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser

1070 1075 1080

Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu

1085 1090 1095

Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser

1100 1105 1110

Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val

1115 1120 1125

Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Leu Gly Leu Asp Val

1130 1135 1140

ProVal

1145

<210> 2

<211> 3441

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221> synthetic

<222> (1)..(3441)

<223> Synthetic nucleotide sequence v1 Her2

<400> 2

atggaactgg ctgctctgtg tagatgggga ctgctgcttg ctctgttgcc tcctggagct 60

gcttctaccc aagtgtgcac aggcaccgac atgaagctga gactgcctgc ttctcctgag 120

acacacctgg acatgctgag acacctgtac cagggatgtc aggtggtgca gggaaatctg 180

gaactgacct acctgcctac caacgccagc ctgagctttc tgcaggacat ccaagaggtg 240

cagggatacg tgctgatcgc tcacaatcaa gtgagacagg tgccactgca gaggctgaga 300

atcgttagag gcacccagct gttcgaggac aactatgctc tggctgtgct ggacaatggc 360

gaccctctga acaacaccac acctgtgaca ggagcttctc ctggtggact gagagaactg 420

cagctgagaa gcctgaccga gatcctgaaa ggaggagtgc tgatccagcg gaaccctcag 480

ctgtgctacc aggacaccat cctgtggaag gacatcttcc acaagaacaa ccagctggct 540

ctgacactga tcgacaccaa cagaagcaga gcctgccatc cttgctctcc catgtgcaag 600

ggctctagat gttggggaga gagcagcgag gattgccaga gcctgaccag aacagtgtgt 660

gctggaggat gtgccagatg caaaggacct ctgcctaccg actgctgcca cgagcaatgt 720

gcagctggat gtacaggacc aaagcactct gattgcctgg cctgcctgca cttcaaccac 780

tctggaatct gcgagctgca ctgtcctgct ctggtcacct acaacacacg gaccttcaag 840

agcatgccta atcctgaagg cagatacacc tttggagcca gctgtgtgac agcctgtcct 900

tacaactacc tgagcaccga cgtgggcagc tgcacactcg tttgtcctgc tgccaatcaa 960

gaagtgacag ccgaggacgg cacccagaga tgcgagaagt gtagcaagcc ttgcgctaga 1020

gtgtgttacg gactcggcat ggaacacctg agagaagtga gagccgtgac cagtgccaac 1080

atccaagagt ttgctggctg caagaagatc tttggcagcc tcgccttcct gcctgagagc 1140

ttcgatggcg atcctgccag caatactgct cctctgcagc ctgaacagct ccaggtgttc 1200

gagacactgg aagagatcac aggctacctg tacatcagcg catggccaga cagcctgcct 1260

gacctgtccg tgttccagaa cctgcaagtg atcagaggca gaatcctgca caacggagcc 1320

1380

cttggatctg gcctggctct gatccaccac aatacccacc tgtgcttcgt gcacaccgtg 1440

ccttgggacc agctgtttcg gaatcctcat caggctctgc tgcacacagc caacagacct 1500

gaggatgagt gtgttggcga aggcctggct tgtcaccagc tctgtgctag aggacactgt 1560

tggggacctg gacctacaca gtgtgtgaac tgtagccagt tcctgagagg ccaagaatgc 1620

1680

ctgccttgtc atcccgagtg ccagcctcag aatggcagcg tgacatgttt tggaccagct 1740

gccgatcagt gcgtggcctg tgctcactat aaggaccctc cagcctgcgt ggccagatgt 1800

cctagcggag tgaagcctga cctgagctac atgcccatct gggcatttcc agatgaggaa 1860

ggagcttgcc agccttgtcc tatcaactgc acccacagct gcgtggacct ggacgataag 1920

ggatgtccag ccgagcagag agcctctcca ctgacctcta tcatctctgc cgtcgtggggc 1980

atcctgctgg tggtggttct gggagttgtg ttcggcatcc tgatcaagag acggcagcag 2040

aagatccgga agtacaccat gcggagactg ctgcaagaga ctgagctggt ggaacctctg 2100

acacccagcg gagctatgcc taaccaggct cagatgcgga ttctgaaaga aaccgagctg 2160

cggaaagtga aggtgctcgg ctctggagcc tttggcacag tgtacaaagg catctggatc 2220

2280

cccaaggcca acaaagagat cctggacgag gcctacgtga tggctggtgt tggcagccct 2340

tatgtgtcta gactgctggg catctgtctg accagcaccg tgcagctggt cactcagctg 2400

atgccttacg gctgcctgct ggatcacgtg agagagaata gaggcagact gggctctcag 2460

gacctgctga actggtgcat gcagatcgcc aagggcatga gctacctcga ggatgtgaga 2520

ctggtccaca gagatctggc tgccagaaac gtgctcgtga agtctcctaa ccacgtgaag 2580

atcaccgact tcggactggc taggctgctg gatatcgacg agacagagta ccacgctgat 2640

ggaggcaagg tgcccatcaa gtggatggct ctggaatcca tcctgagacg gagattcacc 2700

caccagtccg atgtgtggtc ttacggagtg acagtgtggg agctgatgac cttcggagcc 2760

aagccttacg acggcatccc tgccagagag atcccagatc tgctggaaaa gggagagaga 2820

ctgcctcagc ctcctatctg caccatcgac gtgtacatga ttatggtcaa gtgttggatg 2880

atcgacagcg agtgcagacc cagattcaga gaactggtgt ccgagttctc tcggatggcc 2940

agagatcctc agagattcgt ggtcatccag aacgaggatc tgggacctgc cagccctctg 3000

gacagcacct tctacagatc cctgctggaa gatgacgaca tgggtgacct ggtggacgct 3060

gaagaagctc tggttcctca gcagggcttc ttctgccctg atcctgctcc aggagcaggt 3120

ggaatggtgc atcacagaca cagaagctcc agcaccagaa gcggaggcgg agatctgaca 3180

ctgggactcg agccatctga ggaagaggct cctagatctc ctctggctcc ttctgaagga 3240

gctggaagcg acgttttcga cggagatctt ggaatgggag ctgccaaagg actccagtct 3300

3360

ccaagcgaga cagatggata tgtggcacct ctgacctgct ctcctcagcc agaactggga 3420

cttgatgtgc ctgtttgatg a 3441

<210> 3

<211> 427

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221> synthetic

<222> (1)..(427)

