A method for treating cancer by intratumoral and / or intravenous administration of recombinant MVA encoding 4-1BBL (CD137L) and / or CD40L.

Abstract

The present invention relates to compositions and / or related methods for reducing tumor volume and / or increasing survival rates in cancer patients. The compositions comprise a tumor-associated antigen ("TAA") and a recombinant MVA encoding 4-1BBL and / or CD40L, and can be administered to a subject in any suitable manner, including intravenously and / or intratumorally.

Description

[Technical field]

[0001] The present invention relates to a therapy for treating cancer, the therapy comprising intravenous or intratumoral administration of a recombinant modified vaccinia Ankara (MVA) virus comprising a nucleic acid encoding 4-1BBL (CD137L). Recombinant modified vaccinia Ankara (MVA) virus (also referred to as "recombinant MVA" or "rMVA"), as used herein, refers to an MVA comprising at least one polynucleotide encoding a tumor associated antigen (TAA). In a more specific aspect, the present invention comprises intravenous or intratumoral administration of a recombinant MVA comprising a nucleic acid encoding a TAA and a nucleic acid encoding 4-1BBL. In an additional aspect, the present invention comprises intravenous or intratumoral administration of a recombinant MVA comprising a nucleic acid encoding a TAA and a nucleic acid encoding CD40L. In an additional aspect, the present invention comprises intravenous and / or intratumoral administration of a recombinant MVA comprising a nucleic acid encoding a TAA, 4-1BBL (CD137L) and CD40L. [Background technology]

[0002] Recombinant poxviruses have been used as immunotherapeutic vaccines against infectious organisms and, more recently, against tumors (Mastrangelo et al. (2000) J Clin Invest. 105(8):1031-1034).

[0003] One poxvirus strain that has proven useful as an immunotherapeutic vaccine against infectious diseases and cancer is the modified vaccinia Ankara (MVA) virus (sometimes simply referred to as "MVA"). MVA was generated by serial passage of the Ankara strain of vaccinia virus (CVA) in chicken embryo fibroblasts for 516 generations (for review, see Mayr et al. (1975) Infection 3:6-14). These long passages resulted in a genome of the resulting MVA virus that was described as highly host cell restricted for replication in avian cells, since the deleted genomic sequence was approximately 31 kilobases (Meyer et al. (1991) J. Gen. Virol. 72:1031-1038). In various animal models, the resulting MVA was shown to be remarkably non-pathogenic (Mayr & Danner (1978) Dev. Biol. Stand. 41:225-34). MVA strains with improved safety profiles have been described for the development of safer products, such as vaccines or medicines (see WO2002042480; see also, e.g., U.S. Pat. Nos. 6,761,893 and 6,913,752, both of which are incorporated herein by reference). Such variants are capable of productive replication in non-human cells and cell lines, particularly chicken embryo fibroblasts (CEF), but are replication-incompetent in human cell lines, including, inter alia, HeLa, HaCat and 143B cell lines. Such strains are also incapable of productive replication in vivo, e.g., in certain mouse strains, such as the transgenic mouse model AGR129, which is severely immunocompromised and highly susceptible to replicative viruses (see U.S. Pat. No. 6,761,893). Such MVA variants and their derivatives (including recombinant forms) are referred to as "MVA-BN" and have been described (see International Publication No. WO 2002 / 042480; see also, e.g., U.S. Pat. Nos. 6,761,893 and 6,913,752).

[0004] The use of poxvirus vectors encoding tumor-associated antigens (TAAs) has been shown to reduce tumor size and improve overall survival in cancer patients (see, e.g., WO2014 / 062778). Administration of poxvirus vectors encoding TAAs such as HER2, CEA, MUC1, and / or Brachyury to cancer patients has been shown to generate vigorous and specific T cell responses in the patients to combat the cancer (see above; see also Guardino et al. ((2009) Cancer Res. 69(24), doi 10.1158 / 0008-5472.SABCS-09-5089); Heery et al. (2015) JAMA Oncol. 1:1087-95).

[0005] One type of TAAs found to be expressed on many cancer and tumor cells are endogenous retroviral (ERV) proteins. ERVs are remnants of previously foreign retroviruses that have invaded the host germline and have since been transmitted vertically throughout the genetic population (see Bannert et al. (2018) Frontiers in Microbiology, Volume 9, Article 178). ERV-induced genetic recombination events and dysregulation of normal cellular genes have been demonstrated to have a contributory effect on tumor formation (see supra). Furthermore, there is evidence that certain ERV proteins have oncogenic properties (see supra). ERVs have been found to be expressed in a wide variety of cancers, including, for example, breast, ovarian, melanoma, prostate, pancreatic, and lymphoma.(For example, Bannert et al. (2018) Front. Microbiol. 9:178, Cegolon et al. (2013) BMC Cancer 13:4, Wang-Johanning et al. (2003) Oncogene 22:1528-35, Wang-Johanning et al. (2007) Int.J.Cancer 120:81-90, Wang-Johanning et al. (2008) Cancer Res. 68:5869-77, Wang-Johanning et al. (2018) Cancer Res. 78(13 Suppl.), AACR Annual Meeting April 2018, Abstract 1257, Contreras-Galindo et al. (2008) J.Virol. 82:9329-36, Schiavetti et al. (2002) Cancer Res. 62:5510-16, Maliniemi et al.(2013)PLoS One 8:e76281、Fava et al.(2017)Genes Dev.31:34-45、Muster et al.(2003)Cancer Res.63:8735-41、Buscher et al.(2005)Cancer Res.65:4172-80、Serafino et al.(2009)Expt'l. Cell Res. 315:849-62、Iramaneerat et al. (2011) Int. J. Gynecol. Cancer 21:51-7、Ishida et al.(2006)Cancer Sci.97:1139-46、Goering et al.(2011)Carcinogenesis 32:1484-92、Agoni et al.(2013)Front. Oncol.9:180、Li et al.(2017) J.Mol.Diagn.19:4-23

[0006] Poxviruses such as MVA have been shown to have enhanced efficacy when combined with CD40 agonists such as CD40 ligand (CD40L) (see WO2014 / 037124) or 4-1BB agonists such as 4-1BB ligand (4-1BBL) (Spencer et al. (2014) PLoS One 9:e105520), in addition to their effects with TAAs.

[0007] CD40 / CD40L are members of the tumor necrosis factor receptor / tumor necrosis factor ("TNFR / TNF") superfamily. Whereas CD40 is constitutively expressed on many cell types, including B cells, macrophages, and DCs, its ligand, CD40L, is expressed primarily on activated CD4+ T cells (Lee et al. (2002) J. Immunol. 171(11):5707-5717; Ma and Clark (2009) Semin. Immunol. 21(5):265-272). Cognate interactions between DCs and CD4+ T cells early after infection or immunization "license" DCs to prime CD8+ T cell responses (Ridge et al. (1998) Nature 393:474-478). Licensing of DCs upregulates costimulatory molecules, increasing DC survival and improving cross-presentation capacity. This process is primarily mediated by CD40 / CD40L interactions (Bennet et al. (1998) Nature 393:478-480; Schoenberger et al. (1998) Nature 393:480-483), although CD40 / CD40L-independent mechanisms also exist (CD70, LT.beta.R). Interestingly, a direct interaction between CD40L expressed on DCs and CD40 expressed on CD8+ T cells has also been suggested, providing a possible explanation for the generation of helper-independent CTL responses (Johnson et al. (2009) Immunity 30:218-227).

[0008] 4-1BB / 4-1BBL is a member of the TNFR / TNF superfamily. 4-1BBL is a costimulatory ligand expressed on activated B cells, monocytes and DCs. 4-1BB is constitutively expressed by several innate immune cell populations, including natural killer (NK) and natural killer T (NKT) cells, Tregs, as well as DCs, monocytes and neutrophils. Interestingly, 4-1BB is expressed on activated but not resting T cells (Wang et al. (2009) Immunol. Rev. 229:192-215). Binding of 4-1BB induces proliferation and production of interferon gamma (IFN-γ) and interleukin 2 (IL-2) and enhances T cell survival through upregulation of anti-apoptotic molecules such as Bcl-xL (Snell et al. (2011) Immunol. Rev. 244:197-217). Importantly, stimulation of 4-1BB enhances NK cell proliferation, IFN-γ production, and cytolytic activity through enhanced antibody-dependent cellular cytotoxicity (ADCC) (Kohrt et al. (2011) Blood 117:2423-32).

[0009] The 4-1BB / 4-1BBL immune axis is currently being explored by various immunotherapeutic strategies. For example, the transplantation of autologous chimeric antigen receptor (CAR) T cells has shown clinical efficacy in large B-cell lymphoma and was approved by the FDA in 2017. CARs, which combine the extracellular domain derived from a tumor-specific antibody, the intracellular signaling domain CD3ζ, and the costimulatory motif 4-1BB, are introduced into the patient's autologous T cells. The addition of 4-1BB is essential for the in vivo persistence and antitumor cytotoxicity of CAR T cells (Song et al. (2011) Cancer Res. 71:4617e27). Antibodies targeting 4-1BB are currently being investigated.

[0010] Several studies have shown that agonistic antibodies targeting the 4-1BB / 4-1BBL pathway exhibit antitumor activity when used as monotherapy (Palazon et al. (2012) Cancer Discovery 2:608-23). ​​Agonistic antibodies targeting 4-1BB (Urelumab from BMS and Utomilumab from Pfizer) are currently in clinical development. In recent years, studies combining 4-1BBL with other therapies have met with mixed results. For example, when mice bearing pre-existing MC38 (murine adenocarcinoma) tumors, but no B16 melanoma tumors, were treated with antibodies against CTLA-4 and anti-4-1BB, significant CD8+ T cell-dependent tumor regression was observed along with long-lasting immunity against these tumors. In another example, treatment with anti-4-1BB (Bristol-Myers Squibb (BMS)-469492) resulted in only modest regression of M109 tumors, but significantly delayed the growth of EMT6 tumors.

[0011] The tumor microenvironment is composed of various cell types, ranging from infiltrating immune cells to cancer cells, extracellular matrix, endothelial cells, and other involved cells that influence tumor progression. This complex intertwined equilibrium not only varies from patient to patient, but also within the same lesion (Jimenez-Sanchez et al. (2017) Cell 170(5):927-938). Tumor stratification based on tumor-infiltrating lymphocyte (TIL) and programmed death-ligand 1 (PD-L1) expression highlights the importance of the inflammatory environment for objective cancer response (Teng et al. (2015) Cancer Res. 75(11):2139-45). A cross-cancer analysis of gene expression profiles from the Cancer Genome Atlas (TCGA) supports the correlation of tumor inflammatory signatures with objective responses to immunotherapy (Danaher et al. (2018) J. Immunother. Cancer 6(1):63).

[0012] In recent years, attempts to improve the route of vaccine administration for cancer therapy have been expanded from subcutaneous injection to intravenous administration. For example, it has been shown that intravenous administration of an MVA vaccine encoding a heterologous antigen can induce a strong specific immune response against that antigen (see WO2014 / 037124). Furthermore, the immune response was enhanced when CD40L was included in the MVA vaccine.

[0013] Inoculation of tumor lesions with bacterially derived substances (Coley toxins) has long been reported to result in healing responses, highlighting the role of local infection in promoting antitumor responses (Coley (1906) Proc. R. Soc. Med. 3 (Surg Sect): 1-48). Local administration of pathogen-associated molecular patterns (PAMPs), bacterial products, and viruses into tumor lesions induces an antimicrobial program that subsequently initiates a cascade of events including i) secretion of proinflammatory cytokines such as type I interferons, type II interferons, type III interferons, and tumor necrosis factor alpha (TNF-α), ii) danger signals such as alarmins and heat shock proteins, and iii) release of tumor antigens (Aznar et al. (2017) J. Immunol. 198: 31-39). Although regression has been evaluated in untreated tumor lesions ((2018)Cancer Discov. 8(6):67), local administration of immunotherapeutic agents to tumors induces a systemic immune response.

[0014] Intratumoral administration of MVA vaccines has been reported in recent years. Intratumoral injection of MVA expressing GM-CSF and immunization with DNA vaccines were found to prolong survival of mice bearing HPV16 E7 tumors (Nemeckova et al. (2007) Neoplasma 54:4). Other studies of intratumoral injection of MVA failed to show growth inhibition of pancreatic tumors (White et al. (2018) PLoS One 13(2):e0193131). Intratumoral injection of heat-inactivated MVA induced an antitumor immune response that was dependent on the production of the danger signal type I interferon and antigen cross-presentation by dendritic cells (Dai et al. (2017) Sci. Immunol. 2(11):eaal1713).

[0015] It is clear that there is a significant unmet medical need for additional cancer treatments, including active immunotherapies and cancer vaccines. In addition, there is a need for therapies that can induce an enhanced immune response in multiple areas of a patient's immune response. In many aspects, embodiments of the present disclosure address these needs by providing vaccines, therapies, and combination therapies that augment and improve upon currently available cancer treatments. Summary of the Invention

[0016] In various embodiments of the present invention, recombinant MVA encoding a tumor-associated antigen (TAA) and 4-1BB ligand (also referred to herein as 41BBL, 4-1BBL or CD137L) has been shown to enhance and / or augment the treatment of cancer patients when administered intratumorally or intravenously. More specifically, various embodiments of the present disclosure have demonstrated increased inflammation in tumors, decreased regulatory T cells (Tregs) in tumors, reduced T cell exhaustion in tumors, increased tumor-specific T cell and NK cell activation, increased tumor volume reduction, and / or increased survival in subjects with cancer, as compared to administration of the recombinant MVA alone.

[0017] In various embodiments of the present invention, recombinant MVA encoding a tumor-associated antigen (TAA) and CD40 ligand (CD40L) has been shown to enhance treatment of cancer patients when administered intratumorally or intravenously. More specifically, various embodiments of the present disclosure have shown increased inflammation in tumors, decreased regulatory T cells (Tregs) in tumors, reduced T cell exhaustion in tumors, increased tumor-specific T cell and NK cell activation, increased tumor volume reduction, and / or increased survival in subjects with cancer, when compared to administration of recombinant MVA alone.

[0018] In an additional embodiment, the invention includes a recombinant Modified Vaccinia Ankara (MVA) virus comprising a nucleic acid encoding 4-1BBL (CD137L) and a nucleic acid encoding CD40L, the MVA virus enhancing treatment of cancer patients when administered intravenously and / or intratumorally.

[0019] Thus, in one embodiment, the invention includes a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, the method comprising intratumorally administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding 4-1BBL, wherein intratumor administration of the recombinant MVA enhances the inflammatory response in the cancerous tumor of the subject, increases tumor shrinkage, and / or increases overall survival compared to non-intratumor injection of a recombinant MVA virus comprising a first and second nucleic acid encoding a TAA and the antigen 4-1BBL.

[0020] In additional embodiments, the invention includes a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, the method comprising intratumorally administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding CD40L, wherein intratumor administration of the recombinant MVA enhances the inflammatory response in the cancerous tumor of the subject, increases tumor shrinkage, and / or increases overall survival compared to non-intratumor injection of a recombinant MVA virus comprising a first and second nucleic acid encoding a TAA and the antigen CD40L.

[0021] In additional embodiments, the invention includes a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, comprising administering intratumorally and / or intravenously to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor associated antigen (TAA), a second nucleic acid encoding CD40L, and a third nucleic acid encoding 4-1BBL (CD137L), wherein administration of the recombinant MVA enhances the inflammatory response in the cancerous tumor, increases tumor reduction, and / or increases overall survival in the subject, as compared to injection of the recombinant MVA virus comprising the first and second nucleic acids encoding the TAA, the antigen CD40L, and the antigen 4-1BBL by a different injection route (i.e., non-intratumorally or non-intravenously).

[0022] In an additional embodiment, the invention includes a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, the method comprising intravenously administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding 4-1BBL, wherein intravenous administration of the recombinant MVA enhances natural killer (NK) cell responses and enhances CD8 T cell responses specific for the TAA compared to non-intravenous injection of a recombinant MVA virus comprising a first and second nucleic acid encoding the TAA and the antigen 4-1BBL.

[0023] In an additional embodiment, the invention includes a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, the method comprising intravenously administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding CD40L, wherein intravenous administration of the recombinant MVA enhances natural killer (NK) cell responses and enhances CD8 T cell responses specific for the TAA compared to non-intravenous injection of a recombinant MVA virus comprising a first and second nucleic acid encoding the TAA and the antigen CD40L.

[0024] In additional embodiments, the invention includes a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, comprising administering to the subject intravenously and / or intratumorally a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor-associated antigen (TAA), a second nucleic acid encoding CD40L, and a third nucleic acid encoding 4-1BBL, wherein intravenous and / or intratumor administration of the recombinant MVA results in an enhanced natural killer (NK) cell response and an enhanced CD8 T cell response specific for the TAA compared to non-intravenous or non-intratumor injection of a recombinant MVA virus comprising a first nucleic acid encoding a TAA, a second nucleic acid encoding the antigen CD40L, and a third nucleic acid encoding the antigen 4-1BBL.

[0025] In yet another embodiment, the invention includes a method of inducing an enhanced inflammatory response in a cancerous tumor in a subject, the method comprising intratumorally administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a first heterologous tumor-associated antigen (TAA) and a second nucleic acid encoding the antigen 4-1BBL, wherein intratumor administration of the recombinant MVA results in an enhanced inflammatory response in the tumor compared to an inflammatory response produced by non-intratumoral injection of a recombinant MVA virus comprising a first and second nucleic acid encoding a heterologous tumor-associated antigen and the antigen 4-1BBL.

[0026] In yet another embodiment, the invention includes a method of inducing an enhanced inflammatory response in a cancerous tumor in a subject, the method comprising intratumorally administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a first heterologous tumor-associated antigen (TAA) and a second nucleic acid encoding the antigen CD40L, wherein intratumor administration of the recombinant MVA results in an enhanced inflammatory response in the tumor compared to the inflammatory response produced by non-intratumoral injection of a recombinant MVA virus comprising a first and second nucleic acid encoding a heterologous tumor-associated antigen and the antigen CD40L.

[0027] In yet another embodiment, the invention includes a method of inducing an enhanced inflammatory response in a cancerous tumor in a subject, the method comprising intratumorally and / or intravenously administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a first heterologous tumor-associated antigen (TAA), a second nucleic acid encoding the antigen CD40L, and a third nucleic acid encoding the antigen 4-1BBL, wherein intratumorally and / or intravenously administration of the recombinant MVA results in an enhanced inflammatory response in the tumor compared to an inflammatory response produced by non-intratumor or non-intravenous injection of a recombinant MVA virus comprising a first nucleic acid encoding a heterologous tumor-associated antigen, a second nucleic acid encoding the antigen CD40L, and a third nucleic acid encoding the antigen 4-1BBL.

[0028] In various additional embodiments, the present invention provides a recombinant Modified Vaccinia Ankara (MVA) for treating a subject with cancer, the recombinant MVA comprising: a) a first nucleic acid encoding a tumor-associated antigen (TAA); and b) a second nucleic acid encoding 4-1BBL.

[0029] In various additional embodiments, the present invention includes a recombinant Modified Vaccinia Ankara (MVA) for treating a subject with cancer, the recombinant MVA comprising: a) a first nucleic acid encoding a tumor-associated antigen (TAA); and b) a second nucleic acid encoding CD40L.

[0030] In various additional embodiments, the present invention includes a recombinant Modified Vaccinia Ankara (MVA) for treating a subject with cancer, the recombinant MVA comprising: a) a first nucleic acid encoding a tumor associated antigen (TAA); b) a second nucleic acid encoding CD40L; and c) a third nucleic acid encoding 4-1BBL.

[0031] In yet another embodiment, a recombinant MVA encoding the antigen 4-1BBL, when administered intratumorally to a patient in combination with administration of a checkpoint inhibitor antagonist, enhances treatment of a cancer patient, more specifically, increases the reduction in tumor volume and / or increases the survival rate of the cancer patient.

[0032] In yet another embodiment, a recombinant MVA encoding the antigen CD40L, when administered intratumorally to a patient in combination with administration of a checkpoint inhibitor antagonist, enhances treatment of a cancer patient, more specifically, increases the reduction in tumor volume and / or increases the survival rate of the cancer patient.

[0033] In yet another embodiment, a recombinant MVA encoding the antigens CD40L and 4-1BBL, when administered intratumorally and / or intravenously to a patient in combination with administration of a checkpoint inhibitor antagonist, enhances treatment of a cancer patient, more specifically, increases the reduction in tumor volume and / or increases the survival rate of the cancer patient.

[0034] In another embodiment, the recombinant MVA of the invention is administered simultaneously with or after administration of the antibody, hi a more preferred embodiment, the recombinant MVA is administered after the antibody.

[0035] In another embodiment, the recombinant MVA of the invention is administered simultaneously with or after the administration of the antibody by the same route(s) of administration, hi another embodiment, the recombinant MVA is administered by one or more different routes of administration or after the administration of the antibody.

[0036] In yet another embodiment, the invention includes a method of enhancing antibody therapy in a cancer patient, the method comprising administering to the cancer patient a pharmaceutical combination of the invention, where administration of the pharmaceutical combination enhances antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the antibody therapy compared to administration of the antibody therapy alone.

[0037] In a preferred embodiment, the first nucleic acid encodes a TAA that is an endogenous retroviral (ERV) protein. In a more preferred embodiment, the ERV protein is from the human endogenous retroviral protein K (HERV-K) family. In a more preferred embodiment, the ERV protein is selected from HERV-K envelope and HERV-K gag proteins.

[0038] In a preferred embodiment, the first nucleic acid encodes a TAA that is an endogenous retrovirus (ERV) peptide. In a more preferred embodiment, the ERV peptide is from the human endogenous retrovirus protein K (HERV-K) family. In a more preferred embodiment, the ERV peptide is selected from the pseudogene of the HERV-K envelope protein (HERV-K-MEL).

[0039] In other preferred embodiments, the first nucleic acid encodes a TAA selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-related protein 1 (TRP1), tyrosine-related protein 1 (TRP2), Brachyury, melanoma preferentially expressed antigen (PRAME), folate receptor 1 (FOLR1), and combinations thereof.

[0040] In one or more preferred embodiments, the recombinant MVA is MVA-BN or a derivative thereof.

[0041] In various additional embodiments, the recombinant MVA and methods described herein are administered to a subject with cancer in combination with either an immune checkpoint molecule antagonist or an immune checkpoint molecule agonist. In further embodiments, the recombinant MVA and methods described herein are administered to a subject with cancer in combination with an antibody specific for a TAA to treat the subject with cancer. In more preferred embodiments, the recombinant MVA and methods described herein are administered in combination with an immune checkpoint molecule antagonist or agonist selected from CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and ICOS. In the most preferred embodiments, the immune checkpoint molecule antagonist or immune checkpoint molecule agonist comprises an antibody. In the most preferred embodiments, the immune checkpoint molecule antagonist or immune checkpoint molecule agonist comprises a PD-1 antibody or a PD-L1 antibody.

[0042] Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0043] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments of the invention and, together with the description, serve to explain the principles of the invention. [Brief description of the drawings]

