Recombinant MVA viruses for intratumoral and / or intravenous administration to treat cancer

Abstract

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

Description

Field of the Invention

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

[0003] Recombinant poxviruses have been used as immunotherapy vaccines against infectious organisms and, more recently, against tumors (Mastrangelo et al. (2000) J Clin Invest. 105(8): 1031-1034).

[0004] One poxvirus strain that has been shown to be useful as an immunotherapy vaccine against infectious diseases and cancer is the modified vaccinia Ankara (MVA) virus (sometimes referred to simply as "MVA"). MVA was produced in chicken embryo fibroblasts (CVA) by 516 serial passages of the Ankara vaccinia virus strain (reviewed in Mayr et al. (1975) Infection 3: 6-14). As a result of these long-term passages, the genome of the resulting MVA virus has lost approximately 31 kb of genomic sequence and has therefore been described as highly restricting replication to avian host cells (Meyer et al. (1991) J. Gen. Virol. 72: 1031-1038). The resulting MVA has been shown to be remarkably avirulent in various animal models (Mayr and Danner (1978) Dev. Biol. Stand. 41: 225-34). MVA strains with enhanced safety have been described for use in the development of safer products (e.g., vaccines or drugs) (see International PCT Publication WO2002042480; see also, for example, U.S. Patent Nos. 6,761,893 and 6,913,752, all of which are incorporated herein by reference). Such variants are capable of reproductive replication in non-human cells and cell lines, particularly chicken embryo fibroblasts (CEFs), but are unable to replicate in human cell lines, including, in particular, HeLa, HaCat, and 143B cell lines. Such strains are also unable to reproductively replicate in vivo, for example, in certain mouse strains, such as the transgenic mouse model AGR 129, which is severely immunocompromised and highly susceptible to replicating viruses (see U.S. Patent No. 6,761,893). Such MVA variants and derivatives thereof, including recombinants, have been described and are referred to as "MVA-BN" (see International PCT Publication WO 2002 / 042480; see also, eg, US Patent Nos. 6,761,893 and 6,913,752).

[0005] The use of poxvirus vectors encoding tumor-associated antigens (TAAs) has been shown to successfully reduce tumor size and improve overall survival in cancer patients (see, for example, WO 2014 / 062778). It has been demonstrated that when poxvirus vectors encoding TAAs (such as HER2, CEA, MUC1, and / or Brachyury) are administered to cancer patients, the patients develop a robust and specific T cell response to fight the cancer (ibid.; 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).

[0006] A TAA found to be expressed in many cancers and tumor cells is an endogenous retrovirus (ERV) protein. ERV is a remnant of a former exogenous form that invades the host's reproductive system and is thereafter vertically transmitted through genetic populations (see Bannert et al. (2018) Frontiers in Microbiology, Vol. 9, No. 178). ERV-induced genomic recombination events and the dysregulation of normal cell genes have been shown to promote tumor formation (ibid). In addition, there is evidence that certain ERV proteins have oncogenic properties (ibid). ERV has been found to be expressed in a variety of cancers, including, for example, breast cancer, ovarian cancer, melanoma, prostate cancer, pancreatic cancer, and lymphoma. (See, e.g., 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).

[0007] In addition to their effectiveness against TAAs, poxviruses such as MVA have been shown to have enhanced efficacy when combined with CD40 agonists such as CD40 ligand (CD40L) (see WO 2014 / 037124) or with 4-1BB agonists such as 4-1BB ligand (4-1BBL) (Spencer et al. (2014) PLoS One 9:e105520).

[0008] CD40 / CD40L is a member of the tumor necrosis factor receptor / tumor necrosis factor ("TNFR / TNF") superfamily. While CD40 is constitutively expressed on many cell types, including B cells, macrophages, and DCs, its ligand, CD40L, is primarily expressed 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). Early after infection or immunization, homologous interactions between DCs and CD4+ T cells 'permit' DCs to elicit CD8+ T cell responses (Ridge et al. (1998) Nature 393: 474-478). DC permissiveness results in upregulation of co-stimulatory molecules, increased survival, and better DC 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), but 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 proposed, providing a possible explanation for the generation of helper-independent CTL responses (Johnson et al. (2009) Immunity 30: 218-227).

[0009] 4-1BB / 4-1BBL is a member of the TNFR / TNF superfamily. 4-1BBL is a co-stimulatory ligand expressed in activated B cells, monocytes and DCs. 4-1BB is constitutively expressed by natural killer (NK) and natural killer T (NKT) cells, Treg and several innate immune cell populations (including DCs, monocytes and neutrophils). Interestingly, 4-1BB is expressed on activated rather than resting T cells (Wang et al. (2009) Immunol. Rev. 229: 192-215). 4-1BB ligation induces the proliferation and production of interferon gamma (IFN-γ) and interleukin 2 (IL-2), and enhances T cell survival by upregulating anti-apoptotic molecules such as Bcl-xL (Snell et al. (2011) Immunol. Rev. 244: 197-217). Importantly, 4-1BB stimulation enhances NK cell proliferation, IFN-γ production, and cytolytic activity by enhancing antibody-dependent cellular cytotoxicity (ADCC) (Kohrt et al. (2011) Blood 117:2423-32).

[0010] The 4-1BB / 4-1BBL immune axis is currently being explored through different immunotherapy strategies. For example, autologous transfer of chimeric antigen receptor (CAR) T cells has shown clinical benefits in large B-cell lymphoma and was approved by the FDA in 2017. The patient's autologous T cells are transduced with CAR, which combines the extracellular domain derived from a tumor-specific antibody, the CD3ζ intracellular signaling domain, and the 4-1BB co-stimulatory motif. The addition of 4-1BB is crucial for the in vivo persistence and anti-tumor toxicity of CAR T cells (Song et al. (2011) Cancer Res. 71: 4617e27). Antibodies against 4-1BB are currently being studied.

[0011] Several studies have shown that agonistic antibodies targeting the 4-1BB / 4-1BBL pathway exhibit anti-tumor activity when used as a monotherapy (Palazón et al. (2012) Cancer Discovery 2: 608-23). Agonistic antibodies against 4-1BB (Urelumab, BMS; Utolimumab, Pfizer) are currently in clinical development. In recent years, studies combining 4-1BBL with other therapies have achieved varying degrees of success. For example, when CTLA-4 and anti-4-1BB antibodies were administered to mice with pre-existing MC38 (murine adenocarcinoma) tumors rather than B16 melanoma tumors, significant CD8+ T cell-dependent tumor regression was observed, as well as long-term immunity to these tumors. In another example, treatment with anti-4-1BB (Bristol-Myers Squibb (BMS) -469492) only resulted in moderate regression of M109 tumors, but significantly delayed the growth of EMT6 tumors.

[0012] The tumor microenvironment is composed of multiple cell types, from infiltrating immune cells to cancer cells, extracellular matrix, endothelial cells, and other cellular players that influence tumor progression. This complex and entangled balance varies not only from patient to patient but also within lesions of the same subject (Jiménez-Sánchez et al. (2017) Cell 170(5):927-938). Tumor stratification based on expression of tumor-infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) has emphasized the importance of an inflammatory environment in achieving objective responses to cancer (Teng et al. (2015) Cancer Res. 75(11):2139-45). Pan-cancer analysis of gene expression profiles by The Cancer Genome Atlas (TCGA) supports that tumor inflammatory signatures are associated with objective responses to immunotherapy (Danaher et al. (2018) J. Immunother. Cancer 6(1):63).

[0013] In recent years, efforts to improve the administration of cancer therapy vaccines have expanded from subcutaneous injection to intravenous administration. For example, intravenous administration of MVA vaccines encoding heterologous antigens has been shown to induce strong specific immune responses to the antigen (see WO 2014 / 037124). Furthermore, when MVA vaccines include CD40L, an enhanced immune response is produced.

[0014] Inoculation of bacterial-derived materials (Coley's toxins) into tumor lesions has long been reported to achieve a healing response, highlighting the role of local infection in promoting anti-tumor 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, leading to a series of events following administration, including: i) secretion of proinflammatory cytokines, such as type I, II, and 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). Local administration of immunotherapy to tumors induces a systemic immune response, as regression has been assessed in untreated tumor lesions ((2018) Cancer Discov. 8 (6): 67).

[0015] In the past few years, intratumoral administration of MVA vaccines has been reported. Intratumoral injection of GM-CSF-expressing MVA and immunization with a DNA vaccine were found to prolong the survival of mice bearing HPV16 E7 tumors (Nemeckova et al. (2007) Neoplasma 54:4). Other studies of intratumoral injection of MVA failed to demonstrate an inhibitory effect on pancreatic tumor growth (White et al. (2018) PLoS One 13(2):e0193131). Intratumoral injection of heat-killed MVA induced an antitumor immune response that relied on dendritic cells producing danger signals, type I interferon, and antigen cross-presentation (Dai et al. (2017) Sci. Immunol. 2(11):eaal1713).

[0016] The activity of many cancer vaccines involves inducing an adaptive immune response against the tumor. Effective activation of tumor-specific T cells requires, first, exclusive and high expression of the antigen in tumors but not in healthy tissues to minimize tolerance induction and favor a competent T cell repertoire. Second, effective processing of the tumor antigen and intracellular loading of HLA molecules. Finally, presentation of the immunogenic HLA / peptide complex on the cell surface and its recognition by tumor-specific T cells.

[0017] Clearly, there is a significant unmet medical need for other cancer treatments, including active immunotherapy and cancer vaccines. Furthermore, there is a need for therapies that can induce enhanced immune responses in multiple areas of a patient's immune response. In many respects, embodiments of the present disclosure address these needs by providing vaccines, therapies, and combination therapies that augment and improve currently available cancer treatments. Summary of the Invention

[0018] It is determined in multiple embodiments of the present invention that when administered intratumorally or intravenously, recombinant MVA encoding tumor-associated antigens (TAA) and 4-1BB ligands (also referred to herein as 41BBL, 4-1BBL or CD137L) increases the therapeutic effectiveness of cancer patients and / or enhances the treatment of cancer patients. More specifically, it is determined that compared with the administration of recombinant MVA alone, various embodiments of the present disclosure result in increased inflammation in tumors, reduced regulatory T cells (Treg) in tumors and T cell exhaustion, amplification of tumor-specific T cells and activation of NK cells, an increase in tumor volume reduction and / or an increase in the survival rate of cancer subjects.

[0019] In various embodiments of the present invention, it was determined that recombinant MVA encoding a tumor-associated antigen (TAA) and CD40 ligand (CD40L) enhanced treatment of cancer patients when administered intratumorally or intravenously. More specifically, it was determined that various embodiments of the present disclosure resulted in increased inflammation in the tumor, decreased regulatory T cells (Tregs) and T cell depletion in the tumor, expansion of tumor-specific T cells and activation of NK cells, increased reduction in tumor volume, and / or increased survival of cancer subjects compared to administration of the recombinant MVA alone.

[0020] In additional embodiments, the present invention includes recombinant modified vaccinia Ankara (MVA) viruses comprising a nucleic acid encoding 4-1BBL (CD137L) and a nucleic acid encoding CD40L that enhances treatment of cancer patients when administered intravenously and / or intratumorally.

[0021] Thus, in one embodiment, the present 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 intratumoral administration of the recombinant MVA results in enhanced inflammatory response in the cancerous tumor, increased tumor reduction, and / or increased overall survival in the subject, compared to non-intratumoral injection of a recombinant MVA virus comprising first and second nucleic acids encoding TAA and 4-1BBL antigens.

[0022] In another 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 CD40L, wherein intratumoral administration of the recombinant MVA results in enhanced inflammatory response in the cancerous tumor, increased tumor reduction, and / or increased overall survival in the subject compared to non-intratumoral injection of the recombinant MVA virus comprising the first and second nucleic acids encoding the TAA and CD40L antigens.

[0023] In another embodiment, the present invention includes a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, the method 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 results in enhanced inflammatory response in the cancerous tumor, increased tumor reduction, and / or increased overall survival in the subject compared to administration of the recombinant MVA virus comprising the first and second nucleic acids encoding the TAA, CD40L antigen, and 4-1BBL antigen by a different route of injection (i.e., non-intratumoral injection or non-intravenous injection).

[0024] In another embodiment, the present 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 the intravenous administration of the recombinant MVA enhances natural killer (NK) cell responses and enhances CD8 T cell responses specific for TAAs compared to non-intravenous injection of the recombinant MVA virus comprising the first and second nucleic acids encoding the TAA and 4-1BBL antigens.

[0025] In another embodiment, the present 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 the intravenous administration of the recombinant MVA enhances a natural killer (NK) cell response and enhances a CD8 T cell response specific for the TAA compared to non-intravenous injection of the recombinant MVA virus comprising the first and second nucleic acids encoding the TAA and CD40L antigens.

[0026] In another embodiment, the present invention includes a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, the method comprising intravenously and / or intratumorally administering 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, wherein the intravenous and / or intratumoral 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 or non-intratumoral injection of the recombinant MVA virus comprising a first nucleic acid encoding the TAA, a second nucleic acid encoding the CD40L antigen, and a third nucleic acid encoding the 4-1BBL antigen.

[0027] In another embodiment, the present invention includes a method of inducing an enhanced inflammatory response in a cancerous tumor in a subject, the method comprising administering to the subject intratumorally 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 a 4-1BBL antigen, wherein the intratumoral administration of the recombinant MVA produces an enhanced inflammatory response in the tumor as compared to the inflammatory response produced by non-intratumoral injection of a recombinant MVA virus comprising a first and a second nucleic acid encoding a heterologous tumor-associated antigen and a 4-1BBL antigen.

[0028] In another embodiment, the invention includes a method of inducing an enhanced inflammatory response in a cancerous tumor in a subject, the method comprising administering to the subject intratumorally 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 a CD40L antigen, wherein the intratumoral administration of the recombinant MVA produces an enhanced inflammatory response in the tumor as compared to the inflammatory response produced by non-intratumoral injection of a recombinant MVA virus comprising the first and second nucleic acids encoding the heterologous tumor-associated antigen and the CD40L antigen.

[0029] In another embodiment, the present 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 a CD40L antigen, and a third nucleic acid encoding a 4-1BBL antigen, wherein the intratumoral and / or intravenous administration of the recombinant MVA produces an enhanced inflammatory response in the tumor as compared to the inflammatory response produced by non-intratumoral 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 a CD40L antigen, and a third nucleic acid encoding a 4-1BBL antigen.

[0030] In various additional embodiments, the present invention provides 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 for use in treating a subject having cancer.

[0031] In various additional embodiments, the present invention includes 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 CD40L for use in treating a subject having cancer.

[0032] In various additional embodiments, the present invention includes a recombinant modified vaccinia Ankara (MVA) for treating a subject having 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.

[0033] In another embodiment, when recombinant MVA encoding the 4-1BBL antigen is administered intratumorally to a patient in combination with administration of a checkpoint inhibitor antagonist, it can enhance treatment of cancer patients, more specifically increase reduction in tumor volume and / or increase survival of cancer patients.

[0034] In another embodiment, when recombinant MVA encoding the CD40L antigen is administered intratumorally to a patient in combination with administration of a checkpoint inhibitor antagonist, it can enhance treatment of cancer patients, more specifically increase reduction in tumor volume and / or increase survival of cancer patients.

[0035] In another embodiment, recombinant MVA encoding CD40L and 4-1BBL antigens, when administered intratumorally and / or intravenously to a patient in combination with administration of a checkpoint inhibitor antagonist, can enhance treatment of cancer patients, more specifically increase reduction in tumor volume and / or increase survival of cancer patients.

[0036] In another embodiment, the recombinant MVA of the invention is administered simultaneously with or after administration of the antibody. In a more preferred embodiment, the recombinant MVA is administered after the antibody.

[0037] In another embodiment, the recombinant MVA of the invention is administered by the same route of administration and at the same time as or after the administration of the antibody. In another embodiment, the recombinant MVA is administered by a different route of administration or after the administration of the antibody.

[0038] In 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, wherein 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.

[0039] In a preferred embodiment, the first nucleic acid encodes a TAA that is an endogenous retrovirus (ERV) protein. In a more preferred embodiment, the ERV protein is from the human endogenous retrovirus protein K (HERV-K) family. In a more preferred embodiment, the ERV protein is selected from the HERV-K envelope and HERV-K gag proteins.

[0040] 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).

[0041] 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, antigen preferentially expressed in melanoma (PRAME), folate receptor 1 (FOLR1), and combinations thereof.