<223> Brachyury synthetic amino acid sequence

<400> 3

Met Ser Ser Pro Gly Thr Glu Ser Ala Gly Lys Ser Leu Gln Tyr Arg

1 5 10 15

Val Asp His Leu Leu Ser Ala Val Glu Asn Glu Leu Gln Ala Gly Ser

20 25 30

Glu Lys Gly Asp Pro Thr Glu Arg Glu Leu Arg Val Gly Leu Glu Glu

35 40 45

Ser Glu Leu Trp Leu Arg Phe Lys Glu Leu Thr Asn Glu Met Ile Val

50 55 60

Thr Lys Asn Gly Arg Arg Met Phe Pro Val Leu Lys Val Asn Val Ser

65 70 75 80

Gly Leu Asp Pro Asn Ala Met Tyr Ser Phe Leu Leu Asp Phe Val Ala

85 90 95

Ala Asp Asn His Arg Trp Lys Tyr Val Asn Gly Glu Trp Val Pro Gly

100 105 110

Gly Lys Pro Glu Pro Gln Ala Pro Ser Cys Val Tyr Ile His Pro Asp

115 120 125

Ser Pro Asn Phe Gly Ala His Trp Met Lys Ala Pro Val Ser Phe Ser

130 135 140

Lys Val Lys Leu Thr Asn Lys Leu Asn Gly Gly Gly Gln Ile Met Leu

145 150 155 160

Asn Ser Leu His Lys Tyr Glu Pro Arg Ile His Ile Val Arg Val Gly

165 170 175

Gly Pro Gln Arg Met Ile Thr Ser His Cys Phe Pro Glu Thr Gln Phe

180 185 190

Ile Ala Val Thr Ala Tyr Gln Asn Glu Glu Ile Thr Ala Leu Lys Ile

195 200 205

Lys Tyr Asn Pro Phe Ala Lys Ala Phe Leu Asp Ala Lys Glu Arg Ser

210 215 220

Asp His Lys Glu Met Met Glu Glu Pro Gly Asp Ser Gln Gln Pro Gly

225 230 235 240

Tyr Ser Gln Trp Gly Trp Leu Leu Pro Gly Thr Ser Thr Leu Cys Pro

245 250 255

Pro Ala Asn Pro His Pro Gln Phe Gly Gly Ala Leu Ser Leu Pro Ser

260 265 270

Thr His Ser Cys Asp Arg Tyr Pro Thr Leu Arg Ser His Tyr Ala His

275 280 285

Arg Asn Asn Ser Pro Thr Tyr Ser Asp Asn Ser Pro Ala Cys Leu Ser

290 295 300

Met Leu Gln Ser His Asp Asn Trp Ser Ser Leu Gly Met Pro Ala His

305 310 315 320

Pro Ser Met Leu Pro Val Ser His Asn Ala Ser Pro Pro Thr Ser Ser

325 330 335

Ser Gln Tyr Pro Ser Leu Trp Ser Val Ser Asn Gly Ala Val Thr Pro

340 345 350

Gly Ser Gln Ala Ala Ala Val Ser Asn Gly Leu Gly Ala Gln Phe Phe

355 360 365

Arg Gly Ser Pro Ala His Tyr Thr Pro Leu Thr His Pro Val Ser Ala

370 375 380

Pro Ser Ser Ser Gly Ser Pro Leu Tyr Glu Gly Ala Ala Ala Ala Thr

385 390 395 400

Asp Ile Val Asp Ser Gln Tyr Asp Ala Ala Ala Gln Gly Arg Leu Ile

405 410 415

Ala Ser Trp Thr Pro Val Ser Pro Pro Ser Met

420 425

<210> 4

<211> 1287

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221> synthetic

<222> (1)..(1287)

<223> Brachyury synthetic nucleotide sequence

<400> 4

atgtctagcc ctggcacaga gtctgctggc aagagcctcc agtacagagt ggaccatctg 60

ctgagcgctg tggagaatga actgcaggct ggaagcgaga agggagatcc tacagaaaga 120

gagctgagag tcggactgga agagtccgag ctgtggctgc ggttcaaaga actgaccaac 180

gagatgatcg tgaccaagaa cggcagacgg atgttccctg tgctgaaagt gaacgtgtcc 240

ggactggacc ctaacgccat gtacagcttt ctgctggatt tcgtggcagc tgacaaccac 300

agatggaagt acgtgaacgg agagtgggtg ccaggaggaa aacctgaacc tcaggctcct 360

agctgcgtgt acattcaccc tgacagccct aacttcggag cccactggat gaaggctcct 420

gtgtccttca gcaaagtgaa gctgaccaac aagctgaacg gaggaggcca gatcatgctg 480

aacagcctgc acaagtatga gcctaggatc cacatcgtca gagttggagg ccctcagcgg 540

atgatcacca gccactgttt ccctgagaca cagttcatcg cagtgaccgc ttaccagaac 600

gaggaaatca cagccctgaa gatcaagtac aatcccttcg ccaaggcctt cctggacgcc 660

aaagagcgga gcgaccacaa agaaatgatg gaagaacctg gcgacagcca gcagcctggc 720

tattctcaat ggggatggct gctgccaggc acctccacat tgtgccctcc agccaatcct 780

catcctcagt ttggcggagc cctgagcctg cctagcacac acagctgcga cagataccct 840

acactgagaa gccactacgc tcacagaaac aacagcccta cctacagcga caatagccct 900

gcctgtctga gcatgctgca gtcccacgac aattggtcca gcctgggaat gcctgctcac 960

ccttctatgc tgcctgtctc tcacaacgcc tctccaccta caagcagctc tcagtaccct 1020

agccttttgga gcgtgtccaa tggagctgtg acacctggat ctcaggctgc cgctgtgtct 1080

aatggactgg gagcccagtt cttcagaggc agccctgctc actacacacc tctgacacat 1140

ccagtgtctg ctcctagcag cagcggaagc cctctctatg aaggagccgc tgcagccacc 1200

gacatcgtgg attctcagta tgatgctgcc gcacagggca gactgatcgc ctcttggaca 1260

cctgtgagcc caccttccat gtgatga 1287

<210> 5

<211> 435

<212> PROTEIN

<213> Homo sapiens

<220>

<221> Isoform

<222> (1)..(435)