[0044] [Figure 1]We show that 4-1BBL-mediated costimulation of CD8 T cells by tumor cells infected with MVA-OVA-4-1BBL affects cytokine production without the need for DCs. In contrast, MVA-OVA-CD40L enhances cytokine production only in the presence of DCs. Dendritic cells (DCs) were generated after 14 days of culture of bone marrow cells from C57BL / 6 mice in the presence of recombinant Flt3L as described in Example 2. B16.F10 cells were infected with MVA-OVA, MVA-OVA-CD40L or MVA-OVA-4-1BBL, and infected tumor cells were harvested and co-cultured in the presence of DCs, where indicated. Naive OVA(257-264)-specific CD8+ T cells were magnetically purified from OT-I mice and added to the co-cultures described above. Cells were cultured and their supernatants were collected for analysis of cytokine concentrations by Luminex. Supernatant concentrations of IL-6 (Figure 1A), GM-CSF (Figure 1B), IL-2 (Figure 1C) and IFN-γ (Figure 1D) are shown. Data are presented as mean ± SEM. [Diagram 2]We show that tumor cells infected with MVA-OVA-4-1BBL directly, i.e., without the need for DCs, stimulate the differentiation of antigen-specific CD8 T cells into activated effector T cells, whereas CD40L-mediated costimulation of tumor cells infected with MVA-OVA-CD40L is dependent on the presence of DCs. Dendritic cells (DCs) were generated after 14 days of culture of bone marrow cells from C57BL / 6 mice in the presence of recombinant Flt3L as described in Example 3. B16.F10 (melanoma model) cells were infected with MVA-OVA, MVA-OVA-CD40L or MVA-OVA-4-1BBL. The next day, infected tumor cells were harvested and co-cultured in the presence of DCs (where indicated). Naive OVA(257-264)-specific CD8+ T cells were magnetically purified from OT-I mice and added to the co-culture at a ratio of 1:5. Cells were cultured for 48 hours at 37°C and 5% CO2. Cells were subsequently stained and analyzed by flow cytometry. Figure 2A shows the GMFI of T-bet on OT-I CD8+ T cells (indicated as "CD8+" in the figure), and Figure 2B shows the percentage of OT-I CD8+ T cells that are CD44+ Granzyme B+ IFNγ+ TNFα+. Data are presented as mean ± SEM. [Diagram 3] These results show that infection with MVA encoding either CD40L or 4-1BBL induces tumor cell death in tumor cell lines and macrophages. As described in Example 4, tumor cell lines B16.OVA (Figures 3A and 3B), MC38 (Figure 3C), and B16.F10 (Figure 3D) were infected with vectors at the indicated MOI for 20 hours. Cell viability was analyzed by flow cytometry. Figures 3A, 3C, 3D, and 3E show the percentage of dead cells ("Live / Dead+"). Figure 3B: HMGB1 was quantified by ELISA in the supernatants of Figure 3A. Figure 3E: Bone marrow derived macrophages (BMDMs) were infected at the indicated MOI for 20 hours. Cell viability was analyzed by flow cytometry. Data are presented as mean ± SEM. [Figure 4]Figure 1 shows that rMVA-4-1BBL induces NK cell activation in vivo. C57BL / 6 mice (n=5 / group) were immunized intravenously with either saline or 5x107 TCID50 of "rMVA" (=MVA-OVA), "rMVA-4-1BBL" (=MVA-OVA-4-1BBL) or 5x107 TCID50 of rMVA in combination with 200 μg of anti-4-1BBL antibody (clone TKS-1) as described in Example 5. After 24 hours, mice were sacrificed and spleens were processed for flow cytometric analysis. Geometric mean fluorescence intensity (GMFI) of CD69 (A) and CD70 (B) are shown. Data are presented as mean ± SEM. [Diagram 5] This shows that intravenous immunization with rMVA-4-1BBL promotes serum IFN-γ secretion in vivo. As described in Example 6, C57BL / 6 mice (n=5 mice / group) were immunized intravenously with either saline or 5×107 TCID50 of "rMVA" (=MVA-OVA), "rMVA-4-1BBL" (=MVA-OVA-4-1BBL) or 5×107 TCID50 of rMVA in combination with 200 μg of anti-4-1BBL antibody (clone TKS-1). Figure 5A: After 6 hours, mice were bled, serum was isolated from whole blood, and IFN-γ concentration in serum was determined by Luminex. Figure 5B: After 3, 21, and 45 hours, mice were intravenously injected with Brefeldin A to stop protein secretion. At 6, 24 and 48 hours after immunization, mice were sacrificed and splenocytes were analyzed by flow cytometry. Data are presented as mean ± SEM. [Figure 6]This shows that intravenous immunization with "rMVA-4-1BBL" (=MVA-OVA-4-1BBL) promotes serum IFN-γ secretion in B16.OVA tumor-bearing mice. As described in Example 7, B16.OVA tumor-bearing C57BL / 6 mice (n=5 mice / group) were grouped and 7 days after tumor inoculation, PBS or 5×107 TCID50 of rMVA (=MVA-OVA) or rMVA-4-1BBL were administered iv. After 6 hours, the mice were bled, serum was isolated from whole blood, and IFN-γ concentration in serum was determined by Luminex. Data are shown as mean ± standard error. [Figure 7] Figure 1 shows that antigen-specific CD8+ T cells and vector-specific CD8+ T cells are increased after intravenous priming and boosting with "rMVA-4-1BBL" (=MVA-OVA-4-1BBL). As described in Example 8, C57BL / 6 mice (n=4 mice / group) were primed intravenously on day 0 and boosted on day 41 with either saline or 5x107 TCID50 of "rMVA" (=MVA-OVA), rMVA-4-1BBL, or 5x107 TCID50 of rMVA in combination with 200μg of anti-4-1BBL antibody (clone TKS-1). Mice were bled on days 6, 21, 35, 48, and 64 after priming, and peripheral blood was analyzed by flow cytometry. Figure 7A shows the percentage of antigen (OVA)-specific CD8+ T cells among peripheral blood leukocytes (PBL), and Figure 7B shows the percentage of vector (B8R)-specific CD8+ T cells among PBL. On day 70 after prime immunization, mice were sacrificed. Spleens were removed and subjected to flow cytometry analysis. Figure 7C shows the percentage of antigen (OVA)-specific CD8+ T cells among live cells, and Figure 7D shows the percentage of vector (B8R)-specific CD8+ T cells among live cells. Data are shown as mean ± standard error. [Figure 8]This shows that intravenous injection of MVA virus encoding 4-1BBL enhances antitumor activity compared to recombinant MVA not encoding 4-1BBL. As described in Example 9, B16.OVA tumor-bearing C57BL / 6 mice (n=5 / group) were divided into groups and intravenously administered PBS or 5×107 TCID50 of MVA-OVA ("rMVA" in the figure) or MVA-OVA-4-1BBL ("rMVA-4-1BBL" in the figure) on day 7 after tumor inoculation (black dotted line). Tumor growth was measured at regular intervals. [Figure 9] This shows that intratumoral injection of MVA viruses encoding 4-1BBL or CD40L enhanced the antitumor effect. As described in Example 10, B16.OVA tumor-bearing C57BL / 6 mice (n=4-5 mice / group) were divided into groups and intratumorally administered PBS or 5×107 TCID50 of MVA-OVA (shown as "rMVA" in the figure), MVA-OVA-CD40L (shown as "rMVA-CD40L" in the figure), or MVA-OVA-4-1BBL (shown as "rMVA-4-1BBL" in the figure) on days 7 (dotted black line), 12, and 15 (dashed grey line) after tumor inoculation. Tumor growth was measured at regular intervals. [Figure 10] This figure shows the antitumor effect of intratumoral injection of MVA virus encoding CD40L against established colon cancer. As described in Example 11, MC38 tumor-bearing C57BL / 6 mice (n=5 mice / group) were divided into groups and intratumorally (it) administered PBS or 5×107 TCID50 of MVA-TAA (shown as "rMVA" in the figure) or MVA-TAA-CD40L (shown as "rMVA-CD40L" in the figure) on days 14 (dotted black line), 19, and 22 (dashed black line) after tumor inoculation. Tumor growth was measured at regular intervals. In these experiments, the TAA encoded by the recombinant MVA included the antigens AH1A5, p15E, and TRP2. [Figure 11]We show that checkpoint blockade and tumor-targeted antibodies synergize with intratumoral (it) administration of rMVA-4-1BBL (also referred to herein as "MVA-OVA-4-1BBL"). B16.OVA tumor-bearing C57BL / 6 mice (n=5 / group) were grouped and, where indicated (check), administered 200 μg of IgG2a, anti-TRP-1 or anti-PD-1 antibodies intraperitoneally as described in Example 12. On days 13 (black dotted line), 18 and 21 (grey dashed line) after tumor inoculation, mice were immunized intratumorally (it) with either PBS or 5×107 TCID50 of MVA-OVA-4-1BBL. Tumor growth was measured at regular intervals. [Figure 12] This shows that intratumoral injection of MVA-OVA-4-1BBL results in superior antitumor activity compared to treatment with anti-CD137 antibodies. C57BL / 6 mice were injected sc with 5x105 B16.OVA cells as described in Example 13. Seven days later, when tumors measured greater than 5x5mm, mice were divided into groups and injected intratumorally with either PBS, 5x107 TCID50 MVA-OVA-4-1BBL, or 10μg anti-4-1BB(3H3) antibody. Tumor growth was measured at regular intervals. The mean tumor volume is shown in Figure 12A. Figure 12B: On day 12 after priming, peripheral blood lymphocytes were stained with OVA-dextramer and analyzed by FACS. The percentage of OVA-dextramer+ CD44+ T cells among CD8+ T cells is shown. [Figure 13]Figure 13 shows the antitumor effect of intravenous injection of MVA virus encoding the endogenous retroviral antigen Gp70. Balb / c mice were injected sc with 5x105 CT26.wt cells as described in Example 14. When tumors measured greater than 5x5mm, CT26.wt tumor-bearing mice (n=5 per group) were divided into groups and injected iv with PBS or 5x107 TCID50 MVA, rMVA-Gp70 or rMVA-Gp70-CD40L on day 12 after tumor inoculation. Tumor growth was measured at regular intervals. Shown are the mean tumor diameter (Figure 13A) and mean tumor volume (Figure 13B). Figure 13C: Blood cells were restimulated 7 days after immunization, and the percentage of CD8+CD44+IFN-γ+ cells in the blood after stimulation is shown. [Figure 14] Figure 14 shows the antitumor effect of intravenous injection of MVA virus encoding the endogenous retroviral antigens Gp70 and CD40L. C57BL / 6 mice were injected sc with 5x105 B16.F10 cells as described in Example 15. Seven days later, when tumors measured more than 5x5mm, B16.F10 tumor-bearing C57BL / 6 mice (n=5 per group) were divided into groups and injected iv with PBS or 5x107 TCID50 of MVA, rMVA-Gp70, or rMVA-Gp70-CD40L. Tumor growth was measured at regular intervals. Shown are the mean tumor volume (Figure 14A) and the percentage of CD8+CD44+IFN-γ+ cells in the blood after stimulation with p15e peptide 7 days after immunization (Figure 14B). [Figure 15]Cytokine / chemokine backbone response to IT immunization may be increased by the adjuvant effect of 4-1BBL. As used herein, "adjuvant effect" is intended to mean that a particular encoded protein or component of a recombinant MVA increases the immune response caused by other encoded protein(s) or component(s) of the recombinant MVA. In this figure, 5x105 B16.OVA cells were subcutaneously (sc) implanted into C57BL / 6 mice (see Example 23). On day 10, mice were immunized intratumorally (it) with PBS or 2x108 TCID50 of MVA-BN, MVA-OVA, or MVA-OVA-4-1BBL (n=6 mice / group). After 6 hours, tumors were excised and tumor lysates were processed. Cytokine / chemokine profiles were analyzed by Luminex. Figure 15 shows that cytokines / chemokines were upregulated in immunized mice. [Figure 16] MVA-OVA-4-1BBL enhances the cytokine / chemokine proinflammatory response to intratumoral (it) immunization. 5x105 B16.OVA cells were subcutaneously (sc) implanted into C57BL / 6 mice (see Examples 23 and 24). On day 10, mice were intratumoral (it) immunized with PBS or 2x108 TCID50 of MVA-BN, MVA-OVA, or MVA-OVA-4-1BBL (n=6 mice / group). 6 hours later, tumors were excised and tumor lysates were processed. Cytokine / chemokine profiles were analyzed by Luminex. Figure 16 shows upregulated cytokines / chemokines in mice immunized with MVA-OVA-4-1BBL compared to MVA-BN. [Figure 17]Quantitative and qualitative analysis of T cells in the tumor microenvironment (TME) and tumor-draining lymph nodes (TdLN) after intratumoral injection of MVA-OVA-4-1BBL. C57BL / 6 mice were subcutaneously (sc) injected with 5x105 B16.OVA cells. After 9-13 days, when tumors measured greater than 5x5mm, mice were divided into groups and injected intratumorally with either PBS, 2x108 TCID50 of MVA-OVA or MVA-OVA-4-1BBL (see Example 25). At 1, 3 and 7 days after immunization, mice were sacrificed and tumors and tumor-draining lymph nodes (TdLN) were digested with collagenase / DNase and analyzed by flow cytometry. The numbers of CD45+ cells, CD8+ T cells, CD4+ T cells and OVA-specific CD8+ T cells per mg of tumor and per TdLN are shown. [Figure 18] Quantitative and qualitative analysis of T cells in the TME and draining LNs after intratumoral injection of MVA-OVA-4-1BBL. C57BL / 6 mice were subcutaneously (sc) injected with 5x105 B16.OVA cells. 9-13 days later, when tumors measured >5.5x5.5mm, mice were divided into groups and injected intratumorally with either PBD or 2x108 TCID50 of MVA-OVA or MVA-OVA-4-1BBL (see Example 26). 1, 3, and 7 days after immunization, mice were sacrificed and tumors and TdLNs (tumor-draining lymph nodes) were digested with collagenase / DNase and analyzed by flow cytometry. Figure 18A: The percentage of Ki67+ cells among OVA-specific CD8+ T cells in tumors (left panel) and TdLNs (right panel) is shown. Figure 18B: GMFI of PD1 among OVA-specific CD8+ T cells in tumors 7 days after it immunization is shown. Figure 18C: Ratio of OVA-specific Teff / Treg in tumors 7 days after it immunization is shown. [Figure 19]Quantitative and qualitative analysis of NK cells in the TME and tumor-draining lymph nodes (TdLNs) after intratumoral injection of MVA-OVA-4-1BBL. C57BL / 6 mice were subcutaneously (sc) injected with 5x105 B16.OVA cells. After 9-13 days, when tumors measured greater than 5.5x5.5 mm, mice were divided into groups and injected intratumorally with either PBS or 2x108 TCID50 of MVA-OVA or MVA-OVA-4-1BBL (see Example 27). At 1, 3 and 7 days after immunization, mice were sacrificed and tumors and tumor-draining lymph nodes (TdLNs) were digested with collagenase / DNase and analyzed by flow cytometry. The number of NK cells per mg of tumor and per TdLN, as well as the GMFI of CD69, granzyme B and Ki67 surface markers of NK cells in tumors and TdLNs are shown. [Figure 20] CD8 T cell dependence of MVA-OVA-4-1BBL-mediated antitumor activity. C57BL / 6 mice were subcutaneously (sc) injected with 5x105 B16.OVA cells. Seven days later, mice were divided into groups and intratumorally injected with PBS or 2x108 TCID50 of MVA-OVA-4-1BBL (see Example 28). These intratumoral (it) injections were repeated 5 and 8 days after the first injection (dashed vertical lines). In addition, control antibodies of IgG2b isotype (left and middle panels) or anti-CD8 antibodies (2.43, right panel) were injected intraperitoneally (ip) (100 μg / mouse) 1 day before, 1 day, 4 days, 7 days, and 11 days after the first immunization. Tumor growth was measured at regular intervals, and the mean tumor diameter is shown. [Figure 21-1]Dependence of MVA-OVA and MVA-OVA-4-1BBL-mediated antitumor effects on Batf3+ DCs. C57BL / 6 or Batf3- / - mice were subcutaneously (sc) injected with 5x105 B16.OVA cells. Seven days later (vertical dashed lines), mice were divided into groups and intratumorally injected with PBS or 2x108 TCID50 of MVA, MVA-OVA or MVA-OVA-4-1BBL (see Example 29). This it injection was repeated 5 and 8 days after the first intratumoral injection (vertical dashed lines). Tumor growth was measured at regular intervals. Figure 21A: Mean tumor diameter is shown. Figure 21B: 11 days after the first immunization, blood was collected and analyzed for the presence of antigen-specific T cells (i.e., OVA257-264-specific T cells). The percentage of OVA-specific T cells among CD8+ T cells is shown. [Figure 21-2] Dependence of MVA-OVA and MVA-OVA-4-1BBL-mediated antitumor effects on Batf3+ DCs. C57BL / 6 or Batf3- / - mice were subcutaneously (sc) injected with 5x105 B16.OVA cells. Seven days later (vertical dashed lines), mice were divided into groups and intratumorally injected with PBS or 2x108 TCID50 of MVA, MVA-OVA or MVA-OVA-4-1BBL (see Example 29). This it injection was repeated 5 and 8 days after the first intratumoral injection (vertical dashed lines). Tumor growth was measured at regular intervals. Figure 21A: Mean tumor diameter is shown. Figure 21B: 11 days after the first immunization, blood was collected and analyzed for the presence of antigen-specific T cells (i.e., OVA257-264-specific T cells). The percentage of OVA-specific T cells among CD8+ T cells is shown. [Figure 22-1]Role of NK cells in intratumoral administration of MVA-OVA-4-1BBL in B16.OVA melanoma-bearing mice. C57BL / 6 or IL15Rα- / - mice were subcutaneously (sc) administered 5x105 B16.OVA cells. Seven days later, mice were divided into groups and intratumoral injected with PBS or 2x108 TCID50 of MVA-OVA or MVA-OVA-4-1BBL (see Example 30). Treatment was repeated 5 and 8 days after the first injection. Tumor growth was measured at regular intervals. The mean tumor diameter (Figure 22A) and survival rate (%) (Figure 22B) are shown. Eleven days after the first immunization, blood was collected and analyzed for the presence of antigen-specific T cells (Figure 22C). The percentage of OVA257-264 dextramer+ (SIINFEKL+) CD44+ T cells among CD8+ T cells is shown. [Figure 22-2] Role of NK cells in intratumoral administration of MVA-OVA-4-1BBL in B16.OVA melanoma-bearing mice. C57BL / 6 or IL15Rα- / - mice were subcutaneously (sc) administered 5x105 B16.OVA cells. Seven days later, mice were divided into groups and intratumoral injected with PBS or 2x108 TCID50 of MVA-OVA or MVA-OVA-4-1BBL (see Example 30). Treatment was repeated 5 and 8 days after the first injection. Tumor growth was measured at regular intervals. The mean tumor diameter (Figure 22A) and survival rate (%) (Figure 22B) are shown. Eleven days after the first immunization, blood was collected and analyzed for the presence of antigen-specific T cells (Figure 22C). The percentage of OVA257-264 dextramer+ (SIINFEKL+) CD44+ T cells among CD8+ T cells is shown. [Figure 23]Figure 23 shows NK cell-dependent cytokine / chemokine profiles in response to IT immunization with MVA-OVA-4-1BBL. 5x105 B16.OVA cells were subcutaneously (sc) implanted into C57BL / 6 and IL15Rα- / - mice (see Example 31). On day 7, mice were immunized intratumorally (it) with PBS or 2x108 TCID50 of MVA-OVA or MVA-OVA-4-1BBL (n=2-3 mice / group). After 6 hours, tumors were excised and tumor lysates were processed. Cytokine / chemokine profiles were analyzed by Luminex. Figure 23 shows cytokines / chemokines that are decreased in the absence of IL15Rα after intratumor (it) immunization with MVA-OVA-4-1BBL. [Figure 24-1] Figure 24 shows the antitumor effect of intratumoral immunization with MVA-gp70-CD40L compared to MVA-gp70-4-1BBL in B16.F10 melanoma-bearing mice. C57BL / 6 mice were subcutaneously (sc) injected with 5x105 B16.F10 cells. Seven days later, mice were divided into groups and intratumorally injected with PBS or 5x107 TCID50 of MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, MVA-4-1BBL or MVA-CD40L (see Example 32). Treatment was repeated 5 and 8 days after the first injection. Tumor growth was measured at regular intervals. Figure 24A shows the mean tumor diameter, and Figure 24B shows the appearance of white spots in mice treated with MVA-gp70-4-1BBL. Eleven days after the first immunization, blood was collected and analyzed for the presence of antigen-specific T cells. The percentage of IFNγ-producing CD44+ T cells among CD8+ T cells after restimulation with p15E is shown in FIG. 24C. [Figure 24-2]Figure 24 shows the antitumor effect of intratumoral immunization with MVA-gp70-CD40L compared to MVA-gp70-4-1BBL in B16.F10 melanoma-bearing mice. C57BL / 6 mice were subcutaneously (sc) injected with 5x105 B16.F10 cells. Seven days later, mice were divided into groups and intratumorally injected with PBS or 5x107 TCID50 of MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, MVA-4-1BBL or MVA-CD40L (see Example 32). Treatment was repeated 5 and 8 days after the first injection. Tumor growth was measured at regular intervals. Figure 24A shows the mean tumor diameter, and Figure 24B shows the appearance of white spots in mice treated with MVA-gp70-4-1BBL. Eleven days after the first immunization, blood was collected and analyzed for the presence of antigen-specific T cells. The percentage of IFNγ-producing CD44+ T cells among CD8+ T cells after restimulation with p15E is shown in FIG. 24C. [Figure 25-1] Antitumor effect of intratumoral administration of MVA-gp70-4-1BBL-CD40L in B16.F10 melanoma-bearing mice. C57BL / 6 mice were subcutaneously (sc) administered 5x105 B16.F10 cells. Seven days later, mice were divided into groups and intratumorally injected with PBS or 5x107 TCID50 of MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, MVA-gp70-4-1BBL-CD40L, MVA-4-1BBL, MVA-CD40L or MVA-4-1BBL-CD40L (see Example 33). Treatment was repeated 5 and 8 days after the first injection. Tumor growth was measured at regular intervals. The mean tumor diameter is shown in Figure 25A. Eleven days after the first immunization, blood was collected and restimulated with p15e peptide. The percentage of IFNγ+ CD44+ T cells among CD8+ T cells is shown in FIG. 25B. [Figure 25-2]Antitumor effect of intratumoral administration of MVA-gp70-4-1BBL-CD40L in B16.F10 melanoma-bearing mice. C57BL / 6 mice were subcutaneously (sc) administered 5x105 B16.F10 cells. Seven days later, mice were divided into groups and intratumorally injected with PBS or 5x107 TCID50 of MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, MVA-gp70-4-1BBL-CD40L, MVA-4-1BBL, MVA-CD40L or MVA-4-1BBL-CD40L (see Example 33). Treatment was repeated 5 and 8 days after the first injection. Tumor growth was measured at regular intervals. The mean tumor diameter is shown in Figure 25A. Eleven days after the first immunization, blood was collected and restimulated with p15e peptide. The percentage of IFNγ+ CD44+ T cells among CD8+ T cells is shown in FIG. 25B. [Figure 26-1] Antitumor effect of MVA-gp70 adjuvanted with CD40L or 4-1BBL in CT26 tumor-bearing mice. Balb / c mice were subcutaneously (sc) injected with 5x105 Ct26wt cells. Thirteen days later, mice were divided into groups and intratumorally injected with PBS or 5x107 TCID50 of MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, MVA-gp70-4-1BBL-CD40L, MVA-4-1BBL, MVA-CD40L and MVA-4-1BBL-CD40L (see Example 34). Treatment was repeated 5 and 8 days after the first injection. Tumor growth was measured at regular intervals. Figure 26A shows the mean tumor diameter, and Figure 26B shows the survival rate (%). (C) Eleven days after the first immunization, blood was collected and restimulated with AH1 peptide. The percentage of IFNγ+ CD44+ T cells among CD8+ T cells is shown. [Figure 26-2]Antitumor effect of MVA-gp70 adjuvanted with CD40L or 4-1BBL in CT26 tumor-bearing mice. Balb / c mice were subcutaneously (sc) injected with 5x105 Ct26wt cells. Thirteen days later, mice were divided into groups and intratumorally injected with PBS or 5x107 TCID50 of MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, MVA-gp70-4-1BBL-CD40L, MVA-4-1BBL, MVA-CD40L and MVA-4-1BBL-CD40L (see Example 34). Treatment was repeated 5 and 8 days after the first injection. Tumor growth was measured at regular intervals. Figure 26A shows the mean tumor diameter, and Figure 26B shows the survival rate (%). (C) Eleven days after the first immunization, blood was collected and restimulated with AH1 peptide. The percentage of IFNγ+ CD44+ T cells among CD8+ T cells is shown. [Figure 27] Quantitative and qualitative analysis of T cells in the tumor microenvironment (TME) and tumor-draining lymph nodes (TdLN) after intratumoral injection of MVA-gp70 further containing 4-1BBL and / or CD40L. C57BL / 6 mice were subcutaneously (sc) injected with 5x105 B16.F10 cells. Nine days later, when tumors measured greater than 5x5mm, mice were divided into groups and intratumorally injected with either PBS or 5x107 TCID50 of MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, or MVA-gp70-4-1BBL-CD40L (see Example 35). Three days after immunization, mice were sacrificed and tumors and tumor-draining lymph nodes (TdLN) were harvested, digested with collagenase / DNase, and analyzed by flow cytometry. Figure 27 shows the number of CD8+ T cells, p15E-specific CD8+ T cells and Ki67+ p15E-specific CD8+ T cells per mg of tumor and per TdLN. Data are presented as mean ± SEM. [Figure 28]Quantitative and qualitative analysis of T cells in the tumor microenvironment (TME) and tumor-draining lymph nodes (TdLN) after intratumoral injection of MVA-gp70 additionally expressing 4-1BBL and / or CD40L. C57BL / 6 mice were subcutaneously (sc) injected with 5x105 B16.F10 cells (see Example 36). Nine days later, when tumors measured greater than 5.5x5.5mm, mice were divided into groups and intratumorally injected with either PBS or 5x107 TCID50 of MVA-Gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, and MVA-gp70-4-1BBL-CD40L. Three days after immunization, mice were sacrificed, tumors and TdLNs were harvested, digested with collagenase / DNase, and individual cells were analyzed by flow cytometry. The numbers of NK cells, Ki67+ NK cells and Granzyme B+ NK cells per mg of tumor and per TdLN are shown. Data are presented as mean ± SEM. [Figure 29-1] Antitumor effect of intravenous administration of MVA-gp70 adjuvanted with 4-1BBL and / or CD40L in CT26.WT tumor-bearing mice. Balb / c mice were subcutaneously (sc) administered 5x105 CT26.WT cells. Twelve days later, mice were divided into groups and intravenously injected with PBS or 5x107 TCID50 of MVA-Gp70, MVA-Gp70-4-1BBL, MVA-Gp70-CD40L, MVA-Gp70-4-1BBL-CD40L and MVA-4-1BBL-CD40L (see Example 37). Figure 29A shows the mean tumor diameter and Figure 29B shows the survival rate (%). Seven days after the first immunization, blood was collected and restimulated with AH1 peptide. FIG. 29C shows the percentage of IFNγ+ CD44+ T cells among CD8+ T cells as mean ± standard error. [Figure 29-2]Antitumor effect of intravenous administration of MVA-gp70 adjuvanted with 4-1BBL and / or CD40L in CT26.WT tumor-bearing mice. Balb / c mice were subcutaneously (sc) administered 5x105 CT26.WT cells. Twelve days later, mice were divided into groups and intravenously injected with PBS or 5x107 TCID50 of MVA-Gp70, MVA-Gp70-4-1BBL, MVA-Gp70-CD40L, MVA-Gp70-4-1BBL-CD40L and MVA-4-1BBL-CD40L (see Example 37). Figure 29A shows the mean tumor diameter and Figure 29B shows the survival rate (%). Seven days after the first immunization, blood was collected and restimulated with AH1 peptide. FIG. 29C shows the percentage of IFNγ+ CD44+ T cells among CD8+ T cells as mean ± standard error. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[0046] As described and exemplified herein, the recombinant MVA and methods of the invention enhance multiple aspects of the immune response in cancer patients. In various embodiments, the invention shows that a recombinant MVA comprising a tumor-associated antigen (TAA) and the antigen 4-1BBL, when administered intratumorally or intravenously to a subject with cancer, enhances anti-tumor activity in the subject. As described in more detail herein, this enhanced anti-tumor activity includes increased tumor volume reduction, increased overall survival, enhanced CD8 T cell responses to the TAA, and enhanced inflammatory responses (e.g., increased NK cell activity, increased cytokine production, etc.).

[0047] As described and exemplified herein, the recombinant MVA and methods of the invention enhance multiple aspects of the immune response in cancer patients. In various embodiments, the invention shows that a recombinant MVA comprising a tumor-associated antigen (TAA) and the antigen CD40L, administered intratumorally or intravenously to a subject with cancer, enhances anti-tumor activity in the subject. As described in more detail herein, this enhanced anti-tumor activity includes increased tumor volume reduction, increased overall survival, enhanced CD8 T cell responses to the TAA, and enhanced inflammatory responses (e.g., increased NK cell activity, increased cytokine production, etc.).

[0048] In an additional aspect, various embodiments of the present invention have demonstrated that intratumoral administration of a recombinant MVA comprising a tumor-associated antigen (TAA) and the antigen 4-1BBL in combination with at least one immune checkpoint molecule antagonist / agonist increases tumor shrinkage and overall survival in subjects with cancer.

[0049] In a further aspect, various embodiments of the present invention have demonstrated that intratumoral administration of a recombinant MVA comprising a tumor-associated antigen (TAA) and the antigen 4-1BBL in combination with a tumor-specific antibody increases tumor shrinkage and overall survival in subjects with cancer.

[0050] Previously, the antigen 4-1BBL has been encoded in recombinant MVA viruses, but the immunogenic effects of MVA encoding 4-1BBL were unknown (see, e.g., Spencer et al. (2014) PLoS One 9(8):e105520). In Spencer's publication, co-expression of 4-1BBL with transgenic antigens in either MVA or adenoviral vectors increased murine CD8 T cell responses, but no increase in IFN-γ responses was observed in non-human primates after intramuscular administration of adenoviral vectors encoding 4-1BBL (ibid., p. 2, p. 6). Furthermore, the immunogenic effects of using MVA encoding 4-1BBL as part of treating cancer and destroying tumors and / or tumor cells were unknown.

[0051] Various embodiments of the present disclosure have demonstrated that an MVA encoding 4-1BBL and a TAA (referred to herein as MVA-TAA-4-1BBL) can be effective in treating cancer in a subject, such as a human. As shown and described herein, administration of MVA-TAA-4-1BBL can enhance multiple aspects of the immune response in a subject with cancer, effectively reducing and killing tumor cells. One or more of the enhanced anti-tumor effects of various embodiments of the present disclosure are outlined below.