[0042] In one or more preferred embodiments, the recombinant MVA is MVA-BN or a derivative thereof.

[0043] In various other embodiments, the recombinant MVA and methods described herein are administered to cancer subjects in combination with immune checkpoint molecule antagonists or agonists. In further embodiments, the recombinant MVA and methods described herein are administered to cancer subjects in combination with antibodies specific for TAAs to treat subjects with cancer. In a more preferred embodiment, the recombinant MVA and methods described herein are administered in combination with immune checkpoint molecule antagonists or agonists selected from CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and ICOS. In a most preferred embodiment, the immune checkpoint molecule antagonists or agonists include antibodies. In a most preferred embodiment, the immune checkpoint molecule antagonists or agonists include PD-1 or PD-L1 antibodies.

[0044] Other objects and advantages of the present invention will be partially listed in the following description, and in part will become obvious from the description, or can be learned through practice of the present invention. The objects and advantages of the present invention will be realized and achieved by means of the elements and combinations particularly pointed out in the appended claims.

[0045] 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

[0046] Figure 1 A, 1B, 1C and 1D illustrate that MVA-OVA-4-1BBL-infected tumor cells can affect cytokine production by 4-1BBL-mediated CD8 T cell co-stimulation without the need for DC. In contrast, MVA-OVA-CD40L only enhances cytokine production in the presence of DC. As described in Example 2, dendritic cells (DC) were produced after 14 days of culturing bone marrow cells from C57BL / 6 mice in the presence of recombinant Flt3L. B16.F10 cells were infected with MVA-OVA, MVA-OVA-CD40L or MVA-OVA-4-1BBL and harvested and co-cultured with infected tumor cells in the presence of DC when indicated. Initial OVA (257-264)-specific CD8+ T cells were magnetically purified from OT-I mice and added to the co-culture. Cells were cultured and supernatants were collected for cytokine concentration analysis by Luminex. IL-6 ( Figure 1 A), GM-CSF ( Figure 1 B), IL-2 ( Figure 1 C) and IFN-γ( Figure 1 D) Supernatant concentrations. Data are shown as mean ± SEM.

[0047] Figure 2 A and Figure 2B shows that MVA-OVA-4-1BBL-infected tumor cells directly (i.e., without the need for DCs) drive antigen-specific CD8 T cells to differentiate into activated effector T cells, while CD40L-mediated co-stimulation of MVA-OVA-CD40L-infected tumor cells depends on the presence of DCs. As described in Example 3, bone marrow cells from C57BL / 6 mice were cultured for 14 days in the presence of recombinant Flt3L to generate dendritic cells (DCs). 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 (when 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. The cells were cultured at 37°C, 5% CO2, for 48 hours. The cells were then stained and analyzed by flow cytometry. Figure 2 A shows the GMFI of T-bet on OT-I CD8+ T cells (indicated by “CD8+” in the figure); Figure 2 B shows the percentage of OT-I CD8+ T cells that were CD44+ granzyme B+ IFNγ+ TNFα+. Data are shown as mean ± SEM.

[0048] Figure 3 A, 3B, 3C, 3D and 3E illustrate that infection with MVA encoding CD40L or 4-1BBL induces tumor cell death in tumor cell lines and macrophages. As described in Example 4, the tumor cell line B16.OVA ( Figure 3 A and 3B), MC38( Figure 3 C) and B16.F10( Figure 3 D) Cells were infected with the vectors at the indicated MOI for 20 hours. Cell viability was analyzed by flow cytometry. Figure 3 A, 3C, 3D, and 3E show the percentage of dead cells ("live / dead+"). Figure 3 B: From Figure 3 HMGB1 in the supernatant of A was quantified by ELISA. Figure 3 E: Bone marrow-derived macrophages (BMDM) were infected at the indicated MOI for 20 hours. Cell viability was analyzed by flow cytometry. Data are presented as mean ± SEM.

[0049] Figure 4 A and 4B show that rMVA-4-1BBL induces NK cell activation in vivo. As described in Example 5, C57BL / 6 mice (n=5 / group) were treated with physiological saline or 5×10 7TCID50 "rMVA" (=MVA-OVA), "rMVA-4-1BBL" (=MVA-OVA-4-1BBL) or 5×10 7 TCID50 rMVA was combined with 200 μg of anti-4-1BBL antibody (clone TKS-1) for intravenous immunization. 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) is shown. Data are presented as mean ± SEM.

[0050] Figure 5 A and 5B show that intravenous rMVA-4-1BBL immunization promoted serum IFN-γ secretion in vivo. As described in Example 6, C57BL / 6 mice (n=5 / group) were treated with saline or 5×10 7 TCID50 "rMVA" (=MVA-OVA), "rMVA-4-1BBL" (=MVA-OVA-4-1BBL) or 5×10 7 TCID50 rMVA was combined with 200 μg of anti-4-1BBL antibody (clone TKS-1) for intravenous immunization. Figure 5 A: Six hours later, the mice were bled, serum was separated from the whole blood, and the IFN-γ concentration in the serum was measured by Luminex. Figure 5 B: 3, 21, and 45 hours after immunization, mice were intravenously injected with Brefeldin A to stop protein secretion. Mice were sacrificed 6, 24, and 48 hours after immunization and spleen cells were analyzed by flow cytometry. Data are shown as mean ± SEM.

[0051] Figure 6 It was shown that intravenous "rMVA-4-1BBL" (=MVA-OVA-4-1BBL) immunization promoted serum IFN-γ secretion in mice bearing B16.OVA tumors. As described in Example 7, C57BL / 6 mice bearing B16.OVA tumors (n=5 / group) were divided into groups and received iv (intravenous) PBS or 5×10 7 TCID50 of rMVA (=MVA-OVA) or rMVA-4-1BBL. Six hours later, mice were bled, serum was separated from whole blood, and serum IFN-γ concentration was determined by Luminex. Data are shown as mean ± SEM.

[0052] Figure 7A, 7B, 7C and 7D show the expansion of antigen- and vector-specific CD8+ T cells after priming and boosting with intravenous "rMVA-4-1BBL" (=MVA-OVA-4-1BBL). As described in Example 8, C57BL / 6 mice (n=4 / group) received either saline or 5×10 7 TCID50 "rMVA" (=MVA-OVA), rMVA-4-1BBL or 5×10 7 Mice were primed intravenously with TCID50 rMVA combined with 200 μg of anti-4-1BBL antibody (clone TKS-1) and boosted on day 41. Mice were bled on days 6, 21, 35, 48, and 64 after priming, and peripheral blood was analyzed by flow cytometry. Figure 7 A shows the percentage of antigen (OVA)-specific CD8 + T cells in peripheral blood leukocytes (PBL); Figure 7 B shows the percentage of vector (B8R)-specific CD8+ T cells in PBL. Mice were sacrificed on day 70 after priming. Spleens were harvested and analyzed by flow cytometry. Figure 7 C shows the percentage of antigen (OVA)-specific CD8+ T cells in living cells; and Figure 7 D shows the percentage of vector (B8R)-specific CD8+ T cells in living cells. Data are shown as mean ± SEM.

[0053] Figure 8 The antitumor effect of intravenously injected MVA virus encoding 4-1BBL was shown to be enhanced compared to recombinant MVA without 4-1BBL. As described in Example 9, C57BL / 6 mice (n=5 / group) bearing B16.OVA tumors were divided into groups on day 7 after tumor inoculation (black dashed line) and received either PBS or 5×10 7 TCID50 MVA-OVA ("rMVA" in the figures) or MVA-OVA-4-1BBL ("rMVA-4-1BBL" in the figures) was administered intravenously. Tumor growth was measured regularly.

[0054] Figure 9 A, 9B, 9C and 9D show the enhanced antitumor effect of intratumoral injection of MVA virus encoding 4-1BBL or CD40L. As described in Example 10, C57BL / 6 mice (n=4-5 / group) bearing B16.OVA tumors were divided into groups and received PBS or 5×10 7MVA-OVA (labeled "rMVA" in the figure), MVA-OVA-CD40L (labeled "rMVA-CD40L" in the figure), or MVA-OVA-4-1BBL (labeled "rMVA-4-1BBL" in the figure) was intratumorally administered at TCID50. Tumor growth was measured regularly.

[0055] Figure 10 A, 10B, and 10C show the antitumor effects of intratumoral injection of MVA virus encoding CD40L against established colon cancer. As described in Example 11, C57BL / 6 mice (n=5 / group) bearing MC38 tumors were divided into groups and received PBS or 5×10 7 TCID50 MVA-TAA (labeled "rMVA" in the figure) or MVA-TAA-CD40L (labeled "rMVA-CD40L" in the figure) was administered intratumorally (it). Tumor growth was measured regularly. In these experiments, the recombinant MVA encoded TAA containing the antigens AH1A5, p15E, and TRP2.

[0056] Figure 11 The synergistic effect of checkpoint blockade and tumor-targeting antibodies with intratumoral (it) administration of rMVA-4-1BBL (also referred to herein as "MVA-OVA-4-1BBL") is demonstrated. As described in Example 12, C57BL / 6 mice (n=5 / group) bearing B16.OVA tumors were grouped and received 200 μg of IgG2a, anti-TRP-1, or anti-PD-1 antibodies intraperitoneally when indicated (checkmarks). On days 13 (black dashed line), 18, and 21 (grey dashed line) after tumor inoculation, PBS or 5×10 7 Mice were immunized intratumorally (it) with TCID50 MVA-OVA-4-1BBL. Tumor growth was measured regularly.

[0057] Figure 12 The results showed that intratumoral MVA-OVA-4-1BBL injection produced a superior antitumor effect compared to anti-CD137 antibody treatment. As described in Example 13, C57BL / 6 mice received 5×10 5 Seven days later, when the tumors measured more than 5 × 5 mm, the mice were divided into groups and injected intratumorally with PBS, 5 × 10 7 TCID50 MVA-OVA-4-1BBL or 10 μg anti-4-1BB (3H3) antibody. Tumor growth was measured regularly. Figure 12 In A, mean tumor volumes are shown. Figure 12B: Peripheral blood lymphocytes were stained with OVA-dextramer and analyzed by FACS on day 12 after priming. The percentage of OVA-dextramer+CD44+ T cells in CD8+ T cells is shown.

[0058] Figure 13 The antitumor effect of intravenous injection of MVA virus encoding the endogenous retroviral antigen Gp70 was demonstrated. As described in Example 14, Balb / c mice received 5×10 5 On day 12 after tumor inoculation, when tumors measured more than 5×5 mm, mice bearing CT26.wt tumors (n=5 / group) were divided into groups and received iv (intravenous) PBS or 5×10 7 TCID50 of MVA, rMVA-Gp70 or rMVA-Gp70-CD40L. Tumor growth was measured regularly. The mean tumor diameter ( Figure 13 A) and mean tumor volume ( Figure 13 B). Figure 13 C: 7 days after immunization, blood cells were restimulated, and the percentage of CD8+CD44+IFN-γ+ cells in the blood after stimulation is shown.

[0059] Figure 14 The antitumor effect of intravenous injection of MVA virus encoding the endogenous retroviral antigen Gp70 plus CD40L was shown. As described in Example 15, C57BL / 6 mice received 5×10 5 Seven days later, when tumors measured more than 5×5 mm, C57BL / 6 mice bearing B16.F10 tumors (n=5 / group) were divided into groups and received iv (intravenous) PBS or 5×10 7 TCID50 of MVA, rMVA-Gp70, or rMVA-Gp70-CD40L. Tumor growth was measured regularly. The mean tumor volume in the blood after stimulation with p15e peptide 7 days after immunization is shown ( Figure 14 A) and the percentage of CD8+CD44+IFN-γ+ cells ( Figure 14 B).

[0060] Figure 15 :4-1BBL adjuvanting can increase the cytokine / chemokine response of the MVA-BN backbone to IT immunization. In this article, "adjuvant" means that a specific encoded protein or component of recombinant MVA increases the immune response generated by other encoded proteins or components of recombinant MVA. Here, 5×10 5B16.OVA cells were implanted subcutaneously (sc) into C57BL / 6 mice (see Example 23). On day 10, PBS or 2×10 8 Mice were immunized intratumorally (it) with TCID50 MVA-BN, MVA-OVA, or MVA-OVA-4-1BBL (n=6 mice / group). Six hours later, tumors were extracted and tumor lysates were processed. Cytokine / chemokine profiles were analyzed by Luminex. Figure 15 showed upregulation of cytokines / chemokines in immunized mice.

[0061] Figure 16 :MVA-OVA-4-1BBL increased the cytokine / chemokine proinflammatory response to intratumoral (it) immunity. 5 B16.OVA cells were implanted subcutaneously (sc) into C57BL / 6 mice (see Examples 23 and 24). On day 10, PBS or 2×10 8 Mice were immunized intratumorally (it) with TCID50 of MVA-BN, MVA-OVA, or MVA-OVA-4-1BBL (n=6 mice / group). Six hours later, tumors were extracted and tumor lysates were processed. Cytokine / chemokine profiles were analyzed by Luminex. Figure 16 Shown are those cytokines / chemokines that were upregulated in MVA-OVA-4-1BBL immunized mice compared to MVA-BN.

[0062] Figure 17 : Quantitative and qualitative T cell analysis of the tumor microenvironment (TME) and tumor-draining lymph nodes (TdLN) after intratumoral injection of MVA-OVA-4-1BBL. C57BL / 6 mice received 5×10 5 Nine to thirteen days later, when tumors measured more than 5 × 5 mm, mice were divided into groups and injected intratumorally with PBS, 2 × 10 8 TCID50 MVA-OVA or MVA-OVA-4-1BBL (see Example 25). One, three, and seven 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 number of CD45+ cells, CD8+ T cells, CD4+ T cells, and OVA-specific CD8+ T cells per mg of tumor and per TdLN is shown.

[0063] Figure 18 : Quantitative and qualitative T cell analysis of the TME and draining LN after intratumoral injection of MVA-OVA-4-1BBL. C57BL / 6 mice received 5×105 Nine to thirteen days later, when tumors measured more than 5.5 × 5.5 mm, mice were divided into groups and injected intratumorally with PBS or 2 × 10 8 TCID50 MVA-OVA or MVA-OVA-4-1BBL (see Example 26) One, three and seven days after immunization, mice were sacrificed and tumors and TdLN (tumor draining lymph nodes) were digested with collagenase / DNase and analyzed by flow cytometry. Figure 18 A: Shown are the percentages of Ki67+ cells among OVA-specific CD8+ T cells in tumors (left panel) and TdLNs (right panel). Figure 18 B: shows the GMFI of PD1 in OVA-specific CD8+ T cells in tumors seven days after IT immunization. Figure 18 C: shows the OVA-specific Teff / Treg ratio in tumors seven days after it immunization.

[0064] Figure 19 : Quantitative and qualitative NK cell analysis of the TME and tumor-draining lymph nodes (TdLN) after intratumoral injection of MVA-OVA-4-1BBL. C57BL / 6 mice received 5×10 5 Nine to thirteen days later, when tumors measured more than 5.5 × 5.5 mm, mice were divided into groups and injected intratumorally with PBS or 2 × 10 8 TCID50 MVA-OVA or MVA-OVA-4-1BBL (see Example 27). Mice were sacrificed one, three, and seven days after immunization, and tumors and tumor-draining lymph nodes (TdLN) were digested with collagenase / DNase and analyzed by flow cytometry. The number of NK cells per mg of tumor and TdLN and the GMFI of CD69, granzyme B, and Ki67 surface markers of NK cells in tumors and TdLN are shown.

[0065] Figure 20 :The anti-tumor effect mediated by MVA-OVA-4-1BBL is dependent on CD8 T cells. C57BL / 6 mice were subcutaneously (sc) injected with 5×10 5 Seven days later, the mice were divided into groups and injected intratumorally with PBS or 2×10 8 TCID 50MVA-OVA-4-1BBL (see Example 28). These intratumoral (it) injections were repeated on days 5 and 8 after the first injection (vertical dashed lines). In addition, IgG2b isotype control antibody (left and middle figures) or anti-CD8 antibody (2.43; right figure) was injected intraperitoneally (ip) (100 μg / mouse) on day -1 before the first immunization and on days 1, 4, 7, and 11 after the first immunization. Tumor growth was measured regularly, and the average tumor diameter is shown.