<223> Brachyury protein isoform 1 from GenBank accession number O15178.1

<400> 5

Met Ser Ser Pro Gly Thr Glu Ser Ala Gly Lys Ser Leu Gln Tyr Arg

1 5 10 15

Val Asp His Leu Leu Ser Ala Val Glu Asn Glu Leu Gln Ala Gly Ser

20 25 30

Glu Lys Gly Asp Pro Thr Glu Arg Glu Leu Arg Val Gly Leu Glu Glu

35 40 45

Ser Glu Leu Trp Leu Arg Phe Lys Glu Leu Thr Asn Glu Met Ile Val

50 55 60

Thr Lys Asn Gly Arg Arg Met Phe Pro Val Leu Lys Val Asn Val Ser

65 70 75 80

Gly Leu Asp Pro Asn Ala Met Tyr Ser Phe Leu Leu Asp Phe Val Ala

85 90 95

Ala Asp Asn His Arg Trp Lys Tyr Val Asn Gly Glu Trp Val Pro Gly

100 105 110

Gly Lys Pro Glu Pro Gln Ala Pro Ser Cys Val Tyr Ile His Pro Asp

115 120 125

Ser Pro Asn Phe Gly Ala His Trp Met Lys Ala Pro Val Ser Phe Ser

130 135 140

Lys Val Lys Leu Thr Asn Lys Leu Asn Gly Gly Gly Gln Ile Met Leu

145 150 155 160

Asn Ser Leu His Lys Tyr Glu Pro Arg Ile His Ile Val Arg Val Gly

165 170 175

Gly Pro Gln Arg Met Ile Thr Ser His Cys Phe Pro Glu Thr Gln Phe

180 185 190

Ile Ala Val Thr Ala Tyr Gln Asn Glu Glu Ile Thr Ala Leu Lys Ile

195 200 205

Lys Tyr Asn Pro Phe Ala Lys Ala Phe Leu Asp Ala Lys Glu Arg Ser

210 215 220

Asp His Lys Glu Met Met Glu Glu Pro Gly Asp Ser Gln Gln Pro Gly

225 230 235 240

Tyr Ser Gln Trp Gly Trp Leu Leu Pro Gly Thr Ser Thr Leu Cys Pro

245 250 255

Pro Ala Asn Pro His Pro Gln Phe Gly Gly Ala Leu Ser Leu Pro Ser

260 265 270

Thr His Ser Cys Asp Arg Tyr Pro Thr Leu Arg Ser His Arg Ser Ser

275 280 285

Pro Tyr Pro Ser Pro Tyr Ala His Arg Asn Asn Ser Pro Thr Tyr Ser

290 295 300

Asp Asn Ser Pro Ala Cys Leu Ser Met Leu Gln Ser His Asp Asn Trp

305 310 315 320

Ser Ser Leu Gly Met Pro Ala His Pro Ser Met Leu Pro Val Ser His

325 330 335

Asn Ala Ser Pro Pro Thr Ser Ser Ser Gln Tyr Pro Ser Leu Trp Ser

340 345 350

Val Ser Asn Gly Ala Val Thr Pro Gly Ser Gln Ala Ala Ala Val Ser

355 360 365

Asn Gly Leu Gly Ala Gln Phe Phe Arg Gly Ser Pro Ala His Tyr Thr

370 375 380

Pro Leu Thr His Pro Val Ser Ala Pro Ser Ser Ser Gly Ser Pro Leu

385 390 395 400

Tyr Glu Gly Ala Ala Ala Ala Thr Asp Ile Val Asp Ser Gln Tyr Asp

405 410 415

Ala Ala Ala Gln Gly Arg Leu Ile Ala Ser Trp Thr Pro Val Ser Pro

420 425 430

Pro Ser Met

435

<210> 6

<211> 1308

<212> DNA

<213> Homo sapiens

<220>

<221> Isoform

<222> (1)..(1308)

<223> Brachyury protein isoform 1 coding sequence, GenBank accession number

O15178.1

<400> 6

atgagcagcc ctggcacaga gagcgccggc aagagcctgc agtaccggggt ggaccatctg 60

ctgagcgccg tggagaatga gctgcaggcc ggctccgaga agggcgaccc caccgagagg 120

gaactgagag tgggcctgga agagtccgag ctgtggctgc ggttcaaaga actgaccaac 180

gagatgatcg tgaccaagaa cggcagacgg atgttccccg tgctgaaagt gaacgtgtcc 240

ggcctggacc ccaacgccat gtacagcttt ctgctggact tcgtggccgc cgacaaccac 300

aggtggaaat acgtgaacgg cgagtgggtg ccaggcggca aacctgagcc tcaggccccc 360

agctgcgtgt acatccaccc cgacagcccc aatttcggcg cccactggat gaaggccccc 420

gtgtccttca gcaaagtgaa gctgaccaac aagctgaacg gcggaggcca gatcatgctg 480

aacagcctgc acaagtacga gccccggatc cacattgtgc gcgtgggcgg accccagaga 540

atgatcacca gccactgctt ccccgagaca cagtttatcg ccgtgaccgc ctaccagaac 600

gaggaaatca ccgccctgaa gatcaagtac aaccccttcg ccaaggcctt cctggacgcc 660

aaagagcgga gcgaccacaa agaaatgatg gaagaacccg gcgacagcca gcagcctggc 720

tacagccagt ggggctggct gctgccaggc acctccactc tgtgcccccc tgccaaccct 780

caccctcagt tcggcggagc cctgagcctg cctagcacac acagctgcga cagatacccc 840

accctgcgga gccacagaag cagcccctac cccagcccat acgcccaccg gaacaacagc 900

cccacctaca gcgacaactc ccccgcctgc ctgagcatgc tgcagagcca cgacaactgg 960

tccagcctgg gcatgcctgc ccaccctagc atgctgcccg tgtcccacaa tgccagcccc 1020

cctaccagca gctcccagta ccctagcctg tggagcgtgt ccaatggcgc cgtgacacct 1080

ggatctcagg ccgctgccgt gagcaatggc ctgggagccc agttctttag aggcagccct 1140

gcccactaca cccctctgac ccaccctgtg tccgccccta gctccagcgg cagccctctg 1200

1260

ggcagactga tcgccagctg gacccccgtg tctcccccca gcatgtga 1308

<210> 7

<211> 435

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221> Isoform

<222> (1)..(435)

<223> Brachyury protein isoform 1 (L254V)

<400> 7

Met Ser Ser Pro Gly Thr Glu Ser Ala Gly Lys Ser Leu Gln Tyr Arg

1 5 10 15

Val Asp His Leu Leu Ser Ala Val Glu Asn Glu Leu Gln Ala Gly Ser

20 25 30

Glu Lys Gly Asp Pro Thr Glu Arg Glu Leu Arg Val Gly Leu Glu Glu

35 40 45

Ser Glu Leu Trp Leu Arg Phe Lys Glu Leu Thr Asn Glu Met Ile Val

50 55 60

Thr Lys Asn Gly Arg Arg Met Phe Pro Val Leu Lys Val Asn Val Ser

65 70 75 80

Gly Leu Asp Pro Asn Ala Met Tyr Ser Phe Leu Leu Asp Phe Val Ala

85 90 95

Ala Asp Asn His Arg Trp Lys Tyr Val Asn Gly Glu Trp Val Pro Gly

100 105 110

Gly Lys Pro Glu Pro Gln Ala Pro Ser Cys Val Tyr Ile His Pro Asp

115 120 125

Ser Pro Asn Phe Gly Ala His Trp Met Lys Ala Pro Val Ser Phe Ser

130 135 140

Lys Val Lys Leu Thr Asn Lys Leu Asn Gly Gly Gly Gln Ile Met Leu

145 150 155 160

Asn Ser Leu His Lys Tyr Glu Pro Arg Ile His Ile Val Arg Val Gly

165 170 175

Gly Pro Gln Arg Met Ile Thr Ser His Cys Phe Pro Glu Thr Gln Phe

180 185 190

Ile Ala Val Thr Ala Tyr Gln Asn Glu Glu Ile Thr Ala Leu Lys Ile

195 200 205

Lys Tyr Asn Pro Phe Ala Lys Ala Phe Leu Asp Ala Lys Glu Arg Ser

210 215 220

Asp His Lys Glu Met Met Glu Glu Pro Gly Asp Ser Gln Gln Pro Gly

225 230 235 240

Tyr Ser Gln Trp Gly Trp Leu Leu Pro Gly Thr Ser Thr Val Cys Pro

245 250 255

Pro Ala Asn Pro His Pro Gln Phe Gly Gly Ala Leu Ser Leu Pro Ser

260 265 270

Thr His Ser Cys Asp Arg Tyr Pro Thr Leu Arg Ser His Arg Ser Ser

275 280 285

Pro Tyr Pro Ser Pro Tyr Ala His Arg Asn Asn Ser Pro Thr Tyr Ser

290 295 300

Asp Asn Ser Pro Ala Cys Leu Ser Met Leu Gln Ser His Asp Asn Trp

305 310 315 320

Ser Ser Leu Gly Met Pro Ala His Pro Ser Met Leu Pro Val Ser His

325 330 335

Asn Ala Ser Pro Pro Thr Ser Ser Ser Gln Tyr Pro Ser Leu Trp Ser

340 345 350

Val Ser Asn Gly Ala Val Thr Pro Gly Ser Gln Ala Ala Ala Val Ser

355 360 365

Asn Gly Leu Gly Ala Gln Phe Phe Arg Gly Ser Pro Ala His Tyr Thr

370 375 380

Pro Leu Thr His Pro Val Ser Ala Pro Ser Ser Ser Gly Ser Pro Leu

385 390 395 400

Tyr Glu Gly Ala Ala Ala Ala Thr Asp Ile Val Asp Ser Gln Tyr Asp

405 410 415

Ala Ala Ala Gln Gly Arg Leu Ile Ala Ser Trp Thr Pro Val Ser Pro

420 425 430

Pro Ser Met

435

<210> 8

<211> 1308

<212> DNA

<213> Artificial organism

<220>

<223> Artificial sequence

<220>

<221> isoform

<222> (1)..(1308)