[0052] Intravenous administration of recombinant MVA encoding 4-1BBL enhances anti-tumor effects. In at least one embodiment, the present invention includes a recombinant MVA encoding a TAA and antigen 4-1BBL (rMVA-TAA-4-1BBL) that is administered intravenously and that, when administered intravenously, enhances anti-tumor effects compared to intravenous administration of a recombinant MVA that does not encode 4-1BBL or compared to non-intravenous administration of the recombinant MVA encoding 4-1BBL (e.g., subcutaneous administration of the recombinant MVA encoding 4-1BBL). These enhanced anti-tumor effects include enhanced NK cell responses (as shown in FIG. 4), enhanced inflammatory responses as indicated by increased secretion of IFN-γ (as shown in FIGS. 5 and 6), increased recruitment of antigen- and vector-specific CD8 T cells (as shown in FIG. 7), and increased tumor shrinkage (as shown in FIG. 8).

[0053] Intratumoral administration of recombinant MVA encoding 4-1BBL enhances inflammation in the tumor. In another embodiment of the present invention, we confirmed that infection of tumor cells with MVA-OVA-4-1BBL but not with MVA-OVA-CD40L activates antigen-specific CD8+ T cells and produces T cell-derived cytokines (such as GM-CSF, IL-2, and IFN-γ) in the absence of DCs cross-presenting the antigen (Figures 1A-1D). This was unexpected in the case of GM-CSF, a growth factor produced by naive T cells upon activation that induces maturation of dendritic and myeloid cell subsets (Min et al. (2010) J. Immunol. 184:4625-4629). In the presence of DCs cross-presenting antigen, antigen-specific CD8+ T cells stimulated by tumor cells infected with rMVA-CD40L produced IFN-γ but not IL-2 or GM-CSF as in the case of rMVA-4-1BBL (Figures 1A-1D). Interestingly, large amounts of IL-6, an important cytokine produced by DCs, were detected (Figure 1A).

[0054] In one beneficial aspect, enhanced inflammation in tumors can induce large numbers of TILs (tumor infiltrating lymphocytes) to kill tumor cells at the tumor site (see, e.g., Lanitis et al. (2017) Annals Oncol. 28(suppl 12):xii18-xii32). These inflammatory tumors are also known as "hot" tumors, and in light of the increased numbers of TILs, cytokines, and other inflammatory molecules, tumor cell destructiveness can be enhanced.

[0055] Intratumoral administration of a recombinant MVA encoding 4-1BBL reduces tumor volume and increases overall survival. In one aspect, the present invention includes a recombinant MVA encoding the antigen 4-1BBL (MVA-4-1BBL), which is administered intratumorally and which has an enhanced anti-tumor effect in a subject with cancer compared to intratumoral administration of a recombinant MVA that does not encode 4-1BBL.

[0056] Recombinant MVA virus has been administered intratumorally before (see, e.g., White et al. (2018) PLoS One 13:e0193131 and Nemeckova et al. (2007) Neoplasma 54:326-33), but those studies have had mixed results. For example, Nemeckova's study found that intratumoral injection of vaccinia virus MVA expressing GM-CSF and immunization with a DNA vaccine prolonged survival of mice bearing HPV16-induced tumors (see Abstract in Nemeckova). Also, White et al. failed to show growth inhibition of pancreatic tumors after intratumoral injection of MVA (see Abstract in White).

[0057] As part of the present disclosure, a recombinant MVA comprising one or more nucleic acids encoding a TAA and 4-1BBL was administered intratumorally to subjects. As shown in Figure 9, intratumoral injection of MVA-TAA-4-1BBL demonstrated a significant reduction in tumor volume compared to recombinant MVA TAA.

[0058] Intratumoral administration of recombinant MVA encoding 4-1BBL in combination with an immune checkpoint molecule antagonist or agonist increases antitumor activity. In various embodiments, the invention includes administering MVA-TAA-4-1BBL in combination with an immune checkpoint antagonist or agonist. Preferably, administration of MVA-TAA-4-1BBL is intravenous or intratumoral. Combining the MVA of the invention with an immune checkpoint antagonist or agonist is beneficial because this combination provides a more effective cancer treatment. For example, the combination and / or combination therapy of the invention enhances multiple aspects of the immune response in cancer patients. In at least one aspect, the combination synergistically enhances both the innate and adaptive immune responses, and when combined with an immune checkpoint molecule antagonist or agonist, reduces tumor volume and increases survival in cancer patients.

[0059] The data obtained in this application show that combining MVA-TAA-4-1BBL with immune checkpoint antagonists or agonists enhances the antitumor effect. Moreover, as shown in Figure 11, intratumoral administration of MVA-OVA-4-1BBL combined with intraperitoneal administration of PD-1 antibody reduced tumor volume compared to PD-1 alone.

[0060] Intratumoral administration of recombinant MVA encoding 4-1BBL in combination with an antibody specific for a tumor-associated antigen (TAA) increases the antitumor effect. In various embodiments, the present invention includes administering MVA-TAA-4-1BBL in combination with an antibody specific for a TAA. Preferably, the administration of MVA-TAA-4-1BBL is intravenous or intratumoral. It is beneficial to combine the MVA of the present invention with a TAA-specific antibody, which can work together to provide a more effective cancer treatment.

[0061] In an exemplary embodiment, the enhanced NK cell response induced by administration of MVA-TAA-4-1BBL acts synergistically with a TAA-specific antibody to enhance antibody-dependent cellular cytotoxicity (ADCC) in a subject with cancer, resulting in increased tumor cell killing and tumor destruction.

[0062] The data obtained in this application demonstrate that the combination of MVA-TAA-4-1BBL with a TAA-specific antibody enhances the antitumor effect. Moreover, as shown in Figure 11, intratumoral administration of MVA-OVA-4-1BBL combined with intraperitoneal administration of TRP-1 antibody reduced tumor volume compared to TRP-1 antibody alone.

[0063] According to the present invention, administration of MVA-TAA-4-1BBL as part of prime and boost immunization increases the expansion of antigen- and vector-specific CD8+ T cells. In another aspect, the present invention provides a method of administering MVA-TAA-4-1BBL as part of an allogeneic and / or xenogeneic prime-boost regimen. Preferably, administration of MVA-TAA-4-1BBL is intravenous or intratumoral. As shown in FIG. 7, the expansion of antigen- and vector-specific CD8+ T cells was increased upon priming and boosting with intravenous administration of MVA-TAA-4-1BBL.

[0064] definition As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to a "nucleic acid" includes one or more of such nucleic acids, and reference to "the method" includes reference to equivalent steps and methods known to those of skill in the art that may be modified or substituted for the methods described herein.

[0065] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every single element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0066] Throughout this specification and the claims that follow, the word "comprise" and variations such as "comprises" and "comprising" are to be understood as implying the inclusion of the stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps, unless the context requires otherwise. As used herein, the term "comprising" may be replaced with the terms "containing" or "including" or, in some cases, as used herein, the term "having." Less preferably, any of the above terms (comprising, containing, including, having) may be replaced with the term "consisting of" when used herein in the context of any aspect or embodiment of the invention. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0067] As used herein, the conjunction "and / or" between elements shown is understood to include both individual options and combinations of options. For example, when two elements are connected by "and / or", the first option refers to the first element being applicable without the second element. The second option refers to the second element being applicable without the first element. The third option refers to the first and second elements being applicable together. Any one of these options should be understood to fall within the meaning, i.e., to satisfy the requirements of the term "and / or" as used herein. The simultaneous application of two or more of the options should also be understood to fall within the meaning, i.e., to satisfy the requirements of the term "and / or".

[0068] A "mutated" or "modified" protein or antigen as described herein is defined herein as having any modification, such as a deletion, addition, insertion and / or substitution, made to the nucleic acid or amino acid.

[0069] "Percent sequence homology (%) or percent sequence identity (%)" with respect to nucleic acid sequences described herein is defined as the percentage of nucleotides in a candidate sequence that are identical to those in a reference sequence (i.e., the nucleic acid sequence from which it is derived) when the candidate and reference sequences are aligned and, if necessary, gaps are introduced to maximize the percent sequence identity, without any conservative substitutions being considered as sequence identity. Alignment to determine percent sequence identity or percent sequence homology of nucleotides can be performed in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to maximize alignment over the full length of the sequences to be compared.

[0070] For example, suitable alignment of nucleic acid sequences can be obtained by the local homology algorithm of Smith and Waterman, (1981), Advances in Applied Mathematics 2:482-489. This algorithm can be applied to amino acid sequences by using a scoring matrix developed by Dayhoff (Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, DC, USA) and standardized by Gribskov ((1986), Nucl. Acids Res. 14(6):6745-6763). An exemplary implementation of this algorithm for determining percent identity of sequences is provided by the Genetics Computer Group (Madison, Wisconsin, USA) in a utility application called "BestFit". The default parameters for this method are described in the Wisconsin Sequence Analysis Package programs Manual, Version 8 (1995) (available from the Genetics Computer Group, Madison, Wisconsin, USA). In the context of the present invention, the preferred method for determining percent identity is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by Collins and Sturrok, and distributed by IntelliGenetics, Inc., Mountain View, California, USA. From this suite of packages, the Smith-Waterman algorithm can be used, using default parameters in the score table (e.g., gap open penalty: 12, gap extension penalty: 1, gap: 6). From the data generated, the "match" value reflects "sequence identity".Other suitable programs for calculating the 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, strand=both, cutoff=60, expect=10, Matrix=BLOSUM62, Descriptions=50 sequences, sort by=HIGH SCORE, Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found at the internet address blast.ncbi.nlm.nih.gov / .

[0071] The term "prime-boost vaccination" or "prime-boost regimen" refers to a vaccination strategy or regimen that uses a first priming injection of a vaccine targeting a given antigen followed by one or more boosting injections of the same vaccine at intervals. Prime-boost vaccination can be homologous or heterologous. Homologous prime-boost vaccination uses a vaccine containing the same antigen and the same vector for both the priming injection and the one or more boosting injections. Heterologous prime-boost vaccination uses a vaccine containing the same antigen for both the priming injection and the one or more boosting injections, but containing a different vector for the priming injection and the one or more boosting injections. For example, homologous prime-boost vaccination may use a recombinant poxvirus containing a nucleic acid expressing one or more antigens in the priming injection and the same recombinant poxvirus expressing one or more antigens in the one or more boosting injections. In contrast, heterologous prime-boost vaccination may use a priming injection of a recombinant poxvirus containing nucleic acid expressing one or more antigens, and one or more boosting injections of a different recombinant poxvirus expressing one or more antigens.

[0072] The term "recombinant" refers to a polynucleotide, virus, or vector of semi-synthetic or synthetic origin that is not found in nature or is linked to another polynucleotide in a configuration not found in nature. By "recombinant MVA" or "rMVA" as used herein is generally intended a modified vaccinia Ankara (MVA) that includes at least one polynucleotide encoding a tumor-associated antigen (TAA).

[0073] As used herein, reducing tumor volume or tumor volume reduction can be characterized as a reduction in tumor volume and / or size, but can also be characterized in terms of clinical trial endpoints as understood in the art. Some exemplary clinical trial endpoints associated with a reduction in tumor volume and / or size can include, but are not limited to, response rate (RR), objective response rate (ORR), etc.

[0074] As used herein, increased survival rate can be characterized as the increased survival rate of cancer patients, but can also be characterized in terms of clinical trial endpoints that are understood in the art.Some exemplary clinical trial endpoints associated with increased survival rate include, but are not limited to, overall survival rate (OS), progression-free survival rate (PFS), etc.

[0075] As used herein, a "transgene" or "heterologous" gene is understood to be a nucleic acid or amino acid sequence that is not present in the wild-type poxvirus genome (e.g., vaccinia, fowl pox or MVA). The skilled artisan will understand that a "transgene" or "heterologous gene", when present in a poxvirus such as vaccinia virus, has been introduced into the poxvirus genome in such a way that the gene is expressed as the corresponding heterologous gene product, i.e., as a "heterologous antigen" and / or "heterologous protein", after administration of the recombinant poxvirus to a host cell. Expression is usually achieved by operably linking the heterologous gene to regulatory elements that allow expression in the poxvirus-infected cell. Preferably, the regulatory elements include a natural or synthetic poxvirus promoter.

[0076] "Vector" refers to a recombinant DNA plasmid, recombinant RNA plasmid, or recombinant virus that can contain a heterologous polynucleotide. The heterologous polynucleotide can include a sequence of interest for prophylactic or therapeutic purposes, and can optionally be in the form of an expression cassette. As used herein, a vector need not be capable of replicating in the ultimate target cell or subject. The term includes cloning vectors and viral vectors.

[0077] Concomitant Uses and Methods In various embodiments, the invention includes a recombinant MVA comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding 4-1BBL, which upon intratumoral administration induces both an inflammatory response and an enhanced T cell response compared to the inflammatory response and the T cell response induced by non-intratumoral administration of a recombinant MVA virus comprising a first nucleic acid encoding a TAA and a second nucleic acid encoding 4-1BBL.

[0078] In various additional embodiments, the invention comprises a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding 4-1BBL, which upon intratumoral administration induces both an enhanced intratumoral inflammatory response and an enhanced T cell response compared to the intratumoral inflammatory response and T cell response induced by intratumoral administration of a recombinant MVA virus comprising the first nucleic acid encoding the TAA.

[0079] Enhanced Inflammatory Response in Tumors In various embodiments of the present disclosure, it has been determined that intratumoral administration of a recombinant MVA encoding a TAA and 4-1BBL induces an enhanced inflammatory response in tumors compared to administration of the recombinant MVA alone. In at least one embodiment, an "enhanced inflammatory response" in tumors according to the present disclosure is characterized by one or more of 1) increased expression of IFN-γ and / or 2) increased expression of Granzyme B (GraB) in tumors and / or tumor cells. Thus, whether an inflammatory response is enhanced in tumors and / or tumor cells according to the present disclosure can be determined by measuring to determine whether the expression of one or more molecules indicative of an increased inflammatory response is increased, including secretion of chemokines and cytokines, as known in the art. Exemplary inflammatory response markers include one or more of markers useful for measuring the frequency and / or activity of NK cells, including one or more of IFN-γ and / or Granzyme B (GraB). These molecules and their measurements are proven assays understood in the art and can be performed according to known techniques. See, for example, Borrego et al. ((1999) Immunology 7(1):159-165).

[0080] Enhanced NK Cell Responses In various additional embodiments of the present disclosure, it has been determined that intratumoral or intravenous administration of a recombinant MVA encoding a TAA and 4-1BBL induces an enhanced NK cell response in a tumor or tumor environment, as compared to administration of the recombinant MVA alone. In one embodiment, an "enhanced NK cell response" according to the present disclosure is characterized by one or more of: 1) increased NK cell frequency, 2) increased NK cell activation, and / or 3) increased NK cell proliferation. Thus, whether an NK cell response is enhanced according to the present disclosure can be determined by measuring the expression of one or more molecules indicative of increased NK cell frequency, increased NK cell activation, and / or increased NK cell proliferation. Exemplary markers useful for measuring NK cell frequency and / or activity include one or more of NKp46, IFN-γ, CD69, CD70, NKG2D, FasL, granzyme B, CD56, and / or Bcl-XL. Exemplary markers useful for measuring NK cell activation include one or more of IFN-γ, CD69, CD70, NKG2D, FasL, granzyme B, and / or Bcl-XL. Exemplary markers useful for measuring NK cell proliferation include Ki67. These molecules and their measurement are well-established assays understood in the art and can be performed according to known techniques (see, for example, Borrego et al. (1999) Immunology 7(1):159-165). Additionally, assays for measuring these molecules can be found in Examples 5 and 6 of the present disclosure. In at least one aspect, 1) an increase in NK cell frequency can be defined as at least a 2-fold increase in CD3-NKp46+ cells compared to pre-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-XL 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 expression of Ki67 compared to pre-treatment / baseline expression.

[0081] Enhanced T Cell Responses According to the present application, an "enhanced T cell response" is characterized by one or more of 1) an increased frequency of CD8 T cells, 2) increased activation of CD8 T cells, and / or 3) increased proliferation of CD8 T cells. Thus, according to the present application, whether a T cell response is enhanced can be determined by measuring the expression of one or more molecules indicative of 1) an increased frequency of CD8 T cells, 2) increased activation of CD8 T cells, and / or 3) increased proliferation of CD8 T cells. Exemplary markers that are useful for measuring 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. The frequency of antigen-specific T cells can be measured by MHC multimers (such as pentamers, or dextramers as demonstrated by the present application). Such measurements and assays, as well as other measurements and assays suitable for use in evaluating the methods and compositions of the present invention, are well established and understood in the art.

[0082] In one embodiment, increased CD8 T cell frequency is characterized by at least a two-fold increase in IFN-γ and / or dextramer+ CD8 T cells compared to pre-treatment / baseline. Increased CD8 T cell activation is characterized by at least a two-fold increase in CD69 and / or CD44 expression compared to pre-treatment / baseline expression. Increased CD8 T cell proliferation is characterized by at least a two-fold increase in Ki67 expression compared to pre-treatment / baseline expression.

[0083] In alternative embodiments, the enhanced T cell response is characterized by increased expression of effector cytokines and / or increased cytotoxic effector function. Increased effector cytokine expression can be measured by expression of one or more of IFN-γ, TNF-α, and / or IL-2 compared to pre-treatment / baseline. Increased cytotoxic effector function can be measured by expression of one or more of CD107a, granzyme B, and / or perforin, and / or antigen-specific killing of target cells.

[0084] The assays, cytokines, markers and molecules described herein, and their measurement, are established and understood in the art and can be performed according to known techniques. In addition, assays for measuring T cell responses can be found in the Examples analyzing T cell responses, including, but not limited to, Examples 2, 3, 8, 13 and 14.

[0085] The present invention recognizes that enhanced T cell responses are particularly beneficial when combined with enhanced NK cell responses and enhanced inflammatory responses, as enhanced T cells effectively target and kill tumor cells that have evaded and / or survived the initial innate immune response in cancer patients.

[0086] In yet further embodiments, the combinations and methods described herein are for use in treating human cancer patients. In preferred embodiments, the cancer patient is suffering from and / or diagnosed with a cancer selected from the group consisting of breast cancer, lung cancer, head and neck cancer, thyroid cancer, melanoma, gastric cancer, bladder cancer, kidney cancer, liver cancer, melanoma, pancreatic cancer, prostate cancer, ovarian cancer, urothelial cancer, cervical cancer, or colorectal cancer. In yet further embodiments, the combinations and methods described herein are for use in treating human cancer patients suffering from and / or diagnosed with breast cancer, colorectal cancer, or melanoma, preferably melanoma, more preferably colorectal cancer, and most preferably colorectal cancer.

[0087] Certain Exemplary Tumor-Associated Antigens In certain embodiments, the immune response is raised in the subject against a cell-associated polypeptide antigen, hi certain such embodiments, the cell-associated polypeptide antigen is a tumor-associated antigen (TAA).

[0088] The term "polypeptide" refers to a polymer of two or more amino acids linked together by peptide bonds or modified peptide bonds. The amino acids may be natural amino acids, unnatural amino acids, or chemical analogues of natural amino acids. The term also refers to proteins, i.e., functional biomolecules comprising at least one polypeptide, and when comprising at least two polypeptides, the polypeptides may be complexed, covalently linked, or non-covalently linked. The polypeptide(s) in a protein may be glycosylated and / or lipidated, and / or may contain prosthetic groups.

[0089] Endogenous retroviral proteins (ERVs) Preferably, the TAA of the present invention is comprised in an endogenous retroviral protein (ERV). More preferably, the ERV is an ERV of the human HERV-K protein family. Most preferably, the HERV-K protein is selected from the HERV-K envelope (env) protein, the HERV-K group specific antigen (gag) protein, and the HERV-K "melanoma risk marker" (mel) protein (see, e.g., Cegolon et al. (2013) BMC Cancer 13:4).

[0090] ERVs make up 8% of the human genome and originate from germline infections hundreds of years ago. Most of these elements inserted into the human genome are so mutated that they are not transcribed or translated. However, a small proportion of ERVs that were acquired more recently are still functional, translated, and in some cases even produce viral particles. ERV transcription is very limited because their loci are usually highly methylated and, as a result, not translated in somatic cells (Kassiotis (2016) Nat. Rev. Immunol. 16: 207-19). Only under some circumstances, such as cellular stress (chemicals, UV radiation, hormones, cytokines), can ERVs be reactivated. Importantly, ERVs are expressed in a wide variety of cancers but not in normal tissues (Cegolon et al. (2013) BMC Cancer 13:4, Wang-Johanning et al. (2003) Oncogene 22:1528-35). This very restricted expression pattern ensures that ERVs are not or only rarely exposed to immune tolerance mechanisms, likely resulting in a competent ERV-specific T cell repertoire. Thus, ERVs can be used as tumor antigens ("TAA") in MVA.

[0091] In various additional embodiments, the TAA includes, but is not limited to, HER2, PSA, PAP, CEA, MUC-1, FOLR1, PRAME, Survivin, TRP1, TRP2, or Brachyury, alone or in combination. Such exemplary combinations may include, for example, CEA and MUC-1 (also known as CV301) in MVA. Other exemplary combinations may include PAP and PSA.

[0092] In further embodiments, the additional TAAs include 5α-reductase, α-fetoprotein, AM-1, APC, April, BAGE, β-catenin, Bcl12, bcr-abl, 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, farnesyltransferase, FGF8b, FGF8a , FLK-1 / KDR, folate receptor, G250, GAGE ​​family, gastrin-17, gastrin-releasing hormone, GD2 / GD3 / GM2, GnRH, GnTV, GP1, gp100 / Pmel17, gp-100-in4, gp15, gp75 / TRP1, 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 These may include, but are not limited to, A / B, MT-MMP, mucin, NY-ESO-1, osteonectin, p15, p170 / MDR1, p53, p97 / melanotransferrin, PAI-1, PDGF, uPA, PRAME, probasin, progenypoietin, PSA, PSM, RAGE-1, Rb, RCAS1, SART-1, SSX family, STAT3, STn, TAG-72, TGF-α, TGF-β, thymosin-β-15, TNF-α, TRP1, TRP2, tyrosinase, VEGF, ZAG, p16INK4 and glutathione-S-transferase.

[0093] A preferred PSA antigen contains an amino acid change at position 155 from isoleucine to leucine (see US Pat. No. 7,247,615, incorporated herein by reference).

[0094] In one or more preferred embodiments of the present invention, the heterologous TAA is selected from HER2 and / or Brachyury.

[0095] Any TAA may be used, provided that at least one objective or desired result of the present invention (such as stimulating an immune response) is achieved after administration of an MVA containing that TAA. Exemplary sequences of TAAs, including those mentioned herein, are known in the art and are suitable for use in the compositions and methods of the present invention. The sequences of the TAA used in the compositions and methods of the present invention may be identical to sequences known in the art or disclosed herein, or may have less than 100% sequence identity (such as at least 90%, 91%, 92%, 95%, 97%, 98%, 99% or more) with any of the nucleotide or amino acid sequences known in the art or disclosed herein. That is, the sequence of a TAA used in a composition or method of the invention may differ by fewer than 20 nucleotides or amino acids, or fewer than 19, fewer than 18, fewer than 15, fewer than 14, fewer than 13, fewer than 12, fewer than 11, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2, or fewer than 1 nucleotide or amino acid from a reference sequence known in the art and / or disclosed herein, provided that at least one object or desired result of the invention is achieved. Those of skill in the art are familiar with techniques and assays to evaluate TAAs to ensure their suitability for use in an MVA or method of the invention.

[0096] Modified Tumor-Associated Antigens In certain embodiments, the cell-associated polypeptide antigen is modified so that when the epitope derived from the polypeptide antigen is presented in association with class I MHC molecule on the surface of APC, a CTL response is induced against the cell presenting the epitope on its surface.In certain such embodiments, at least one first foreign TH epitope is presented in association with class II MHC molecule on the surface of APC.In certain such embodiments, the cell-associated antigen is a tumor-associated antigen.

[0097] Exemplary APCs capable of presenting epitopes include dendritic cells and macrophages. Additional exemplary APCs include any pinocytic or phagocytic APC that can simultaneously present 1) a CTL epitope bound to a class I MHC molecule, and 2) a TH epitope bound to a class II MHC molecule.

[0098] In certain embodiments, modifications to one or more of the TAAs, including but not limited to HERV-K env, HERV-K gag, HERV-K mel, CEA, MUC-1, PAP, PSA, PRAME, FOLR1, HER2, survivin, TRP1, TRP2, or Brachyury, are made such that polyclonal antibodies that react primarily with one or more of the TAAs described herein are elicited following administration to a subject. Such antibodies can attack and eliminate tumor cells and prevent metastatic cells from developing and leading to metastasis. The effector mechanism of this anti-tumor effect will be mediated by complement-dependent and antibody-dependent cellular cytotoxicity. In addition, the induced antibodies can also inhibit the growth of cancer cells through inhibition of growth factor-dependent oligodimerization and receptor internalization. In certain embodiments, such modified TAAs can induce CTL responses against known and / or putative TAA epitopes presented by tumor cells.

[0099] In certain embodiments, the modified TAA polypeptide antigen comprises a CTL epitope of the cell-associated polypeptide antigen and a variation, the variation comprising at least one CTL epitope or a foreign TH epitope. Certain such modified TAAs can, in one non-limiting example, comprise one or more polypeptide antigens HER2 comprising at least one CTL epitope and a variation comprising at least one CTL epitope of a foreign TH epitope, the method of making this TAA being described in U.S. Patent No. 7,005,498, and U.S. Patent Application Publication Nos. 2004 / 0141958 and 2006 / 0008465.

[0100] Certain such modified TAAs can include, in one non-limiting example, one or more polypeptide antigens MUC-1 that include at least one CTL epitope and variations that include at least one CTL epitope of a foreign epitope, methods for making such TAAs are described in U.S. Patent Application Publication No. 2014 / 0363495.

[0101] Additional promiscuous T cell epitopes include peptides capable of binding to most HLA-DR molecules encoded by various HLA-DRs. See, e.g., WO98 / 23635 (Frazer IH et al., assigned to The University of Queensland), Southwood et al. (1998) J. Immunol. 160:3363 3373, Sinigaglia et al. (1988) Nature 336:778 780, Rammensee et al. (1995) Immunogenetics 41:178 228, Chicz et al. (1993) J. Exp. Med. 178:27 47, Hammer et al. (1993) Cell 74:197 203, and Falk et al. (1994) Immunogenetics 39:230 242. This last reference also discusses HLA-DQ and HLA-DP ligands, all of the epitopes listed in these references are relevant as potential natural epitopes as described herein, since they are epitopes that share common motifs with these potential epitopes.

[0102] In certain other embodiments, the promiscuous T cell epitope is an artificial T cell epitope capable of binding to a large proportion of haplotypes. In certain such embodiments, the artificial T cell epitope is a pan DR epitope peptide ("PADRE") as described in WO95 / 07707 and the corresponding article Alexander et al. (1994) Immunity 1:751 761.

[0103] 4-1BBL (also referred to herein as "41BBL" or "4-1BB ligand") As exemplified by the present disclosure, the inclusion of 4-1BBL as part of a recombinant MVA and related methods induces an increased and enhanced anti-tumor effect upon intratumoral or intravenous administration in subjects with cancer. Thus, in various embodiments, in addition to encoding a TAA, there is a recombinant MVA that encodes the antigen 4-1BBL.