[0066] Figure 21 : The anti-tumor effects of MVA-OVA and MVA-OVA-4-1BBL are dependent on Batf3+ DCs. C57BL / 6 mice or Batf3- / - mice were subcutaneously (sc) injected with 5×10 5 Seven days later (vertical dashed line), mice were divided into groups and injected intratumorally with PBS or 2×10 8 TCID50 of MVA, MVA-OVA or MVA-OVA-4-1BBL (see Example 29). On days 5 and 8 after the first intratumoral injection, repeated it injections (vertical dashed lines) were performed. Tumor growth was measured regularly. Figure 21 A: The mean tumor diameter is shown. Figure 21 B: Blood was drawn 11 days after the first immunization and antigen-specific T cells (i.e., OVA 257-264 The percentage of OVA-specific T cells among CD8+ T cells is shown.

[0067] Figure 22 : Effect of NK cells on intratumoral administration of MVA-OVA-4-1BBL in mice bearing B16.OVA melanoma. C57BL / 6 or IL15Ra- / - mice received 5×10 5 Seven days later, the mice were divided into groups and injected intratumorally with PBS or 2×10 8 TCID50 of MVA-OVA or MVA-OVA-4-1BBL (see Example 30). Treatment was repeated on days 5 and 8 after the first injection. Tumor growth was measured regularly. The mean tumor diameter ( Figure 22 A) and survival percentage ( Figure 22 B). Eleven days after the first immunization, blood was drawn and analyzed for the presence of antigen-specific T cells ( Figure 22 C) shows OVA expression in CD8+ T cells 257-264 -Percentage of dextramer+ (SIINFEKL+) CD44+ T cells.

[0068] Figure 23The NK cell-dependent cytokine / chemokine profile in response to IT immunization with MVA-OVA-4-1BBL is shown. 5 B16.OVA cells were implanted subcutaneously (sc) into C57BL / 6 and IL15Ra- / - mice (see Example 31). On day 7, cells were treated with PBS or 2×10 8 Mice were immunized intratumorally (it) with TCID50 MVA-OVA or MVA-OVA-4-1BBL (n=2-3 mice / group). Six hours later, tumors were extracted and tumor lysates were processed. Cytokine / chemokine profiles were analyzed by Luminex. Figure 23 Shown are those cytokines / chemokines that were reduced in the absence of IL15Ra following MVA-OVA-4-1BBL intratumoral (it) immunization.

[0069] Figure 24 The antitumor efficacy of intratumoral immunization with MVA-gp70-CD40L compared with MVA-gp70-4-1BBL was shown in mice bearing B16.F10 melanoma. C57BL / 6 mice received 5×10 5 Seven days later, the mice were divided into groups and injected intratumorally with PBS or 5×10 7 TCID50 of MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, MVA-4-1BBL or MVA-CD40L (see Example 32). Treatment was repeated on days 5 and 8 after the first injection. Tumor growth was measured regularly. Figure 24 A shows the average tumor diameter, and Figure 24 B shows the appearance of vitiligo in mice treated with MVA-gp70-4-1BBL. Eleven days after the first immunization, blood was drawn and analyzed for the presence of antigen-specific T cells. The percentage of IFNγ-producing CD44+ T cells within CD8+ T cells after p15E restimulation was shown in Figure 2. Figure 24 As shown in C.

[0070] Figure 25 : Antitumor efficacy of intratumoral administration of MVA-gp70-4-1BBL-CD40L in mice bearing B16.F10 melanoma. C57BL / 6 mice received 5×10 5 Seven days later, the mice were divided into groups and injected intratumorally with PBS or 5×10 7TCID50 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 on days 5 and 8 after the first injection. Tumor growth was measured regularly. The mean tumor diameter is shown in Figure 25 In A. Eleven days after the first immunization, blood was drawn and restimulated with p15e peptide. The percentage of IFNγ+CD44+T cells in CD8+T cells was as follows: Figure 25 As shown in B.

[0071] Figure 26 : Antitumor efficacy of MVA-gp70 supplemented with CD40L or 4-1BBL in mice bearing CT26 tumors. Balb / c mice received 5×10 5 Thirteen days later, the mice were divided into groups and injected intratumorally with PBS or 5×10 7 TCID50MVA-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 on days 5 and 8 after the first injection. Tumor growth was measured regularly. Figure 26 A shows the average tumor diameter, and Figure 26 B shows the survival percentage. Figure 26 C: Eleven days after the first immunization, blood was drawn and restimulated with AH1 peptide; the percentage of IFNγ+CD44+T cells in CD8+T cells is shown.

[0072] Figure 27 Quantitative and qualitative T cell analysis of 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 received 5×10 5 Nine days later, when tumors measured more than 5 × 5 mm, mice were divided into groups and injected intratumorally with PBS or 5 × 10 7 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 collected, digested with collagenase / DNase, and analyzed by flow cytometry. Figure 27 CD8 per mg tumor and per TdLN are shown + T cells, p15E-specific CD8 + T cells and Ki67 + p15E-specific CD8 + The number of T cells is shown in Table 1. The data represent the mean ± SEM.

[0073] Figure 28 Quantitative and qualitative T cell analysis of the tumor microenvironment (TME) and tumor-draining lymph nodes (TdLN) after intratumoral injection of MVA-gp70 further expressing 4-1BBL and / or CD40L. C57BL / 6 mice received 5×10 5 Nine days later, when the tumors measured more than 5.5×5.5 mm, the mice were divided into groups and injected intratumorally with PBS or 5×10 7 TCID50 of MVA-Gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, and MVA-gp70-4-1BBL-CD40L. Three days after immunization, mice were sacrificed, and tumors and TdLNs were harvested and digested with collagenase / DNase, and the resulting single cells were analyzed by flow cytometry. The NK cells, Ki67, and NK cells per mg of tumor and TdLN are shown. + NK cells and granzyme B + The number of NK cells was shown as mean ± SEM.

[0074] Figure 29 : Antitumor efficacy of intravenously administered MVA-gp70 adjuvanted with 4-1BBL and / or CD40L in mice bearing CT26.WT tumors. Balb / c mice received 5×10 5 Twelve days later, the mice were divided into groups and intravenously injected with PBS or 5×10 7 TCID 50 MVA-Gp70, MVA-Gp70-4-1BBL, MVA-Gp70-CD40L, MVA-Gp70-4-1BBL-CD40L and MVA-4-1BBL-CD40L (see Example 37). Figure 29 A shows the average tumor diameter, and Figure 29 B shows the survival percentage. Seven days after the first immunization, blood was drawn and restimulated with AH1 peptide; Figure 29 C will CD8 + IFNγ in T cells + CD44 + The percentages of T cells are shown as mean ± SEM.

[0075] Figure 30 Description of the MVA-based vector MVA-HERV-FOLR1-PRAME-h4-1-BBL ("MVA-mBN494" or "MVA-BN-4IT") ( Figure 30 A), and additionally demonstrated the ability of the vector to load TAAs into the HLA of infected cells ( Figure 30 B) and the ability to express h4-1-BBL in a functional, i.e., h4-1-BB receptor-binding, form ( Figure 30 C). See Examples 38 and 39 for more details.

[0076] Figure 31 Description of the MVA-based vector "MVA-mBN502" ( Figure 31 C), and additionally shows ERVK-env / MEL( Figure 31 A; as used in MVA-mBN494) and ERVK-env / MEL_03 ( Figure 31 B; Schematic diagram of (as used in MVA-mBN502). DETAILED DESCRIPTION

[0077] It is to be understood that both the foregoing summary and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[0078] Described and illustrated herein are multiple aspects of the recombinant MVA and methods of the present invention that enhance immune responses in cancer patients. In various aspects, the present invention demonstrates that when a recombinant MVA comprising a tumor-associated antigen (TAA) and a 4-1BBL antigen is administered intratumorally or intravenously to a cancer subject, an increased anti-tumor effect is achieved in the subject. As described in more detail herein, this increased anti-tumor effect includes a greater reduction in tumor volume, increased overall survival, enhanced CD8 T cell responses to TAAs, and enhanced inflammatory responses, such as increased NK cell activity, increased cytokine production, and the like.

[0079] Described and illustrated herein are various aspects of the invention's recombinant MVAs and methods for enhancing immune responses in cancer patients. In various aspects, the invention demonstrates that when a recombinant MVA comprising a tumor-associated antigen (TAA) and a CD40L antigen is administered intratumorally or intravenously to a cancer subject, an enhanced anti-tumor effect is achieved in the subject. As described in greater detail herein, this enhanced anti-tumor effect includes greater reductions in tumor volume, increased overall survival, enhanced CD8 T cell responses to TAAs, and enhanced inflammatory responses, such as increased NK cell activity, increased cytokine production, and the like.

[0080] In additional aspects, various embodiments of the present invention demonstrate that when a recombinant MVA comprising a tumor-associated antigen (TAA) and a 4-1BBL antigen is administered intratumorally in combination with at least one immune checkpoint molecule antagonist / agonist, tumor reduction is increased and overall survival of cancer subjects is improved.

[0081] In a still further aspect, various embodiments of the present invention demonstrate that when recombinant MVA comprising a tumor associated antigen (TAA) and a 4-1BBL antigen is administered intratumorally in combination with a tumor specific antibody, tumor reduction is increased and overall survival of cancer subjects is improved.

[0082] Although recombinant MVA viruses have previously encoded the 4-1BBL antigen, the immunogenic benefits of MVA encoding 4-1BBL are unknown (see, for example, Spencer et al. (2014) PLoS One 9(8): e105520). In Spencer, co-expression of 4-1BBL and a transgenic antigen in an MVA vector or an adenoviral vector resulted in increased CD8 T cell responses in mice; however, there was no increase in IFN-γ responses in non-human primates after intramuscular administration of an adenoviral vector encoding 4-1BBL (ibid., pp. 2, 6). Furthermore, the immunogenic benefits of using MVA encoding 4-1BBL as part of a treatment for cancer and destruction of tumors and / or tumor cells are unknown.

[0083] Various embodiments of the present disclosure demonstrate that MVA encoding 4-1BBL and TAA (referred to herein as MVA-TAA-4-1BBL) can effectively treat cancer in subjects (e.g., humans). It is shown and described herein that administration of MVA-TAA-4-1BBL can enhance multiple aspects of the immune response in cancer subjects and can effectively reduce and kill tumor cells. One or more enhanced anti-tumor effects of various embodiments of the present disclosure are summarized below.

[0084] Intravenous administration of a recombinant MVA encoding 4-1BBL produces enhanced anti-tumor effects. In at least one aspect, the present invention includes intravenously administered recombinant MVA encoding TAA and 4-1BBL antigens (rMVA-TAA-4-1BBL), wherein intravenous administration enhances the anti-tumor effect compared to intravenous administration of a recombinant MVA without 4-1BBL, or compared to non-intravenous administration of a recombinant MVA encoding 4-1BBL (e.g., subcutaneous administration of a recombinant MVA encoding 4-1BBL). These enhanced anti-tumor effects include enhanced NK cell responses (e.g., Figure 4 ), as shown by increased IFN-γ secretion (as shown by Figure 5 and 6 ), increased antigen- and vector-specific CD8 T cell expansion (as shown in Figure 7 ) and increased tumor reduction (as shown in Figure 8 ).

[0085] Intratumoral administration of recombinant MVA encoding 4-1BBL can enhance inflammation in tumors. In another aspect of the present invention, it was determined that infection of tumor cells with MVA-OVA-4-1BBL, but not with MVA-OVA-CD40L, in the absence of antigen cross-presenting DCs, activated antigen-specific CD8+ T cells to produce T cell-derived cytokines such as GM-CSF, IL-2, and IFN-γ. Figure 1 A-1D). This is unexpected in the context of GM-CSF, a growth factor produced by naive T cells upon activation that induces maturation of dendritic cells and myeloid cell subsets (Min et al. (2010) J. Immunol. 184:4625-4629). In the presence of antigen cross-presenting DCs, antigen-specific CD8+ T cells stimulated by tumor cells infected with rMVA-CD40L produced IFN-γ, but did not produce IL-2 or GM-CSF as with rMVA-4-1BBL ( Figure 1 Interestingly, high levels of IL-6, a key cytokine produced by DCs ( Figure 1 A).

[0086] On the one hand, increased inflammation in tumors can lead to a large number of TILs (tumor-infiltrating lymphocytes) at the tumor site that kill tumor cells (see, for example, Lanitis et al. (2017) Annals Oncol. 28 (suppl 12): xii18-xii32). Given the increased number of TILs, cytokines, and other inflammatory molecules, these inflamed tumors, also known as "hot" tumors, are able to enhance tumor cell destruction.

[0087] Intratumoral administration of a recombinant MVA encoding 4-1BBL can reduce tumor volume and improve overall survival. In one aspect, the present invention comprises an intratumorally administered recombinant MVA encoding the 4-1BBL antigen (MVA-4-1BBL), wherein the intratumoral administration enhances the anti-tumor effect in a subject with cancer compared to intratumoral administration of a recombinant MVA without 4-1BBL.

[0088] Although recombinant MVA viruses have been administered intratumorally before (see, for example, White et al. (2018) PLoS One 13:e0193131 and Nemeckova et al. (2007) Neoplasma 54:326-33), studies have yielded mixed results. For example, Nemeckova et al. found that intratumoral injection of GM-CSF-expressing vaccinia virus MVA and immunization with a DNA vaccine prolonged the survival of mice bearing HPV16-induced tumors (see Nemeckova, Abstract). In addition, White et al. were unable to demonstrate inhibition of pancreatic tumor growth after intratumoral injection of MVA (see White, Abstract).

[0089] As part of the present disclosure, a recombinant MVA comprising one or more nucleic acids encoding TAA and 4-1BBL is administered intratumorally to a subject. Figure 9 As shown in , intratumoral injection of MVA-TAA-4-1BBL showed a significant reduction in tumor volume compared to recombinant MVA TAA.

[0090] Intratumoral administration of a recombinant MVA encoding 4-1BBL administered in combination with an immune checkpoint molecule antagonist or agonist produces an enhanced anti-tumor effect. In various embodiments, the present invention includes administering MVA-TAA-4-1BBL in combination with an immune checkpoint antagonist or agonist. Preferably, the administration of MVA-TAA-4-1BBL is intravenous or intratumoral administration. The MVA of the present invention is advantageous in combination with an immune checkpoint antagonist or agonist because the combination provides a more effective cancer treatment. For example, the combination and / or combination therapy of the present invention enhances multiple aspects of the immune response of cancer patients. In at least one aspect, the combination synergistically enhances both innate and adaptive immune responses, and when combined with an antagonist or agonist of an immune checkpoint molecule, reduces tumor volume and increases the survival rate of cancer patients.

[0091] The data presented in this application demonstrate that MVA-TAA-4-1BBL produces enhanced anti-tumor effects when combined with immune checkpoint antagonists or agonists. Figure 11 As shown in , tumor volume was reduced when intratumoral administration of MVA-OVA-4-1BBL was combined with intraperitoneally administered PD-1 antibody compared to PD-1 alone.

[0092] Intratumoral administration of a recombinant MVA encoding 4-1BBL administered in combination with a tumor-associated antigen (TAA)-specific antibody produces an enhanced anti-tumor effect. In various embodiments, the present invention includes administering MVA-TAA-4-1BBL in combination with an antibody specific for TAA. Preferably, the administration of MVA-TAA-4-1BBL is intravenous or intratumoral. The MVA of the present invention is advantageously combined with a TAA-specific antibody and can work together to provide a more effective cancer treatment.

[0093] In one exemplary aspect, the enhanced NK cell response induced by administration of MVA-TAA-4-1BBL synergizes with TAA-specific antibodies to enhance antibody-dependent cellular cytotoxicity (ADCC) in a subject. This enhanced ADCC in a subject with cancer results in increased tumor cell killing and tumor destruction.

[0094] The data presented in this application indicate that MVA-TAA-4-1BBL produces enhanced anti-tumor effects when combined with TAA-specific antibodies. Figure 11 As shown in , tumor volume was reduced when intratumoral administration of MVA-OVA-4-1BBL was combined with intraperitoneal TRP-1 antibody compared to TRP-1 antibody alone.

[0095] According to the present invention, administration of MVA-TAA-4-1BBL as part of a prime and boost immunization increases antigen- and vector-specific CD8+ T cell expansion. In other aspects, the present invention provides a method wherein MVA-TAA-4-1BBL is administered as part of a homologous and / or heterologous prime-boost regimen. Preferably, administration of MVA-TAA-4-1BBL is intravenous or intratumoral. Figure 7 As shown in , antigen- and vector-specific CD8+ T cell expansion was increased during priming and boosting by intravenous administration of MVA-TAA-4-1BBL.