<223> Coding sequence for Brachyury protein isoform 1 with L254V

<400> 8

atgagctccc ctggcaccga gagcgcggga aagagcctgc agtaccgagt ggaccacctg 60

ctgagcgccg tggagaatga gctgcaggcg ggcagcgaga agggcgaccc cacagagcgc 120

gaactgcgcg tgggcctgga ggagagcgag ctgtggctgc gcttcaagga gctcaccaat 180

gagatgatcg tgaccaagaa cggcaggagg atgtttccgg tgctgaaggt gaacgtgtct 240

ggcctggacc ccaacgccat gtactccttc ctgctggact tcgtggcggc ggacaaccac 300

cgctggaagt acgtgaacgg ggaatgggtg ccggggggca agccggagcc gcaggcgccc 360

agctgcgtct acatccaccc cgactcgccc aacttcgggg cccactggat gaaggctccc 420

gtctccttca gcaaagtcaa gctcaccaac aagctcaacg gagggggcca gatcatgctg 480

aactccttgc ataagtatga gcctcgaatc cacatagtga gagttggggg tccacagcgc 540

atgatcacca gccactgctt ccctgagacc cagttcatag cggtgactgc ttatcagaac 600

gaggagatca cagctcttaa aattaagtac aatccatttg caaaggcttt ccttgatgca 660

aaggaaagaa gtgatcacaa agagatgatg gaggaacccg gagacagcca gcaacctggg 720

tactcccaat gggggtggct tcttcctgga accagcaccg tttgtccacc tgcaaatcct 780

catcctcagt ttggaggtgc cctctccctc ccctccacgc acagctgtga caggtaccca 840

accctgagga gccaccggtc ctcaccctac cccagcccct atgctcatcg gaacaattct 900

ccaacctatt ctgacaactc acctgcatgt ttatccatgc tgcaatccca tgacaattgg 960

tccagccttg gaatgcctgc ccatcccagc atgctccccg tgagccacaa tgccagccca 1020

cctaccagct ccagtcagta ccccagcctg tggtctgtga gcaacggcgc cgtcaccccg 1080

ggctcccagg cagcagccgt gtccaacggg ctgggggccc agttcttccg gggctccccc 1140

gcgcactaca cacccctcac ccatccggtc tcggcgccct cttcctcggg atccccactg 1200

tacgaagggg cggccgcggc cacagacatc gtggacagcc agtacgacgc cgcagcccaa 1260

ggccgcctca tagcctcatg gacacctgtg tcgccacctt ccatgtga 1308

<210> 9

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221> synthetic

<222> (1)..(449)

<223> Brachyury-I3 fusion protein

<400> 9

Met Lys Asn Asn Leu Tyr Glu Glu Lys Met Asn Met Ser Lys Lys Ser

1 5 10 15

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

20 25 30

His Leu Leu Ser Ala Val Glu Asn Glu Leu Gln Ala Gly Ser Glu Lys

35 40 45

Gly Asp Pro Thr Glu Arg Glu Leu Arg Val Gly Leu Glu Glu Ser Glu

50 55 60

Leu Trp Leu Arg Phe Lys Glu Leu Thr Asn Glu Met Ile Val Thr Lys

65 70 75 80

Asn Gly Arg Arg Met Phe Pro Val Leu Lys Val Asn Val Ser Gly Leu

85 90 95

Asp Pro Asn Ala Met Tyr Ser Phe Leu Leu Asp Phe Val Ala Ala Asp

100 105 110

Asn His Arg Trp Lys Tyr Val Asn Gly Glu Trp Val Pro Gly Gly Lys

115 120 125

Pro Glu Pro Gln Ala Pro Ser Cys Val Tyr Ile His Pro Asp Ser Pro

130 135 140

Asn Phe Gly Ala His Trp Met Lys Ala Pro Val Ser Phe Ser Lys Val

145 150 155 160

Lys Leu Thr Asn Lys Leu Asn Gly Gly Gly Gln Ile Met Leu Asn Ser

165 170 175

Leu His Lys Tyr Glu Pro Arg Ile His Ile Val Arg Val Gly Gly Pro

180 185 190

Gln Arg Met Ile Thr Ser His Cys Phe Pro Glu Thr Gln Phe Ile Ala

195 200 205

Val Thr Ala Tyr Gln Asn Glu Glu Ile Thr Ala Leu Lys Ile Lys Tyr

210 215 220

Asn Pro Phe Ala Lys Ala Phe Leu Asp Ala Lys Glu Arg Ser Asp His

225 230 235 240

Lys Glu Met Met Glu Glu Pro Gly Asp Ser Gln Gln Pro Gly Tyr Ser

245 250 255

Gln Trp Gly Trp Leu Leu Pro Gly Thr Ser Thr Val Cys Pro Pro Ala

260 265 270

Asn Pro His Pro Gln Phe Gly Gly Ala Leu Ser Leu Pro Ser Thr His

275 280 285

Ser Cys Asp Arg Tyr Pro Thr Leu Arg Ser His Arg Ser Ser Pro Tyr

290 295 300

Pro Ser Pro Tyr Ala His Arg Asn Asn Ser Pro Thr Tyr Ser Asp Asn

305 310 315 320

Ser Pro Ala Cys Leu Ser Met Leu Gln Ser His Asp Asn Trp Ser Ser

325 330 335

Leu Gly Met Pro Ala His Pro Ser Met Leu Pro Val Ser His Asn Ala

340 345 350

Ser Pro Pro Thr Ser Ser Ser Gln Tyr Pro Ser Leu Trp Ser Val Ser

355 360 365

Asn Gly Ala Val Thr Pro Gly Ser Gln Ala Ala Ala Val Ser Asn Gly

370 375 380

Leu Gly Ala Gln Phe Phe Arg Gly Ser Pro Ala His Tyr Thr Pro Leu

385 390 395 400

Thr His Pro Val Ser Ala Pro Ser Ser Ser Gly Ser Pro Leu Tyr Glu

405 410 415

Gly Ala Ala Ala Ala Thr Asp Ile Val Asp Ser Gln Tyr Asp Ala Ala

420 425 430

Ala Gln Gly Arg Leu Ile Ala Ser Trp Thr Pro Val Ser Pro Pro Ser

435 440 445

Met

<210> 10

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221> fusion protein

<222> (1)..(1350)