[0104] 4-1BB / 4-1BBL is a member of the TNFR / TNF superfamily. 4-1BBL is a costimulatory ligand expressed on activated B cells, monocytes and DCs. 4-1BB is constitutively expressed by several innate immune cell populations, including natural killer (NK) and natural killer T (NKT) cells, Tregs, as well as DCs, monocytes and neutrophils. Interestingly, 4-1BB is expressed on activated but not resting T cells (Wang et al. (2009) Immunol. Rev. 229:192-215). Binding of 4-1BB induces proliferation and production of interferon gamma (IFN-γ) and interleukin 2 (IL-2) and enhances T cell survival through upregulation of anti-apoptotic molecules such as Bcl-xL (Snell et al. (2011) Immunol. Rev. 244:197-217). Importantly, stimulation of 4-1BB enhances NK cell proliferation, IFN-γ production, and cytolytic activity through enhanced antibody-dependent cellular cytotoxicity (ADCC) (Kohrt et al. (2011) Blood 117:2423-32).

[0105] In one or more preferred embodiments, 4-1BBL is encoded by the MVA of the present invention. In one or more other preferred embodiments, 4-1BBL is human 4-1BBL. In a further preferred embodiment, the 4-1BBL comprises a nucleic acid encoding an amino acid sequence having a sequence that is at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:3, i.e., a sequence that differs from the amino acid sequence shown in SEQ ID NO:3 by less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2 or less than 1 amino acid. In a further preferred embodiment, the 4-1BBL comprises a nucleic acid encoding an amino acid sequence comprising SEQ ID NO:3. In additional embodiments, the nucleic acid encoding 4-1BBL comprises a nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:4, i.e., the sequence has fewer than 20, fewer than 10, fewer than 5, fewer than 4, fewer than 3, fewer than 2 or fewer than 1 nucleic acid that differs from the nucleic acid sequence set forth in SEQ ID NO:4. In a more preferred embodiment, the 4-1BBL comprises a nucleic acid comprising SEQ ID NO:4.

[0106] CD40L As demonstrated by the present disclosure, including CD40L as part of the combinations and associated methods further increases tumor volume reduction, progression free survival, and increases survival rates achieved by the present invention. Thus, in various embodiments, the combination treatments of the present invention further include administering CD40L to the cancer patient. In a preferred embodiment, the CD40L is encoded as part of a recombinant MVA as described herein.

[0107] CD40 is constitutively expressed on many cell types, including B cells, macrophages, and dendritic cells, whereas its ligand CD40L is expressed primarily on activated helper T cells. Cognate interactions between dendritic cells and helper T cells early after infection or immunization "license" dendritic cells to prime CTL responses. Licensing of dendritic cells results in upregulation of costimulatory molecules, increased survival, and improved cross-presentation capacity. This process is primarily mediated by CD40 / CD40L interactions. However, various configurations of CD40L have been described, ranging from membrane-bound to soluble (monomeric or trimeric) configurations that induce diverse stimuli and induce or inhibit APC activation, proliferation, and differentiation.

[0108] In one or more preferred embodiments, the CD40L is encoded by the MVA of the present invention. In one or more other preferred embodiments, the CD40L is human CD40L. In further preferred embodiments, the CD40L comprises a nucleic acid encoding an amino acid having a sequence that is at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1, i.e., a sequence that differs from the amino acid sequence shown in SEQ ID NO: 1 by fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2 or fewer than 1 amino acid. In further preferred embodiments, the CD40L comprises a nucleic acid encoding an amino acid comprising SEQ ID NO: 1. In additional embodiments, the nucleic acid encoding CD40L comprises a nucleic acid sequence that is at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:2, i.e., the sequence has fewer than 20, fewer than 10, fewer than 5, fewer than 4, fewer than 3, fewer than 2 or fewer than 1 nucleic acid that differs from the nucleic acid sequence set forth in SEQ ID NO:2. In a more preferred embodiment, the CD40L comprises a nucleic acid comprising SEQ ID NO:2.

[0109] Immune checkpoint molecular antagonists As described herein, in at least one aspect, the invention includes the use of immune checkpoint antagonists. Such immune checkpoint antagonists function to interfere with and / or block the function of immune checkpoint molecules. Some preferred immune checkpoint antagonists include cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), lymphocyte activation gene 3 (LAG-3), and T cell immunoglobulin and mucin domain 3 (TIM-3).

[0110] Additionally, exemplary immune checkpoint antagonists can include, but are not limited to, CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, T cell immunoreceptor with an Ig domain and an ITIM domain (TIGIT), and V domain Ig suppressor of T cell activation (VISTA).

[0111] Such immune checkpoint molecule antagonists can include antibodies that specifically bind to an immune checkpoint molecule and inhibit and / or block the biological activity and function of the immune checkpoint molecule.

[0112] Other immune checkpoint molecule antagonists can include antisense nucleic acid RNA that interferes with the expression of immune checkpoint molecules, and small interfering RNA that interferes with the expression of immune checkpoint molecules.

[0113] In addition, the antagonist can be in the form of a small molecule that inhibits or blocks the function of immune checkpoints. Some non-limiting examples of these small molecules include NP12 (Aurigene), (D)PPA-1 by Tsinghua Univ, high affinity PD-1 (Stanford), BMS-202 and BMS-8 (Bristol Myers Squibb (BMS)), CA170 / CA327 (Curis / Aurigene), and small molecule inhibitors of CTLA-4, PD-1, PD-L1, LAG-3, and TIM-3.

[0114] In addition, the antagonist can be in the form of Anticalin®, which inhibits or blocks the function of immune checkpoint molecules. See, e.g., Rothe et al. ((2018) BioDrugs 32(3):233-243).

[0115] In addition, it is contemplated that the antagonist may be in the form of an Affimer®. Affimers are Fc fusion proteins that inhibit or block the function of immune checkpoint molecules. Other fusion proteins that can function as immune checkpoint antagonists are immune checkpoint fusion proteins (e.g., anti-PD-1 protein AMP-224) and anti-PD-L1 proteins, such as those described in US2017 / 0189476.

[0116] Candidate immune checkpoint molecule antagonists can be screened for function (such as the ability to interfere with the function of an immune checkpoint molecule) in vitro or in mouse models by a variety of techniques known in the art and / or disclosed herein.

[0117] ICOS Agonists The present invention further includes ICOS agonists. ICOS agonists activate ICOS. ICOS is a positive costimulatory molecule expressed on activated T cells, and upon binding to its ligand, promotes the proliferation of activated T cells (Dong (2001) Nature 409:97-101).

[0118] In one embodiment, the agonist is ICOS-L, the natural ligand of ICOS. The agonist can be a mutant form of ICOS-L that retains its binding and activation properties. The mutant form of ICOS-L can be screened for ICOS stimulating activity in vitro.

[0119] Immune checkpoint antagonist or immune checkpoint agonist antibodies. In a preferred embodiment, the immune checkpoint molecule antagonist and / or immune checkpoint molecule agonist each comprise an antibody. As described herein, in various embodiments, the antibody can be a synthetic monoclonal or polyclonal antibody and can be produced by techniques well known in the art. Such an antibody specifically binds (as opposed to non-specifically) to the immune checkpoint molecule via the antigen-binding site of the antibody. In producing an antibody that is immunoreactive with an immunogen, a peptide, fragment, variant, fusion protein, etc. of the immune checkpoint can be used as the immunogen. More specifically, the polypeptide, fragment, variant, fusion protein, etc. comprises an antigenic determinant, i.e., an epitope, that induces the formation of an antibody.

[0120] In a more preferred embodiment, the antibodies of the invention are antibodies that have been approved, or are in the process of being approved, by a sovereign government for the treatment of human cancer patients. Some non-limiting examples of these antibodies that are already approved or are in the approval process include CTLA-4 (ipilimumab® and tremelimumab), PD-1 (pembrolizumab, lambrolizumab, Amplimune-224 (AMP-224)), Amplimune-514 (AMP-514), nivolumab, MK-3475 (Merck), BI754091 (Boehringer Ingelheim)), PD-L1 (atezolizumab, avelumab, durvalumab, MPDL3280A (Roche), MED14736 (AZN), MSB0010718C (Merck)), LAG-3 (IMP321, BMS-986016, BI754111 (Boehringer Ingelheim), LAG525 (Novartis), MK-4289 (Merck), and TSR-033 (Tesaro).

[0121] In an exemplary embodiment, the immune checkpoint molecules CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and ICOS, and peptides based on the amino acid sequences of CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and ICOS, can be used to prepare antibodies that specifically bind to CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, or ICOS. The term "antibody" is intended to include polyclonal antibodies, monoclonal antibodies, fragments thereof (such as F(ab')2 and Fab fragments, single chain variable fragments (scFv), single domain antibody fragments (VHH or nanobodies), bivalent antibody fragments (diabodies), and the like), as well as any recombinantly and synthetically produced binding partners.

[0122] Antibodies specific for tumor-associated antigens (TAA) In various embodiments of the present invention, the recombinant MVA and methods described herein are combined with or administered in combination with an antibody specific for a TAA. In more specific embodiments, the recombinant MVA and methods described herein are combined with or administered in combination with an antibody specific for an antigen expressed on the cell membrane of a tumor cell. It is understood in the art that in many cancers, one or more antigens are expressed or overexpressed on the tumor cell membrane. See, e.g., Durig et al. (2002) Leukemia 16:30-5; Mocellin et al. (2013) Biochim. Biophys. Acta 1836:187-96; Arteaga (2011) Nat.Rev.Clin.Oncol., doi:10.1038 / nrclinonc.2011.177; Finn (2017) Cancer Immunol. Res.5:347-54; Ginaldi et al. (1998) J.Clin.Pathol.51:364-9. Assays to determine whether an antigen is expressed or overexpressed on tumor cells are readily understood in the art, as are methods for generating antibodies against specific antigens (see above).

[0123] In a more specific embodiment, the pharmaceutical combinations and related methods of the present invention comprise an antibody that a) is specific for an antigen expressed on the cell membrane of a tumor and b) comprises an Fc domain. In at least one aspect, the characteristics of the antibody (e.g., a) and b)) allow the antibody to bind to and interact with effector cells, such as NK cells, macrophages, basophils, neutrophils, eosinophils, monocytes, mast cells and / or dendritic cells, and allow the antibody to bind to a tumor antigen expressed on tumor cells. In a preferred embodiment, the antibody comprises an Fc domain. In an additional preferred embodiment, the antibody is capable of binding to and interacting with NK cells.

[0124] Some exemplary antibodies against antigens expressed on tumor cells that are contemplated by the present disclosure include anti-CD20 (e.g., rituximab, ofatumumab, tositumomab), anti-CD52 (e.g., alemtuzumab Campath®), anti-EGFR (e.g., cetuximab Erbitux®, panitumumab), anti-CD2 (e.g., siplizumab), anti-CD37 (e.g., BI836826), anti-CD123 (e.g., JNJ-56022473), anti-CD30 (e.g., XmAb2513), anti-CD38 (e.g., daratumumab Darzalex®), anti-PDL1 (e.g., avelumab ... Antibodies that may be used include, but are not limited to, anti-HER2 (e.g., trastuzumab, hajuma, ABP980, and / or pertuzumab), anti-CEA, anti-MUC1, anti-FLT3, anti-CD19, anti-CD40, anti-SLAMF7, anti-CCR4, anti-B7-H3, anti-ICAM1, anti-CSF1R, anti-CA125 (e.g., oregovomab), anti-FRα (e.g., MOv18-IgG1, mirvetuximab soravtansine (IMGN853), MORAb-202), anti-mesothelin (e.g., MORAb-009), anti-TRP2, and anti-HER2 (e.g., trastuzumab, hajuma, ABP980, and / or pertuzumab).

[0125] In a more preferred embodiment, the antibodies included as part of the invention include antibodies that bind to corresponding antigens on tumor cells and induce antibody-dependent cell-mediated cytotoxicity (ADCC) when administered to a patient, hi a further preferred embodiment, the antibodies include antibodies that are approved or pre-approved for the treatment of cancer.

[0126] In a further preferred embodiment, the antibody is an anti-HER2 antibody, an anti-EGFR antibody and / or an anti-CD20 antibody.

[0127] In the most preferred embodiments, the anti-HER2 antibody is selected from pertuzumab, trastuzumab, Hajuma, ABP980 and ado-trastuzumab emtansine.

[0128] In a most preferred embodiment, the anti-EGFR antibody is cetuximab and the anti-CD20 antibody is rituximab.

[0129] As described herein, in various embodiments, the antibody can be a synthetic monoclonal or polyclonal antibody and can be produced by techniques well known in the art. Such an antibody specifically binds (as opposed to non-specifically) to a TAA via the antigen-binding site of the antibody. In producing an antibody that is immunoreactive with an immunogen, a peptide, fragment, variant, fusion protein, etc. of a TAA can be used as the immunogen. More specifically, the polypeptide, fragment, variant, fusion protein, etc. contains an antigenic determinant, i.e., an epitope, that elicits the formation of an antibody.

[0130] antibody In various embodiments of the present invention, the recombinant MVA and methods described herein are administered in combination with and / or in combination with either 1) an antibody that is an immune checkpoint antagonist or immune checkpoint agonist, or 2) a TAA-specific antibody.

[0131] It is contemplated that the antibody may be a synthetic monoclonal or polyclonal antibody and may be produced by techniques well known in the art. Such an antibody specifically binds (as opposed to non-specifically) to an immune checkpoint molecule or TAA via the antigen-binding site of the antibody. In producing an antibody that is immunoreactive with an immunogen, a peptide, fragment, variant, fusion protein, etc. of the immune checkpoint and / or TAA may be used as the immunogen. More specifically, the polypeptide, fragment, variant, fusion protein, etc. comprises an antigenic determinant, i.e., an epitope, that induces the formation of an antibody.

[0132] These antigenic determinants, or epitopes, can be either linear epitopes or conformational (discontinuous) epitopes. A linear epitope is composed of a single amino acid section of a polypeptide, whereas a conformational or discontinuous epitope is composed of multiple amino acid sections from different regions of the polypeptide chain that become adjacent due to protein folding (Janeway, Jr. and Travers, Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Because folded proteins have complex surfaces, the number of available epitopes is very large. However, due to protein conformation and steric hindrance, the number of antibodies that actually bind to those epitopes is less than the number of available epitopes (Janeway, Jr. and Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nd ed. 1996)). Epitopes can be identified by any method known in the art.

[0133] Antibodies that specifically bind to and block the function of TAAs or immune checkpoint molecules such as CTLA-4, PD-1, PD-L1, LAG-3, TIM-3 or ICOS ("antagonist antibodies") or enhance / activate their function ("agonist antibodies"), including scFV fragments, are included in the present invention. Such antibodies can be produced by conventional means.

[0134] In one embodiment, the invention includes monoclonal antibodies against a TAA or immune checkpoint molecule that block the function of the TAA or immune checkpoint molecule (an "antagonist antibody") or enhance / activate its function (an "agonist antibody").

[0135] Antibodies can bind to their targets with high avidity and high specificity. They are relatively large molecules (approximately 150 kDa) that can sterically block the interaction between two proteins (e.g., PD-1 and its target ligand) when their antibody binding site is in close proximity to the interaction site between the proteins. The present invention further includes antibodies that bind to epitopes in close proximity to the immune checkpoint molecule ligand binding site.

[0136] In various embodiments, the invention includes antibodies that interfere with intermolecular interactions (e.g., protein-protein interactions) and antibodies that prevent intramolecular interactions (e.g., conformational changes within a molecule). Antibodies can be screened for their ability to block or enhance / activate the biological activity of immune checkpoint molecules. Both polyclonal and monoclonal antibodies can be prepared by conventional techniques.

[0137] In an exemplary embodiment, the TAA or immune checkpoint molecules CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and ICOS, and peptides based on the amino acid sequences of the TAA or CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and ICOS, can be used to prepare antibodies that specifically bind to the TAA or CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, or ICOS. The term "antibody" is intended to include polyclonal antibodies, monoclonal antibodies, fragments thereof (F(ab')2 and Fab fragments, single chain variable fragments (scFv), single domain antibody fragments (VHH or nanobody), bivalent antibody fragments (diabody), etc.), and any recombinantly and synthetically produced binding partners. In another exemplary embodiment, the antibody binds to an immune checkpoint molecule with a Kd of about 10. 7 M -1 Binding to the above is defined as specifically binding to an immune checkpoint molecule. The affinity of a binding partner or antibody can be readily determined using conventional techniques, such as those described by Scatchard et al. ((1949) Ann. NY Acad. Sci. 51:660).

[0138] Polyclonal antibodies can be easily produced from various sources, such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, or rats, using procedures well known in the art. In general, purified TTA or CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and ICOS, or peptides based on the amino acid sequences of CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and ICOS, which are appropriately conjugated, are administered to a host animal, typically by parenteral injection. After booster immunization, a small amount of serum sample is taken and tested for reactivity against the CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and ICOS polypeptides. Examples of various assays useful for such measurements include those described in Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, as well as procedures such as countercurrent immunoelectrophoresis (CIEP), radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot assay, and sandwich assay. See U.S. Patent Nos. 4,376,110 and 4,486,530.

[0139] Monoclonal antibodies can be readily prepared using well-known procedures, see, for example, those described in U.S. Pat. Reissue No. 32,011, U.S. Pat. Nos. 4,902,614, 4,543,439, and 4,411,993, and Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKeam, and Bechtol (eds.) (1980).

[0140] For example, a host animal such as a mouse can be injected intraperitoneally with isolated and purified immune checkpoint molecules at least once, preferably at least twice at intervals of about three weeks. Mouse serum is then assayed by conventional dot blot or antibody capture (ABC) to determine which mouse is best for fusion. After about two to three weeks, the mouse is given an intravenous boost of immune checkpoint molecules. The mouse is then sacrificed and the spleen cells are fused with commercially available myeloma cells (such as Ag8.653 (ATCC)) according to established protocols. Briefly, the myeloma cells are washed several times in medium and fused to mouse spleen cells at a ratio of about three spleen cells to one myeloma cell. The fusing agent can be any suitable fusing agent used in the art, such as polyethylene glycol (PEG). The fused cells are seeded onto plates containing a medium that selectively grows the fused cells. The fused cells are then allowed to grow for about eight days. The resulting hybridoma supernatants are harvested and added to plates that are first coated with goat anti-mouse Ig. After washing, a label, such as a labeled immune checkpoint molecule polypeptide, is added to each well and then incubated. Positive wells can then be detected. Positive clones can be grown in bulk culture, and then the supernatants are purified on a Protein A column (Pharmacia).

[0141] Monoclonal antibodies of the invention can be made using alternative techniques such as those described in Alting-Mees et al. ((1990) Strategies in Mol. Biol. 3:1-9, "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas"), which is incorporated herein by reference. Similarly, binding partners can be constructed using recombinant DNA techniques to introduce the variable regions of a gene encoding a specific binding antibody. Such techniques are described in Larrick et al. ((1989) Biotechnology 7:394).

[0142] Antigen-binding fragments of such antibodies, which can be produced by conventional techniques, are also included in the present invention. Examples of such fragments include, but are not limited to, Fab fragments and F(ab')2 fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided.

[0143] The monoclonal antibodies of the present invention include chimeric antibodies, e.g., humanized versions of mouse monoclonal antibodies. Such humanized antibodies can be prepared by known techniques and can offer the advantage of reduced immunogenicity when the antibody is administered to humans. In one embodiment, the humanized monoclonal antibody comprises the variable region (or only the antigen-binding site) of a mouse antibody and the constant region derived from a human antibody. Alternatively, the humanized antibody fragment can comprise the antigen-binding site of a mouse monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for making chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. ((1988) Nature 332:323), Liu et al. ((1987) Proc. Nat'l. Acad. Sci. 84:3439), Larrick et al. ((1989) Bio / Technology 7:934), and Winter and Harris ((1993) TIPS 14:139). Procedures for making antibodies transgenically can be found in GB 2,272,440, and U.S. Patent Nos. 5,569,825 and 5,545,806, both of which are incorporated herein by reference.

[0144] Genetically engineered antibodies can be used, including chimeric and humanized monoclonal antibodies that contain both human and non-human portions, which can be produced using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be prepared by genetic engineering using standard DNA techniques known in the art, e.g., as described in Robinson et al., International Publication No. WO 87 / 02671; Akira et al., European Patent Publication No. 0184187; Taniguchi, M., European Patent Publication No. 0171496; Morrison et al., European Patent Publication No. 0173494; Neuberger et al., International Publication No. WO 86 / 01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Publication No. 0125023; Better et al., (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Nat'l. Acad. Sci. 84:3439-3443; Liu et al. al. (1987) J. Immunol. 139:3521-3526, Sun et al. (1987) Proc. Nat'l. Acad. Sci. 84:214-218, Nishimura et al. (1987) Cancer Res. 47:999-1005, Wood et al. (1985) Nature 314:446-449, Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559), Morrison (1985) Science 229:1202-1207, Oi et al. (1986) BioTechniques 4:214, Winter U.S. Patent No. 5,225,539, Jones et al.(1986)Nature 321:552 525, Verhoeyan et al. (1988) Science 239:1534, and Beidler et al. (1988) J. Immunol. 141:4053-4060.

[0145] With respect to synthetic and semi-synthetic antibodies, such terms are intended to cover, but are not limited to, antibody fragments, isotype-switched antibodies, humanized antibodies (e.g., mouse-human antibodies, human-mouse antibodies), hybrids, antibodies with multiple specificities, and completely synthetic antibody-like molecules.

[0146] For therapeutic applications, "human" monoclonal antibodies having human constant and variable regions are often preferred to minimize the patient's immune response to the antibody. Such antibodies can be generated by immunizing transgenic animals containing human immunoglobulin genes. See Jakobovits et al. Ann NY Acad Sci 764:525-535(1995).

[0147] Human monoclonal antibodies against TAAs or immune checkpoint molecules can also be prepared by constructing combinatorial immunoglobulin libraries, such as Fab or scFv phage display libraries, using immunoglobulin light and heavy chain cDNAs prepared from mRNA derived from lymphocytes of a subject. See, for example, McCafferty et al., International Publication No. WO 92 / 01047, Marks et al. (1991) J. Mol. Biol. 222:581-597, and Griffths et al. (1993) EMBO J. 12:725-734. In addition, combinatorial libraries of antibody variable regions can be generated by mutating known human antibodies. For example, the variable regions of human antibodies known to bind immune checkpoint molecules can be mutated, for example, using randomly modified mutagenized oligonucleotides, to generate a library of mutated variable regions, which can then be screened for binding to the immune checkpoint molecules. Methods for inducing random mutagenesis within the CDR regions of immunoglobulin heavy and / or light chains, mating and pairing randomized heavy and light chains, and screening methods can be found, for example, in Barbas et al., International Publication No. WO 96 / 07754; Barbas et al. (1992) Proc. Nat'l Acad. Sci. USA 89:4457-4461.

[0148] The immunoglobulin library can be expressed by a population of display packages, preferably a population of display packages derived from filamentous phage, to form an antibody display library. Examples of methods and reagents particularly suitable for use in generating antibody display libraries are described, for example, in U.S. Pat. No. 5,223,409 to Ladner et al., International Publication No. WO 92 / 18619 to Kang et al., International Publication No. WO 91 / 17271 to Dower et al., International Publication No. WO 92 / 20791 to Winter et al., International Publication No. WO 92 / 15679 to Markland et al., International Publication No. WO 93 / 01288 to Breitling et al., International Publication No. WO 92 / 01047 to McCafferty et al., International Publication No. WO 92 / 09690 to Garrard et al., International Publication No. WO 90 / 02809 to Ladner et al., Fuchs et al. (1991) Bio / Technology 9:1370 1372, Hay et al. (1992) Hum Antibod Hybridomas 3:81-85, Huse et al. (1989) Science 246:1275-1281, Griffiths et al. (1993) supra, Hawkins et al. (1992) J.Mol.Biol.226:889-896, Clackson et al. (1991) Nature 352:624-628, Gram et al. al. (1992) Proc. Nat'l. Acad. Sci. 89:3576-3580, Garrad et al. (1991) Bio / Technology 9:1373-1377, Hoogenboom et al. (1991) Nucl. Acid Res. 19:4133-4137 and Barbas et al. al. (1991) Proc. Nat'l. Acad. Sci. 88:7978-7982. Once displayed on the surface of the display package (e.g., filamentous phage), the antibody library is screened to identify and isolate packages that express antibodies that bind to the TAA or immune checkpoint molecule.

[0149] Recombinant MVA In a more preferred embodiment of the present invention, one or more of the proteins and nucleotides disclosed herein are contained in a recombinant MVA. As described and exemplified by the present disclosure, in various aspects, intravenous administration of the recombinant MVA of the present disclosure induces an enhanced immune response in cancer patients. Thus, in one or more preferred embodiments, the present invention includes a recombinant MVA comprising a first nucleic acid encoding one or more of the TAAs described herein and a second nucleic acid encoding CD40L.

[0150] Examples of MVA virus strains which are useful in practicing the invention and which have been deposited under the provisions of the Budapest Treaty are the MVA572 strain, deposited on 27 January 1994 at the European Collection of Animal Cell Cultures (ECACC), Vaccine Research and Production Laboratory, Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom, under the deposit number ECACC94012707, and the MVA575 strain, deposited on 7 December 2000 under ECACC00120707; the MVA-BN strain and its derivatives, deposited on 30 August 2000 at the European Collection of Cell Cultures (ECACC), under the number V00083008, are further exemplary strains.

[0151] A "derivative" of MVA-BN refers to a virus that exhibits essentially the same replication characteristics as MVA-BN as described herein, but differs in one or more portions of its genome. MVA-BN and its derivatives are replication incompetent, meaning that they are unable to productively replicate in vivo and in vitro. More specifically, in vitro MVA-BN or its derivatives have been described as being able to productively replicate in chicken embryo fibroblasts (CEF), but unable to productively replicate in the human keratinocyte cell line HaCat (Boukamp et al. (1988) J. Cell Biol. 106:761-771), the human osteosarcoma cell line 143B (ECACC Accession No. 91112502), the human embryonic kidney cell line 293 (ECACC Accession No. 85120602), and the human cervical carcinoma cell line HeLa (ATCC Accession No. CCL-2). In addition, MVA-BN or its derivatives have a viral amplification rate at least 2-fold lower, and more preferably 3-fold lower, than MVA-575 in HeLa and HaCaT cell lines. Tests and assays for these properties of MVA-BN and its derivatives are described in WO02 / 42480 (U.S. Patent Application Publication No. 2003 / 0206926) and WO03 / 048184 (U.S. Patent Application Publication No. 2006 / 0159699).

[0152] As described in the paragraph above, the term "unable to reproductively replicate" or "reproductively incompetent" in human cell lines in vitro is explained, for example, in WO02 / 42480, which also teaches how to obtain MVA with the desired properties as described above. This term applies to viruses that have an in vitro viral amplification factor of less than 1 at 4 days post-infection using the assays described in WO02 / 42480 or US Patent No. 6,761,893.

[0153] The term "reproductive replication failure" refers to a virus having a viral amplification rate in an in vitro human cell line, as described in the previous paragraph, of less than 1 at 4 days post-infection. The assays described in WO02 / 42480 or U.S. Patent No. 6,761,893 are applicable to determine the viral amplification rate.