[0096] definition

[0097] Unless the context clearly dictates otherwise, as used herein, the singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "a nucleic acid" includes one or more nucleic acids, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that can modify or substitute for the methods described herein.

[0098] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain, using only routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0099] Throughout the specification and claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" should be understood to include the stated integer or step or group of integers or steps but not to exclude any other integer or step or group of integers or steps. When used herein, the term "comprise" may be replaced with the terms "contain" or "include," or sometimes when used herein, with the term "have." Any of the above terms (comprise, contain, include, have), although less preferred, whenever used in the context of an aspect or embodiment of the invention, may be replaced with the term "consisting of." When used herein, "consisting of excludes any element, step, or ingredient not specified in the claim elements. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0100] As used herein, the term "and / or" connecting multiple listed elements is understood to include both individual and combined options. For example, where two elements are connected by "and / or," the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any of these options is understood to fall within the meaning and therefore meets the requirements of the term "and / or" as used herein. The simultaneous applicability of more than one option is also understood to fall within the meaning and therefore meets the requirements of the term "and / or."

[0101] A "mutated" or "modified" protein or antigen as described herein is any modification of the nucleic acid or amino acids, such as a deletion, addition, insertion and / or substitution, as defined herein.

[0102] " Percentage (%) of sequence homology or identity" with respect to nucleic acid sequences as described herein is defined as the percentage of nucleotides in the candidate sequence that are identical with the nucleotides in the reference sequence (i.e., the nucleic acid sequence from which they are derived) after the sequences are aligned and spaces are introduced (if necessary) to achieve maximum percentage sequence identity, and without considering any conservative substitutions as part of the sequence identity. Comparisons for determining nucleotide sequence identity or homology percentages can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring comparisons, including any algorithm for achieving maximum comparison over the full length of the compared sequences.

[0103] For example, the local homology algorithm of Smith and Waterman ((1981) Advances in Applied Mathematics 2: 482-489) provides suitable alignments of nucleic acid sequences. This algorithm can be applied to amino acid sequences using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, ed. MO Dayhoff, 5th Supplement 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 sequence identity is provided by the Genetics Computer Group (Madison, Wisconsin, USA) in the "BestFit" utility application. The default parameters of this method are described in Wisconsin Sequence Analysis Package Program Manual, 8th edition (1995) (available from Genetics Computer Group, Madison, Wisconsin, USA). A preferred method for establishing identity percentage in the context of the present invention is to use the MPSRCH program package distributed by IntelliGenetics, Inc. (Mountain View, California, USA) and copyright owned by the University of Edinburgh, developed by Collins and Sturrok. From this software package, the Smith-Waterman algorithm can be used, wherein default parameters are used for the score sheet (for example, a gap open penalty of 12, a gap extension penalty of 1, and a gap of 6). From the data generated, a "Match" value reflects "sequence identity." Other suitable programs for calculating the percentage identity or similarity between sequences are generally known in the art, and for example, another alignment program is the BLAST program using default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strands = two; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort by = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + Swiss protein + Spupdate + PIR. Details of these programs can be found at the following website: blast.ncbi.nlm.nih.gov / .

[0104] The term "prime-boost vaccination" or "prime-boost regimen" refers to a vaccination strategy or regimen that uses a first prime injection of a vaccine targeting a specific antigen, followed by one or more booster injections of the same vaccine at intervals. Prime-boost vaccinations can be homologous or heterologous. Homologous prime-boost vaccination uses a vaccine that includes the same antigen and vector for the prime injection and one or more booster injections. Heterologous prime-boost vaccination uses a vaccine that includes the same antigen for the prime injection and one or more booster injections but different vectors for the prime injection and one or more booster injections. For example, homologous prime-boost vaccination can use a recombinant poxvirus that includes a nucleic acid expressing one or more antigens for the prime injection, and the same recombinant poxvirus that expresses one or more antigens for one or more booster injections. In contrast, heterologous prime-boost vaccination can use a recombinant poxvirus that includes a nucleic acid expressing one or more antigens for the prime injection, and a different recombinant poxvirus that expresses one or more antigens for one or more booster injections.

[0105] The term "recombinant" means a polynucleotide, virus, or vector of semisynthetic or synthetic origin that does not occur in nature or is linked to another polynucleotide in an arrangement that does not occur in nature. As used herein, "recombinant MVA" or "rMVA" generally refers to a modified vaccinia Ankara (MVA) that includes at least one polynucleotide encoding a tumor-associated antigen (TAA).

[0106] As used herein, reduction in tumor volume or a decrease in tumor volume can be characterized as a decrease in tumor volume and / or size, but can also be characterized according to clinical trial endpoints understood in the art. Some exemplary clinical trial endpoints related to reduction in tumor volume and / or size may include, but are not limited to, response rate (RR), objective response rate (ORR), etc.

[0107] As used herein, an improvement in survival rate can be characterized as an improvement in the survival rate of cancer patients, but can also be characterized according to clinical trial endpoints understood in the art. Some exemplary clinical trial endpoints related to the improvement in survival rate include, but are not limited to, overall survival (OS), progression-free survival (PFS), etc.

[0108] 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 genome of a wild-type poxvirus (e.g., cowpox, fowlpox, or MVA). Those skilled in the art understand that a "transgene" or "heterologous gene," when present in a poxvirus, such as a vaccinia virus, is incorporated into the poxvirus genome in such a manner that, upon administration of the recombinant poxvirus to a host cell, it is expressed as the corresponding heterologous gene product, i.e., a "heterologous antigen" and / or a "heterologous protein." Expression is typically achieved by operably linking the heterologous gene to regulatory elements that permit expression in poxvirus-infected cells. Preferably, the regulatory elements include a natural or synthetic poxvirus promoter.

[0109] "Vector" refers to a recombinant DNA or RNA plasmid or virus that may include a heterologous polynucleotide. The heterologous polynucleotide may include a sequence of interest for prophylactic or therapeutic purposes and may optionally be in the form of an expression cassette. As used herein, a vector need not be capable of replication in the ultimate target cell or subject. The term includes cloning vectors and viral vectors.

[0110] Combinations and Methods

[0111] In various embodiments, the present invention comprises a recombinant MVA comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding 4-1BBL, which, when administered intratumorally, induces an inflammatory response and an enhanced T cell response compared to the inflammatory response and 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.

[0112] 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, wherein the recombinant MVA induces an enhanced intratumoral inflammatory response and an enhanced T cell response when administered intratumorally, compared to the intratumoral inflammatory response and T cell response induced by intratumoral administration of a recombinant MVA virus comprising a first nucleic acid encoding the TAA.

[0113] Enhanced inflammatory response in tumors. In various aspects of the present disclosure, it was determined that intratumoral administration of a recombinant MVA encoding TAA and 4-1BBL induces an enhanced inflammatory response in the tumor compared to administration of the recombinant MVA alone. In at least one aspect, an "enhanced inflammatory response" in a tumor according to the present disclosure is characterized by one or more of the following: 1) an increase in IFN-γ expression and / or 2) an increase in the expression of granzyme B (GraB) in the tumor and / or tumor cells. Therefore, whether the inflammatory response in a tumor and / or tumor cells according to the present disclosure is enhanced can be determined by measuring whether the expression of one or more molecules indicative of an increased inflammatory response is increased, including the secretion of chemokines and cytokines known in the art. Exemplary inflammatory response markers include one or more markers that can be used to measure NK cell frequency and / or activity, including one or more of the following: IFN-γ and / or granzyme B (GraB). These molecules and their measurement are understood in the art and can be performed according to validated assays known in the art. See, for example, Borrego et al. ((1999) Immunology 7(1): 159-165).

[0114] Enhanced NK cell response. In various other aspects of the present disclosure, it was determined that intratumoral or intravenous administration of a recombinant MVA encoding TAA and 4-1BBL induced an enhanced NK cell response in a tumor or tumor environment compared to administration of the recombinant MVA alone. In one aspect, an “enhanced NK cell response” according to the present disclosure is characterized by one or more of the following: 1) an increase in NK cell frequency, 2) an increase in NK cell activation, and / or 3) an increase in NK cell proliferation. Therefore, whether an NK cell response is enhanced according to the present disclosure can be determined by measuring the expression of one or more molecules that indicate increased NK cell frequency, increased NK cell activation, and / or increased NK cell proliferation. Exemplary markers that can be used to measure NK cell frequency and / or activity include one or more of the following: NKp46, IFN-γ, CD69, CD70, NKG2D, FasL, granzyme B, CD56, and / or Bcl-XL. Exemplary markers that can be used to measure NK cell activation include one or more of the following: IFN-γ, CD69, CD70, NKG2D, FasL, granzyme B and / or Bcl-XL. Exemplary markers that can be used to measure NK cell proliferation include: Ki67. These molecules and their measurements are understood in the art and can be analyzed according to validated techniques (see, for example, Borrego et al. (1999) Immunology 7(1): 159-165). In addition, assays for measuring 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 pretreatment / baseline; 2) an increase in NK cell activation can be defined as at least a 2-fold increase in IFN-γ, CD69, CD70, NKG2D, FasL, granzyme B and / or Bcl-XL expression compared to pretreatment / baseline expression; and / or 3) an increase in NK cell proliferation can be defined as at least a 1.5-fold increase in Ki67 expression compared to pretreatment / baseline expression.

[0115] Enhanced T cell response. According to the present application, an “enhanced T cell response” is characterized by one or more of the following: 1) an increase in CD8 T cell frequency; 2) an increase in CD8 T cell activation; and / or 3) an increase in CD8 T cell proliferation. Therefore, whether the T cell response is enhanced according to the present application can be determined by measuring the expression of one or more molecules, which indicate 1) an increase in CD8 T cell frequency, 2) an increase in CD8 T cell activation; and / or 3) an increase in CD8 T cell proliferation. Exemplary markers that can be used to measure CD8 T cell frequency, activation, and proliferation include CD3, CD8, IFN-γ, TNF-α, IL-2, CD69, and / or CD44, and Ki67, respectively. As shown in the present application, measuring antigen-specific T cell frequency can also be measured by MHC multimers, such as pentamers or dextramers. Such measurements and analyses, as well as other measurements and analyses suitable for evaluating the methods and compositions of the present invention, are verified and understood in the art.

[0116] In one aspect, the increase in CD8 T cell frequency is characterized by at least a 2-fold increase in IFN-γ and / or dextramer+ CD8 T cells compared to pretreatment / baseline. The increase in CD8 T cell activation is characterized by at least a 2-fold increase in CD69 and / or CD44 expression compared to pretreatment / baseline expression. The increase in CD8 T cell proliferation is characterized by at least a 2-fold increase in Ki67 expression compared to pretreatment / baseline expression.

[0117] In another aspect, the enhanced T cell response is characterized by increased CD8 T cell expression of effector cytokines and / or increased cytotoxic effector function. The increase in effector cytokine expression can be measured by expression of one or more of IFN-γ, TNF-α, and / or IL-2 compared to pretreatment / baseline. The increase in 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.

[0118] The assays, cytokines, markers, and molecules described herein, and their measurement, are validated and understood in the art and can be performed according to known techniques. Additionally, assays for measuring T cell responses can be found in the working examples, where T cell responses are analyzed, including but not limited to Examples 2, 3, 8, 13, and 14.

[0119] The enhanced T cell response achieved by the present invention is particularly advantageous in combination with an enhanced NK cell response and an enhanced inflammatory response because the enhanced T cells effectively target and kill tumor cells that have evaded and / or survived beyond the cancer patient's initial innate immune response.

[0120] In further embodiments, the combinations and methods described herein are used to treat a human cancer patient. In preferred embodiments, the cancer patient suffers from and / or has been diagnosed with a cancer selected from the group consisting of breast cancer, lung cancer, head and neck cancer, thyroid cancer, melanoma, stomach cancer, bladder cancer, kidney cancer, liver cancer, melanoma, pancreatic cancer, prostate cancer, ovarian cancer, urothelial cancer, cervical cancer, or colorectal cancer. In further embodiments, the combinations and methods described herein are used to treat a human cancer patient suffering from and / or diagnosed with breast cancer, colorectal cancer, or melanoma, preferably melanoma, more preferably colorectal cancer, or most preferably colorectal cancer.

[0121] Certain exemplary tumor-associated antigens In certain embodiments, an immune response is generated in a subject against a cell-associated polypeptide antigen. In certain such embodiments, the cell-associated polypeptide antigen is a tumor-associated antigen (TAA).

[0122] The term "polypeptide" refers to a polymer of two or more amino acids joined to one another by peptide bonds or modified peptide bonds. The amino acids can be naturally occurring as well as non-naturally occurring, or chemical analogs of naturally occurring amino acids. The term also refers to a protein, i.e., a functional biomolecule comprising at least one polypeptide; when comprising at least two polypeptides, the polypeptides may form a complex, be covalently linked, or be non-covalently linked. The polypeptides within a protein may be glycosylated and / or lipidated and / or include prosthetic groups.

[0123] Endogenous retroviral proteins (ERV) Preferably, the TAA is contained in an endogenous retroviral protein (ERV). More preferably, the ERV is an ERV from the human HERV-K protein family. Most preferably, the HERV-K protein is selected from the group consisting of a HERV-K envelope (env) protein, a HERV-K group-specific antigen (gag) protein, and a HERV-K "melanoma risk marker" (mel) protein (see, e.g., Cegolon et al. (2013) BMC Cancer 13:4).

[0124] ERVs make up 8% of the human genome and are derived from germline infections millions of years ago. Most of the elements that have been inserted into our genome are severely mutated and are therefore not transcribed or translated. However, a small number of recently acquired ERVs are still functional and translated, and in some cases even produce viral particles. Transcription of ERVs is very limited because the locus is usually highly methylated and is therefore not transcribed in somatic cells (Kassiotis (2016) Nat. Rev. Immunol. 16: 207-19). Only under certain circumstances, such as cellular stress (chemicals, UV radiation, hormones, cytokines), can ERVs be reactivated. Importantly, ERVs are also expressed in many different types of cancer, but not in normal tissues (Cegolon et al. (2013) BMC Cancer 13: 4; Wang-Johanning et al. (2003) Oncogene 22: 1528-35). This very limited expression pattern ensures that ERVs are not or rarely exposed to immune tolerance mechanisms, which may generate a competent ERV-specific T cell pool. In this manner, ERVs can be used as tumor antigens ("TAAs") in MVA.

[0125] In various additional embodiments, TAAs include, but are 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 CEA and MUC-1, for example, in MVA also known as CV301. Other exemplary combinations may include PAP and PSA.

[0126] In still further embodiments, additional TAAs may include, but are not limited to, 5α-reductase, alpha-fetoprotein, AM-1, APC, April, BAGE, β-catenin, Bcl12, bcr-abl, CA-125, CASP-8 / FLICE, cathepsin, 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, GP 1. 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 A / B, MT-MMPs, mucin, NY-ESO-1, osteonectin, p15, P170 / MDR1, p53, p97 / melanotransferrin, PAI-1, PDGF, uPA, PRAME, probasin, progenipoietin, 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.

[0127] A preferred PSA antigen includes an amino acid change from isoleucine to leucine at position 155 (see US Patent 7,247,615, which is incorporated herein by reference).

[0128] In one or more preferred embodiments of the present invention, the heterologous TAA is selected from HER2 and / or Brachyury.

[0129] Any TAA can be used, so long as it achieves at least one of the goals or desired purposes of the present invention, for example, stimulating an immune response after administration of an MVA containing it. TAAs, including the exemplary sequences of TAAs mentioned herein, are known in the art and are suitable for use in the compositions and methods of the present invention. TAA sequences used in the compositions and methods of the present invention can be identical to sequences known in the art or disclosed herein, or they can share less than 100% identity, for example, at least 90%, 91%, 92%, 95%, 97%, 98% or 99% or more sequence identity with nucleotide or amino acid sequences known in the art or disclosed herein. Thus, the TAA sequences used in the compositions or methods of the present invention can differ from a reference sequence known in the art and / or disclosed herein by less than 20, or less than 19, 18, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides or amino acids, so long as it achieves at least one of the goals or desired purposes of the present invention. Those skilled in the art are familiar with techniques and assays for evaluating TAAs to ensure their suitability for use in the MVA or methods of the present invention.

[0130] Modified tumor-associated antigens In certain embodiments, the cell-associated polypeptide antigen is modified so that, when presented in association with an MHC class I molecule on the surface of an APC, a CTL response is induced against cells presenting epitopes derived from the polypeptide antigen on their surface. In certain such embodiments, at least one first exogenous TH epitope is presented in association with an MHC class II molecule on the surface of the APC. In certain such embodiments, the cell-associated antigen is a tumor-associated antigen.