<223> Coding sequence encoding an I3-Brachyury fusion protein

with SEQ ID NO 9

<400> 10

atgaaaaata acttgtatga agaaaaaatg aacatgagta agaaaagctc ccctggcacc 60

gagagcgcgg gaaagagcct gcagtaccga gtggaccacc tgctgagcgc cgtggagaat 120

gagctgcagg cgggcagcga gaagggcgac cccacagagc gcgaactgcg cgtgggcctg 180

gaggagagcg agctgtggct gcgcttcaag gagctcacca atgagatgat cgtgaccaag 240

aacggcagga ggatgtttcc ggtgctgaag gtgaacgtgt ctggcctgga ccccaacgcc 300

atgtactcct tcctgctgga cttcgtggcg gcggacaacc accgctggaa gtacgtgaac 360

ggggaatggg tgccgggggg caagccggag ccgcaggcgc ccagctgcgt ctacatccac 420

cccgactcgc ccaacttcgg ggcccactgg atgaaggctc ccgtctcctt cagcaaagtc 480

aagctcacca acaagctcaa cggagggggc cagatcatgc tgaactcctt gcataagtat 540

gagcctcgaa tccacatagt gagagttggg ggtccacagc gcatgatcac cagccactgc 600

ttccctgaga cccagttcat agcggtgact gcttatcaga acgaggagat cacagctctt 660

aaaattaagt acaatccatt tgcaaaggct ttccttgatg caaaggaaag aagtgatcac 720

aaagagatga tggaggaacc cggagacagc cagcaacctg ggtactccca atgggggtgg 780

cttcttcctg gaaccagcac cgtttgtcca cctgcaaatc ctcatcctca gtttgggaggt 840

gccctctccc tcccctccac gcacagctgt gacaggtacc caaccctgag gagccaccgg 900

tcctcaccct accccagccc ctatgctcat cggaacaatt ctccaaccta ttctgacaac 960

tcacctgcat gtttatccat gctgcaatcc catgacaatt ggtccagcct tggaatgcct 1020

gcccatccca gcatgctccc cgtgagccac aatgccagcc cacctaccag ctccagtcag 1080

taccccagcc tgtggtctgt gagcaacggc gccgtcaccc cgggctccca ggcagcagcc 1140

gtgtccaacg ggctgggggc ccagttcttc cggggctccc ccgcgcacta cacacccctc 1200

acccatccgg tctcggcgcc ctcttcctcg ggatccccac tgtacgaagg ggcggccgcg 1260

gccacagaca tcgtggacag ccagtacgac gccgcagccc aaggccgcct catagcctca 1320

tggacacctg tgtcgccacc ttccatgtga 1350

<210> 11

<211> 261

<212> PROTEIN

<213> Homo sapiens

<220>

<221> hCD40L

<222> (1)..(261)

<223> hCD40L from NCBI RefSeq NP_000065.1.

<400> 11

Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly

1 5 10 15

Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu

20 25 30

Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg

35 40 45

Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val

50 55 60

Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser

65 70 75 80

Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys

85 90 95

Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu

100 105 110

Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser

115 120 125

Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly

130 135 140

Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln

145 150 155 160

Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr

165 170 175

Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser

180 185 190

Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala

195 200 205

Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His

210 215 220

Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn

225 230 235 240

Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe

245 250 255

Gly Leu Leu Lys Leu

260

<210> 12

<211> 789

<212> DNA

<213> Homo sapiens

<220>

<221> hCD40L

<222> (1)..(789)

<223> hCD40L from NCBI RefSeq NP_000065.1.

<400> 12

atgatcgaga catacaacca gacaagccct agaagcgccg ccacaggact gcctatcagc 60

atgaagatct tcatgtacct gctgaccgtg ttcctgatca cccagatgat cggcagcgcc 120

ctgtttgccg tgtacctgca cagacggctg gacaagatcg aggacgagag aaacctgcac 180

gaggacttcg tgttcatgaa gaccatccag cggtgcaaca ccggcgagag aagtctgagc 240

ctgctgaact gcgaggaaat caagagccag ttcgaggggct tcgtgaagga catcatgctg 300

aacaaagagg aaacgaagaa agagaactcc ttcgagatgc agaagggcga ccagaatcct 360

cagatcgccg ctcacgtgat cagcgaggcc agcagcaaga caacaagcgt gctgcagtgg 420

gccgagaagg gctactacac catgagcaac aacctggtca ccctggagaa cggcaagcag 480

ctgacagtga agcggcaggg cctgtactac atctacgccc aagtgacctt ctgcagcaac 540

agagaggcca gctctcaggc tcctttcatc gccagcctgt gcctgaagtc tcctggcaga 600

ttcgagcgga ttctgctgag agccgccaac acacacagca gcgccaaacc ttgtggccag 660

cagtctattc acctcggcgg agtgtttgag ctgcagcctg gcgcaagcgt gttcgtgaat 720

gtgacagacc ctagccaggt gtcccacggc accggcttta catctttcgg actgctgaag 780

ctgtgatga 789

<210> 13

<211> 1145

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221> synthetic

<222> (1)..(1145)

<223> Synthetic amino acid sequence v2 Her2

<400> 13

Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu

1 5 10 15

Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys

20 25 30

Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His

35 40 45

Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr

50 55 60

Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val

65 70 75 80

Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu

85 90 95

Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr

100 105 110

Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Pro

115 120 125

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

130 135 140

Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln

145 150 155 160

Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn

165 170 175

Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys

180 185 190

His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser

195 200 205

Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys

210 215 220

Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys

225 230 235 240

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

245 250 255

His Phe Asn His Ser Gly Ile Cys Glu Leu Ala Cys Pro Ala Leu Val

260 265 270

Thr Tyr Asn Thr Arg Thr Ala Lys Ser Met Pro Asn Pro Glu Gly Arg

275 280 285

Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu

290 295 300

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

305 310 315 320

Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Ala Cys Ser Lys

325 330 335

Ala Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu

340 345 350

Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys

355 360 365

Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp

370 375 380

Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe

385 390 395 400

Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro

405 410 415

Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg

420 425 430

Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu

435 440 445

Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly

450 455 460

Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val

465 470 475 480

Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr

485 490 495

Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His

500 505 510

Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys

515 520 525

Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys

530 535 540

Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys

545 550 555 560

Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys

565 570 575

Phe Gly Pro Ala Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp

580 585 590

Pro Pro Ala Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu

595 600 605

Ser Tyr Met Pro Ile Trp Ala Phe Pro Asp Glu Glu Gly Ala Cys Gln

610 615 620

Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys

625 630 635 640

Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser

645 650 655

Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly

660 665 670

Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg

675 680 685

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

690 695 700

Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu

705 710 715 720

Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys

725 730 735

Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile

740 745 750

Met Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu

755 760 765

Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg

770 775 780

Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu

785 790 795 800

Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg

805 810 815

Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly

820 825 830

Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala

835 840 845

Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe

850 855 860

Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp

865 870 875 880

Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg

885 890 895

Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val

900 905 910

Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala

915 920 925

Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro

930 935 940

Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met

945 950 955 960

Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe

965 970 975

Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu

980 985 990

Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu

995 1000 1005

Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Ala

1010 1015 1020

Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly

1025 1030 1035

Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg

1040 1045 1050

Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu

1055 1060 1065

Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser

1070 1075 1080

Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu

1085 1090 1095

Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser

1100 1105 1110

Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val

1115 1120 1125

Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Leu Gly Leu Asp Val

1130 1135 1140

ProVal

1145

<210> 14

<211> 3441

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221> synthetic

<222> (1)..(3441)