[0154] As explained in the previous paragraph, viral amplification or replication in human cell lines in vitro is usually expressed as the ratio of virus produced from an infected cell (output) to the amount originally used to infect the cell at the initial location (input), referred to as the "amplification ratio". An amplification ratio of "1" defines an amplification state in which the amount of virus produced from an infected cell is the same as the amount originally used to infect the cell, i.e., the infected cell is permissive for virus infection and reproduction. In contrast, an amplification ratio below 1, i.e., less virus produced than the amount entered, indicates a non-productively replicating virus, i.e., attenuation of the virus.

[0155] As used herein, "adjuvant effect" is intended to mean the augmentation of the immune response produced by a particular encoded protein or component of a recombinant MVA caused by other encoded protein(s) or component(s) of that recombinant MVA.

[0156] Expression Cassettes / Control Sequences In various embodiments, one or more of the nucleic acids described herein are incorporated into one or more expression cassettes, in which the one or more nucleic acids are operably linked to an expression control sequence. "Operably linked" means that the described components are in a relationship that allows them to function in the intended manner, for example, a relationship that allows a promoter to transcribe and express the nucleic acid. An expression control sequence operably linked to a coding sequence is linked such that expression of the coding sequence is under conditions compatible with the expression control sequence. Expression control sequences include, but are not limited to, a suitable promoter, enhancer, transcription terminator, a start codon at the beginning of an open reading frame encoding a protein, a splicing signal in an intron, and an in-frame stop codon. Suitable promoters include, but are not limited to, SV40 early promoter, RSV promoter, retroviral LTR, adenovirus major late promoter, human CMV immediate early I promoter, and various poxvirus promoters (including, but not limited to, vaccinia virus or MVA-derived promoters and FPV-derived promoters such as 30K promoter, I3 promoter, PrS promoter, PrS5E promoter, Pr7.5K, PrHyb promoter, Pr13.5 long promoter, 40K promoter, MVA-40K promoter, FPV 40K promoter, 30k promoter, PrSynIIm promoter, PrLE1 promoter, and PR1238 promoter). Additional promoters are further described in WO2010 / 060632, WO2010 / 102822, WO2013 / 189611, WO2014 / 063832 and WO2017 / 021776, which are incorporated by reference in their entireties.

[0157] Additional expression control sequences include, but are not limited to, leader sequences, stop codons, polyadenylation signals, and any other sequences necessary for proper transcription and subsequent translation of a nucleic acid sequence encoding a desired recombinant protein (e.g., HER2, Brachyury, and / or CD40L) in a desired host system. The poxvirus vectors of the present invention may also contain additional elements necessary for the transfer and subsequent replication of an expression vector containing a nucleic acid sequence in a desired host system. Those skilled in the art will further appreciate that such vectors are readily constructed using conventional methods (Ausubel et al. (1987) in "Current Protocols in Molecular Biology," John Wiley and Sons, New York, NY) and are commercially available.

[0158] Methods and regimens for administering the combination drug of the present invention In one or more aspects, the combination of the present invention can be administered as part of an allogeneic and / or xenogeneic prime-boost regimen. As shown in part by the data shown in Figure 7, the allogeneic prime-boost regimen increases the specific CD8 T cell response and CD4 T cell response in the subject. Thus, in one or more embodiments, there are combinations and / or methods for reducing tumor size and / or increasing survival in cancer patients, comprising administering to the cancer patient a combination of the present disclosure, where the combination is administered as part of an allogeneic or xenogeneic prime-boost regimen.

[0159] Generation of recombinant MVA viruses containing transgenes The recombinant MVA virus provided by the present invention can be produced by conventional methods known in the art. Methods for obtaining recombinant poxviruses or inserting foreign coding sequences into poxvirus genomes are well known to those skilled in the art. For example, methods for standard molecular biology techniques such as DNA cloning, DNA isolation, RNA isolation, Western blot analysis, RT-PCR and PCR amplification techniques are described in Molecular Cloning, A Laboratory Manual (2nd ed., J. Sambrook et al., Cold Spring Harbor Laboratory Press (1989)), and techniques for handling and manipulating viruses are described in Virology Methods Manual (Mahy et al. (eds.), Academic Press (1996)). Similarly, techniques and know-how for handling, manipulating and genetically modifying MVA are described in Molecular Virology: A Practical Approach (Davison & Elliott (eds.), The Practical Approach Series, IRL Press at Oxford University Press, Oxford, UK (1993) (see, e.g., Chapter 9: Expression of genes by Vaccinia viral vectors) and Current Protocols in Molecular Biology (John Wiley & Son, Inc. (1998) (see, e.g., Chapter 16, Section IV: Expression of proteins in mammalian cells using vaccinia viral vectors)).

[0160] A variety of methods may be applicable for the generation of the various recombinant MVA viruses disclosed in the present invention. The DNA sequence to be inserted into the virus may be placed in an E. coli plasmid construct in which DNA homologous to a section of poxvirus DNA is inserted. Alternatively, the DNA sequence to be inserted can be ligated to a promoter. This promoter-gene linkage can be placed in a plasmid construct such that both ends of the promoter-gene linkage are flanked by DNA homologous to DNA sequences flanking the region of poxvirus DNA containing the non-essential locus. The resulting plasmid construct can be amplified by growth in E. coli and isolated. The isolated plasmid containing the DNA gene sequence to be inserted may be transfected into a cell culture, for example, of chicken embryo fibroblasts (CEF), at the same time that the culture is infected with the MVA virus. Recombination between the homologous MVA virus DNA in the plasmid and the viral genome, respectively, can generate a poxvirus modified by the presence of the foreign DNA sequence.

[0161] According to a preferred embodiment, a suitable cell culture of cells, for example CEF cells, can be infected with the MVA virus. The infected cells can then be transfected with a first plasmid vector (preferably under the transcriptional control of a poxvirus expression control element) that contains a foreign or heterologous gene(s), such as one or more of the nucleic acids provided in the present disclosure. As explained above, the plasmid vector also contains a sequence that can direct the insertion of the foreign sequence into a predetermined part of the MVA virus genome. Optionally, the plasmid vector also contains a cassette that contains a marker gene and / or a selection gene operably linked to a poxvirus promoter. Suitable marker or selection genes are, for example, genes that code for green fluorescent protein, β-galactosidase, neomycin-phosphoribosyltransferase or other markers. The use of a selection or marker cassette simplifies the identification and isolation of the recombinant poxviruses that have been produced. However, recombinant poxviruses can also be identified by PCR techniques. The recombinant poxvirus thus obtained can then be used to infect further cells, which can then be transfected with a second vector containing one or more second foreign or heterologous genes. To be sure, this gene should be introduced into a different insertion site of the poxvirus genome, and the second vector also differs in the sequences that are homologous to the poxvirus and that direct the integration of the second foreign gene or genes into the genome of the poxvirus. After homologous recombination has taken place, 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 by using the recombinant virus isolated in the previous infection step and by using a further vector containing one or more additional foreign genes for transfection.

[0162] Alternatively, the infection and transfection steps as described above can be interchanged, i.e., suitable cells can first be transfected with a plasmid vector containing the foreign genes and then infected with the poxvirus. As a further alternative, each foreign gene can be introduced into a different virus, and all the resulting recombinant viruses can be co-infected into cells and screened for recombinants containing all the foreign genes. A third alternative is to ligate the DNA genome and the foreign sequence in vitro and reconstruct the recombinant vaccinia virus DNA genome using a helper virus. A fourth alternative is to carry out homologous recombination in E. coli or another bacterial species between the MVA virus genome cloned as a bacterial artificial chromosome (BAC) and a linear foreign sequence flanked by DNA sequences that are homologous to sequences flanking the desired integration site in the MVA virus genome.

[0163] One or more of the nucleic acids of the present disclosure may be inserted into any suitable portion of the MVA virus or MVA virus vector. The suitable portion of the MVA virus is a non-essential portion of the MVA genome. The non-essential portion of the MVA genome may be an intergenic region or a known deletion site 1-6 of the MVA genome. Alternatively or additionally, the non-essential portion of the recombinant MVA may be a coding region of the MVA genome that is not essential for viral growth. However, the insertion site is not limited to these preferred insertion sites in the MVA genome. It is within the scope of the present invention that a nucleic acid of the present invention (e.g., HER2, Brachyury, HERV-K-env, HERV-K-gag, PRAME, FOLR1, CD40L and / or 4-1BBL), as well as any associated promoter as described herein, may be inserted into any location of the viral genome, as long as a recombinant that can be amplified and propagated in at least one cell culture system, such as chicken embryo fibroblasts (CEF cells), can be obtained.

[0164] Preferably, the nucleic acid of the invention may be inserted into one or more of the intergenic regions (IGRs) of the MVA virus. The term "intergenic region" preferably refers to a portion of the viral genome located between two adjacent open reading frames (ORFs) of the MVA virus genome, preferably between two essential ORFs of the MVA virus genome. In MVA, in a particular embodiment, the IGRs are selected from IGR07 / 08, IGR44 / 45, IGR64 / 65, IGR88 / 89, IGR136 / 137 and IGR148 / 149.

[0165] Additionally or alternatively, in the MVA virus, the nucleotide sequence may be inserted into one or more of the known deletion sites of the MVA genome, i.e., deletion sites I, II, III, IV, V or VI. The term "known deletion site" refers to a portion of the MVA genome that has been deleted through serial passages in CEF cells and that has been characterized at passage 516 relative to the genome of the parent virus from which the MVA is derived, in particular the parent chorioallantoic vaccinia virus Ankara (CVA), as described, for example, in Meisinger-Henschel et al. ((2007) J. Gen. Virol. 88:3249-3259).

[0166] vaccine In certain embodiments, the recombinant MVA of the present disclosure can be formulated as part of a vaccine, in which the MVA virus can be converted into a physiologically acceptable form.

[0167] An exemplary preparation is as follows: Purified virus is added to 5×10 8 The titer is TCID50 / ml, and the mixture is stored at -80°C in 10 mM Tris, 140 mM NaCl (pH 7.4). 8 ~1×10 9The virus particles can be lyophilized in phosphate buffered saline (PBS) in the presence of 2% peptone and 1% human albumin in ampoules, preferably glass ampoules. Alternatively, the vaccine injection can be prepared by step-lyophilizing the virus in the formulation. In certain embodiments, the formulation contains additional additives such as mannitol, dextran, sugars, glycine, lactose, polyvinylpyrrolidone, or other additives including, but not limited to, antioxidants, inert gases, stabilizers, or recombinant proteins (e.g., human serum albumin) suitable for in vivo administration. The ampoules can then be sealed and stored at a suitable temperature, for example, between 4°C and room temperature, for several months. However, unless necessary, the ampoules are preferably stored at a temperature below -20°C, most preferably at about -80°C.

[0168] In various embodiments involving vaccination or therapy, the lyophilisate is dissolved in 0.1-0.5 ml of an aqueous solution, preferably saline or a Tris buffer such as 10 mM Tris, 140 mM NaCl, pH 7.7. The recombinant MVA, vaccine or pharmaceutical composition of the present disclosure may be dissolved in a solution of 10 4 ~10 10 TCID50 / ml, 10 5 ~5×10 9 TCID50 / ml, 10 6 ~5×10 9 TCID50 / ml or 10 7 ~5×10 9 It is anticipated that the compound can be formulated in a range of concentrations from 10 to 1000TCID50 / ml. 6 ~10 10 Contains TCID50, 10 6 TCID50, 10 7 TCID50, 10 8 TCID50, 5×10 8 TCID50, 10 9 TCID50, 5×10 9 TCID50 or 10 10Optimization of dosage and number of doses is within the skill and knowledge of one of ordinary skill in the art.

[0169] In one or more preferred embodiments, the recombinant MVA of the invention is administered intravenously to a cancer patient, as defined herein. In other embodiments, the recombinant MVA of the invention is administered intratumorally to a cancer patient. In other embodiments, the recombinant MVA of the invention is administered both intravenously and intratumorally to a cancer patient, either simultaneously or at different times.

[0170] In additional embodiments, the immune checkpoint antagonists or immune checkpoint agonists, or preferably antibodies, of the present invention can be administered systemically or locally, i.e., by intraperitoneal, parenteral, subcutaneous, intravenous, intramuscular, intranasal, intradermal or any other route of administration known to the skilled practitioner.

[0171] Kits, Compositions and Methods of Use In various embodiments, the invention includes kits, pharmaceutical combinations, pharmaceutical compositions and / or immunogenic combinations comprising a) a recombinant MVA comprising a nucleic acid as described herein, and / or b) one or more antibodies as described herein.

[0172] It is contemplated that the kits and / or compositions may include one or more containers or vials of a recombinant poxvirus of the present disclosure, one or more containers or vials of an antibody of the present disclosure, along with instructions for administering the recombinant MVA and antibody. In a more specific embodiment, it is contemplated that the kits may include instructions for administering an initial priming dose of the recombinant MVA and antibody, followed by one or more boosting doses of the recombinant MVA and antibody.

[0173] Kits and / or compositions provided herein may generally include one or more pharma- ceutically acceptable and / or approved carriers, additives, antibiotics, preservatives, diluents and / or stabilizers. Such auxiliary substances can be water, saline, glycerol, ethanol, wetting agents, emulsifying agents, pH buffering substances, and the like. Suitable carriers are typically large molecules that are slowly metabolized, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and the like.

[0174] Specific Exemplary Embodiments Embodiment 1 is a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, the method comprising intratumorally administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding 4-1BBL, wherein intratumor administration of the recombinant MVA enhances an inflammatory response in the cancerous tumor, increases tumor shrinkage, and / or increases overall survival in the subject compared to non-intratumor injection of a recombinant MVA virus comprising a first and second nucleic acid encoding a TAA and the antigen 4-1BBL.

[0175] Embodiment 2 is a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, comprising intravenously administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding 4-1BBL, wherein intravenous administration of the recombinant MVA enhances natural killer (NK) cell responses and enhances CD8 T cell responses specific for the TAA compared to non-intravenous injection of a recombinant MVA virus comprising a first and second nucleic acid encoding the TAA and the antigen 4-1BBL.

[0176] Embodiment 3 is a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, comprising administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding 4-1BBL, wherein administration of the recombinant MVA increases tumor reduction and / or increases overall survival in the subject compared to administration of the recombinant MVA and the antigen 4-1BBL alone.

[0177] Embodiment 4 is a method of inducing an enhanced inflammatory response in a cancerous tumor of a subject, comprising intratumorally administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a first heterologous tumor-associated antigen (TAA) and a second nucleic acid encoding the antigen 4-1BBL, wherein intratumor administration of the recombinant MVA enhances the inflammatory response in the tumor compared to the inflammatory response caused by non-intratumoral injection of a recombinant MVA virus comprising a heterologous tumor-associated antigen and a first and second nucleic acid encoding the antigen 4-1BBL. Such enhanced inflammatory responses are discussed elsewhere herein and may include, for example, induction of NK cells and T cells.

[0178] Embodiment 5 is a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, comprising administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding an endogenous retroviral antigen (ERV) and a second nucleic acid encoding 4-1BBL, wherein administration of the recombinant MVA increases tumor reduction and / or increases overall survival in the subject compared to administration of the recombinant MVA and the antigen 4-1BBL alone.

[0179] Embodiment 6 is a method according to any one of embodiments 1 to 5, wherein the subject is a human.

[0180] Embodiment 7 is the method according to any one of embodiments 1 to 4, wherein the TAA is an endogenous retroviral (ERV) protein.

[0181] Embodiment 8 is the method according to embodiment 7, wherein the ERV is an ERV protein expressed in a tumor cell.

[0182] Embodiment 9 is the method according to any one of embodiments 7 to 8, wherein the ERV is derived from the human endogenous retrovirus protein K (HERV-K) family.

[0183] Embodiment 10 is the method according to embodiment 9, wherein the HERV-K protein is selected from a HERV-K envelope protein, a HERV-K gag protein, and a HERV-K mel protein.

[0184] Embodiment 11 is the method according to embodiment 9, wherein the HERV-K protein is selected from the group consisting of HERV-K envelope protein, HERV-K gag protein, HERV-K mel protein and immunogenic fragments thereof.

[0185] Embodiment 12 is a method according to any one of embodiments 1 to 6, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-related protein 1 (TRP1), tyrosine-related protein 1 (TRP2), Brachyury, FOLR1, PRAME, p15, and combinations thereof.

[0186] Embodiment 13 is a method according to any one of embodiments 1 to 6 and 12, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA) and mucin 1 cell surface associated (MUC-1).

[0187] Embodiment 14 is the method according to any one of embodiments 1 to 6 and 12, wherein the TAA is selected from the group consisting of PAP or PSA.

[0188] Embodiment 15 is the method according to any one of embodiments 1 to 6, 12 and 14, wherein the TAA is PSA.

[0189] Embodiment 16 is the method of any one of embodiments 1 to 6, wherein the TAA is selected from the group consisting of 5-alpha-reductase, alpha-fetoprotein (AFP), AM-1, APC, April, B-melanoma antigen gene (BAGE), beta-catenin, Bcl12, bcr-abl, Brachyury, CA-125, caspase-8 (CASP-8, also known as FLICE), cathepsin, 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 / decay accelerating factor (DAF), CD59 / Protectin, CDC27, CDK4, carcinoembryonic antigen (CEA), c-myc, cyclooxygenase-2 (cox-2), deleted in colorectal cancer gene ("DCC"), DcR3, E6 / E7, CGFR, EMBP, Dna78, farnesyltransferase, fibroblast growth factor-8a (FGF8a), fibroblast growth factor-8b (FGF8b), FLK-1 / KDR, folate receptor body, G250, G melanoma 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→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 mammary tumor virus (HMTV), heat shock protein of 70 kilodaltons ("HSP70"), human telomerase reverse transcriptase (hTERT), insulin-like growth factor receptor-1 (IGFR-1), interleukin-13 receptor (IL-13R), inducible iNOS, Ki67, KIAA0205, K-ras, H-ras, N-ras, KSA, LKLR-FUT, melanoma antigen-encoding gene 1 (MAGE-1), melanoma antigen-encoding gene 2 (MAGE-2), melanoma antigen-encoding gene 3 (MAGE-3), melanoma antigen-encoding gene 4 (MAGE-4), mammaglobin, MAP17, melan-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-α (TGF-α), transforming growth factor-β (TGF-β), thymosin-β-15, tumor necrosis factor The method is selected from the group consisting of tumor necrosis factor-alpha (TNF-α), TP1, TRP-2, tyrosinase, vascular endothelial growth factor (VEGF), ZAG, p16INK4 and glutathione-S-transferase (GST), the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-related protein 1 (TRP1), tyrosine-related protein 1 (TRP2), Brachyury, and combinations thereof.

[0190] Embodiment 17 is the method according to any one of embodiments 1 to 16, wherein the recombinant MVA further comprises a third nucleic acid encoding the antigen CD40L.

[0191] Embodiment 18 is a method according to any one of embodiments 1 to 17, further comprising administering to the subject at least one immune checkpoint molecule antagonist or immune checkpoint molecule agonist.

[0192] Embodiment 19 is the method of embodiment 18, wherein the immune checkpoint molecule antagonist or immune checkpoint molecule agonist is selected from CTLA-4, PD-1, PD-L1, LAG-3, TIM-3 and ICOS.

[0193] Embodiment 20 is the method according to any one of embodiments 18 to 19, wherein the immune checkpoint molecule antagonist is PD-1 and / or PD-L1.

[0194] Embodiment 21 is the method of embodiment 20, wherein the immune checkpoint molecule antagonist further comprises LAG-3.

[0195] Embodiment 22 is a method according to any one of embodiments 18 to 21, wherein the immune checkpoint molecule antagonist comprises an antibody.

[0196] Embodiment 23 is the method of any one of embodiments 1 to 17, further comprising administering to the subject an antibody specific for the second TAA.

[0197] Embodiment 24 is the method of embodiment 23, wherein the antibody specific for the second TAA is specific for an antigen expressed on the cell membrane of a tumor.

[0198] Embodiment 25 is a method according to embodiment 23, wherein the antibody specific for the second TAA a) is specific for an antigen expressed on the cell membrane of a tumor, and b) comprises an Fc domain.

[0199] Embodiment 26 is a pharmaceutical composition for use in the method according to any one of embodiments 1 to 25.

[0200] Embodiment 27 is a vaccine for use in the method according to any one of embodiments 1 to 25.

[0201] Embodiment 28 is a recombinant Modified Vaccinia Ankara (MVA) for treating a subject with cancer, the recombinant MVA comprising: a) a first nucleic acid encoding a tumor-associated antigen (TAA); and b) a second nucleic acid encoding 4-1BBL.

[0202] Embodiment 29 is a recombinant MVA as described in embodiment 28, wherein the TAA is an endogenous retroviral (ERV) protein.

[0203] Embodiment 30 is a recombinant MVA according to embodiment 29, wherein the ERV protein is derived from the human endogenous retroviral protein K (HERV-K) family.

[0204] Embodiment 31 is a recombinant MVA according to embodiment 30, wherein the retroviral protein K is selected from HERV-K envelope protein, HERV-K gag protein and HERV-K mel protein.

[0205] Embodiment 32 is the recombinant MVA according to any one of embodiments 28 to 31, further comprising a third nucleic acid encoding CD40L.

[0206] Embodiment 33 is a combination drug comprising a) a recombinant MVA according to any one of embodiments 28 to 32, and b) at least one immune checkpoint molecule antagonist or immune checkpoint molecule agonist.

[0207] Embodiment 34 is the combination medicine of embodiment 33, wherein the immune checkpoint molecule antagonist or immune checkpoint molecule agonist is selected from CTLA-4, PD-1, PD-L1, LAG-3, TIM-3 and ICOS.

[0208] Embodiment 35 is the combination medicine according to embodiment 34, wherein the immune checkpoint molecule antagonist is PD-1 and / or PD-L1.

[0209] Embodiment 36 is a combination pharmaceutical according to embodiment 35, wherein the immune checkpoint molecule antagonist further comprises LAG-3.

[0210] Embodiment 37 is a combination pharmaceutical according to any one of embodiments 33 to 36, wherein the immune checkpoint molecule antagonist comprises an antibody.

[0211] Embodiment 38 is a pharmaceutical combination comprising a) a recombinant MVA according to any one of embodiments 28 to 32 and b) an antibody specific for a second TAA.

[0212] Embodiment 39 is the pharmaceutical combination of embodiment 38, wherein the antibody specific for the second TAA is specific for an antigen expressed on the cell membrane of a tumor.

[0213] Embodiment 40 is a combination pharmaceutical described in embodiment 39, wherein the antibody specific for the second TAA a) is specific for an antigen expressed on the cell membrane of a tumor, and b) comprises an Fc domain.

[0214] Embodiment 41 is a recombinant MVA according to any one of embodiments 28 to 32, a vaccine according to embodiment 27, a pharmaceutical composition according to embodiment 26, or a combination medicine according to any one of embodiments 33 to 40, which is used to reduce tumor size and / or increase survival rate in a subject having a cancerous tumor.

[0215] Embodiment 42 is a recombinant MVA according to any one of embodiments 28-32, a vaccine according to embodiment 27, a pharmaceutical composition according to embodiment 26, or a pharmaceutical combination according to any one of embodiments 33-40 for use in a method for reducing tumor size and / or increasing survival in a subject having a cancerous tumor, comprising intratumorally administering to said subject the recombinant MVA according to embodiments 28-32, the vaccine according to embodiment 27, the pharmaceutical composition according to embodiment 26, or the pharmaceutical combination according to any one of embodiments 33-40, which intratumorally administering enhances the inflammatory response in said cancerous tumor, increases tumor shrinkage, and / or increases overall survival in said subject compared to non-intratumor injection of a recombinant MVA virus comprising a TAA and a first and second nucleic acid encoding the antigen 4-1BBL.

[0216] Embodiment 43 is a recombinant MVA according to any one of embodiments 28 to 32, a vaccine according to embodiment 27, a pharmaceutical composition according to embodiment 26, or a combination drug according to any one of embodiments 33 to 40 for use in a method for reducing tumor size and / or increasing survival rate in a subject having a cancerous tumor, comprising intravenously administering to the subject the recombinant MVA according to embodiments 28 to 32, the vaccine according to embodiment 27, the pharmaceutical composition according to embodiment 26, or the combination drug according to any one of embodiments 33 to 40, which intravenous administration results in increased tumor reduction and / or increased overall survival rate in the subject compared to non-intravenous administration of a recombinant MVA virus comprising a TAA and a first and second nucleic acid encoding the antigen 4-1BBL.

[0217] Embodiment 44 is a recombinant MVA according to any one of embodiments 28 to 32, a vaccine according to embodiment 27, a pharmaceutical composition according to embodiment 26, or a combination drug according to any one of embodiments 33 to 40, for use in a method for inducing an enhanced inflammatory response in a cancerous tumor of a subject having cancer, the method comprising intratumorally administering to the subject the recombinant MVA according to embodiments 28 to 32, the vaccine according to embodiment 27, the pharmaceutical composition according to embodiment 26, or the combination drug according to any one of embodiments 33 to 40, wherein the intratumor administration enhances the inflammatory response in the cancerous tumor of the subject compared to non-intratumor injection of a recombinant MVA virus comprising a TAA and a first and second nucleic acid encoding the antigen 4-1BBL.

[0218] Embodiment 45 is a recombinant MVA according to any one of embodiments 28 to 32, a vaccine according to embodiment 27, a pharmaceutical composition according to embodiment 26, or a combination medicine according to any one of embodiments 33 to 40, for use in a method for treating cancer in a subject.

[0219] Embodiment 46 is a recombinant MVA according to any one of embodiments 28 to 32, a vaccine according to embodiment 27, a pharmaceutical composition according to embodiment 26, or a combination medicine according to any one of embodiments 33 to 40, for use in a method for treating cancer, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, head and neck cancer, thyroid cancer, melanoma, gastric cancer, bladder cancer, kidney cancer, liver cancer, melanoma, pancreatic cancer, prostate cancer, ovarian cancer, urothelial, cervical or colorectal cancer.

[0220] Embodiment 47 is a recombinant MVA as described in embodiment 44, wherein the enhanced inflammatory response is localized to the tumor.

[0221] Embodiment 48 is a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, comprising intratumorally administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding CD40L, wherein intratumor administration of the recombinant MVA enhances an inflammatory response in the cancerous tumor, increases tumor shrinkage, and / or increases overall survival in the subject compared to non-intratumor injection of a recombinant MVA virus comprising a first and second nucleic acid encoding a TAA and CD40L.

[0222] Embodiment 49 is a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, comprising intravenously administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding CD40L, wherein intravenous administration of the recombinant MVA enhances natural killer (NK) cell responses and enhances CD8 T cell responses specific for the TAA compared to non-intravenous injection of a recombinant MVA virus comprising a first and second nucleic acid encoding the TAA and the antigen CD40L.

[0223] Embodiment 50 is a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, comprising administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding CD40L, wherein administration of the recombinant MVA increases tumor reduction and / or increases overall survival in the subject compared to administration of the recombinant MVA and the antigen CD40L alone.