[0131] Exemplary APCs capable of presenting epitopes include dendritic cells and macrophages. Other exemplary APCs include any pinocytic or phagocytic APC capable of simultaneously presenting: 1) a CTL epitope bound to an MHC class I molecule; and 2) a TH epitope bound to an MHC class II molecule.

[0132] In certain embodiments, one or more TAAs, such as 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 modified so that after administration to a subject, polyclonal antibodies reacting primarily with one or more TAAs described herein are elicited. Such antibodies can attack and eliminate tumor cells and prevent metastatic cells from developing into metastatic tumors. The effector mechanism of this anti-tumor effect will be mediated by complement and antibody-dependent cellular toxicity. In addition, the induced antibodies can also inhibit cancer cell growth by inhibiting growth factor-dependent oligodimerization and internalization of receptors. In certain embodiments, such modified TAAs can induce CTL responses to known and / or predicted TAA epitopes displayed by tumor cells.

[0133] In certain embodiments, the modified TAA polypeptide antigen comprises a CTL epitope and a variation of a cell-associated polypeptide antigen, wherein the variation comprises at least one CTL epitope or an exogenous TH epitope. In a non-limiting example, certain such modified TAAs may comprise one or more HER2 polypeptide antigens comprising at least one CTL epitope and a variation of at least one CTL epitope comprising an exogenous TH epitope, and methods for producing the same are described in U.S. Patent No. 7,005,498 and U.S. Patent Publication Nos. 2004 / 0141958 and 2006 / 0008465.

[0134] In one non-limiting example, certain such modified TAAs may include one or more MUC-1 polypeptide antigens including at least one CTL epitope and a variation of at least one CTL epitope including a foreign epitope, and methods of producing the same are described in U.S. Patent Publication No. 2014 / 0363495.

[0135] Other promiscuous T cell epitopes include peptides that bind to most HLA-DR molecules encoded by different HLA-DR. See, for example, WO 98 / 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. The latter reference also relates to HLA-DQ and -DP ligands. All epitopes listed in these references are related to the candidate natural epitopes described herein, as are epitopes that share common sequence motifs with these epitopes.

[0136] In certain other embodiments, the promiscuous T cell epitope is an artificial T cell epitope that is capable of binding to a majority of haplotypes. In certain such embodiments, the artificial T cell epitope is a pan-DR epitope peptide ("PADRE") as described in WO 95 / 07707 and corresponding paper Alexander et al. (1994) Immunity 1:751-761.

[0137] 4-1BBL (Also referred to herein as "41BBL" or "4-1BB ligand"). As described herein, including 4-1BBL as part of a recombinant MVA and related methods induces increased and enhanced anti-tumor effects when administered intratumorally or intravenously to a subject with cancer. Thus, in various embodiments, in addition to encoding a TAA, there is also a recombinant MVA encoding the 4-1BBL antigen.

[0138] 4-1BB / 4-1BBL is a member of the TNFR / TNF superfamily. 4-1BBL is a co-stimulatory ligand expressed in activated B cells, monocytes and DCs. 4-1BB is constitutively expressed by natural killer (NK) and natural killer T (NKT) cells, Treg and several innate immune cell populations (including DCs, monocytes and neutrophils). Interestingly, 4-1BB is expressed on activated rather than resting T cells (Wang et al. (2009) Immunol. Rev. 229: 192-215). 4-1BB ligation induces the proliferation and production of interferon gamma (IFN-γ) and interleukin 2 (IL-2), and enhances T cell survival by upregulating anti-apoptotic molecules such as Bcl-xL (Snell et al. (2011) Immunol. Rev. 244: 197-217). Importantly, 4-1BB stimulation enhances NK cell proliferation, IFN-γ production, and cytolytic activity by enhancing antibody-dependent cellular cytotoxicity (ADCC) (Kohrt et al. (2011) Blood 117:2423-32).

[0139] In one or more preferred embodiments, 4-1BBL is encoded by an MVA of the present invention. In one or more other preferred embodiments, 4-1BBL is human 4-1BBL. In more preferred embodiments, 4-1BBL comprises a nucleic acid encoding an amino acid sequence having a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 3, i.e., differing by fewer than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid sequence from the amino acid sequence set forth in SEQ ID NO: 3. In more preferred embodiments, 4-1BBL comprises a nucleic acid encoding an amino acid sequence comprising SEQ ID NO: 3. In further embodiments, the nucleic acid encoding 4-1BBL comprises a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 4, i.e., differing by fewer than 20, 10, 5, 4, 3, 2, or 1 nucleic acid sequence from the nucleic acid sequence set forth in SEQ ID NO: 4. In a more preferred embodiment, 4-1BBL comprises a nucleic acid comprising SEQ ID NO:4.

[0140] CD40L As described herein, including CD40L as part of the combinations and related methods further enhances the reduction in tumor volume, prolongs progression-free survival, and improves the survival rates achieved by the present invention. Thus, in various embodiments, the method further comprises administering CD40L to the cancer patient. In preferred embodiments, CD40L is encoded as part of a recombinant MVA as described herein.

[0141] While CD40 is constitutively expressed on many cell types, including B cells, macrophages, and dendritic cells, its ligand, CD40L, is primarily expressed on activated T helper cells. Early after infection or immunization, homologous interactions between dendritic cells and T helper cells 'license' dendritic cells to initiate CTL responses. Dendritic cell licensing leads to upregulation of co-stimulatory molecules, increased survival, and improved cross-presentation capacity. This process is primarily mediated by the CD40 / CD40L interaction. However, various configurations of CD40L have been described, ranging from membrane-bound to soluble (monomer to trimer), which induce different stimuli, either inducing or inhibiting activation, proliferation, and differentiation of APCs.

[0142] In one or more preferred embodiments, CD40L is encoded by an MVA of the present invention. In one or more other preferred embodiments, CD40L is human CD40L. In more preferred embodiments, CD40L comprises a nucleic acid encoding an amino acid sequence having a sequence at least 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 1, i.e., differing by fewer than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid sequence from the amino acid sequence set forth in SEQ ID NO: 1. In more preferred embodiments, CD40L comprises a nucleic acid encoding an amino acid sequence comprising SEQ ID NO: 1. In further embodiments, the nucleic acid encoding CD40L comprises a nucleic acid sequence having at least 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 2, i.e., differing by fewer than 20, 10, 5, 4, 3, 2, or 1 amino acid sequence from the nucleic acid sequence set forth in SEQ ID NO: 2. In more preferred embodiments, CD40L comprises a nucleic acid comprising SEQ ID NO: 2.

[0143] Immune checkpoint molecule antagonists As described herein, at least in one aspect, the present invention includes the use of immune checkpoint antagonists. Such immune checkpoint antagonists are used 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).

[0144] In addition, exemplary immune checkpoint antagonists may include, but are not limited to, CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, T cell immunoreceptor with Ig and ITIM domains (TIGIT), and V-domain Ig inhibitor of T cell activation (VISTA).

[0145] Such immune checkpoint molecule antagonists may include antibodies that specifically bind to immune checkpoint molecules and inhibit and / or block the biological activity and function of the immune checkpoint molecules.

[0146] Other immune checkpoint molecule antagonists may 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.

[0147] Antagonists can also be in the form of small molecules that inhibit or block immune checkpoint function. Some non-limiting examples of these include NP12 (Aurigene), (D)PPA-1 from Tsinghua Univ, high-affinity PD-1 (Stanford); BMS-202 and BMS-8 (Bristol Myers Squibb (BMS) and CA170 / CA327 (Curis / Aurigene); and small molecule inhibitors of CTLA-4, PD-1, PD-L1, LAG-3, and TIM-3.

[0148] Antagonists can also inhibit or block the function of immune checkpoint molecules. See, for example, Rothe et al. ((2018) BioDrugs 32(3):233–243).

[0149] It is expected that antagonists may additionally present Affimers are Fc fusion proteins that inhibit or block the function of immune checkpoint molecules. Other fusion proteins that can act as immune checkpoint antagonists are immune checkpoint fusion proteins (such as the anti-PD-1 protein AMP-224) and anti-PD-L1 proteins, such as those described in US2017 / 0189476.

[0150] Candidate immune checkpoint molecule antagonists can be screened for function by a variety of techniques known in the art and / or disclosed herein, such as the ability to interfere with immune checkpoint molecule function in vitro or in mouse models.

[0151] ICOS agonists The present invention further includes ICOS agonists. ICOS agonists activate ICOS. ICOS is a positive co-stimulatory molecule expressed on activated T cells and promotes their proliferation upon binding to its ligand (Dong (2001) Nature 409: 97-101).

[0152] In one embodiment, the agonist is ICOS-L, a natural ligand of ICOS. The agonist may be a mutant form of ICOS-L that retains binding and activation properties. Mutated forms of ICOS-L may be screened for their activity in stimulating ICOS in vitro.

[0153] Antibodies that are immune checkpoint antagonists or agonists . In a preferred embodiment, the immune checkpoint molecule antagonist and / or agonist each comprises an antibody. As described herein, in various embodiments, the antibody can be synthetic, monoclonal or polyclonal and can be prepared by techniques well known in the art. Such antibodies specifically bind to the immune checkpoint molecule through the antibody's antigen binding site (as opposed to non-specific binding). Immune checkpoint peptides, fragments, variants, fusion proteins, etc. can be used as immunogens to produce antibodies that immunoreact with them. More specifically, polypeptides, fragments, variants, fusion proteins, etc. contain antigenic determinants or epitopes that trigger antibody formation.

[0154] In a more preferred embodiment, the antibodies of the present invention are those that are approved by a sovereign government for use in treating human cancer patients or are in the process of being approved. Some non-limiting examples of such antibodies that have been approved or are in the process of being approved include the following antibodies: CTLA-4 ( and tremelimumab); PD-1 (pembrolizumab, lambrolizumab, amplimmune-224 (AMP-224), amplimmune-514 (AMP-514), nivolumab, MK-3475 (Merck), BI 754091 (Boehringer Ingelheim)) and PD-L1 (atezolizumab, avelulmab, durvalumab, MPDL3280A (Roche), MED14736 (AZN), MSB0010718C (Merck); LAG-3 (IMP321, BMS-986016, BI754111 (Boehringer Ingelheim)). Ingelheim), LAG525 (Novartis), MK-4289 (Merck), TSR-033 (Tesaro).

[0155] In one exemplary aspect, 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 any recombinant and synthetically produced binding partners.

[0156] Tumor-associated antigen (TAA)-specific antibodies . In various embodiments of the present invention, the recombinant MVA and methods described herein are combined or administered in combination with TAA-specific antibodies. In more specific embodiments, the recombinant MVA and methods described herein are combined or administered in combination with antibodies specific for antigens expressed on the cell membrane of tumor cells. It is understood in the art that in many cancers, one or more antigens are expressed or overexpressed on the tumor cell membrane. See, for example, 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 for determining whether an antigen is expressed or overexpressed on tumor cells are well understood in the art (supra), as are methods for generating antibodies to specific antigens.

[0157] In a more specific embodiment, drug combinations and related methods include antibodies, wherein the antibody a) is specific for antigens expressed on tumor cell membranes and b) includes an Fc domain. In at least one aspect, the characteristics of the antibody (e.g., a) and b)) enable the antibody to be bound to and interact with effector cells, such as NK cells, macrophages, basophils, neutrophils, eosinophils, monocytes, mast cells and / or dendritic cells, and enable the antibody to be bound to tumor antigens expressed on tumor cells. In a preferred embodiment, the antibody includes an Fc domain. In another preferred embodiment, the antibody can bind to and interact with NK cells.

[0158] Some exemplary antibodies against antigens expressed on tumor cells contemplated by the present disclosure include, but are not limited to, anti-CD20 (e.g., rituximab; ofatumumab; tositumomab), anti-CD52 (e.g., alemtuzumab), ), anti-EGFR (such as cetuximab) 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), ), anti-PD-1 (e.g., avelumab, atezolizumab, durvalumab), anti-GD2 (e.g., 3F8, ch14.18, KW-2871, dinutuximab), anti-CEA, anti-MUC1, anti-FLT3, anti-CD19, anti-CD40, anti-SLAMF7, anti-CCR4, anti-B7-H3, anti-ICAM1, anti-CSF1R, anti-CA125 (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, Herzuma, ABP 980, and / or pertuzumab).

[0159] In a more preferred embodiment, the antibodies encompassed as part of the present invention include antibodies that, when administered to a patient, bind to a corresponding antigen on a tumor cell and induce antibody-dependent cell-mediated cytotoxicity (ADCC). In a more preferred embodiment, the antibodies include antibodies that are approved or pre-approved for the treatment of cancer.

[0160] In a more preferred embodiment, the antibody is an anti-HER2 antibody, an anti-EGFR antibody and / or an anti-CD20 antibody.

[0161] In a most preferred embodiment, the anti-HER2 antibody is selected from the group consisting of Pertuzumab, Trastuzumab, Herzuma, ABP980, and Ado-trastuzumab emtansine.

[0162] In a most preferred embodiment, the anti-EGFR antibody and anti-CD20 antibody are cetuximab and rituximab, respectively.

[0163] As described herein, in various embodiments, antibodies can be synthetic, monoclonal or polyclonal and can be prepared by techniques well known in the art. Such antibodies specifically bind to TAAs through the antibody's antigen-binding site (as opposed to non-specific binding). TAA peptides, fragments, variants, fusion proteins, etc. can be used as immunogens to generate antibodies that immunoreact therewith. More specifically, polypeptides, fragments, variants, fusion proteins, etc. contain antigenic determinants or epitopes that trigger antibody formation.

[0164] Antibody In various embodiments of the invention, the recombinant MVAs and methods described herein are combined and / or administered in combination with 1) an immune checkpoint antagonist or agonist antibody or 2) a TAA-specific antibody.

[0165] Antibodies are expected to be synthetic, monoclonal or polyclonal and can be prepared by techniques well known in the art. Such antibodies specifically bind to immune checkpoint molecules or TAA (in contrast to non-specific binding) through the antigen binding site of the antibody. Immune checkpoint and / or TAA peptides, fragments, variants, fusion proteins, etc. can be used as immunogens to produce antibodies with their immunoreactions. More specifically, polypeptides, fragments, variants, fusion proteins, etc. contain antigenic determinants or epitopes that trigger antibody formation.

[0166] These antigenic determinants or epitopes can be linear or conformational (discontinuous). Linear epitopes are composed of single amino acid portions of a polypeptide, while conformational or discontinuous epitopes are composed of amino acid portions from different regions of the polypeptide chain that are in close proximity when the protein folds (Janeway, Jr. and Travers, Immuno Biology 3:9 (Garland Publishing Inc., 2nd edition, 1996)). Because folded proteins have complex surfaces, the number of available epitopes is quite large; however, due to the conformation and steric hindrance of the protein, the number of antibodies that actually bind to the epitope is less than the number of available epitopes (Janeway, Jr. and Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nd edition, 1996)). Epitopes can be identified by any method known in the art.

[0167] The present invention includes antibodies that specifically bind to TAA or immune checkpoint molecules such as CTLA-4, PD-1, PD-L1, LAG-3, TIM-3 or ICOS and block their function ("antagonist antibodies") or enhance / activate their function ("agonist antibodies"), including scFV fragments. Such antibodies can be produced by conventional means.

[0168] In one embodiment, the invention includes monoclonal antibodies directed against a TAA or immune checkpoint molecule or that block ("antagonist antibodies") or enhance / activate ("agonist antibodies") the function of an immune checkpoint molecule or TAA.

[0169] Antibodies are able to bind to their targets with high affinity and specificity. They are relatively large molecules (about 150kDa) and when the antibody binding site is located near the protein-protein interaction site, they can spatially inhibit the interaction between two proteins (such as PD-1 and its target ligand). The present invention further includes antibodies that bind to epitopes in close proximity to the ligand binding sites of immune checkpoint molecules.

[0170] In various embodiments, the present invention includes antibodies that interfere with intermolecular interactions (e.g., protein-protein interactions), and antibodies that interfere with intramolecular interactions (e.g., conformational changes within a molecule). Antibodies can be screened for the ability to block or enhance / activate the biological activity of immune checkpoint molecules. Both polyclonal and monoclonal antibodies can be prepared by conventional techniques.