<223> Synthetic nucleotide sequence v2 Her2

<400> 14

atggaactgg ctgctctgtg tagatgggga ctgctgcttg ctctgttgcc tcctggagct 60

gcttctaccc aagtgtgcac aggcaccgac atgaagctga gactgcctgc ttctcctgag 120

acacacctgg acatgctgag acacctgtac cagggatgtc aggtggtgca gggaaatctg 180

gaactgacct acctgcctac caacgccagc ctgagctttc tgcaggacat ccaagaggtg 240

cagggatacg tgctgatcgc tcacaatcaa gtgagacagg tgccactgca gaggctgaga 300

atcgttagag gcacccagct gttcgaggac aactatgctc tggctgtgct ggacaatggc 360

gaccctctga acaacaccac acctgtgaca ggagcttctc ctggtggact gagagaactg 420

cagctgagaa gcctgaccga gatcctgaaa ggaggagtgc tgatccagcg gaaccctcag 480

ctgtgctacc aggacaccat cctgtggaag gacatcttcc acaagaacaa ccagctggct 540

ctgacactga tcgacaccaa cagaagcaga gcctgccatc cttgctctcc catgtgcaag 600

ggctctagat gttggggaga gagcagcgag gattgccaga gcctgaccag aacagtgtgt 660

gctggaggat gtgccagatg caaaggacct ctgcctaccg actgctgcca cgagcaatgt 720

gcagctggat gtacaggacc aaagcactct gattgcctgg cctgcctgca cttcaaccac 780

tctggaatct gcgagctcgc ctgtcctgct ctggtcacct acaacacacg gaccgccaag 840

agcatgccta atcctgaagg cagatacacc tttggagcca gctgtgtgac agcctgtcct 900

tacaactacc tgagcaccga cgctggagcc tgcacactcg tttgtcctgc tgccaatcaa 960

gaagtgacgg ccgaggacgg cacccagaga tgcgaggcct gtagcaaggc ttgcgctaga 1020

gtgtgttacg gactcggcat ggaacacctg agagaagtga gagccgtgac cagtgccaac 1080

atccaagagt ttgctggctg caagaagatc tttggcagcc tcgccttcct gcctgagagc 1140

ttcgatggcg atcctgccag caatactgct cctctgcagc ctgaacagct ccaggtgttc 1200

gagacactgg aagagatcac aggctacctg tacatcagcg catggccaga cagcctgcct 1260

gacctgtccg tgttccagaa cctgcaagtg atcagaggca gaatcctgca caacggagcc 1320

1380

cttggatctg gcctggctct gatccaccac aatacccacc tgtgcttcgt gcacaccgtg 1440

ccttgggacc agctgtttcg gaatcctcat caggctctgc tgcacacagc caacagacct 1500

gaggatgagt gtgttggcga aggcctggct tgtcaccagc tctgtgctag aggacactgt 1560

tggggacctg gacctacaca gtgtgtgaac tgtagccagt tcctgagagg ccaagaatgc 1620

1680

ctgccttgtc atcccgagtg ccagcctcag aatggcagcg tgacatgttt tggaccagct 1740

gccgatcagt gcgtggcctg tgctcactat aaggaccctc cagcctgcgt ggccagatgt 1800

cctagcggag tgaagcctga cctgagctac atgcccatct gggcatttcc agatgaggaa 1860

ggagcttgcc agccttgtcc tatcaactgc acccacagct gcgtggacct ggacgataag 1920

ggatgtccag ccgagcagag agcctctcca ctgacctcta tcatctctgc cgtcgtggggc 1980

atcctgctgg tggtggttct gggagttgtg ttcggcatcc tgatcaagag acggcagcag 2040

aagatccgga agtacaccat gcggagactg ctgcaagaga ctgagctggt ggaacctctg 2100

acacccagcg gagctatgcc taaccaggct cagatgcgga ttctgaaaga aaccgagctg 2160

cggaaagtga aggtgctcgg ctctggagcc tttggcacag tgtacaaagg catctggatc 2220

2280

cccaaggcca acaaagagat cctggacgag gcctacgtga tggctggtgt tggcagccct 2340

tatgtgtcta gactgctggg catctgtctg accagcaccg tgcagctggt cactcagctg 2400

atgccttacg gctgcctgct ggatcacgtg agagagaata gaggcagact gggctctcag 2460

gacctgctga actggtgcat gcagatcgcc aagggcatga gctacctcga ggatgtgaga 2520

ctggtccaca gagatctggc tgccagaaac gtgctcgtga agtctcctaa ccacgtgaag 2580

atcaccgact tcggactggc taggctgctg gatatcgacg agacagagta ccacgctgat 2640

ggaggcaagg tgcccatcaa gtggatggct ctggaatcca tcctgagacg gagattcacc 2700

caccagtccg atgtgtggtc ttacggagtg acagtgtggg agctgatgac cttcggagcc 2760

aagccttacg acggcatccc tgccagagag atcccagatc tgctggaaaa gggagagaga 2820

ctgcctcagc ctcctatctg caccatcgac gtgtacatga ttatggtcaa gtgttggatg 2880

atcgacagcg agtgcagacc cagattcaga gaactggtgt ccgagttctc tcggatggcc 2940

agagatcctc agagattcgt ggtcatccag aacgaggatc tgggacctgc cagccctctg 3000

gacagcacct tctacagatc cctgctggaa gatgacgaca tgggtgacct ggtggacgct 3060

gaagaagctc tggttcctca gcagggcttc ttctgccctg atcctgctcc aggagcaggt 3120

ggaatggtgc atcacagaca cagaagctcc agcaccagaa gcggaggcgg agatctgaca 3180

ctgggactcg agccatctga ggaagaggct cctagatctc ctctggctcc ttctgaagga 3240

gctggaagcg acgttttcga cggagatctt ggaatgggag ctgccaaagg actccagtct 3300

3360

ccaagcgaga cagatggata tgtggcacct ctgacctgct ctcctcagcc agaactggga 3420

cttgatgtgc ctgtttgatg a 3441

<210> 15

<211> 205

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221> synthetic

<222> (1)..(205)

<223> Synthetic amino acid sequence Twist

<400> 15

Met Gln Asp Val Ser Ser Ser Pro Val Ser Pro Ala Asp Asp Ser Leu

1 5 10 15

Ser Asn Ser Glu Glu Glu Pro Asp Arg Gln Gln Pro Ala Ser Gly Lys

20 25 30

Arg Gly Ala Arg Lys Arg Arg Ser Ser Arg Arg Ser Ala Gly Gly Ser

35 40 45

Ala Gly Pro Gly Gly Ala Thr Gly Gly Gly Ile Gly Gly Gly Asp Glu

50 55 60

Pro Gly Ser Pro Ala Glyn Gly Lys Arg Gly Lys Lys Ser Ala Gly Gly

65 70 75 80

Gly Gly Gly Gly Gly Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser

85 90 95

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

100 105 110

Arg Val Met Ala Asn Val Arg Glu Arg Gln Arg Thr Gln Ser Leu Asn

115 120 125

Glu Ala Phe Ala Ala Leu Arg Lys Ile Ile Pro Thr Leu Pro Ser Asp

130 135 140

Lys Leu Ser Lys Ile Gln Thr Leu Lys Leu Ala Ala Arg Tyr Ile Asp

145 150 155 160

Phe Leu Tyr Gln Val Leu Gln Ser Asp Glu Leu Asp Ser Lys Met Ala

165 170 175

Ser Cys Ser Tyr Val Ala His Glu Arg Leu Ser Tyr Ala Phe Ser Val

180 185 190

Trp Arg Met Glu Gly Ala Trp Ser Met Ser Ala Ser His

195 200 205

<210> 16

<211> 618

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221> synthetic

<222> (1)..(618)