[0224]

[0046] Embodiment 51 is a method for reducing tumor size and / or increasing survival rate in a subject having a cancerous tumor, comprising administering intravenously and / or intratumorally to the subject the recombinant MVA of any one of embodiments 28 to 32, the vaccine of embodiment 27, the pharmaceutical composition of embodiment 26, or the combination of embodiments 33 to 40, wherein the intravenous administration is performed intravenously and / or intratumorally to the subject, The intravenous and / or intratumoral administration of any MVA selected from the group consisting of: 1) a recombinant MVA virus comprising a first nucleic acid encoding a TAA and a second nucleic acid encoding the antigen 4-1BBL; 2) a recombinant MVA virus comprising a first nucleic acid encoding a TAA and a second nucleic acid encoding the antigen CD40L; or 3) a recombinant MVA virus comprising a first nucleic acid encoding a TAA, a second nucleic acid encoding the antigen 4-1BBL, and a third nucleic acid encoding the antigen CD40L, results in increased tumor shrinkage and / or increased overall survival in the subject compared to non-intravenous or non-intratumoral administration of any MVA selected from the group consisting of: 1) a recombinant MVA virus comprising a first nucleic acid encoding a TAA and a second nucleic acid encoding the antigen 4-1BBL;

[0225]

[0046] Embodiment 52 is a method for reducing tumor size and / or increasing survival rate in a subject having a cancerous tumor, comprising administering intravenously and intratumorally to the subject the recombinant MVA of any one of embodiments 28 to 32, the vaccine of embodiment 27, the pharmaceutical composition of embodiment 26, or the combination of embodiments 33 to 40, wherein the intravenous administration is performed intratumorally. and intratumoral administration of the MVA selected from the group consisting of: 1) a recombinant MVA virus comprising a first nucleic acid encoding a TAA and a second nucleic acid encoding the antigen 4-1BBL, 2) a recombinant MVA virus comprising a first nucleic acid encoding a TAA and a second nucleic acid encoding the antigen CD40L, or 3) a recombinant MVA virus comprising a first nucleic acid encoding a TAA, a second nucleic acid encoding the antigen 4-1BBL, and a third nucleic acid encoding the antigen CD40L, which results in increased tumor shrinkage and / or increased overall survival in the subject compared to non-intravenous or non-intratumoral administration of the MVA. The intravenous and intratumoral administrations may be performed simultaneously or at different times, as will be apparent to one skilled in the art. In addition, the present application relates to the invention described in the claims, but may also include the following as other aspects. 1. A method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, said method comprising intratumorally administering to said subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding 4-1BBL, wherein intratumor administration of said recombinant MVA enhances an inflammatory response in said cancerous tumor, increases tumor reduction and / or increases overall survival of said subject compared to non-intratumor injection of a recombinant MVA virus comprising a first and second nucleic acid encoding a TAA and the antigen 4-1BBL. 2. A method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, said method comprising intravenously administering to said subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor associated antigen (TAA) and a second nucleic acid encoding 4-1BBL, wherein intravenous administration of said recombinant MVA results in an enhanced natural killer (NK) cell response and an enhanced CD8 T cell response specific for said TAA compared to non-intravenous injection of a recombinant MVA virus comprising a first and second nucleic acid encoding a TAA and the antigen 4-1BBL. 3. A method for reducing tumor size and / or increasing survival in a subject having a cancerous tumor, said method comprising administering to said subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor associated antigen (TAA) and a second nucleic acid encoding 4-1BBL, wherein administration of the recombinant MVA results in increased tumor reduction and / or increased overall survival in said subject compared to administration of the recombinant MVA and the antigen 4-1BBL alone. 4. A method for inducing an enhanced inflammatory response in a cancerous tumor in a subject, the method comprising intratumorally administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a first heterologous tumor-associated antigen (TAA) and a second nucleic acid encoding an antigen 4-1BBL, wherein intratumor administration of the recombinant MVA results in an enhanced inflammatory response in the tumor compared to the inflammatory response produced by non-intratumoral injection of a recombinant MVA virus comprising a heterologous tumor-associated antigen and a first and second nucleic acid encoding the antigen 4-1BBL. 5. The method according to claim 1, wherein the subject is a human. 6. The method according to claim 1, wherein the TAA is an endogenous retroviral (ERV) protein expressed in tumor cells. 7. The method according to claim 6, wherein the ERV protein is derived from the human endogenous retrovirus K (HERV-K) family and is selected from the HERV-K envelope protein and the HERV-K gag protein. 8. The method according to claim 1, wherein said TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-related protein 1 (TRP1), tyrosine-related protein 1 (TRP2), Brachyury, PRAME, FOLR1, HERV-K-env, HERV-K-gag, p15, MEL, and combinations thereof. 9. A method for reducing tumor size and / or increasing survival in a subject having a cancerous tumor, said method comprising administering to said subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding an endogenous retroviral (ERV) protein and a second nucleic acid encoding 4-1BBL, wherein administration of said recombinant MVA results in increased tumor reduction and / or increased overall survival in said subject compared to administration of the recombinant MVA and the antigen 4-1BBL alone. 10. The method according to claim 9, wherein the ERV protein is from the human endogenous retrovirus protein K (HERV-K) family. 11. The method according to claim 10, wherein the retroviral protein K is selected from the group consisting of HERV-K envelope protein and HERV-K-gag protein. 12. The method according to claim 1, wherein the recombinant MVA further comprises a third nucleic acid encoding the antigen CD40L. 13. The method of claim 1, further comprising administering to the subject at least one immune checkpoint molecule antagonist or immune checkpoint molecule agonist. 14. The method according to claim 13, wherein the immune checkpoint molecule antagonist or the immune checkpoint molecule agonist is selected from antagonists or agonists of CTLA-4, PD-1, PD-L1, LAG-3, TIM-3 and ICOS. 15. The method according to claim 14, wherein the immune checkpoint molecule antagonist is an antibody. 16. A recombinant Modified Vaccinia Ankara (MVA) for use in the treatment of cancer, comprising: a) a first nucleic acid encoding a tumor-associated antigen (TAA); and b) a second nucleic acid encoding 4-1BBL, wherein intratumoral administration of the recombinant MVA enhances the inflammatory response in a cancerous tumor, reduces the size of the cancerous tumor, and / or increases overall survival in a human patient compared to non-intratumoral injection of the recombinant MVA. 17.c) A recombinant MVA for the use according to claim 16, further comprising a third nucleic acid encoding CD40L. 18. A recombinant MVA for use according to claim 16, wherein said TAA is an endogenous retroviral (ERV) protein. 19. A recombinant MVA for use according to claim 18, wherein the ERV protein is derived from the human endogenous retrovirus K (HERV-K) family and is selected from HERV-K-env and HERV-K-gag. 20. The recombinant MVA for use according to claim 16, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-related protein 1 (TRP1), tyrosine-related protein 1 (TRP2), Brachyury, FOLR1, PRAME, HERV-K-env, HERV-K-gag, p15, MEL and combinations thereof. 21. A recombinant Modified Vaccinia Ankara (MVA) for treating a subject with cancer, the recombinant MVA comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); and (b) a second nucleic acid encoding 4-1BBL. 22. The recombinant MVA according to claim 21, wherein said TAA is an ERV protein of the human endogenous retrovirus K (HERV-K) family. 23. The recombinant MVA according to claim 22, wherein the ERV protein is selected from the group consisting of HERV-K env protein and HERV-K gag protein. 24.c) A recombinant MVA according to claim 23, further comprising a third nucleic acid encoding CD40L. 25. A combination pharmaceutical comprising (a) a recombinant MVA as described in 24 above, and (b) at least one immune checkpoint molecule antagonist or immune checkpoint molecule agonist. 26. The combination drug according to claim 25, wherein the immune checkpoint molecule antagonist or the immune checkpoint molecule agonist is selected from antagonists or agonists of CTLA-4, PD-1, PD-L1, LAG-3, TIM-3 and ICOS. 27. A method for reducing tumor size and / or increasing survival in a subject having a cancerous tumor, said method comprising administering to said subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a tumor associated antigen (TAA), a second nucleic acid encoding 4-1BBL, and a third nucleic acid encoding CD40L, wherein administration of said recombinant MVA results in increased tumor reduction and / or increased overall survival in said subject compared to administration of the recombinant MVA, antigen 4-1BBL and antigen CD40L alone. 28. A method for inducing an enhanced inflammatory response in a cancerous tumor in a subject, the method comprising intratumorally administering to the subject a recombinant Modified Vaccinia Ankara (MVA) comprising a first nucleic acid encoding a first heterologous tumor-associated antigen (TAA), a second nucleic acid encoding the antigen 4-1BBL, and a third nucleic acid encoding the antigen CD40L, wherein intratumor administration of the recombinant MVA results in an enhanced inflammatory response in the tumor compared to an inflammatory response produced by non-intratumoral injection of a recombinant MVA virus comprising a first nucleic acid encoding a heterologous tumor-associated antigen, a second nucleic acid encoding the antigen 4-1BBL, and a third nucleic acid encoding the antigen CD40L. 29. The method according to claim 27, comprising administering the recombinant Modified Vaccinia Ankara (MVA) intravenously to the subject. 30. The method according to claim 27, comprising administering the recombinant modified vaccinia Ankara (MVA) intratumorally to the subject. 31. The method according to claim 27, comprising administering the recombinant Modified Vaccinia Ankara (MVA) intravenously and intratumorally to the subject. 32. The method according to claim 31, wherein the intravenous administration and the intratumoral administration are carried out at different times. EXAMPLES

[0226] The following examples illustrate the invention, but should not be construed in any way as limiting the scope of the claims.

[0227] Example 1: Construction of recombinant MVA-TAA-4-1BBL and MVA-TAA-CD40L The construction of recombinant MVA viruses embodying the elements of the invention was carried out by inserting the indicated transgenes together with their promoters into the vector MVA-BN. The transgenes were inserted using a recombinant plasmid containing the transgene and a selection cassette and sequences homologous to the target locus in MVA-BN. Homologous recombination between the viral genome and the recombinant plasmid was caused by transfecting the recombinant plasmid into CEF cells infected with MVA-BN. The selection cassette was then deleted in a second step with the aid of a plasmid expressing a CRE-recombinase that specifically targets the loxP sites flanking the selection cassette, thereby excising the intervening sequences.

[0228] For the construction of MVA-OVA and MVA-OVA-4-1BBL, the recombinant plasmids contained the transgene OVA or OVA and 4-1BBL, respectively, preceded by a promoter sequence, and a sequence identical to the target insertion site in MVA-BN for homologous recombination into the viral genome.

[0229] For the construction of MVA-OVA-CD40L, the recombinant plasmid contained the transgenes OVA and CD40L (each preceded by a promoter sequence) and sequences identical to the target insertion site in MVA-BN for homologous recombination into the viral genome.

[0230] For the construction of MVA-gp70-4-1BBL, the recombinant plasmid contained two transgenes, gp70 and 4-1BBL, each preceded by a promoter sequence, and sequences identical to the target insertion site in MVA-BN for homologous recombination into the viral genome. Similarly, for the construction of MVA-gp70-CD40L, the recombinant plasmid contained two transgenes, gp70 and CD40L, each preceded by a promoter sequence, and the appropriate modifications were made to the recombinant plasmid when construction of MVA-gp70-4-1-BBL-CD40L was desired.

[0231] For the construction of MVA-HERV-K, the recombinant plasmid contained the transgene HERV-K, for the construction of MVA-HERV-K-4-1BBL, the recombinant plasmid contained the transgenes HERV-K and 4-1BBL, and for the construction of MVA-HERV-K-4-1BBL-CD40L, the recombinant plasmid contained the transgenes HERV-K, 4-1BBL, and CD40L. Each transgene or group of transgenes was preceded by a promoter sequence and a sequence identical to the target insertion site in MVA-BN for homologous recombination into the viral genome.

[0232] In the construction of the mBN MVAs described above, CEF cell cultures were inoculated with MVA-BN and transfected with the respective recombinant plasmids. Samples from these cell cultures were then inoculated into CEF cultures in medium containing the selective pressure agent, and fluorescent expressing virus clones were isolated by plaque purification. In a second step, the selection cassette containing the fluorescent protein was deleted from these virus clones by CRE-mediated recombination (with two loxP sites in each construct flanking the selection cassette). After the second recombination step, only the transgene sequence (e.g., OVA, 4-1BBL, gp70, HERV-K and / or CD40L) was retained, with the promoter inserted in the target locus of MVA-BN. Stocks of plaque-purified viruses with the selection cassette deleted were prepared.

[0233] Cells inoculated with the constructs described demonstrate expression of a given transgene.

[0234] The constructs described herein were generated by using the clonal form of MVA-BN in bacterial artificial chromosomes (BAC). The recombinant plasmids contained the transgene sequences described below, each downstream of a promoter. The plasmids contained sequences that allow specific targeting of the integration site, as they are also present in MVA. Briefly, infectious virus was reconstituted from the BAC by transfecting BHK-21 cells with BAC DNA and superinfecting the cells with Shope fibroma virus as a helper virus. After three additional passages in CEF cell culture, the constructs were obtained in a helper virus-free state. An exemplary MVA generation method can also be found in Baur et al. ((2010) Virol. 84:8743-52, "Immediate-early expression of recombinant antigen by modified vaccinia virus Ankara breaks the immunodominance of strong vector-specific B8R antigen in acute and memory CD8 T-cell responses").

[0235] Example 2: 4-1BBL-mediated costimulation of CD8 T cells by MVA-OVA-4-1BBL-infected tumor cells affects cytokine production without the requirement for DCs Dendritic cells (DCs) were generated after 14 days of culture of bone marrow cells from C57BL / 6 mice in the presence of recombinant Flt3L. B16.F10 (melanoma model) cells were infected with MVA-OVA, MVA-OVA-CD40L, or MVA-OVA-4-1BBL at an MOI of 10 and cultured overnight at 37°C and 5% CO2. The next day, infected tumor cells were harvested and, where indicated, co-cultured for 4 hours at 37°C and 5% CO2 in the presence of DCs at a 1:1 ratio. Naive OVA(257-264)-specific CD8+ T cells were magnetically purified from OT-I mice and added to the above co-cultures at a ratio of 1:5. Cells were cultured for 48 hours at 37°C and 5% CO2. Culture supernatants were then collected for analysis of cytokine concentrations by Luminex. The results are shown in Figure 1 as supernatant concentrations of IL-6 (Figure 1A), GM-CSF (Figure 1B), IL-2 (Figure 1C) and IFN-γ (Figure 1D). Data are presented as mean ± SEM.

[0236] Consistent with previous reports, MVA-OVA-CD40L significantly affected DC activation and their antigen-presenting ability. That is, large amounts of IL-6 were produced by FLDC infected with MVA-OVA-CD40L (Fig. 1A). Importantly, OVA-specific T cell responses could be induced only in the presence of DC, but not directly by MVA-CD40L-infected B16.F10 cells themselves (Fig. 1B and 1C). These results indicate that DC are clearly required for the effect of MVA-OVA-CD40L. In contrast, MVA-OVA-4-1BBL did not induce IL-6 production in DC, whereas MVA-OVA-4-1BBL-infected B16.F10 cells induced the secretion of T cell activating cytokines IFN-γ, IL-2, and GM-CSF in a DC-independent manner (Fig. 1A-1D).

[0237] Example 3: Tumor cells infected with MVA-OVA-4-1BBL directly (i.e., without the need for DCs) promote the differentiation of antigen-specific CD8 T cells into activated effector T cells Dendritic cells (DCs) were generated after 14 days of culture of bone marrow cells from C57BL / 6 mice in the presence of recombinant Flt3L. B16.F10 (melanoma model) cells were infected with MVA-OVA, MVA-OVA-CD40L, or MVA-OVA-4-1BBL at an MOI of 10 and cultured overnight at 37°C and 5% CO2. The next day, infected tumor cells were harvested and, where indicated, co-cultured for 4 h at 37°C and 5% CO2 in the presence of DCs at a 1:1 ratio. Meanwhile, naive OVA(257-264)-specific CD8+ T cells were magnetically purified from OT-I mice and added to the above co-cultures at a 1:5 ratio. Cells were cultured for 48 h at 37°C and 5% CO2. Cells were subsequently stained and analyzed by flow cytometry. The results are shown in Figure 2 as the GMFI of T-bet on OT-I CD8+ T cells (Figure 2A) and the percentage of CD44+ Granzyme B+ IFN-γ+ TNFα+ OT-I CD8+ T cells (Figure 2B). Data are shown as the mean ± SEM.

[0238] The results show that in the absence of cross-presenting DCs, induction of granzyme B+ and IFNγ+ cytotoxic effector T cells was dependent on 4-1BBL (Figure 2B). Together with the results shown in Figure 1, these findings indicate that MVA-encoded 4-1BBL acts directly on T cells in a DC-independent manner, in contrast to MVA-encoded CD40L, which acts via DC activation.

[0239] Example 4: Infection with MVA encoding either CD40L or 4-1BBL induces tumor cell death in tumor cell lines and macrophages Tumor cell lines B16.OVA (Figures 3A and 3B), MC38 (Figure 3C) and B16.F10 (Figure 3D) were infected with the indicated MOI for 20 h. Cell viability was then analyzed by flow cytometry. Serum HMGB1 was quantified by ELISA in the samples of Figure 3A (Figure 3B). Bone marrow derived macrophages (BMDMs) were infected with the indicated MOI for 20 h. Cell viability was then analyzed by flow cytometry. Results are shown in Figures 3A-3E. Data are presented as mean ± SEM.

[0240] As shown in Figures 3A and 3B, infection with MVA-OVA or MVA-OVA-CD40L moderately induced cell death compared to PBS-treated tumor cells. Interestingly, infection with MVA-OVA-4-1BBL significantly enhanced tumor cell death 18 hours after infection.

[0241] To further confirm these results in non-antigenic cell lines, we performed similar assays using MC38 (Figure 3C) and B16.F10 (Figure 3D) tumor cells infected with MVA, MVA-CD40L, and MVA-4-1BBL (none of which encoded a TAA). Consistently, infection with the MVA induced cell death and efficiently killed bone marrow-derived macrophages (BMDMs) in these tumor cell lines (Figure 3E). Taken together, these data indicate that MVA infection kills tumor cells and macrophages, and that this killing is increased by recombinant MVA expression of CD40L or 4-1BBL.

[0242] Infection of tumor cells with oncolytic viruses induces so-called immunogenic cell death (ICD) (Workenhe et al. (2014) Mol. Ther. 22:251-56). ICD involves the release of intracellular proteins such as calreticulin, ATP, or HMGB1, which act as alarmins to the immune system and induce antitumor immunity by enhancing antigen presentation. We investigated whether infection with MVA induces ICD through secreted HMGB1. We unexpectedly found that MVA-OVA-4-1BBL and MVA-OVA-CD40L induced a significant increase in HMGB1 compared to MVA-OVA (Figure 3B).

[0243] Example 5: MVA encoding 4-1BBL induces NK cell activation in vivo Saline, or 5 × 10 7 TCID50 of MVA-OVA ("rMVA" in Figure 4), 5 × 10 7 TCID50 of MVA-OVA-4-1BBL ("rMVA-4-1BBL" in Figure 4) or 5 × 10 7 C57BL / 6 mice (n=5 / group) were immunized intravenously with either TCID50 of MVA-OVA combined with 200 μg of anti-4-1BBL antibody (clone TKS-1). After 24 hours, mice were sacrificed and spleens were processed for flow cytometric analysis. Results are shown in Figures 4A and 4B. Geometric mean fluorescence intensity (GMFI) of CD69 (Figure 4A) and CD70 (Figure 4B) are shown. Data are shown as mean ± SEM and are representative of two independent experiments.

[0244] The results showed that the addition of 4-1BBL to MVA-OVA enhanced the quality of the NK cell response compared to IV administration of MVA-OVA that does not encode 4-1BBL, and both NK cell activation markers CD69 and CD70 were significantly upregulated compared to MVA-OVA (Figures 4A and B). Co-injection of 4-1BBL blocking antibody demonstrated that MVA-OVA-induced NK cell activation was completely independent of 4-1BBL, but could be enhanced by delivery of excess 4-1BBL signaling by MVA-OVA-4-1BBL.

[0245] Example 6: Intravenous immunization with MVA encoding 4-1BBL enhances serum IFN-γ secretion in vivo Saline, or 5 × 10 7 TCID50 of "rMVA" (=MVA-OVA), 5 × 10 7 TCID50 of "rMVA-4-1BBL" (=MVA-OVA-4-1BBL) or 5 × 10 7 C57BL / 6 mice (n=5 / group) were immunized intravenously with either TCID50 of MVA-OVA combined with 200 μg of anti-4-1BBL antibody (clone TKS-1). Results are shown in Figures 5A and 5B. Data are shown as mean ± SEM. Figure 5A: After 6 hours, mice were bled, serum was isolated from whole blood, and IFN-γ concentrations in serum were determined by Luminex. Figure 5B: After 3, 21, and 45 hours, mice were injected intravenously with Brefeldin A to stop protein secretion. Mice were sacrificed 6, 24, and 48 hours after immunization, and splenocytes were analyzed by flow cytometry.

[0246] 4-1BB-mediated NK cell activation was consistent with increased serum levels of the NK effector cytokine IFNγr (Figure 5A). NK cells are known to produce large amounts of IFN-γ upon activation. To determine whether the increased serum IFN-γ levels could be derived from NK cells, the percentage of IFN-γ-producing NK cells was determined at various time points after intravenous injection of the indicated recombinant MVA vectors. Six hours after injection, when the measured IFN-γ serum levels were high, the percentage of IFN-γ+ NK cells was highest, and then slowly decreased (Figure 5B). The highest frequency of IFN-γ-positive NK cells was observed with MVA-OVA-4-1BBL. Taken together, these data indicate that intravenous immunization with rMVA-4-1BBL potently activates NK cells and increases the production of the NK cell effector cytokine IFN-γ.

[0247] Example 7: Intravenous immunization with rMVA-4-1BBL enhances serum IFN-γ secretion in B16.OVA tumor-bearing mice B16.OVA tumor-bearing C57BL / 6 mice (n=5 / group) were divided into groups and administered PBS or 5×10 7 Mice were administered iv (intravenously) with TCID50 of MVA-OVA (in the figure, "rMVA") or MVA-OVA-4-1BBL (in the figure, "rMVA-4-1BBL"). Six hours later, the mice were bled, serum was isolated from the whole blood, and IFN-γ concentrations in the serum were determined by Luminex. The results are shown in Figure 6. Data are presented as mean ± SEM.

[0248] The data presented in Figure 6 show that a similar effect on NK cells as reported in other experiments could be obtained in the melanoma tumor model. Six hours after immunization, tumor-bearing mice immunized with MVA-OVA-4-1BBL had a greater increase in serum IFN-γ levels, indicating a significant activation of NK cells (Figure 6).

[0249] Example 8: Intravenous prime and boost immunization with rMVA-4-1BBL enhances the expansion of antigen-specific and vector-specific CD8+ T cells 7A-7D show antigen-specific and vector-specific responses after intravenous prime and boost immunizations with rMVA-4-1BBL. C57BL / 6 mice (n=4 / group) were administered saline or 5×10 7 TCID50 of rMVA (=MVA-OVA), 5 × 10 7 TCID50 of rMVA-4-1BBL (=MVA-OVA-4-1BBL) or 5 × 10 7 Mice were primed intravenously on day 0 with either TCID50 of rMVA in combination with 200 μg of anti-4-1BBL antibody (clone TKS-1) and boosted on day 41. Blood was collected from mice on days 6, 21, 35, 48, and 64 after priming, and peripheral blood was analyzed by flow cytometry. Mice were sacrificed on day 70 after priming. Spleens were removed and analyzed by flow cytometry.

[0250] The results are shown in Figures 7A-7D. Figure 7A shows the percentage of antigen (OVA)-specific CD8+ T cells among peripheral blood leukocytes (PBL), and Figure 7B shows the percentage of vector (B8R)-specific CD8+ T cells among PBL. Figure 7C shows the percentage of antigen (OVA)-specific CD8+ T cells among live cells. Figure 7D shows the percentage of vector (B8R)-specific CD8+ T cells among live cells. Data are shown as mean ± standard error.

[0251] The results showed that the maximum number of B8- and OVA-specific CD8 T cells was reached on day 7 after the first immunization, and further increased on day 41 after the second immunization (Figures 7A and B). At day 41, the effect of rMVA-4-1BBL was evident compared to rMVA for both B8 and OVA in terms of antigen-specific T cell responses. Interestingly, co-injection of 4-1BBL blocking antibody showed that the T cell responses induced by rMVA were completely independent of 4-1BBL, but could be enhanced when excess 4-1BBL signals were delivered by MVA-OVA-4-1BBL (Figures 7A and B). Consistent with these results, prime / boost immunization with rMVA-4-1BBL improved OVA- and B8-specific T cell responses in the spleen 70 days after the first immunization (Figures 7C and D).

[0252] Example 9: Enhancement of antitumor activity by intravenous injection of MVA virus encoding TAA and 4-1BBL B16.OVA tumor-bearing C57BL / 6 mice (n=5 / group) were divided into groups and administered PBS or 5 × 10 7 TCID50 of MVA-OVA or 5 × 10 7 TCID50 of MVA-OVA-4-1BBL was administered iv. Tumor growth was measured at regular intervals. As shown in Figure 8, intravenous administration of MVA virus encoding 4-1BBL reduced tumor volume compared to MVA or control (PBS) by prolonging tumor growth delay.

[0253] Example 10: Enhancement of antitumor effects by intratumoral injection of MVA virus encoding 4-1BBL or CD40L B16.OVA tumor-bearing C57BL / 6 mice (n = 4-5 mice / group) were divided into groups and inoculated with PBS or 5 × 10 7TCID50 of MVA-OVA ("rMVA" in the figure), MVA-OVA-CD40L ("rMVA-CD40L" in the figure) or MVA-OVA-4-1BBL ("rMVA-4-1BBL" in the figure) was administered intratumorally (it). Tumor growth was measured at regular intervals. As shown in Figures 9A-9D, intratumoral injection of MVA virus encoding TAA and either 4-1BBL or CD40L resulted in enhanced antitumor activity. More specifically, as shown in Figure 9D, MVA virus encoding 4-1BBL significantly increased tumor growth reduction. Although the present invention is not limited to any particular mechanism or pathway of action, one hypothesis for the observed differences between 4-1BBL and CD40L is that 4-1BBL attempts to activate NK cells and T cells, whereas CD40L attempts to activate DCs. Because B16 melanoma tumors have a higher T cell infiltration (Mosely et al. (2016) Cancer Immunol. Res. 5(1):29-41), MVA encoding 4-1BBL is more effective in this condition than MVA encoding CD40L.

[0254] Regardless of the precise mechanism or pathway by which 4-1BBL and CD40L exert their effects on tumor growth or diameter, the data presented in Figure 9 demonstrate that intratumoral injection of MVA encoding 4-1BBL prolonged tumor growth control and, in some cases, led to complete tumor rejection.