[0171] In one exemplary aspect, 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 TAA or CTLA-4, PD-1, PD-L1, LAG-3, TIM-3 and ICOS can be used to prepare antibodies that specifically bind to 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, such as F(ab')2 and Fab fragments, single-chain variable fragments (scFv), single-domain antibody fragments (VHH or nanobodies), bivalent antibody fragments (bivalent antibodies), and any recombinant and synthetically produced binding partners. In another exemplary aspect, if the antibody is expressed in an amount greater than or equal to about 10 7 M -1 The antibody is defined as specifically binding to an immune checkpoint molecule if it binds with a Kd of 0.05. 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).

[0172] Polyclonal antibodies can be readily produced from a variety of sources, such as horses, cattle, goats, sheep, dogs, chickens, rabbits, mice, or rats, using procedures well known in the art. Generally, purified TAAs or CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and ICOS, or peptides based on the amino acid sequences of appropriately bound CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and ICOS, are typically administered to a host animal by parenteral injection. Following booster immunization, a small serum sample is collected and tested for reactivity to CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and ICOS polypeptides. Examples of various assays that can be used for such determinations include those described in Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, and procedures such as countercurrent immunoelectrophoresis (CIEP), radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot analysis, and sandwich analysis. See U.S. Patent Nos. 4,376,110 and 4,486,530.

[0173] Monoclonal antibodies can be readily prepared using well-known methods. See, for example, the procedures described in U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKeam, and Bechtol (eds.) (1980).

[0174] For example, a host animal, such as a mouse, can be injected intraperitoneally with isolated and purified immune checkpoint molecules at least once, and preferably at least twice at intervals of about 3 weeks. The mouse serum is then analyzed by traditional dot blot techniques or antibody capture (ABC) to determine which animal is most suitable for fusion. After about two to three weeks, a large amount of immune checkpoint molecules are administered intravenously to the mouse. The mouse is then sacrificed according to a predetermined protocol, and the spleen cells are fused with commercially available myeloma cells, such as Ag8.653 (ATCC). In short, the myeloma cells are washed several times in culture medium and fused with mouse spleen cells at a ratio of about three spleen cells to one myeloma cell. The fusion agent can be any suitable reagent used in the art, such as polyethylene glycol (PEG). The fusion is plated onto a plate containing a culture medium that allows the fused cells to selectively grow. The fused cells can then be grown for about eight days. The supernatant of the resulting hybridoma is collected and added to a plate first coated with goat anti-mouse Ig. After washing, a marker, 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 large cultures and the supernatant subsequently purified on a Protein A column (Pharmacia).

[0175] The monoclonal antibodies of the present invention can be produced 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 technology to incorporate the variable regions of genes encoding specific binding antibodies. Such techniques are described in Larrick et al. ((1989) Biotechnology 7:394).

[0176] Antigen-binding fragments of such antibodies that can be produced by conventional techniques are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab and F(ab')2 fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided.

[0177] The monoclonal antibodies of the present invention include chimeric antibodies, such as humanized forms of murine monoclonal antibodies. Such humanized antibodies can be prepared by known techniques and have the advantage of reducing immunogenicity when the antibody is administered to humans. In one embodiment, the humanized monoclonal antibody includes the variable region (or only its antigen binding site) of a murine antibody and the constant region derived from a human antibody. Alternatively, the humanized antibody fragment may include the antigen binding site of a murine monoclonal antibody and the variable region fragment (lacking the antigen binding site) derived from a human antibody. The procedures for producing chimeric and further engineered monoclonal antibodies include those described in the following documents: 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 transgenic production of antibodies can be found in GB 2,272,440, US Pat. Nos. 5,569,825 and 5,545,806, all of which are incorporated herein by reference.

[0178] Antibodies produced by genetic engineering methods can be used, such as chimeric and humanized monoclonal antibodies comprising human and non-human parts, which can be prepared using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be produced by genetic engineering using standard DNA techniques known in the art, for example, using the methods described by Robinson et al. International Publication No. WO 87 / 02671; Akira et al. European Patent Application 0184187; Taniguchi, M., European Patent Application 0171496; Morrison et al. European Patent Application 0173494; Neuberger et al. PCT International Publication No. WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application 0125023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Nat'l. Acad. Sci. 84:3439-3443; Liu et 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; and 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.

[0179] With respect to synthetic and semisynthetic antibodies, such terms are intended to encompass, but are not limited to, antibody fragments, isotype-switched antibodies, humanized antibodies (e.g., mouse-human, human-mouse), hybrids, antibodies with multiple specificities, and fully synthetic antibody-like molecules.

[0180] For therapeutic applications, "human" monoclonal antibodies with human constant and variable regions are generally preferred to minimize the patient's immune response to the antibodies. Such antibodies can be produced by immunizing transgenic animals containing human immunoglobulin genes. See Jakobovits et al. Ann NY Acad Sci 764:525-535 (1995).

[0181] Human monoclonal antibodies against TAAs or immune checkpoint molecules can also be prepared by constructing a combinatorial immunoglobulin library, such as a Fab phage display library or a scFv phage display library, using immunoglobulin light and heavy chain cDNA prepared from mRNA of lymphocytes derived from a subject. See, for example, McCafferty et al. PCT Publication 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, a combinatorial library of antibody variable regions can be produced by mutating known human antibodies. For example, the variable regions of human antibodies known to bind to immune checkpoint molecules can be mutated, such as by using randomly altered mutagenic oligonucleotides to produce a mutated variable region library, which can then be screened for binding to immune checkpoint molecules. Methods for inducing random mutagenesis within the CDR regions of immunoglobulin heavy and / or light chains, methods for crossing random heavy and light chains to form pairs, and screening methods can be found in, for example, Barbas et al. PCT Publication WO 96 / 07754; Barbas et al. (1992) Proc. Nat'l Acad. Sci. USA 89:4457-4461.

[0182] Immunoglobulin libraries can be expressed by display packaging, preferably derived from a population of filamentous phage, to form antibody display libraries. Examples of particularly suitable methods and reagents for generating antibody display libraries can be found, for example, in Ladner et al. U.S. Patent No. 5,223,409; Kang et al. PCT Publication WO 92 / 18619; Dower et al. PCT Publication WO 91 / 17271; Winter et al. PCT Publication WO 92 / 20791; Markland et al. PCT Publication WO 92 / 15679; Breitling et al. PCT Publication WO 93 / 01288; McCafferty et al. PCT Publication WO 92 / 01047; Garrard et al. PCT Publication WO 92 / 09690; Ladner et al. PCT Publication WO 90 / 02809; 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; Griffths 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. (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. (1991) Proc. Nat'l. Acad. Sci. 88:7978-7982. Once displayed on the surface of a display package (e.g., a filamentous phage), the antibody library is screened to identify and isolate packages that express antibodies that bind to the TAA or immune checkpoint molecule.

[0183] Recombinant MVA In more preferred embodiments of the present invention, one or more of the proteins and nucleotides disclosed herein are included in a recombinant MVA. As described and illustrated in this disclosure, intravenous administration of the recombinant MVA of the present disclosure induces an enhanced immune response in cancer patients in various aspects. Thus, in one or more preferred embodiments, the present invention includes a recombinant MVA comprising a first nucleic acid encoding one or more TAAs described herein and a second nucleic acid encoding CD40L.

[0184] Examples of MVA virus strains that can be used to practice the present invention and that have been deposited in accordance with the requirements of the Budapest Treaty are strain MVA 572, deposited on January 27, 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, with the deposit number ECACC 94012707, and MVA 575, deposited on December 7, 2000, under the number ECACC 00120707, MVA-BN, deposited on August 30, 2000, under the number V00083008, at the European Collection of Cell Cultures (ECACC), and derivatives thereof are further exemplary strains.

[0185] As used herein, a "derivative" of MVA-BN refers to a virus that exhibits substantially the same replication characteristics as MVA-BN but exhibits differences in one or more portions of its genome. MVA-BN and its derivatives are replication-incompetent, meaning they are incapable of reproductive replication both in vivo and in vitro. More specifically, in vitro, MVA-BN or its derivatives have been described as capable of reproductive replication in chicken embryo fibroblasts (CEFs), but not in the human keratinocyte cell line HaCat (Boukamp et al. (1988) J. Cell Biol. 106:761-771), the human bone osteosarcoma cell line 143B (ECACC deposit number 91112502), the human embryonic kidney cell line 293 (ECACC deposit number 85120602), and the human cervical adenocarcinoma cell line HeLa (ATCC deposit number CCL-2). In addition, the viral amplification rate of MVA-BN or its derivatives in HeLa cells and HaCaT cell lines is at least two-fold less, and more preferably three-fold less, than that of MVA-575. Testing and analysis of these properties of MVA-BN and its derivatives are described in WO 02 / 42480 (US Patent Application No. 2003 / 0206926) and WO 03 / 048184 (US Patent Application No. 2006 / 0159699).

[0186] The terms "incapable of reproductive replication" or "incompetent of reproductive replication" as described in the previous paragraph in human cell lines in vitro are described, for example, in WO 02 / 42480, which also teaches how to obtain MVAs having the desired properties as described above. The terms apply to viruses that have an in vitro viral amplification rate of less than 1 4 days after infection using the assays described in WO 02 / 42480 or U.S. Patent No. 6,761,893.

[0187] The term "incapable of reproductive replication" means that the virus has a viral amplification rate of less than 1 in an in vitro human cell line as described in the previous paragraph 4 days after infection. The assay described in WO 02 / 42480 or US Patent No. 6,761,893 can be adapted for determination of viral amplification rate.

[0188] The amplification or replication of viruses in vitro human cell lines as described in the previous paragraph is usually expressed as the ratio of the virus produced by infected cells (output) to the amount of virus originally used to infect cells (input), which is called the "amplification rate". An amplification rate of "1" defines an amplification state in which the amount of virus produced from infected cells is the same as the amount originally used to infect cells, meaning that infected cells allow viral infection and propagation. In contrast, an amplification rate of less than 1, i.e., a reduction in output compared to the input level, indicates a lack of propagation and replication, and therefore the virus is weakened.

[0189] By "adjuvant" herein is meant that a particular encoded protein or component of the recombinant MVA increases the immune response produced by other encoded proteins or components of the recombinant MVA.

[0190] Expression cassette / control sequence. In various aspects, one or more nucleic acids described herein are contained in one or more expression cassettes, wherein the one or more nucleic acids are operably linked to expression control sequences. "Operably linked" means that the components described are related, allowing them to function in their intended manner, such as a promoter that transcribes the nucleic acid to be expressed. The expression control sequences operably linked to the coding sequence are joined so that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. Expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, start codons at the beginning of the open reading frame encoding the protein, splicing signals for introns, and in-frame stop codons. Suitable promoters include, but are not limited to, the SV40 early promoter, the RSV promoter, the retroviral LTR, the adenovirus major late promoter, the human CMV immediate early I promoter, and various poxvirus promoters, including, but not limited to, the following vaccinia virus or MVA-derived and FPV-derived promoters: 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 WO 2010 / 060632, WO 2010 / 102822, WO 2013 / 189611, WO 2014 / 063832 and WO 2017 / 021776, which are herein incorporated by reference in their entirety.

[0191] Additional expression control sequences include, but are not limited to, a leader sequence, a stop codon, a polyadenylation signal, and any other sequences necessary for proper transcription and subsequent translation of the nucleic acid sequence encoding the desired recombinant protein (e.g., HER2, Brachyury, and / or CD40L) in the desired host system. Poxvirus vectors may also contain additional elements necessary for transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the desired host system. Those skilled in the art will further appreciate that such vectors are readily constructed using conventional methods (Ausubel et al., (1987), "Current Protocols in Molecular Biology," John Wiley and Sons, New York, NY) and are commercially available.

[0192] Methods of administering the combination and dosing regimens In one or more aspects, the combinations of the invention may be administered as part of a homologous and / or heterologous prime-boost regimen. Figure 7The data shown in part illustrate that a homologous prime-boost regimen increases specific CD8 and CD4 T cell responses in a 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 a combination of the present disclosure to a cancer patient, wherein the combination is administered as part of a homologous or heterologous prime-boost regimen.

[0193] Generation of recombinant MVA viruses containing transgenes

[0194] The recombinant MVA viruses provided herein can be produced by conventional methods known in the art. Methods for obtaining recombinant poxviruses or inserting exogenous coding sequences into poxvirus genomes are well known to those skilled in the art. For example, standard molecular biology techniques, such as DNA cloning, DNA and RNA isolation, Western blot analysis, RT-PCR and PCR amplification techniques are described in Molecular Cloning, A Laboratory Manual (2nd edition, 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)). Likewise, techniques and expertise for the handling, manipulation and genetic engineering of MVA are described in Molecular Virology: A Practical Approach (Davison and 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 virus vectors)) and Current Protocols in Molecular Biology (John Wiley & Son, Inc. (1998) (see, e.g., Chapter 16, Part IV: "Expression of proteins in mammalian cells using vaccinia viral vectors")).

[0195] For the production of various recombinant MVA viruses disclosed herein, different methods can be applied. The DNA sequence to be inserted into the virus can be placed in an E. coli plasmid construct, into which DNA homologous to a poxvirus DNA fragment is inserted. In addition, the DNA sequence to be inserted can be connected to a promoter. The promoter-gene connection can be positioned in the plasmid construct so that both ends of the promoter-gene connection are flanked by DNA homologous to a DNA sequence flanked by regions of poxvirus DNA containing non-essential loci. The resulting plasmid construct can be amplified and isolated by propagation in E. coli. The isolated plasmid containing the DNA gene sequence to be inserted can be transfected into, for example, chicken embryo fibroblast (CEF) cell cultures and the cultures infected with MVA virus simultaneously. Recombination between the homologous MVA viral DNA in the plasmid and the viral genome can produce poxviruses modified by the presence of exogenous DNA sequences.

[0196] According to a preferred embodiment, cells of a suitable cell culture, such as CEF cells, can be infected with the MVA virus. The infected cells can then be transfected with a first plasmid vector comprising one or more exogenous or heterologous genes, such as one or more nucleic acids provided herein; preferably under the transcriptional control of poxvirus expression control elements. As described above, the plasmid vector also includes a sequence capable of directing the insertion of the exogenous sequence into a selected portion of the MVA viral genome. Optionally, the plasmid vector also contains a cassette comprising a marker and / or selection gene operably linked to a poxvirus promoter. Suitable markers or selection genes are, for example, genes encoding 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 poxvirus produced. However, the recombinant poxvirus can also be identified by PCR technology. Subsequently, another cell can be infected with the recombinant poxvirus obtained as described above and transfected with a second vector comprising one or more second exogenous or heterologous genes. If the gene is introduced into a different insertion site in the poxvirus genome, the second vector also differs in the poxvirus homologous sequence that directs integration of the second exogenous gene or genes into the poxvirus genome. After homologous recombination occurs, a recombinant virus containing two or more exogenous or heterologous genes can be isolated. To introduce additional exogenous genes into the recombinant virus, the infection and transfection steps can be repeated by infecting with the recombinant virus isolated in the previous step and transfecting with another vector containing another exogenous gene or genes.

[0197] Alternatively, the infection and transfection steps described above are interchangeable, i.e., suitable cells can be first transfected with a plasmid vector comprising the exogenous gene and then infected with the poxvirus. As another alternative, each exogenous gene can be introduced into a different virus, cells can be co-infected with all the recombinant viruses obtained, and recombinants comprising all the exogenous genes can be screened. A third alternative is to connect the DNA genome and the exogenous sequence in vitro and use a helper virus to reconstruct the recombinant vaccinia virus DNA genome. A fourth alternative is to perform homologous recombination between an MVA virus genome cloned as a bacterial artificial chromosome (BAC) and a linear exogenous sequence in Escherichia coli or another bacterial species, wherein the linear exogenous sequence is flanked by DNA sequences homologous to the sequences of the desired integration site in the flanking MVA virus genome.

[0198] One or more nucleic acids of the present disclosure can be inserted into any suitable part of the MVA virus or MVA viral vector. A suitable part of the MVA virus is a non-essential part of the MVA genome. The non-essential part of the MVA genome can be an intergenic region or known deletion sites 1-6 of the MVA genome. Alternatively or in addition, the non-essential part of the recombinant MVA can be a coding region of the MVA genome, which is non-essential for viral growth. However, the insertion site is not limited to these preferred insertion sites in the MVA genome, because it is within the scope of the present invention that the nucleic acids of the present invention (e.g., HER2, Brachyury, HERV-K-env, HERV-K-gag, PRAME, FOLR1 and CD40L and / or 4-1BBL) and any accompanying promoters described herein can be inserted into any position of the viral genome, as long as it is possible to obtain a recombinant that can be amplified and propagated in at least one cell culture system, such as chicken embryo fibroblasts (CEF cells).