<223> Synthetic nucleotide sequence Twist

<400> 16

atgcaggacg tgtccagcag ccctgtgtct cctgccgacg acagcctgag caacagcgag 60

gaagaacccg acagacagca gcccgcctct ggcaagagag gcgccagaaa gagaagaagc 120

tccagaagaa gcgctggcgg ctctgctgga cctggcggag ctacaggcgg aggaattgga 180

ggcggagatg agcctggctc tccagcccag ggcaagaggg gcaagaaatc tgctggcgga 240

ggcggcggag gaggagctgg aggcggagga ggaggcggcg gaggatcaag ttctggcgga 300

ggaagccctc agagctacga ggaactgcag acccagcgcg tgatggccaa cgtgcgcgag 360

agacagagaa cccagagcct gaacgaggcc ttcgccgccc tgagaaagat catccccacc 420

ctgcccagcg acaagctgag caagatccag accctgaagc tggccgccag atatatcgac 480

ttcctgtatc aagtgctgca gagcgacgag ctggacagca agatggccag ctgctcctac 540

600

atgagcgcca gccactga 618

<210> 17

<211> 260

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221> synthetic

<222> (1)..(260)

<223> Synthetic amino acid sequence of mouse CD40L

<400> 17

Met Ile Glu Thr Tyr Ser Gln Pro Ser Pro Arg Ser Val Ala Thr Gly

1 5 10 15

Leu Pro Ala Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu

20 25 30

Ile Thr Gln Met Ile Gly Ser Val Leu Phe Ala Val Tyr Leu His Arg

35 40 45

Arg Leu Asp Lys Val Glu Glu Glu Val Asn Leu His Glu Asp Phe Val

50 55 60

Phe Ile Lys Lys Leu Lys Arg Cys Asn Lys Gly Glu Gly Ser Leu Ser

65 70 75 80

Leu Leu Asn Cys Glu Glu Met Arg Arg Gln Phe Glu Asp Leu Val Lys

85 90 95

Asp Ile Thr Leu Asn Lys Glu Glu Lys Lys Glu Asn Ser Phe Glu Met

100 105 110

Gln Arg Gly Asp Glu Asp Pro Gln Ile Ala Ala His Val Val Ser Glu

115 120 125

Ala Asn Ser Asn Ala Ala Ser Val Leu Gln Trp Ala Lys Lys Gly Tyr

130 135 140

Tyr Thr Met Lys Ser Asn Leu Val Met Leu Glu Asn Gly Lys Gln Leu

145 150 155 160

Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val Tyr Thr Gln Val Thr Phe

165 170 175

Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg Pro Phe Ile Val Gly Leu

180 185 190

Trp Leu Lys Pro Ser Ser Gly Ser Glu Arg Ile Leu Leu Lys Ala Ala

195 200 205

Asn Thr His Ser Ser Ser Gln Leu Cys Glu Gln Gln Ser Val His Leu

210 215 220

Gly Gly Val Phe Glu Leu Gln Ala Gly Ala Ser Val Phe Val Asn Val

225 230 235 240

Thr Glu Ala Ser Gln Val Ile His Arg Val Gly Phe Ser Ser Phe Gly

245 250 255

Leu Leu Lys Leu

260

<210> 18

<211> 786

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<220>

<221> synthetic

<222> (1)..(786)

<223> Synthetic nucleotide sequence of mouse CD40L

<400> 18

atgatcgaga catacagcca gcccagcccc agaagcgtgg ccacaggact gcctgccagc 60

atgaagatct ttatgtacct gctgaccgtg ttcctgatca cccagatgat cggcagcgtg 120

ctgttcgccg tgtacctgca cagacggctg gacaaggtgg aagaggaagt gaacctgcac 180

gaggacttcg tgttcatcaa gaaactgaag cggtgcaaca agggcgagg cagcctgagc 240

ctgctgaact gcgaggaaat gagaaggcag ttcgaggacc tcgtgaagga catcaccctg 300

aacaaagagg aaaagaaaga aaactccttc gagatgcaga ggggcgacga ggaccctcag 360

atcgctgctc acgtggtgtc cgaggccaac agcaacgccg cttctgtgct gcagtgggcc 420

aagaaaggct actacaccat gaagtccaac ctcgtgatgc tggaaaacgg caagcagctg 480

acagtgaagc gcgaggggcct gtactatgtg tacacccaag tgacattctg cagcaacaga 540

gagcccagca gccagaggcc cttcatcgtg ggactgtggc tgaagcctag cagcggcagc 600

gagagaatcc tgctgaaggc cgccaacacc cacagcagct ctcagctgtg cgagcagcag 660

agcgtgcacc tgggcggagt gttcgagctg caagctggcg cctccgtgtt cgtgaacgtg 720

acagaggcca gccaagtgat ccacagagtg ggcttcagca gctttggact gctgaaactg 780

taatga 786

<---

Claims (36)