[0255] Example 11: Enhancement of antitumor activity by intratumoral injection of MVA virus encoding TAA and CD40L in established colon cancer MC38 tumor-bearing C57BL / 6 mice (n=5 / group) were divided into groups and administered PBS or 5×10 7TCID50 of MVA-TAA ("rMVA" in figure) or MVA-TAA-CD40L ("rMVA-CD40L" in figure) was administered intratumorally (it). Tumor growth was measured at regular intervals. Results are shown in Figures 10A, 10B, 10C for an established non-antigenic MC38 colon carcinoma. In this experiment, TAAs expressed by MVA included the antigens AH1A5, p15E, and TRP2.

[0256] These results demonstrate that intratumoral injection of MVA encoding CD40L in addition to one or more TAAs can significantly delay tumor growth in the MC38 colon cancer model.

[0257] Example 12: Immune checkpoint blockade and tumor antigen-specific antibodies synergize with intratumoral administration of MVA-OVA-4-1BBL B16.OVA melanoma cells (5 × 10 5 5 × 10 cells / group) were subcutaneously injected into C57BL / 6 mice. When tumors reached approximately 5 mm in diameter, mice were divided into groups (n = 5 / group) and, where indicated (check), received 200 μg of IgG2a, anti-TRP-1 or anti-PD-1 intraperitoneally (ip). On days 13 (black dotted line), 18 and 21 (grey dashed line) after tumor inoculation, mice were treated with PBS or 5 × 10 cells / group. 7 Mice were immunized intratumorally (it) with either TCID50 of MVA-OVA-4-1BBL or 100 mM MVA-OVA-4-1BBL. Tumor growth was measured at regular intervals. The results are shown in Figure 11. Combining an antibody specific for the tumor-associated antigen (TAA) Trp1 (anti-Trp1) with intratumoral administration of MVA-OVA-4-1BBL enhanced tumor volume reduction compared to anti-PD-1 alone (Figure 11, middle row). Combining an antibody against the immune checkpoint molecule PD-1 with intratumoral administration of MVA-OVA-4-1BBL enhanced tumor volume reduction compared to anti-PD-1 alone (Figure 11, bottom row).

[0258] These experiments show that anti-PD-1 and anti-TRP-1 antibodies, as single agents, enhanced tumor growth control, while combining either antibody with MVA-OVA-4-1BBL improved the therapeutic effect provided by MVA-OVA-4-1BBL. In this example, combination therapy of intratumoral MVA 4-1BBL with either checkpoint blockade or TAA-targeting antibodies had greater therapeutic activity than either monotherapy. The data also show that tumor-specific antibodies that may induce ADCC may be combined with intratumoral injection of MVA expressing 4-1BBL to achieve synergistic effects.

[0259] Example 13: Intratumoral injection of MVA-OVA-4-1BBL has a superior antitumor effect to treatment with anti-CD137 agonist antibody B16.OVA tumor-bearing C57BL / 6 mice (n=5 / group) were divided into groups and administered PBS, 5×10 7 Either TCID50 of MVA-OVA-4-1BBL or 10 μg of anti-4-1BB (3H3, BioXcell) was injected intratumorally. Tumor growth was measured at regular intervals.

[0260] Figure 12A shows that the antitumor effect of MVA-OVA-4-1BBL is superior to that of the anti-4-1BBL agonist antibody (3H3). Figure 12B shows that only intratumoral immunization with MVA-OVA-4-1BBL induced OVA-specific T cell responses in the blood, whereas anti-4-1BBL agonist antibody did not induce any OVA-specific T cells in the blood.

[0261] Thus, these data indicate that intratumoral treatment with MVA-OVA-4-1BBL is more potent than an anti-CD137 agonist antibody in terms of both tumor-specific T cell responses and tumor growth control.

[0262] Example 14: Intravenous injection of MVA encoding CD40L and the endogenous retroviral (ERV) antigen Gp70 enhances antitumor activity in the CT26 tumor model CT26 tumor-bearing Balb / c mice (n=5 / group) were divided into groups, and on day 12 (black dotted line) after tumor introduction into the mice, PBS or 5×10 7 TCID50 of MVA-BN, MVA-Gp70, or MVA-Gp70-CD40L was administered intravenously (iv). Tumor growth was measured at regular intervals. As shown in Figures 13A and 13B, intravenous administration of MVA virus encoding the endogenous retroviral antigen Gp70 reduced tumor volume compared to MVA or control (PBS). In addition, encoding CD40L by MVA-Gp70-CD40L further enhanced its antitumor activity.

[0263] FIG. 13C shows that intravenous injection of MVA-Gp70 or MVA-Gp70-CD40L induced Gp70-specific CD8 T cells in the blood.

[0264] Thus, in these experiments, MVA was constructed to encode a model ERV, the murine protein gp70 (envelope protein of murine leukemia virus) ("MVA-gp70"). MVA was also generated that further contained the costimulatory molecule CD40L ("MVA-gp70-CD40L"). The potential antitumor effects of these novel constructs were tested using a CT26.wt colon cancer model. CT26.wt cells have been shown to express high levels of gp70 (see, e.g., Scrimieri (2013) Oncoimmunol. 2:e26889). CT26.wt tumor-bearing mice were generated and, once tumors were at least 5 mm x 5 mm, immunized intravenously as described above. Immunization with MVA alone induced only moderate tumor growth retardation. In contrast, immunization with MVA-gp70 led to complete rejection of 3 out of 5 tumors (Figures 13A and B). Immunization with MVA-Gp70-CD40L gave even more striking results, with 4 out of 5 tumors being rejected (FIGS. 13A and B).

[0265] To determine whether these antitumor responses correlated with the induction of gp70-specific T cells after immunization, blood was restimulated with the H-2Kd-restricted gp70 epitope AH1, and the results (Fig. 13C) show that gp70-specific CD8 T cell responses were potently induced in mice treated with MVA-Gp70 and MVA-Gp70-CD40L (Fig. 13C).

[0266] Example 15: Intravenous injection of MVA encoding CD40L and the endogenous retroviral antigen Gp70 enhances antitumor activity in the B16.F10 tumor model On the seventh day after tumor inoculation (black dotted line), when tumor measurements reached approximately 5 × 5 mm, B16.F10 tumor-bearing C57BL / 6 mice (n = 5 mice / group) were divided into groups and administered PBS or 5 × 10 7TCID50 of MVA-BN, MVA-Gp70, or MVA-Gp70-CD40L was administered intravenously (iv). Tumor growth was measured at regular intervals. As shown in Figure 14A, intravenous administration of MVA virus encoding the endogenous retroviral antigens Gp70 and CD40L reduced tumor volume compared to MVA or control (PBS).

[0267] FIG. 14B shows that intravenous injection of MVA-Gp70 or MVA-Gp70-CD40L induced Gp70-specific CD8 T cells in the blood.

[0268] Thus, in these experiments, the efficacy of MVA-Gp70 and MVA-Gp70-CD40L treatment was demonstrated in additional independent tumor models. B16.F10 is a C57BL / 6-derived melanoma cell line that expresses high levels of Gp70 (Scrimieri (2013) Oncoimmunol 2:e26889). Treatment with MVA alone ("MVA-BN") delayed the growth of B16.F10 tumors to a certain extent, comparable to the effect of unadjuvanted MVA-Gp70 (Figure 14A). However, MVA-Gp70-CD40L had a stronger antitumor effect than the control MVA scaffold alone (Figure 14A). Additional experiments showed that all groups receiving MVA encoding the Gp70 antigen induced CD8 T cell responses specific for the H-2Kb-restricted gp70 epitope p15e, but no significant increase in peripheral T cell responses was observed when the MVA also encoded CD40L (Figure 14B).

[0269] Example 16: Enhanced antitumor activity by intravenous injection of MVA virus encoding gp70 and 4-1BBL [hypothetical example] B16.OVA tumor-bearing C57BL / 6 mice (n=5 / group) were divided into groups and administered PBS or 5 × 10 7TCID50 of MVA-OVA or MVA-gp70-4-1BBL is administered intravenously. Tumor growth is measured at regular intervals. Mouse homologs of human endogenous retrovirus (ERV) proteins are not highly expressed in normal mouse tissues, nor are they significantly expressed in mouse tumor tissues, so the efficacy of human ERVs cannot be effectively studied in mouse models. Gp70 is a well-studied mouse ERV protein (see, for example, Bronte et al. (2003) J Immunol. 171(12):6396-6405, Bashratyan et al. (2017) Eur. J.Immunol. 47:575-584, and Nilsson et al. (1999) Virus Genes 18:115-120). Thus, testing of gp70-specific cancer vaccines in mice is very likely to provide strong predictive value for the efficacy of ERV-specific cancer vaccines in humans.

[0270] Example 17: Enhanced antitumor activity by intratumoral injection of MVA viruses encoding gp70 and either 4-1BBL or CD40L [hypothetical example] B16.OVA tumor-bearing C57BL / 6 mice (n = 4-5 mice / group) were divided into groups and inoculated with PBS or 5 × 10 7 TCID50 of MVA-OVA, MVA-OVA-CD40L, or MVA-OVA-4-1BBL was administered intratumorally (it). Tumor growth was measured at regular intervals.

[0271] Example 18: Administration of rMVA-HERV-K-4-1BBL affects cytokine production by direct antigen presentation of infected tumor cells [hypothetical example] Dendritic cells (DCs) are generated after 14 days of culture of bone marrow cells from C57BL / 6 mice in the presence of recombinant Flt3L. B16.F10 cells are infected overnight with MVA-HERV-K, MVA-HERV-K-CD40L, MVA-HERV-K-4-1BBL or MVA-HERV-K-4-1BBL-CD40L at an MOI of 10. The next day, infected tumor cells are harvested and, where indicated, co-cultured for 4 hours at 37°C and 5% CO2 in the presence of DCs at a 1:1 ratio. HERV-K-specific CD8+ T cells are magnetically purified from HERV-K-immunized mice and added to the above co-cultures at a 1:5 ratio. Cells are cultured for 48 hours at 37°C and 5% CO2. Culture supernatants are then harvested for analysis of cytokine concentrations by Luminex. Cytokine levels measured include (A) IL-6, (B) GM-CSF, (C) IL-2, and (D) IFNγ. Data are presented as mean ± SEM.

[0272] Example 19: Administration of rMVA-HERV-K-4-1BBL induces antigen-specific CD8+ T cells into activated effector T cells by direct antigen presentation from infected tumor cells [hypothetical example] Dendritic cells (DCs) are generated after 14 days of culture of bone marrow cells from C57BL / 6 mice in the presence of recombinant Flt3L. B16.F10 cells are infected with MVA-HERV-K, MVA-HERV-K-CD40L, MVA-HERV-K-4-1BBL or MVA-HERV-K-4-1BBL-CD40L at an MOI of 10 overnight. The next day, infected tumor cells are harvested and, where indicated, co-cultured for 4 hours at 37°C and 5% CO2 in the presence of DCs at a 1:1 ratio. Meanwhile, HERV-K-specific CD8+ T cells are magnetically purified from HERV-K-immune mice and added to the above co-cultures at a 1:5 ratio. Cells are cultured for 48 hours at 37°C and 5% CO2. Cells are then stained and analyzed by flow cytometry. Cytokine analysis is performed for (A) GMFI of T-bet on OT-I CD8+ T cells and (B) percentage of CD44+ Granzyme B+ IFNγ+ TNFα+ OT-I CD8+ T cells. Data are shown as mean ± SEM.

[0273] Example 20: Infection with rMVA-HERV-K encoding either CD40L or 4-1BBL induces tumor cell death in tumor cell lines and macrophages [hypothetical examples] Tumor cell lines B16.OVA (A and B), MC38 (C), and B16.F10 (D) are infected with the indicated MOI for 20 hours. Cell viability is then analyzed by flow cytometry. Serum HMGB1 is quantified by ELISA in samples from (A). Bone marrow derived macrophages (BMDMs) are infected with the indicated MOI for 20 hours. Cell viability is then analyzed by flow cytometry. Data are presented as mean ± SEM.

[0274] Example 21: Intratumoral administration of recombinant MVA encoding 4-1BBL reduces Treg cells and attenuates T cell exhaustion in tumors [hypothetical example] B16.OVA tumor-bearing C57BL / 6 mice (n=5 / group) were divided into groups and administered PBS or 5 × 107 TCID50 of MVA-OVA or MVA-OVA-4-1BBL was administered intratumorally (it). Five days later, mice were sacrificed and spleens and tumors were harvested and stained to assess Treg infiltration and T cell exhaustion with fluorochrome-conjugated antibodies. (A) Percentage of CD4+ FoxP3+ T cells among CD45+ leukocytes infiltrating the tumor, (B) Geometric mean fluorescence intensity of PD-1 on tumor-infiltrating CD8 T cells, and (C) Geometric mean fluorescence intensity of Lag-3 on tumor-infiltrating CD8 T cells. Data are shown as mean ± SEM.

[0275] Example 22: Immune checkpoint blockade and tumor antigen-specific antibodies synergize with intratumoral administration of rMVA gp-70-4-1BBL [hypothetical example] B16.OVA tumor-bearing C57BL / 6 mice (n=5 / group) were divided into groups and administered 200 μg of IgG2a, anti-TRP-1, or anti-PD-1 where indicated (checked). On days 13 (black dotted line), 18, and 21 (grey dashed line) after tumor inoculation, mice were treated with PBS or 5 × 10 7 Tumors are immunized intratumorally with either TCID50 of MVA-gp70-4-1BBL. Tumor growth is measured at regular intervals.

[0276] Example 23: Cytokine / chemokine MVA-BN scaffold responses to IT immunization can be augmented by 4-1BBL adjuvant action To assess the potential of recombinant MVA to induce inflammation in the tumor microenvironment (TME), cytokines and chemokines were analyzed in tissues derived from B16.OVA tumors. 5 B16.OVA cells were implanted subcutaneously (sc) into C57BL / 6 mice. On day 10, mice were treated with PBS or 2 × 10 8 TCID 50 Mice were immunized intratumorally (it) with MVA-BN, MVA-OVA, or MVA-OVA-4-1BBL (n = 5-6 mice / group).

[0277] Six hours after injection, cytokine and chemokine expression was measured (Figure 15). Cytokine / chemokine expression in PBS-treated tissues represents a basal inflammatory profile induced by the insertion of the needle into the tumor and the shear pressure of the saline solution. Cytokines including IL-6, IFN-α, IL-15 and TNF-α, and chemokines such as CXCL1, CCL2 and MIP2 were upregulated (Figure 15). IL-25 (also known as IL-17E), which is induced by the activation of NF-κβ in humans and stimulates the production of IL-8, was also detected (Lee et al. (2001) J. Biol. Chem. 276:1660-64). Interestingly, a significant increase in proinflammatory cytokines such as IL-6, IFN-α, or IL-15 / IL15Rα was observed in tumors injected with MVA-OVA-4-1BBL compared with tumor lesions injected with MVA-BN or MVA-OVA.

[0278] Example 24: MVA-OVA-4-1BBL enhances the proinflammatory cytokine / chemokine response to intratumoral (it) immunity Mice and tumors were treated as described in Example 23. Strikingly, several proinflammatory cytokines, including IFN-γ and GM-CSF, were produced only after intratumoral immunization with MVA-OVA-4-1BBL ( FIG. 16 ). Production of other proinflammatory cytokines, including IL-18, CCL5, CCL3, and IL-22, was enhanced upon intratumoral (it) immunization with either MVA-OVA or MVA-OVA-4-1BBL, but not MVA-BN or PBS alone.

[0279] Taken together, this data indicates that intratumoral (it) immunization with MVA can enhance the inflammatory response by inducing a proinflammatory cytokine / chemokine shift in the tumor microenvironment (TME). An enhanced effect was observed when intratumoral immunization was performed with MVA-OVA-4-1BBL compared to MVA or MVA-OVA. Thus, it can be said that the addition of 4-1BBL exerts an "adjuvant effect" on recombinant MVA.

[0280] Example 25: Quantitative and qualitative analysis of T cells in the TME and draining LNs after intratumoral injection of MVA-OVA-4-1BBL To gain a deeper understanding of the cellular processes induced by inflammation after intratumoral (it) injection of MVA-OVA and MVA-OVA-4-1BBL, we performed a detailed analysis of the innate and adaptive immune infiltration at various time points after it injection. B16.OVA tumor-bearing mice were treated with PBS or 2 × 10 8 TCID 50 Mice were injected intratumorally (it) with either MVA-OVA or MVA-OVA-4-1BBL. Mice were sacrificed 1, 3, and 7 days after prime immunization. Tumors and tumor-draining lymph nodes (TdLNs) were removed, treated with collagenase and DNase, and single cells were analyzed by flow cytometry. Immune cell populations were analyzed to determine their size, proliferative behavior, and functional status.

[0281] Results showed that injection of either MVA-OVA or MVA-OVA-4-1BBL into B16.OVA tumors resulted in increased expression of CD45 + It was shown that intratumoral infiltration of leukocytes was induced (Figure 17, top row, left histogram). Interestingly, especially after injection of MVA expressing 4-1BBL, CD45 expression in TdLNs was already observed 3 days after it (intratumoral) immunization. +An increase in leukocyte counts was observed (FIG. 17, top row, right histograms). In TdLNs, this difference was further amplified 7 days after intratumoral (it) immunization, suggesting that in TdLNs, the antitumor effect mediated by immunization with MVA is rapidly initiated on day 3 after immunization.

[0282] One aspect of vaccination-based antitumor therapy is the administration of tumor-specific CD8 + T cells and tumor-specific CD4 + The increase and reactivation of T cells and their enrichment in the tumor. One week after immunization, CD4 + T cells and CD8 + After it immunization with MVA-OVA-4-1BBL, CD4+ T cells increased by day 7 in the tumor, whereas in the TdLN, the increase started on day 3 and peaked on day 7. CD8 + T cells predominantly expressed CD45 + Injection of MVA-OVA-4-1BBL contributed to an increase in CD8 cells compared with injection of MVA-OVA. + The T cell population was further expanded in both the tumor (day 7) and the dLN (days 3 and 7).

[0283] OVA-specific CD8 + Quantification of T cells revealed that they were increased within the tumor microenvironment 7 days after intratumoral (it) immunization, especially in the MVA-OVA-4-1BBL-treated group (Figure 17, bottom left). Impressively, OVA-specific CD8 + The increase in T cells peaked on day 3 after immunization, and the increase was greater in the MVA-OVA-4-1BBL-treated group (Figure 17, bottom right). Taken together, these data suggest that intratumoral immunization with MVA-OVA, and especially MVA-OVA-4-1BBL, enhances the development of adaptive immune responses, beginning 3 days after treatment in the tumor-draining lymph nodes and increasing antigen-specific CD8 T cells by day 7 in the tumor microenvironment.+ A significant increase in T cells has been shown.

[0284] Example 26: Induction of antigen-specific CD8+ T cells by intratumoral injection of MVA-OVA-4-1BBL OVA-specific CD8 cells in tumor-draining lymph nodes (TdLNs) induced by intratumoral injection of MVA-OVA-4-1BBL + T cells showed high proliferation potential. OVA-specific CD8 T cells expressing Ki67 (an indicator of cell proliferation) + The percentage of T cells was higher in TdLNs after treatment with MVA-OVA than with PBS and was further increased in mice immunized with MVA-OVA-4-1BBL (Figure 18A). Furthermore, the exhaustion marker PD-1 was downregulated by OVA-specific CD8 T cells in the tumor by day 7 after immunization with MVA-OVA and MVA-OVA-4-1BBL, suggesting a restoration of functionality (Figure 18B).

[0285] Treg cells (also called "regulatory T cells") are potent inhibitors of antitumor immune responses (see, e.g., Tanaka et al. (2017) Cell Res. 27:109-118). Intratumoral injection of MVA-OVA increased the ratio of OVA-specific Teff / Treg (i.e., the ratio of "Teff" cells, i.e., "effector T cells," to Treg cells) in tumors, with a further increase observed 7 days after treatment with MVA-OVA-4-1BBL (Figure 18C). Thus, intratumoral treatment with MVA-OVA, and especially MVA-OVA-4-1BBL, reduced the frequency of intratumoral Tregs in favor of CD8+ T effector cells, which are beneficial for antitumor immune responses.

[0286] Example 27: Quantitative and qualitative analysis of NK cells in the TME and draining LNs after intratumoral injection of MVA-OVA-4-1BBL NK cell quantification after it immunization with MVA-OVA showed a decrease in NK cells in the tumor 1 day after intratumoral immunization (Figure 19, top row, left histogram). These changes were more pronounced when MVA-OVA-4-1BBL was used. At the same time, both immunization with MVA-OVA and immunization with MVA-OVA-4-1BBL increased NK cells in the tumor-draining lymph nodes (TdLN) 3 and 7 days after immunization (Figure 19, top row, right histogram), but MVA-OVA-4-1BBL induced the greatest increase in NK cells in TdLN.

[0287] CD69 is a marker of early activation of NK cells. Both MVA-OVA and MVA-OVA-4-1BBL viral vectors rapidly upregulated the activation marker CD69 in tumors and draining lymph nodes (TdLNs, FIG. 19, line 2). Furthermore, it immunization induced granzyme B in NK cells in both tumors and TdLNs at various time points, indicating enhanced function of cytotoxic NK cells (FIG. 19, line 3).

[0288] The proliferation ability of NK cells was then analyzed by the expression of Ki67. Ki67 expression on NK cells was significantly increased on day 3 in the tumors and TdLNs of mice intratumorally treated with either MVA-OVA or MVA-OVA-4-1BBL (Figure 19, bottom row).

[0289] These results show that 4-1BBL-adjuvanted MVA-OVA (i.e., MVA-OVA-4-1BBL) further increased the expression of CD69, granzyme B, and Ki67 surface markers on NK cells after intratumoral injection compared with MVA-OVA. These experiments also reveal a prominent role for the draining lymph nodes (TdLNs) in generating antitumor T-cell and antitumor NK-cell responses after intratumoral immunotherapy.

[0290] Although the present invention is not limited to any particular mechanism of action, the expansion of T cells in the TdLNs on day 3 and delayed intratumoral infiltration of T cells on day 7 (see FIG. 17) support a scenario in which tumor-specific T cells are primed and expanded in the TdLNs and then migrated to the tumor to kill tumor cells. It is also possible that intratumoral injection of viral vectors directly activates NK cells in the TdLNs, which further induces DC activation.

[0291] Example 28: Role of CD8 T cells in intratumoral MVA cancer therapy Analysis of T cell responses in tumors and TdLNs (e.g., FIG. 17) showed an increase in tumor-specific T cells at both sites after intratumoral (it) treatment. Experiments were performed to investigate the contribution of T cells to the antitumor effects mediated by MVA-OVA-4-1BBL. In these experiments, C57BL / 6 mice were inoculated with B16.OVA melanoma cells (5×10 5 Mice were injected with CD8 T cells (cells) and tumor growth was monitored after one of several treatments. Treatments included intratumoral (it) injections of PBS or MVA-OVA-4-1BBL in the presence or absence of 100 μg of CD8-T cell depleting antibody ("αCD8", clone 2.43) or isotype control antibody. When tumors reached a diameter of 5 mm, MVA-OVA-4-1BBL was injected (it) and the injections were repeated twice within one week. One day before the first injection of MVA-OVA-4-BBL, mice were injected ip with either anti-CD8 or IgG2b antibody, and the treatment was then repeated four times within two weeks. The data shown in FIG. 20 indicate that CD8 T cells were essential for effective MVA tumor therapy. Taken together, these data indicate that MVA-induced activation and expansion of tumor-specific CD8 T cells in tumors and TdLNs is a critical event for controlling tumor growth.

[0292] Example 29: Dependence of MVA-OVA and MVA-OVA-4-1BBL-mediated antitumor effects on Batf3+ DCs We investigated the role of various participating immune cells to elucidate the underlying cellular and molecular entities that contribute to the antitumor immune response induced by MVA-OVA-4-1BBL. Dendritic cells (DCs), which have the ability to potently phagocytose and present antigens and costimulatory signals to cells of the adaptive immune system, are considered as critical factors in antitumor immunity. Various subtypes of DCs, including CD8α+ DCs (also known as "cDC1"), are involved in activating a potent immune response against tumors. This DC subset has a unique ability to cross-present antigens during immune responses, and CD8α+ DCs are the primary producers of IL-12 in response to infection (Hochrein et al. (2001) J. Immunol. 166:5448-55; Martinez-Lopez et al. (2014) Eur. J. Immunol. 45:119-29) and cancer (Broz et al. (2014) Cancer Cell 26:638-52). CD8α+ DCs are also potent inducers of antitumor CD8+ T cells by cross-presenting tumor-associated antigens (Sanchez-Paulete et al., (2015) Cancer Discovery 6:71-79; Salmon et al. (2016) Immunity 44:924-38). Critically, the development of CD8α+ DCs depends on the transcription factor Batf3 (Hildner et al. (2008) Science 322:1097-1100).

[0293] We used wild-type and Batf3-deficient (Batf3- / -) B16.OVA tumor-bearing mice to assess the importance of this DC subset in intratumoral MVA cancer therapy. Figure 21A shows that B16.OVA tumors grew significantly faster in the absence of cross-presenting DCs (Batf3- / -), indicating a critical role for this antigen-presenting cell (APC) subset in inducing immune responses against tumors. Consistent with previous experiments, intratumoral injection of MVA-OVA delayed tumor growth in wild-type mice and, in one case, completely eliminated the tumor. Injection of mice with MVA-OVA-4-1BBL enhanced this effect, with tumors rejected in three of five mice treated with MVA-OVA-4-1BBL (Figure 21A). Interestingly, in the absence of cross-presenting DCs (Batf3- / -), intratumoral MVA immunotherapy was not impaired at all compared to the wild-type group (Fig. S21A). However, Batf3-DCs appear to be involved in the antitumor response induced by 4-1BBL (Fig. S21A, lower panel).

[0294] Eleven days after the first immunization, peripheral blood CD8 + Flow cytometry analysis of T lymphocyte populations (Figure 21B) revealed that Batf3 - / - In tumor-bearing mice, OVA-specific CD8 + It was shown that T cell frequencies were only slightly decreased compared to wild-type counterparts. Although the invention is not limited to or dependent on any particular mechanism of action, these data suggest that Batf3-dependent DCs play a redundant role in intratumoral cancer therapy using MVA.

[0295] Example 30: Role of NK cells in intratumoral administration of MVA-OVA-4-1BBL NK cells are known to express 4-1BB, and binding of 4-1BB on NK cells has been shown to increase the proliferation and cytotoxicity of these cells (Muntasell et al. (2017) Curr. Opin. Immunol. 45:73-81). In previous experiments (see FIG. 19), we found that intratumoral injection of MVA-OVA-4-1BBL significantly upregulated the activation marker CD69 and the cytotoxicity marker granzyme B on NK cells, which was accompanied by enhanced proliferation.

[0296] To investigate the role of NK cells in the antitumor immune response induced by 4-1BBL, we investigated the role of IL15Rα - / - Mice were used. The IL-15 receptor α subunit (IL-15Rα) mediates high affinity binding of IL-15, a multifunctional cytokine shown to be essential for NK cell development (Lodolce et al. (1998) Immunity 9:669-76). Wild-type, B16.OVA tumor-bearing mice and IL15Rα-deficient (IL15Rα - / - ) B16.OVA tumor-bearing mice were generated and immunized intratumorally with either MVA-OVA or MVA-OVA-4-1BBL. Mice treated with MVA-OVA showed similar therapeutic effects with or without IL-15Rα (Figure 22A). Interestingly, the benefit observed in wild-type mice with MVA-OVA-4-1BBL (tumor rejection in 3 out of 5 mice) was completely lost in IL15Rα-deficient tumor-bearing mice treated with MVA-OVA-4-1BBL (tumor rejection in 1 out of 5 mice, see Figure 22A). These results are also reflected in the survival rate of mice after tumor inoculation (Figure 22B).