[0199] Preferably, the nucleic acid of the present invention can be inserted into one or more intergenic regions (IGR) of the MVA virus. The term "intergenic region" preferably refers to those portions of the viral genome located between two adjacent open reading frames (ORFs) of the MVA viral genome, preferably between two essential ORFs of the MVA viral genome. For MVA, in certain embodiments, the IGR is selected from IGR07 / 08, IGR 44 / 45, IGR 64 / 65, IGR 88 / 89, IGR 136 / 137, and IGR 148 / 149.

[0200] For MVA virus, the nucleotide sequence may additionally or alternatively be inserted into one or more known deletion sites, i.e., deletion site I, II, III, IV, V, or VI of the MVA genome. The term "known deletion site" refers to a portion of the MVA genome that has been deleted by serial passage on CEF cells characterized at generation 516 relative to the genome of the parent virus from which MVA was 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).

[0201] vaccine

[0202] In certain embodiments, the recombinant MVA of the present disclosure can be formulated as part of a vaccine.For the preparation of a vaccine, the MVA virus can be converted into a physiologically acceptable form.

[0203] The following is an exemplary preparation. The purified virus was stored at -80°C with a titer of 5 × 10 8 TCID50 / ml, prepared in 10mM Tris, 140mM NaCl, pH 7.4. For the preparation of vaccine injection, for example, 1×10 8 -1×10 9 Individual virus particles can be lyophilized in phosphate buffered saline (PBS) in the presence of 2% peptone and 1% human albumin in an ampoule (preferably a glass ampoule). Alternatively, the vaccine injection can be prepared by gradually freeze-drying the virus in the preparation. In certain embodiments, the preparation contains additional additives, such as mannitol, dextran, sugar, glycine, lactose, polyvinyl pyrrolidone or other additives, for example, including but not limited to antioxidants or inert gases, stabilizers or recombinant proteins (such as human serum albumin) suitable for in vivo administration. The ampoule is then sealed and can be stored at a suitable temperature, for example, between 4°C and room temperature for several months. However, as long as it is not needed, the ampoule is preferably stored at a temperature below -20°C, most preferably at about -80°C.

[0204] In various embodiments involving vaccination or therapy, the lyophilisate is dissolved in 0.1 to 0.5 ml of an aqueous solution, preferably normal saline or Tris buffer, e.g., 10 mM Tris, 140 mM NaCl pH 7.7. It is contemplated that the recombinant MVA, vaccine, or pharmaceutical composition of the present disclosure can be administered in 10 4 to 10 10 TCID50 / ml, 10 5 to 5×10 9 TCID50 / ml, 10 6 to 5×10 9 TCID50 / ml or 107 to 5×10 9 The concentration range of TCID50 / ml is prepared in solution. Preferred doses for humans include 10 6 to 10 10 TCID50, including 10 6 TCID50,10 7 TCID50,10 8 TCID50, 5×10 8 TCID50,10 9 TCID50, 5×10 9 TCID50 or 10 10 Optimization of the dosage and frequency of administration is within the skill and knowledge of those skilled in the art.

[0205] In one or more preferred embodiments, recombinant MVA is administered intravenously to a cancer patient, as described herein. In other embodiments, recombinant MVA is administered intratumorally to a cancer patient. In other embodiments, recombinant MVA is administered intravenously and intratumorally to a cancer patient, either simultaneously or at different times.

[0206] In some embodiments, the MVA is designed to contain a TAA as well as a co-stimulatory molecule and is intended to be suitable for intravenous or intratumoral administration, or for administration by both routes of administration. Such MVAs may express one or more TAAs, including proteins of the human endogenous retrovirus K superfamily (HERV-K), such as HERV-K-env, HERV-K-gag, or HERV-K-mel, or synthetic variants thereof, such as those described in Example 38.

[0207] In additional embodiments, the recombinant MVA is administered to the patient and the immune checkpoint antagonist or agonist, or preferably an antibody, may also be administered systemically or locally, i.e., intraperitoneally, parenterally, subcutaneously, intravenously, intramuscularly, intranasally, intradermally, or any other route of administration known to those skilled in the art.

[0208] Kits, compositions, and methods of use In various embodiments, the present invention encompasses kits, pharmaceutical combinations, pharmaceutical compositions and / or immunogenic combinations comprising a) a recombinant MVA comprising a nucleic acid described herein and / or b) one or more antibodies described herein.

[0209] It is contemplated that the kits and / or compositions may comprise 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, and instructions for administering the recombinant MVA and the antibody. It is contemplated that in more specific embodiments, the kit may comprise instructions for administering the recombinant MVA and the antibody in a first priming administration, followed by one or more subsequent boosting administrations of the recombinant MVA and the antibody.

[0210] The kits and / or compositions provided herein can generally include one or more pharmaceutically acceptable and / or approved carriers, additives, antibiotics, preservatives, diluents and / or stabilizers. Such auxiliary substances can be water, saline, glycerol, ethanol, wetting agents or emulsifiers, pH buffer substances, etc. Suitable carriers are typically large, slowly metabolized molecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates, etc.

[0211] Certain exemplary embodiments

[0212] 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 intratumoral administration of the recombinant MVA results in enhanced inflammatory response in the cancerous tumor, increased tumor reduction, and / or increased overall survival in the subject, compared to non-intratumoral injection of a recombinant MVA virus comprising first and second nucleic acids encoding TAA and 4-1BBL antigens.

[0213] Embodiment 2 is a method of reducing tumor size and / or increasing survival of 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 the intravenous administration of the recombinant MVA enhances natural killer (NK) cell responses and enhances CD8 T cell responses specific to TAAs compared to non-intravenous injection of the recombinant MVA virus comprising the first and second nucleic acids encoding the TAA and 4-1BBL antigens.

[0214] Embodiment 3 is a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, the method 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 results in increased tumor reduction and / or increased overall survival in the subject compared to administration of the recombinant MVA and the 4-1BBL antigen alone.

[0215] Embodiment 4 is a method of inducing an enhanced inflammatory response in a cancerous tumor of a subject, the method comprising administering to the subject intratumorally 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 a 4-1BBL antigen, wherein the intratumoral administration of the recombinant MVA produces an enhanced inflammatory response in the tumor compared to the inflammatory response produced by non-intratumoral injection of a recombinant MVA virus comprising the first and second nucleic acids encoding the heterologous tumor-associated antigen and the 4-1BBL antigen. This enhanced inflammatory response is discussed elsewhere herein and may include, for example, the induction of NK cells and T cells.

[0216] Embodiment 5 is a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, the method 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 size and / or increases overall survival in the subject compared to administration of the recombinant MVA and the 4-1BBL antigen alone.

[0217] Embodiment 6 is a method according to any one of embodiments 1 to 5, wherein the subject is a human.

[0218] Embodiment 7 is a method according to any one of embodiments 1 to 4, wherein the TAA is an endogenous retroviral (ERV) protein.

[0219] Embodiment 8 is a method according to embodiment 7, wherein the ERV is an ERV protein expressed in tumor cells.

[0220] Embodiment 9 is a method according to any one of embodiments 7 to 8, wherein the ERV is from the human endogenous retrovirus protein K (HERV-K) family.

[0221] Embodiment 10 is a 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 and HERV-K mel protein.

[0222] Embodiment 11 is a 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 peptide, and immunogenic fragments thereof.

[0223] 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.

[0224] 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), or is a TAA that is a complex or combination of AH1A5, p15E and TRP2, e.g., as described in Example 1.

[0225] Embodiment 14 is a 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.

[0226] Embodiment 15 is the method of any one of embodiments 1 to 6, 12, and 14, wherein the TAA is PSA.

[0227] Embodiment 16 is a method according to 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, G250, G melanoma antigen gene family (GAGE-family), gastrin 17, gastrin-releasing hormone, ganglioside 2 (GD2) / ganglioside 3 (GD3) / ganglioside-monosialyl-2 ("GM2"), gonadotropin-releasing hormone (GnRH), UDP-GlcNAc:R1Man(α1-6)R2[GlcNAc:Man(α1-6)]β1,6-N -acetylglucosamine transferase 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), 70-kilodalton heat shock protein ("HSP70"), human telomerase reverse transcriptase (hTERT), insulin-like growth factor receptor-1 (IGFR-1), interleukin-13 receptor (IL-13R), inducible oxidative stress 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-MMPs, mucin, testis-specific antigen NY-ESO-1, osteonectin, p15, P170 / MDR1, p53, p97 / melanotransferrin, PAI-1, platelet-derived growth factor (PDGF), μPA, PRAME, prostaglandin, 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-α (TNF-α), TP1, TRP-2, tyrosinase, vascular endothelial growth factor (VEGF), ZAG, p16INK4, and glutathione-S-transferase (GST). A group consisting of: carcinoembryonic antigen (CEA), mucin 1 cell surface-related (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine composition-related protein 1 (TRP1), tyrosine-related protein 1 (TRP2), Brachyury, and combinations thereof.

[0228] Embodiment 17 is a method according to any one of embodiments 1 to 16, wherein the recombinant MVA further comprises a third nucleic acid encoding a CD40L antigen.

[0229] 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 agonist.

[0230] Embodiment 19 is a method according to embodiment 18, wherein the immune checkpoint molecule is selected from CTLA-4, PD-1, PD-L1, LAG-3, TIM-3 and ICOS.

[0231] Embodiment 20 is a method according to any one of embodiments 18 to 19, wherein the immune checkpoint molecule is PD-1 and / or PD-L1.

[0232] Embodiment 21 is a method according to embodiment 20, wherein the immune checkpoint molecule antagonist further includes an antagonist of LAG-3.

[0233] Embodiment 22 is a method according to any one of embodiments 18 to 21, wherein the immune checkpoint molecule antagonist comprises an antibody.

[0234] Embodiment 23 is a method according to any one of embodiments 1 to 17, further comprising administering to the subject an antibody specific for a second TAA.

[0235] Embodiment 24 is a method according to embodiment 23, wherein the antibody specific for the second TAA is specific for an antigen expressed on a tumor cell membrane.

[0236] 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 a tumor cell membrane and b) comprises an Fc domain.

[0237] Embodiment 26 is a pharmaceutical composition for use in the method according to any one of embodiments 1 to 25.

[0238] Embodiment 27 is a vaccine for use in the method according to any one of embodiments 1 to 25.

[0239] Embodiment 28 is a recombinant modified vaccinia Ankara (MVA) for treating a subject having 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.

[0240] Embodiment 29 is a recombinant MVA according to embodiment 28, wherein TAA is an endogenous retroviral (ERV) protein.

[0241] Embodiment 30 is a recombinant MVA according to embodiment 29, wherein the ERV protein is from the human endogenous retrovirus protein K (HERV-K) family.

[0242] Embodiment 31 is a recombinant MVA according to embodiment 30, wherein the retroviral protein K is selected from the group consisting of HERV-K envelope protein, HERV-K gag protein and HERV-Kmel protein.

[0243] Embodiment 32 is a recombinant MVA according to any one of embodiments 28 to 31, further comprising a third nucleic acid encoding CD40L.

[0244] Embodiment 33 is a pharmaceutical combination comprising a) the recombinant MVA according to any one of embodiments 28 to 32 and b) at least one of an immune checkpoint molecule antagonist or agonist.

[0245] Embodiment 34 is a drug combination according to embodiment 33, wherein the immune checkpoint molecule antagonist or agonist is selected from antagonists or agonists of CTLA-4, PD-1, PD-L1, LAG-3, TIM-3 and ICOS.

[0246] Embodiment 35 is a pharmaceutical combination according to embodiment 34, wherein the immune checkpoint molecule antagonist is an antagonist of PD-1 and / or PD-L1.

[0247] Embodiment 36 is a pharmaceutical combination according to embodiment 35, wherein the immune checkpoint molecule antagonist further includes an antagonist of LAG-3.

[0248] Embodiment 37 is a drug combination according to any one of embodiments 33 to 36, wherein the immune checkpoint molecule antagonist comprises an antibody.

[0249] Embodiment 38 is a pharmaceutical combination comprising a) the recombinant MVA according to any one of embodiments 28 to 32, and b) an antibody specific for a second TAA.

[0250] Embodiment 39 is the pharmaceutical combination according to embodiment 38, wherein the antibody specific for the second TAA is specific for an antigen expressed on the tumor cell membrane.

[0251] Embodiment 40 is the pharmaceutical combination according to embodiment 39, wherein the antibody specific for the second TAA a) is specific for an antigen expressed on a tumor cell membrane and b) comprises an Fc domain.

[0252] Embodiment 41 is the recombinant MVA according to any one of embodiments 28 to 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 to 40, for use in reducing tumor size and / or increasing survival of a subject having a cancerous tumor.

[0253] Embodiment 42 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 pharmaceutical combination according to any one of embodiments 33 to 40, for use in 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 MVA according to embodiments 28 to 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 to 40, wherein the intratumoral administration results in enhanced inflammatory response in the cancerous tumor, increased tumor reduction, and / or increased overall survival in the subject compared to non-intratumoral injection of a recombinant MVA virus comprising a first and a second nucleic acid encoding TAA and a 4-1BBL antigen.

[0254] 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 pharmaceutical combination according to any one of embodiments 33 to 40, for use in 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 the recombinant MVA according to embodiments 28 to 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 to 40, wherein the intravenous administration increases tumor reduction and / or increases overall survival in the subject compared to non-intravenous administration of the recombinant MVA virus comprising first and second nucleic acids encoding TAA and 4-1BBL antigens.

[0255] 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 pharmaceutical combination according to any one of embodiments 33 to 40, for use in a method of inducing an enhanced inflammatory response in a cancerous tumor of a subject with cancer, the method comprising administering the recombinant MVA according to embodiments 28 to 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 to 40 intratumorally to the subject, wherein the intratumoral administration enhances the inflammatory response in the cancerous tumor of the subject compared to non-intratumoral injection of a recombinant MVA virus comprising a first and a second nucleic acid encoding a TAA and a 4-1BBL antigen.

[0256] Embodiment 45 is the recombinant MVA of any one of embodiments 28 to 32, the vaccine of embodiment 27, the pharmaceutical composition of embodiment 26, or the pharmaceutical combination of any one of embodiments 33 to 40, for use in a method of treating cancer in a subject.

[0257] Embodiment 46 is the recombinant MVA of any one of embodiments 28 to 32, the vaccine of embodiment 27, the pharmaceutical composition of embodiment 26, or the pharmaceutical combination of any one of embodiments 33 to 40, for use in a method of treating cancer, wherein the cancer is selected from 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.

[0258] Embodiment 47 is a recombinant MVA according to embodiment 44, wherein the enhanced inflammatory response is localized to the tumor.

[0259] Embodiment 48 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 CD40L, wherein intratumoral administration of the recombinant MVA results in enhanced inflammatory response in the cancerous tumor, increased tumor reduction, and / or increased overall survival in the subject compared to non-intratumoral injection of the recombinant MVA virus comprising the first and second nucleic acids encoding the TAA and CD40L.

[0260] Embodiment 49 is 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 the 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 the recombinant MVA virus comprising the first and second nucleic acids encoding the TAA and CD40L antigens.

[0261] Embodiment 50 is a method of reducing tumor size and / or increasing survival in a subject having a cancerous tumor, the method 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 results in increased tumor reduction and / or increased overall survival in the subject compared to administration of the recombinant MVA and the CD40L antigen alone.

[0262] Embodiment 51 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 pharmaceutical combination according to any one of embodiments 33 to 40, for use in a method of reducing tumor size and / or increasing survival of a subject having a cancerous tumor, the method comprising administering to the subject intravenously and / or intratumorally the recombinant MVA according to embodiments 28 to 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 to 40, wherein the intravenous and / or intratumoral administration increases tumor reduction and / or increases overall survival in the subject compared to non-intravenous or non-intratumoral administration of any MVA selected from the following groups: 1) a recombinant MVA virus comprising a first nucleic acid encoding a TAA and a second nucleic acid encoding a 4-1BBL antigen; 2) a recombinant MVA virus comprising a first nucleic acid encoding a TAA and a second nucleic acid encoding a CD40L antigen; or 3) a recombinant MVA virus comprising a first nucleic acid encoding a TAA, a second nucleic acid encoding a 4-1BBL antigen, and a third nucleic acid encoding a CD40L antigen.