1. Pharmaceutical combination to reduce tumor size and/or increase survival of a cancer patient, comprising: (a) a recombinant modified vaccinia virus Ankara (MVA) containing: (i) a first nucleic acid encoding a first heterologous tumor associated antigen (TAA), and (ii) a second nucleic acid encoding CD40L; And (b) an antibody containing an Fc domain and is specific for an antigen that is expressed on the cell membrane of the tumor cell. 2. Pharmaceutical combination according to claim 1, characterized in that the recombinant MVA additionally contains a second nucleic acid encoding a second heterologous TAA. 3. Pharmaceutical combination according to claim 1 or 2, characterized in that the first and/or second OAA are selected from the group consisting of: 5-α-reductase, α-fetoprotein ("AFP"), AM-1, APC, April , melanoma B antigen gene ("BAGE"), β-catenin, Bcl12, bcr-abl, Brachyury, CA-125, caspase-8 ("CASP-8", also known as "FLICE"), cathepsins, CD19, CD20 , CD21/Complement Receptor 2 ("CR2"), CD22/BL-CAM, CD23/FcεRII, CD33, CD35/Complement Receptor 1 ("CR1"), CD44/PGP-1, CD45/Leukocyte Common Antigen ("LCA" ), CD46/membrane cofactor protein ("MCP"), CD52/CAMPATH-1, CD55/degradation accelerator factor ("DAF"), CD59/protectin, CDC27, CDK4, carcinoembryonic antigen ("CEA"), c-myc, cyclooxygenase-2 ("cox-2"), colorectal cancer deleted gene ("DCC"), DcR3, E6/E7, CGFR, EMBP, Dna78, farnesyl transferase, fibroblast growth factor-8a ("FGF8a"), fibroblast growth factor -8b ("FGF8b"), FLK-1/KDR, folic acid receptor, G250, melanoma G antigen gene family ("GAGE family"), gastrin 17, gastrin-releasing hormone, ganglioside 2 ("GD2")/ganglioside 3 ("GD3")/ganglioside-monosialic acid-2 ("GM2"), gonadotropin-releasing hormone ("GnRH"), UDP-GlcNAc:R1Man(α1-6)R2 [GlcNAc to Man(α1-6)] β1,6-N-acetylglucosaminyltransferase V ("GnT V"), GP1, gp100/Pme117, gp-100-in4, gp15, gp75/tyrosine-related protein-1 ("gp75/TRP-1"), human chorionic gonadotropin ("hCG"), heparanase, HER2, human breast tumor virus ("HMTV"), 70 kDa heat shock protein ("HSP70"), human telomerase reverse transcriptase ("hTERT"), insulin-like growth factor-1 receptor ("IGFR-1"), interleukin-13 receptor ("IL-13R"), inducible nitric oxide synthase ("iNOS"), Ki67, KIAA0205, K-ras, H-ras, N-ras, KSA, LKLR- FUT, melanoma antigen 1 ("MAGE-1"), melanoma antigen 2 ("MAGE-2"), melanoma antigen 3 ("MAGE-3"), melanoma antigen 4 ("MAGE-4"), mammaglobin, MAP17, melanoma/melanoma antigen recognized by T-cells 1 ("MART-1"), mesothelin, MIC A/B, MT-MMP, mucin, NY testis-specific antigen -ESO-1, osteonectin, p15, P170/MDR1, p53, p97/melanotransferrin, PAI-1, platelet-derived growth factor ("PDGF"), μPA, PRAME, probasin, progenipoietin, prostate specific antigen ("PSA"), prostate -specific membrane antigen ("PSMA"), RAGE-1, Rb, RCAS1, SART-1, SSX-family, STAT3, STn, TAG-72, transforming growth factor alpha ("TGF-α"), transforming growth factor beta ("TGF-β"), thymosin-beta-15, tumor necrosis factor alpha ("TNF-α"), TP1, TRP-2, tyrosinase, vascular endothelial growth factor ("VEGF"), ZAG, p16INK4 and glutathione- S-transferases ("GST"). 4. Pharmaceutical combination according to any one of paragraphs. 1-3, characterized in that the first TAA is a HER2 antigen and the antibody is an antibody to HER2. 5. Pharmaceutical combination according to any one of paragraphs. 1-4, characterized in that the HER2 antigen contains at least one mutation: (a) to prevent binding of the HER2 antibody to the HER2 antigen; (b) preventing extracellular dimerization of the HER2 antigen; (c) preventing the tyrosine kinase activity of the HER2 antigen; and/or (d) preventing phosphorylation of the HER2 antigen. 6. Pharmaceutical combination according to claim 4 or 5, characterized in that the HER2 antigen contains at least one mutation in the trastuzumab binding domain selected from the group consisting of: E580, D582, P594, F595, K615 and Q624. 7. Pharmaceutical combination according to claim 4 or 5, characterized in that the HER2 antigen contains at least one mutation in the pertuzumab binding domain selected from the group consisting of: H267, Y274, F279, V308, S310, L317, H318, K333 and P337. 8. Pharmaceutical combination according to any one of paragraphs. 1-7, characterized in that the first nucleic acid encodes a HER2 antigen having at least 90%, 95%, 97%, 98% or 99% identity with SEQ ID NO:1. 9. Pharmaceutical combination according to any one of paragraphs. 1-7, characterized in that the first nucleic acid encodes a HER2 antigen having at least 90%, 95%, 97%, 98% or 99% identity with SEQ ID NO: 13. 10. Pharmaceutical combination according to any one of paragraphs. 1-7, characterized in that the first nucleic acid encodes SEQ ID NO: 1. 11. Pharmaceutical combination according to any one of paragraphs. 1-7, characterized in that the first nucleic acid encodes SEQ ID NO: 13. 12. Pharmaceutical combination according to any one of paragraphs. 1-7, characterized in that the first nucleic acid contains a nucleic acid having at least 90%, 95%, 97%, 98% or 99% identity with SEQ ID NO: 2. 13. Pharmaceutical combination according to any one of paragraphs. 1-7, characterized in that the first nucleic acid contains a nucleic acid having at least 90%, 95%, 97%, 98% or 99% identity with SEQ ID NO: 14. 14. Pharmaceutical combination according to any one of paragraphs. 1-7, characterized in that the first nucleic acid contains SEQ ID NO:2. 15. Pharmaceutical combination according to any one of paragraphs. 1-7, characterized in that the first nucleic acid contains SEQ ID NO: 14. 16. Pharmaceutical combination according to any one of paragraphs. 1-15, characterized in that the second TAA is a Brachyury antigen. 17. Pharmaceutical combination according to claim 16, characterized in that the Brachyury antigen contains one or more mutations to prevent nuclear localization. 18. Pharmaceutical combination according to any one of paragraphs. 1-17, characterized in that the second nucleic acid encodes a Brachyury antigen having at least 90%, 95%, 97%, 98% or 99% identity with SEQ ID NO:3. 19. Pharmaceutical combination according to any one of paragraphs. 16-18, characterized in that the second nucleic acid encodes a Brachyury antigen containing SEQ ID NO:3. 20. Pharmaceutical combination according to any one of paragraphs. 16-19, characterized in that the second nucleic acid contains a nucleic acid having at least 90%, 95%, 97%, 98% or 99% identity with SEQ ID NO:4. 21. Pharmaceutical combination according to any one of paragraphs. 16-20, characterized in that the second nucleic acid contains SEQ ID NO:4. 22. Pharmaceutical combination according to any one of paragraphs. 1-3, characterized in that the antibody is selected from the group consisting of: anti-CD20 (for example, rituximab; ofatumumab; tositumomab), anti-CD52 (for example, alemtuzumab Campat®), anti-EGFR (for example, cetuximab Erbitux®¸ panitumumab), anti-CD2 (eg, ciplizumab), anti-CD37 (eg, BI836826), anti-CD123 (eg, JNJ-56022473), anti-CD30 (eg, XmAb2513), anti-CD38 (eg, daratumumab Darzalex® ), anti-PDL1 (eg, avelumab, atezolizumab, durvalumab), anti-GD2 (eg, 3F8, ch14.18, KW-2871, dinutuximab), anti-CEA, anti-MUC1, anti-FLT3, anti-CD19, anti-CD40, anti-SLAMF7, anti-CCR4, anti-B7-H3, anti-ICAM1, anti-CSF1R, anti-CA125 (eg, oregovomab), anti-FRα (eg, MOv18-IgG1, mirvetuximab soravtansine (IMGN853), MORAb -202), anti-mesothelin (eg MORAb-009) and anti-HER2. 23. A pharmaceutical combination according to claim 4 or 5, characterized in that the antibody is specific for the HER2 antigen. 24. Pharmaceutical combination according to claim 4 or 5, characterized in that the antibody is selected from pertuzumab, trastuzumab, Gerzuma, ABP 980 and ado-trastuzumab emtansine. 25. Pharmaceutical combination according to any one of paragraphs. 1-24, characterized in that MVA is MVA-BN or a derivative of MVA-BN. 26. Pharmaceutical combination according to any one of paragraphs. 1-25, characterized in that (a) is administered simultaneously or after (b). 27. Pharmaceutical combination according to any one of paragraphs. 1-26, characterized in that (a) and (b) are administered to the cancer patient as a priming administration followed by one or more booster administrations of (a) and (b) to the cancer patient. 28. Pharmaceutical combination according to any one of paragraphs. 1-27, characterized in that the cancer patient has and/or is diagnosed with a cancer selected from the group consisting of: breast cancer, lung cancer, head and neck cancer, thyroid cancer, melanoma, stomach cancer, bladder cancer , kidney cancer, liver cancer, melanoma, pancreatic cancer, prostate cancer, ovarian cancer, or colorectal cancer. 29. Pharmaceutical combination according to claim 28, characterized in that the breast cancer is a breast cancer with overexpression of HER2. 30. Pharmaceutical combination according to any one of paragraphs. 1-29, characterized in that (a) is administered intravenously to an oncological patient. 31. A method for reducing tumor size and/or improving the survival of a cancer patient, comprising administering to the patient a pharmaceutical combination according to any one of paragraphs. 1-29. 32. The method of claim 31, wherein (a) the pharmaceutical combination is administered intravenously to a cancer patient.

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