[0297] It is known that IL15Rα deficiency not only affects NK cell development in mice, but also reduces T cell homeostasis and migration to LNs, and selectively reduces CD8 memory T cells (Lodolce et al. (1998) Immunity 9:669-76). Therefore, we also examined the T cell response to the above treatment. Consistent with our previous data, we observed that intratumoral (it) immunization with MVA-OVA induced OVA-specific CD8 T cells in wild-type mice, and this induction was further enhanced by MVA-OVA-4-1BBL (Figure 22C). However, IL15Rα - / - OVA-specific T cell responses in mice were similar to those seen in wild-type mice.

[0298] Although the present invention is not limited to any particular mechanism or pathway of action, these findings suggest that IL15Rα - / - It has been shown that tumor-bearing mice are capable of inducing tumor-specific T cell responses, thus supporting the notion that 4-1BBL-enhanced NK cell activation and function contributes to the therapeutic effect of intratumoral treatment with MVA-OVA-4-1BBL.

[0299] Example 31: NK cell-dependent cytokine / chemokine profile in response to intratumoral immunization with MVA-OVA-4-1BBL To identify cytokines selectively induced by 4-1BBL-4-1BB interactions on NK cells, PBS, or 5 × 10 7 Mice bearing wild-type B16.OVA tumors or IL15Rα treated intratumorally with TCID50 of MVA-OVA or MVA-OVA-4-1BBL - / - Cytokines and chemokines were analyzed in tumor tissues obtained from B16.OVA tumor-bearing mice.

[0300] Previous experiments showed that multiple cytokines and chemokines were increased 6 hours after intratumoral injection of recombinant MVA (Figures 15 and 16). In these experiments, when MVA-OVA-4-1BBL was injected into tumors, stimulation with 4-1BBL significantly increased the production of proinflammatory cytokines or chemokines, such as IFN-γ, CCL3, and CCL5, which are known to be produced by NK cells, compared to when MVA-OVA was injected (Figure 23). This increase induced by 4-1BBL was due to the upregulation of IL15Rα. - / - The tumor was completely abrogated in mice, indicating that intratumoral injection of rMVA-OVA-4-1BBL induced a distinct NK cell-driven cytokine and chemokine profile in the tumor microenvironment 6 hours after injection.

[0301] Example 32: Comparison of the antitumor effect of intratumoral immunization with MVA-gp70-CD40L and MVA-gp70-4-1BBL Gp70 is a self tumor antigen expressed in many syngeneic tumor models (B16.F10, CT26, MC38, 4T1, EL4, etc.), all of which display distinct tumor microenvironments (TMEs) in terms of stromal and immune cell composition. In this example, we tested the efficacy of MVA encoding the tumor antigen gp70 in addition to either CD40L or 4-1BBL in intratumoral immunization of B16.F10 tumor-bearing mice.

[0302] B16.F10 melanoma cells were injected subcutaneously into C57BL / 6 mice. The tumor size was approximately 50 mm 3 Once the tumor had reached 100%, mice were immunized intratumorally with PBS, MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, MVA-4-1BBL, or MVA-CD40L. The results are shown in FIG.

[0303] Immunization with MVA-gp70 induced a transient and moderate growth control of tumors, and this antitumor effect could be enhanced by expression of CD40L in the virus. However, intratumoral immunization with MVA-gp70-4-1BBL produced the most potent therapeutic effect, leading to complete tumor regression in two of five treated mice (Fig. 24A).

[0304] Strikingly, mice that were cured of their tumors after treatment with MVA-gp70-4-1BBL lost pigment in the areas where the tumors were found (Figure 24B). This depigmentation is indicative of the autoimmune pathology vitiligo and is the result of melanocyte destruction by autoreactive T cells. This melanocyte destruction suggests that immune system activation by recombinant MVA is not limited to the TAA encoded by the MVA (gp70 in this example). Rather, this expansion of immune system activation to other antigens (a phenomenon known as epitope spreading) results in a broader immune response that may improve treatment outcomes.

[0305] To evaluate the antigen-specific T cell responses induced by immunization, blood was collected 11 days after the first immunization and analyzed for the presence of antigen-specific T cells. Immunization with both MVA-gp70 and MVA-gp70-CD40L, as well as MVA-CD40L and MVA-4-1BBL, induced measurable p15E-specific T cell responses, ranging from 1 to 2% (Figure 24C). Importantly, this response was significantly (>5-fold) augmented in mice receiving MVA-gp70-4-1BBL. This antigen-specific T cell response to restimulation with p15E peptide correlated with the therapeutic efficacy in the different treatment groups.

[0306] Example 33: Antitumor effect of intratumor immunization with MVA-gp70-4-1BBL-CD40L A recombinant MVA expressing the tumor antigen gp70 together with 4-1BBL and CD40L was produced and tested intratumorally in a B16 melanoma model. B16.F10 melanoma cells were injected subcutaneously into C57BL / 6 mice. When tumors reached approximately 50 mm 3Once the tumor had reached 100% CI, mice were immunized intratumorally with PBS, MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, MVA-gp70-4-1BBL-CD40L, or the corresponding MVA constructs not expressing gp70.

[0307] Immunization with MVA-gp70 induced a transient and significant growth control of the tumor (Figure 25A). This antitumor effect could be enhanced by viral expression of CD40L or 4-1BBL. However, intratumoral immunization with MVA-gp70-4-1BBL-CD40L produced the most potent therapeutic effect, resulting in complete disappearance of tumors in four of five treated mice (Figure 25A). Strikingly, three of four cured mice treated with MVA-gp70-4-1BBL-CD40L showed a loss of pigment at the site of the tumor, which, as discussed in Example 32 above, is indicative of the vitiligo of the autoimmune pathology.

[0308] In addition, gp70-specific T cell responses were measured in blood 11 days after the first immunization. Immunization with MVA-gp70 and MVA-gp70-CD40L, as well as MVA-CD40L and MVA-4-1BBL, induced measurable tumor-specific T cell responses, ranging from 1 to 2%. This response was significantly (>5-fold) enhanced in mice receiving MVA-gp70-4-1BBL (Figure 25B).

[0309] Considering this, in the B16.F10 melanoma model, the antitumor effect of MVA-gp70 could be enhanced when adjuvanted with either CD40L or 4-1BBL, but an even stronger effect was observed when 4-1BBL and CD40L were co-expressed in MVA-gp70-4-1BBL-CD40L.

[0310] Example 34: Intratumoral immunotherapy with MVA-gp70-4-1BBL-CD40L in CT26.WT tumors The constructs were then tested using the CT26 colon cancer model, which has been described as being enriched for T cells and myeloid cells and deemed immunogenic (see, e.g., Mosely et al. (2016) Cancer Immunol. Res. 5:29-41). Balb / c mice were injected subcutaneously (sc) with CT26.wt colon cancer cells. When tumors reached approximately 60 mm3, mice were immunized intratumorally with PBS, MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, MVA-gp70-4-1BBL-CD40L, or MVA-4-1BBL-CD40L.

[0311] Immunization with MVA-gp70 it induced a transient and significant growth control of the tumor. Expression of CD40L on MVA did not enhance this antitumor effect, but strikingly, immunization with MVA-gp70-4-1BBL produced the strongest therapeutic effect, with tumors disappearing completely in all treated mice (Fig. 26A). However, treatment with MVA-gp70-4-1BBL-CD40L did not improve the therapeutic effect. Of note, a virus that does not code for gp70, but only for costimulatory molecules, also produced a significant tumor growth delay, but did not achieve results comparable to those of MVA-gp70-4-1BBL. These findings were reflected in the overall survival of treated mice (Fig. 26B).

[0312] Gp70-specific T cell responses against the H2-Ld CD8+ T cell epitope AH-1 were readily detected in the blood of mice treated with MVA-gp70 and MVA-gp70-CD40L (Figure 26C). This response was significantly (>10-fold) increased in mice receiving MVA-gp70-4-1BBL, correlating with the therapeutic effect shown in Figures 26A and 26B. Treatment with MVA-gp70-4-1BBL-CD40L also enhanced AH-1-specific T cell responses in the blood (Figure 26C).

[0313] Example 35: Comprehensive analysis of the tumor microenvironment and tumor-draining LNs following IT injection of MVA-gp70-4-1BBL-CD40L into B16.F10 tumor-bearing mice The above data showed that when B16.F10 tumor-bearing mice were treated intratumorally with MVA-gp70-4-1BBL-CD40L, tumors were rejected in 80% of treated mice (see FIG. 26). To examine the tumor microenvironment (TME) and TdLN in this tumor model, B16.F10 tumor-bearing mice were injected with PBS or 5×10 7 TCID50 of either MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L or MVA-gp70-4-1BBL-CD40L was administered intratumorally (it). Mice were sacrificed 3 days after prime immunization. Day 3 was chosen based on previous experiments with the OVA system, where changes in both innate and adaptive immune system components were observed at this time point (see FIG. 17). Tumors and TdLNs were removed and digested with collagenase / DNase for single cell analysis using flow cytometry. The abundance of immune cell populations, as well as their proliferative behavior and functional status, were evaluated.

[0314] Intratumoral injection of 4-1BBL-adjuvanted MVA and CD40L-adjuvanted MVA did not produce significant results in the number of CD8 T cells or p15E-specific T cells in the tumor at day 3, as measured by pentamer staining. However, in TdLN, MVA-gp70 and MVA-gp70-CD40L increased CD8 T cells, with an even greater effect when 4-1BBL was added (Figure 27, top right). The increase obtained by adding 4-1BBL was even more pronounced for p15E-specific CD8 T cells in TdLN, so that it immunization with either MVA-gp70-4-1BBL or MVA-gp70-4-1BBL-CD40L increased tumor-specific CD8 T cells (Figure 27, center right). The number of p15E-specific CD8 T cells also correlated with the proliferation status of these cells. For example, the addition of gp70 and optionally CD40L to MVA together with 4-1BBL induced the most abundant Ki67+ gp70-p15E CD8 T cells in TdLNs (Figure 27, bottom right).

[0315] These data indicate that intratumoral (it) immunization with MVA-gp70 enhanced the development of adaptive immune responses in tumors and tumor-draining lymph nodes 3 days after treatment, while the adjuvant effect of 4-1BBL or 4-1BBL plus CD40L specifically increased p15E-specific CD8 T cell responses in TdLNs.

[0316] Example 36: Induction of NK cells in tumors and TdLNs after intratumoral injection of MVA Intratumoral (it) injection of MVA-OVA activated and expanded NK cells on days 1 and 3, respectively (Figure 19). We then examined the infiltration, activation, and expansion of NK cells on day 3 after injection of various MVA constructs. NK cell quantification after it immunization with recombinant MVA showed an increase in NK cells infiltrating the tumor (Figure 28, top left) and TdLN (Figure 28, top right). Infiltration was increased when MVA was encoded with 4-1BBL (e.g., MVA-gp70-4-1BBL and MVA-gp70-4-1BBL-CD40L). Intratumoral (it) injection of MVA-gp70 induced proliferation of NK cells (Ki67+) in tumors (see FIG. 28, center left) and TdLNs (FIG. 28, center right), and adjuvanting with 4-1BBL or 4-1BBL and CD40L enhanced this effect in TdLNs.

[0317] Granzyme B is a marker of NK cell cytotoxicity (see, e.g., Ida et al. (2005) Mod. Rheumatol. 15:315-22). Granzyme B+ NK cells were induced in tumors and TdLNs after intratumoral injection of recombinant MVA (Figure 28, bottom left). Also in this example, the number of cytotoxic NK cells in TdLNs was moderately increased when 4-1BBL or 4-1BBL-CD40L was added to recombinant MVA (Figure 28, bottom right).

[0318] Taken together, these data highlight a critical role for MVA-encoded 4-1BBL-CD40L in the expansion and function of NK cells and TAA-specific T cells following intratumoral (it) immunotherapy: intratumoral treatment with recombinant MVA encoding gp70 and 4-1BBL or gp70, 4-1BBL and CD40L can enhance T cell responses against endogenous retroviral self-antigens such as gp70.

[0319] Example 37: Intravenous immunotherapy with MVA-gp70-4-1BBL-CD40L in CT26.WT tumor-bearing mice The above experiments showed that the novel MVA constructs encoding the tumor antigen gp70 together with the costimulatory molecules 4-1BBL and CD40L were highly potent when applied intratumorally (Figures 25 and 26). In addition, Lauterbach et al. ((2013) Front. Immunol. 4:251) found that MVA encoding CD40L enhanced innate and adaptive immune responses when administered intravenously. In this example, we asked whether intravenous (iv) immunization with MVA-gp70-4-1BBL-CD40L could also control tumor growth.

[0320] CT26.WT colon cancer cells were injected subcutaneously into Balb / c mice. When tumors reached approximately 60 mm3, mice were immunized intravenously with PBS or MVA-Gp70, MVA-Gp70-4-1BBL, MVA-Gp70-CD40L, MVA-gp70-4-1BBL-CD40L, and MVA-4-1BBL-CD40L (no gp70). Two out of five mice immunized iv with MVA-gp70 disappeared from the tumor (Figure 29A). Mice treated with gp70-expressing viruses containing either 4-1BBL or CD40L exhibited a significantly improved antitumor response, with 3 out of 5 mice cured with 4-1BBL and 4 out of 5 mice cured with CD40L. Importantly, iv treatment with MVA-gp70-4-1BBL-CD40L prolonged tumor growth control in all treated mice, with tumors rejected in 3 of 5 mice (Figure 29A). Of note, recombinant MVA containing only costimulatory molecules but no gp70 also significantly delayed tumor growth, but did not result in tumor rejection similar to that observed with MVA-gp70-4-1BBL, MVA-gp70-CD40L, or MVA-gp70-4-1BBL-CD40L (Figure 29A). These findings were reflected in the overall survival of treated mice (Figure 29B).

[0321] Analysis of tumor-directed CD8 T cell responses in the blood by peptide restimulation of PBL revealed that AH1-specific CD8 T cells were significantly induced in all MVA-treated groups, which could be further augmented in the presence of CD40L (i.e., MVA-gp70-CD40L and MVA-gp70-4-1BBL-CD40L) (Figure 29C).

[0322] Example 38: Recombinant MVA containing HERV-K antigen An MVA-based vector (also referred to as "MVA-mBN489", "MVA-HERV-Prame-FOLR1-4-1-BBL-CD40L") is designed that contains a TAA that is a human endogenous retroviral K superfamily protein (HERV-K), specifically ERV-K-env and ERV-K-gag. The MVA is also designed to encode human FOLR1 and PRAME, and to express h4-1BBL, hCD40L, or both h4-1BBL and hCD40L.

[0323] Example 39: Intratumoral immunization with MVA encoding Brachyury antigen The highly attenuated non-replicating vaccinia virus MVA-BN-Brachyury is designed to contain four human transgenes that induce specific and vigorous immune responses against various cancers. The vector co-expresses the human TAA Brachyury and three human costimulatory molecules, namely B7.1 (also known as CD80), intercellular adhesion molecule-1 (ICAM-1, also known as CD54), and leukocyte function-associated antigen-3 (LFA-3, also known as CD58). The three costimulatory molecules, i.e., TRIad of COstimulatory Molecules (TRICOM™), are included to maximize the immune response against the human TAA Brachyury.

[0324] Brachyury is a transcription factor within the T-box family and a driver of EMT, a process associated with cancer progression. Brachyury is overexpressed in cancer cells compared to normal tissues and has been associated with resistance of cancer cells to several therapies and metastatic potential. Cancers known to express Brachyury include lung, breast, ovarian, chordoma, prostate, colorectal and pancreatic adenocarcinoma.

[0325] In vitro and clinical studies have been conducted to demonstrate the safety and potential therapeutic efficacy of MVA encoding Brachyury. See, e.g., Hamilton et al. (2013) Oncotarget 4:1777-90 ("Immunological targeting of tumor cells undergoing an epithelial-mesenchymal transition via a recombinant brachyury-yeast vaccine"), Heery et al. (2015a) J. Immunother. Cancer 3:132 ("Phase I, dose escalation, clinical trial of MVA-brachyury-TRICOM vaccine demonstrating safety and brachyury-specific T cell responses"), and Heery et al. (2015b) Cancer Immunol. Res.3:1248-56 ("Phase I trial of a yeast-based therapeutic cancer vaccine (GI-6301) targeting the transcription factor brachyury")).

[0326] To support the use of the intravenous route during Phase 1 clinical development, a GLP-compliant repeat-dose toxicity study will be conducted in NHPs (cynomolgus monkeys) to evaluate any potential toxicity of MVA-BN-Brachyury (MVA-mBN240B), including a biodistribution part to evaluate the spatial and temporal distribution of MVA-BN-Brachyury in NHPs.

[0327] MVA-BN-Brachyury will be used in Phase III trials to treat cancer patients by intratumoral injection of MVA, optionally in combination with other treatments such as radiation and / or checkpoint inhibitors.

[0328] It will be apparent that the exact details of the methods or compositions described herein may be changed or modified without departing from the spirit of the described invention, and we claim all such modifications or variations that come within the scope and spirit of the following claims.

[0329] Sequence Listing The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard abbreviations for nucleotide bases and either one-letter or three-letter codes for amino acids, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but any reference to the depicted strand should be understood to include the complementary strand.

[0330] Sequences in the sequence listing: SEQ ID NO: 1: Amino acid sequence of hCD40L (261 amino acids) from NCBI RefSeq NP_000065.1 SEQ ID NO:2: hCD40L (792 nucleotides) from NCBI RefSeq NP_000065.1 SEQ ID NO: 3: h4-1BBL (254 amino acids) from NCBI RefSeq NP_003802.1 SEQ ID NO: 4: h4-1BBL in NCBI RefSeq NP_003802.1

[0331] SEQ ID NO:1 hCD40L (261 amino acids) from NCBI RefSeq NP_000065.1 MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEA SSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

[0332] SEQ ID NO:2 hCD40L (792 nucleotides) from NCBI RefSeq NP_000065.1 nt sequence: atgatcgagacatacaaccagacaagccctagaagcgccgccacaggactgcctatcagcatgaagatcttcatgtacctgctgaccgtgttcctgatcacccagatgatcggcagcgccctgtttgccgtgtacctgcacagacggctggacaagatcgaggacgagagaaacctgcacgaggacttcgtgttcatgaagaccatccagcggtgcaacaccggcgagagaagtctgagcctgctgaactgcgaggaaatcaagagccagttcgagggcttcgtgaaggacatcatgctgaacaaagaggaaacgaagaaagagaactccttcgagatgcagaagggcgaccagaatcctcagatcgccgctcacgtgatcagcgaggccagcagcaagacaacaagcgtgctgcagtgggccgagaagggctactacaccatgagcaacaacctggtcaccctggagaacggcaagcagctgacagtgaagcggcagggcctgtactacatctacgcccaagtgaccttctgcagcaacagagaggccagctctcaggctcctttcatcgccagcctgtgcctgaagtctcctggcagattcgagcggattctgctgagagccgccaacacacacagcagcgccaaaccttgtggccagcagtctattcacctcggcggagtgtttgagctgcagcctggcgcaagcgtgttcgtgaatgtgacagaccctagccaggtgtcccacggcaccggctttacatctttcggactgctgaagctgtgatgatag

Claims

1. - a recombinant modified vaccinia Ankara (MVA), comprising (a) a first nucleic acid encoding a tumor associated antigen (TAA) and (b) a second nucleic acid encoding 4-1BBL, the recombinant modified vaccinia Ankara (MVA) being administered intratumorally to a subject; and - optionally a carrier and / or a pharma- ceutically acceptable diluent, and administered intratumorally to a subject.

2. a recombinant modified vaccinia Ankara (MVA) comprising (a) a first nucleic acid encoding a tumor associated antigen (TAA) and (b) a second nucleic acid encoding 4-1BBL, which is administered intratumorally to a subject; - at least one immune checkpoint molecule antagonist or immune checkpoint molecule agonist, and - optionally in combination with a carrier and / or a pharma- ceutically acceptable diluent, and administered intratumorally to a subject.

3. The recombinant modified vaccinia Ankara virus (MVA), (c) a third nucleic acid encoding CD40L.

3. The pharmaceutical composition of claim 1 or 2, further comprising:

4. A pharmaceutical composition described in any one of claims 1 to 3, wherein the first nucleic acid encoding the TAA encodes an endogenous retrovirus (ERV) protein.

5. The pharmaceutical composition of claim 4, wherein the ERV protein is derived from the human endogenous retrovirus K (HERV-K) family and is selected from HERV-K-env and HERV-K-gag.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-related protein 1 (TRP1), tyrosine-related protein 1 (TRP2), brachyury, FOLR1, PRAME, HERV-K-env, HERV-K-gag, endogenous retrovirus (ERV) antigens Gp70, p15, MEL, and combinations thereof.

7. The pharmaceutical composition according to claim 1, wherein the MVA used to obtain the recombinant MVA is MVA-BN deposited at the European Collection of Cell Cultures (ECACC) under the number V00083008.

8. 3. The pharmaceutical composition of claim 2, wherein the immune checkpoint molecule antagonist or the immune checkpoint molecule agonist is selected from an antagonist or agonist of CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and ICOS.

9. 3. The pharmaceutical composition of claim 2, for use in the manufacture of a medicament for treating a tumor in a subject by intratumoral administration.

10. A recombinant Modified Vaccinia Ankara (MVA), comprising (a) a first nucleic acid encoding a tumor associated antigen (TAA) and (b) a second nucleic acid encoding 4-1BBL, administered intratumorally to a subject, and use of the recombinant Modified Vaccinia Ankara (MVA) in the manufacture of a medicament for enhancing inflammatory response in a cancerous tumor, reducing tumor size and / or increasing survival rate in a subject having a cancerous tumor, by intratumoral administration.

11. The recombinant modified vaccinia Ankara virus (MVA), (c) a third nucleic acid encoding CD40L. The use of claim 10, further comprising:

12. The use described in claim 10 or 11, wherein the first nucleic acid encoding the TAA encodes an endogenous retroviral (ERV) protein.

13. The use of claim 12, wherein the ERV protein is derived from the human endogenous retrovirus K (HERV-K) family and is selected from HERV-K-env and HERV-K-gag.

14. The use according to any one of claims 10 to 13, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-related protein 1 (TRP1), tyrosine-related protein 1 (TRP2), brachyury, FOLR1, PRAME, HERV-K-env, HERV-K-gag, endogenous retrovirus (ERV) antigens Gp70, p15, MEL, and combinations thereof.

15. The use according to any one of claims 10 to 14, wherein the MVA used to obtain the recombinant MVA is MVA-BN deposited at the European Collection of Cell Cultures (ECACC) under number V00083008.

16. 2. Use of the pharmaceutical composition of claim 1 for use in the manufacture of a medicament for enhancing inflammatory response in cancerous tumors, reducing tumor size and / or increasing survival rate in a subject having a cancerous tumor by intratumoral administration.

17. 3. Use of the pharmaceutical composition of claim 2 for use in the manufacture of a medicament for enhancing inflammatory response in cancerous tumors, reducing tumor size and / or increasing survival rate in subjects with cancerous tumors by intratumoral administration. Claim 18: A recombinant Modified Vaccinia Ankara (MVA), comprising: (a) a first nucleic acid encoding a tumor associated antigen (TAA); (b) a second nucleic acid encoding 4-1BBL; and (c) a third nucleic acid encoding CD40L, wherein the recombinant Modified Vaccinia Ankara (MVA) is administered intratumorally to a subject; and - optionally a carrier and / or a pharma- ceutically acceptable diluent, and administered intratumorally to a subject. Claim 19: A recombinant Modified Vaccinia Ankara (MVA), comprising: (a) a first nucleic acid encoding a tumor associated antigen (TAA); (b) a second nucleic acid encoding 4-1BBL; and (c) a third nucleic acid encoding CD40L, wherein the recombinant Modified Vaccinia Ankara (MVA) is administered intratumorally to a subject, and wherein the first nucleic acid encoding the TAA encodes an endogenous retrovirus (ERV) protein. - at least one immune checkpoint molecule antagonist or immune checkpoint molecule agonist, and - optionally a carrier and / or a pharma- ceutically acceptable diluent, and administering the same intratumorally to a subject.

20. The pharmaceutical composition of claim 19, wherein the ERV protein is derived from the human endogenous retrovirus K (HERV-K) family and is selected from HERV-K-env and HERV-K gag.

21. The pharmaceutical composition of claim 19 or 20, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-related protein 1 (TRP1), tyrosine-related protein 1 (TRP2), brachyury, FOLR1, PRAME, HERV-K-env, HERV-K-gag, endogenous retrovirus (ERV) antigens Gp70, p15, MEL, and combinations thereof.

22. The pharmaceutical composition according to any one of claims 19 to 21, wherein the MVA used to obtain the recombinant MVA is MVA-BN deposited at the European Collection of Cell Cultures (ECACC) under number V00083008.

23. The pharmaceutical composition of any one of claims 19 to 22, wherein the immune checkpoint molecule antagonist or the immune checkpoint molecule agonist is selected from an antagonist or agonist of CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and ICOS.

24. The pharmaceutical composition of any one of claims 19 to 23, for use in the manufacture of a medicament for treating a tumor in a subject by intratumoral administration.

25. Use of a recombinant Modified Vaccinia Ankara (MVA), comprising (a) a first nucleic acid encoding a tumor associated antigen (TAA), (b) a second nucleic acid encoding 4-1BBL, and (c) a third nucleic acid encoding CD40L, administered intratumorally to a subject, wherein the first nucleic acid encoding the TAA encodes an endogenous retrovirus (ERV) protein, in the manufacture of a medicament for enhancing inflammatory response in a cancerous tumor, reducing tumor size and / or increasing survival rate in a subject having a cancerous tumor, upon intratumoral administration of the recombinant Modified Vaccinia Ankara (MVA).

26. The use of claim 25, wherein the ERV protein is derived from the human endogenous retrovirus K (HERV-K) family and is selected from HERV-K-env and HERV-K gag.

27. The use of claim 25 or 26, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-related protein 1 (TRP1), tyrosine-related protein 1 (TRP2), brachyury, FOLR1, PRAME, HERV-K-env, HERV-K-gag, endogenous retrovirus (ERV) antigens Gp70, p15, MEL, and combinations thereof.

28. The use according to any one of claims 25 to 27, wherein the MVA used to obtain the recombinant MVA is MVA-BN deposited at the European Collection of Cell Cultures (ECACC) under number V00083008.

29. 20. Use of the pharmaceutical composition of claim 18 in the manufacture of a medicament for enhancing an inflammatory response in a cancerous tumor, reducing tumor size and / or increasing survival rate in a subject having a cancerous tumor by intratumoral administration.

30. 20. Use of the pharmaceutical composition of claim 19 in the manufacture of a medicament for enhancing an inflammatory response in a cancerous tumor, reducing tumor size and / or increasing survival rate in a subject having a cancerous tumor by intratumoral administration.

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