[0263] Embodiment 52 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 pharmaceutical combination according to any one of embodiments 33 to 40, for use in a method of reducing tumor size and / or increasing survival of a subject having a cancerous tumor, the method comprising intravenously and intratumorally administering to the subject a recombinant MVA according to embodiments 28 to 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 to 40, wherein the intravenous and intratumoral administration increases tumor reduction and / or increases overall survival in the subject compared to non-intravenous or non-intratumoral administration of any MVA selected from the following groups: 1) a recombinant MVA virus comprising a first nucleic acid encoding a TAA and a second nucleic acid encoding a 4-1BBL antigen; 2) a recombinant MVA virus comprising a first nucleic acid encoding a TAA and a second nucleic acid encoding a CD40L antigen; or 3) a recombinant MVA virus comprising a first nucleic acid encoding a TAA, a second nucleic acid encoding a 4-1BBL antigen, and a third nucleic acid encoding a CD40L antigen. It will be apparent to one skilled in the art that the intravenous and intratumoral administrations can be performed simultaneously or at different times.

[0264] Further implementation plans

[0265] In one aspect, the present invention provides a recombinant modified vaccinia virus Ankara (MVA) comprising:

[0266] (a) a first nucleic acid encoding a tumor-associated antigen (TAA);

[0267] (b) a second nucleic acid encoding 4-1BB ligand (4-1BBL); and

[0268] (c) at least one additional nucleic acid encoding a TAA.

[0269] In one embodiment, the recombinant MVA further comprises:

[0270] (d) Nucleic acid encoding CD40 ligand (CD40L).

[0271] In one embodiment, the recombinant MVA comprises two, three, four, five, six or more nucleic acids, each encoding a different TAA.

[0272] In one embodiment of the recombinant MVA, the TAA is selected from the group consisting of: endogenous retroviral (ERV) proteins, endogenous retroviral (ERV) peptides, 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, p15, AH1A5, folate receptor alpha (FOLR1), melanoma preferentially expressed antigen (PRAME) and MEL; and combinations thereof.

[0273] In one embodiment of the recombinant MVA, the ERV protein is from the human endogenous retrovirus K (HERV-K) family, preferably selected from the group consisting of HERV-K envelope (HERV-K-env) protein and HERV-K gag protein.

[0274] In one embodiment of the recombinant MVA, the ERV peptide is from the human endogenous retrovirus K (HERV-K) family, preferably selected from the pseudogene of the HERV-K envelope protein (HERV-K-env / MEL).

[0275] In another aspect, the present invention provides a recombinant modified vaccinia virus Ankara (MVA) comprising:

[0276] (i) a nucleic acid encoding HERV-K-env / MEL;

[0277] (ii) a nucleic acid encoding HERV-K gag;

[0278] (iii) a nucleic acid encoding FOLR1 and PRAME, preferably expressed as a fusion protein; and (iv) a nucleic acid encoding 4-1BBL.

[0279] In one embodiment, the recombinant MVA further comprises:

[0280] (v) Nucleic acid encoding CD40L.

[0281] In one embodiment, the nucleic acid in (i) encodes a HERV-K-env / MEL comprising a HERV-K-env surface (SU) and a transmembrane (TM) unit, wherein the TM unit is mutated, preferably wherein the TM unit is mutated to inactivate the immunosuppressive domain. Preferably, the HERVK-MEL is inserted into the mutated TM unit. More preferably, the HERVK-MEL replaces a portion of the immunosuppressive domain of the TM unit.

[0282] In one embodiment, the nucleic acid sequence in (i) encodes an amino acid sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO:7.

[0283] In one embodiment, the nucleic acid sequence in (i) comprises or consists of the nucleic acid sequence shown in SEQ ID NO:8.

[0284] In one embodiment, the nucleic acid in (i) encodes a HERVK-env / MEL comprising a HERV-K-env surface (SU) and a transmembrane (TM) unit, wherein the TM unit is shortened to less than 20 amino acids, preferably less than 10 amino acids, more preferably less than 8 amino acids, and most preferably 6 amino acids.

[0285] In one embodiment, the nucleic acid encoding in (i) comprises a HERV-K-env surface (SU) unit HERVK-env / MEL, wherein the RSKR furin protease cleavage site of the HERV-K-env SU unit is deleted. Preferably, HERVK-MEL is connected to the C-terminus of the HERV-Kenv SU unit.

[0286] In one embodiment, the nucleic acid in (i) encodes HERVK-env / MEL comprising a heterologous membrane anchor, preferably derived from human PDGF (platelet-derived growth factor) receptor.

[0287] In one embodiment, the nucleic acid sequence in (i) encodes an amino acid sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11.

[0288] In one embodiment, the nucleic acid sequence in (i) comprises or consists of the nucleic acid sequence shown in SEQ ID NO: 12.

[0289] In one embodiment, the recombinant MVA is derived from MVA-BN.

[0290] In another aspect, the present invention provides a pharmaceutical formulation or composition comprising the recombinant MVA of the present invention.

[0291] In one embodiment, the pharmaceutical formulation or composition is suitable for intratumoral and / or intravenous administration, preferably intratumoral administration.

[0292] In another aspect, the present invention provides recombinant MVA for use as a medicament or vaccine.

[0293] In another aspect, the present invention provides a recombinant MVA for use in treating cancer, preferably melanoma, breast cancer, colon cancer or ovarian cancer.

[0294] In another aspect, the present invention provides a recombinant MVA of the present invention for use in enhancing the inflammatory response of a cancerous tumor, reducing the size of a cancerous tumor, slowing or preventing the growth of a cancerous tumor and / or increasing the overall survival rate of a subject, preferably a human.

[0295] In one embodiment, the recombinant MVA used is administered intratumorally and / or intravenously, preferably intratumorally.

[0296] 23. In one embodiment, the recombinant MVA used is used in combination with a TAA-specific antibody.

[0297] 24. In one embodiment, the recombinant MVA is used in combination with an immune checkpoint molecule antagonist or agonist.

[0298] In another aspect, the present invention provides a method of treatment, wherein the recombinant MVA administered is a recombinant MVA according to the present invention.

[0299] Example

[0300] The following examples illustrate the invention but should not be construed as limiting the scope of the claims in any way.

[0301] Example 1: Construction of recombinant MVA-TAA-4-1BBL and MVA-TAA-CD40L

[0302] Recombinant MVA viruses containing elements of the present disclosure are generated by inserting a designated transgene with its promoter into the vector MVA-BN. The transgene is inserted using a recombinant plasmid containing the transgene and a selection cassette, along with sequences homologous to the target locus within MVA-BN. Homologous recombination between the viral genome and the recombinant plasmid is achieved by transfecting the recombinant plasmid into MVA-BN-infected CEF cells. In a second step, the selection cassette is then deleted with the help of a plasmid expressing the CRE recombinase, which is specific for the loxP sites flanking the selection cassette, thereby excising the intervening sequences. Alternatively, deletion of the selection cassette is achieved by MVA-mediated recombination using MVA-derived internal repeats.

[0303] For the construction of MVA-OVA and MVA-OVA-4-1BBL, the recombinant plasmids included the transgenes OVA or OVA and 4-1BBL, each preceded by a promoter sequence, and sequences identical to the target insertion site within MVA-BN to allow homologous recombination into the viral genome.

[0304] For the construction of MVA-OVA-CD40L, the recombinant plasmid included the transgenes OVA and CD40L, each preceded by a promoter sequence, and sequences identical to the target insertion site within MVA-BN to allow homologous recombination into the viral genome.

[0305] For the construction of MVA-gp70-4-1BBL, the recombinant plasmid included two transgenes, gp70 and 4-1BBL, each preceded by a promoter sequence and a sequence identical to the target insertion site within MVA-BN to allow homologous recombination into the viral genome.

[0306] For the construction of MVA-HERV-K, MVA-HERV-K-4-1BBL, and MVA-HERV-K-4-1BBL-CD40L, the recombinant plasmids included the HERV-K, HERV-K, and 4-1BBL, and HERV-K, 4-1BBL, and CD40L transgenes, respectively. Each transgene or group of transgenes was preceded by a promoter sequence and sequences identical to the target insertion site within MVA-BN to allow homologous recombination into the viral genome.

[0307] For the construction of MVA-AH1A5-p15E-TRP2 and MVA-AH1A5-p15E-TRP2-CD40L, the recombinant plasmids included the transgenes AH1A5-p15E-TRP2 or AH1A5-p15E-TRP2 and CD40L, each preceded by a promoter sequence, and sequences identical to the target insertion site within MVA-BN to allow homologous recombination into the viral genome.

[0308] To produce the above-mentioned mBN MVA, CEF cell cultures were inoculated with MVA-BN and transfected with the corresponding recombinant plasmids. Samples from these cell cultures were inoculated into CEF cultures in a medium containing a drug that induces selective pressure, and viral clones expressing fluorescence were isolated by plaque purification. The loss of the selection cassette containing the fluorescent protein in these viral clones was mediated by CRE-mediated recombination (involving two loxP sites on both sides of the selection cassette in each construct) or MVA-mediated internal recombination in the second step. After the second recombination step, only the transgenic sequence (e.g., OVA, 4-1BBL, gp70, HERV-K and / or CD40L) in which the promoter was inserted into the targeting site of MVA-BN was retained. A stock solution of plaque-purified virus lacking the selection cassette was prepared.

[0309] Expression of the identified transgenes was confirmed in cells inoculated with the constructs.

[0310] The production of the constructs described herein was performed using a cloned version of MVA-BN in a bacterial artificial chromosome (BAC). The recombinant plasmid contained the transgenic sequences described, each located downstream of a promoter. The plasmid included sequences that are also present in MVA and therefore allow specific targeting of the integration site. Briefly, infectious virus was recombined from the BAC by transfecting BAC DNA into BHK-21 cells and repeatedly infecting them with Shope fibroid virus as a helper virus. After three additional passages on CEF cell cultures, a version of the construct free of helper virus was obtained. Exemplary MVA production was also found in Baur et al. ((2010) Virol. 84: 8743-52, "Immediate-early expression of a recombinant antigen by modified vaccinia virus Ankara breaks the immunodominance of strong vector-specific B8R antigen in acute and memory CD8 T-cell responses").

[0311] Example 2: Effect of 4-1BBL-mediated MVA-OVA-4-1BBL-infected tumor cells on co-stimulation of CD8 T cells Influence of cytokine production without the need for DCs

[0312] 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 with 5% CO2. The next day, infected tumor cells were harvested and co-cultured in the presence of DCs at a 1:1 ratio for 4 hours at 37°C with 5% CO2 when 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. The cells were cultured for 48 hours at 37°C with 5% CO2. The culture supernatant was then collected for cytokine concentration analysis by Luminex. The results are shown in Figure 5. Figure 1 The supernatant concentrations of IL-6 ( Figure 1 A); GM-CSF( Figure 1 B); IL-2( Figure 1 C); and IFN-γ ( Figure 1 D). Data are expressed as mean ± SEM.

[0313] Consistent with previous reports, MVA-OVA-CD40L has a significant effect on DC activation and its antigen presentation ability. Therefore, MVA-OVA-CD40L-infected FLDCs produce a large amount of IL-6 ( Figure 1 A). Importantly, OVA-specific T cell responses could be fully induced in the presence of DCs, but not directly by MVA-CD40L-infected B16.F10 cells themselves ( Figure 1 B and 1C). These results indicate that DCs are explicitly required to exert the benefits of MVA-OVA-CD40L. In contrast, MVA-OVA-4-1BBL did not induce IL-6 production in DCs, but MVA-OVA-4-1BBL-infected B16.F10 cells elicited secretion of the T cell-activating cytokines IFN-γ, IL-2, and GM-CSF in a DC-independent manner ( Figure 1 A-1D).

[0314] Example 3: MVA-OVA-4-1BBL-infected tumor cells drive antigen-specific CD8 T cells differentiate into activated effector T cells

[0315] Dendritic cells (DCs) were generated after culturing bone marrow cells from C57BL / 6 mice in the presence of recombinant Flt3L for 14 days. 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 with 5% CO2. The next day, infected tumor cells were harvested and co-cultured in the presence of DCs at a 1:1 ratio for 4 hours at 37°C with 5% CO2 when indicated. At the same time, 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. The cells were cultured for 48 hours at 37°C with 5% CO2. The cells were then stained and analyzed by flow cytometry. The results are shown in Figure 5. Figure 2 The figure shows the GMFI of T-bet on OT-I CD8+ T cells ( Figure 2 A) and the percentage of CD44+ granzyme B+ IFN-γ+ TNFα+ OT-I CD8+ T cells ( Figure 2 B). Data are shown as mean ± SEM.

[0316] The results showed that in the absence of cross-presenting DCs, the induction of granzyme B+ and IFNγ+ cytotoxic effector T cells was dependent on 4-1BBL ( Figure 2 B) with Figure 1 Together with the results presented in , these findings suggest that, in contrast to MVA-encoded CD40L, which acts via DC activation, MVA-encoded 4-1BBL acts directly on T cells in a DC-independent manner.

[0317] Example 4: Infection with MVA encoding CD40L or 4-1BBL induces tumors in tumor cell lines and macrophages cell death

[0318] Tumor cell line B16.OVA ( Figure 3 A and 3B), MC38( Figure 3 C) and B16.F10( Figure 3 D) Infected with the indicated MOI for 20 hours. Then, the cell viability was analyzed by flow cytometry. Figure 3 Serum HMGB1 in samples of A was quantified by ELISA ( Figure 3 B). Bone marrow-derived macrophages (BMDM) were infected at the indicated MOI for 20 hours. Cell viability was then analyzed by flow cytometry. Figure 3 Shown in A-3E. Data are presented as mean ± SEM.

[0319] like Figure 3As shown in A and 3B, infection with MVA-OVA or MVA-OVA-CD40L resulted in a mild induction of cell death compared to PBS-treated tumor cells. Interestingly, infection with MVA-OVA-4-1BBL significantly enhanced tumor cell death at 18 hours post-infection.

[0320] To further confirm these results in non-antigenic cell lines, we used MC38 cells infected with MVA, MVA-CD40L, and MVA-4-1BBL (none of which encodes a TAA). Figure 3 C) and B16.F10( Figure 3 D) Tumor cells were similarly analyzed. Consistently, infection with these MVAs induced cell death in these tumor cell lines and effectively killed bone marrow-derived macrophages (BMDM) ( Figure 3 E) Taken together, these data demonstrate that MVA infection leads to tumor cell and macrophage cell death, which is increased when recombinant MVA expresses CD40L or 4-1BBL.

[0321] Oncolytic viral infection of tumor cells leads to the induction of 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 an alarm to the immune system, leading to enhanced antigen presentation and, thereby, induction of antitumor immunity. We tested whether MVA infection could induce ICD via secreted HMGB1. Unexpectedly, we found that MVA-OVA-4-1BBL and MVA-OVA-CD40L induced a significant increase in HMGB1 compared to MVA-OVA ( Figure 3 B).

[0322] Example 5: MVA encoding 4-1BBL induces NK cell activation in vivo

[0323] C57BL / 6 mice (n=5 / group) were treated with normal saline or 5×10 7 TCID50 MVA-OVA( Figure 4 "rMVA") in the 7 TCID50 MVA-OVA-4-1BBL( Figure 4 "rMVA-4-1BBL" in the sample) or 5 × 10 7 TCID50 MVA-OVA was combined with 200 μg of anti-4-1BBL antibody (clone TKS-1) for intravenous immunization. After 24 hours, the mice were sacrificed and the spleens were processed for flow cytometric analysis. Figure 4 A and Figure 4 As shown in B. CD69 ( Figure 4A) and CD70( Figure 4 B) Geometric mean fluorescence intensity (GMFI). Data are shown as mean ± SEM and are representative of two independent experiments.

[0324] The results showed that the quality of the NK cell response was enhanced by the addition of 4-1BBL to MVA-OVA compared with IV administration of MVA-OVA in the absence of 4-1BBL, and both NK cell activation markers, CD69 and CD70, were strongly upregulated compared with MVA-OVA ( Figure 4 A and B). Co-injection of a 4-1BBL-blocking antibody showed that MVA-OVA-induced NK cell activation was completely independent of 4-1BBL but could be enhanced when MVA-OVA-4-1BBL delivered excessive 4-1BBL signaling.

[0325] Example 6: Intravenous immunization with MVA encoding 4-1BBL promotes serum IFN-γ secretion in vivo

[0326] C57BL / 6 mice (n=5 / group) were treated with normal saline or 5×10 7 TCID50 "rMVA" (=MVA-OVA), 5×10 7 TCID50 "rMVA-4-1BBL" (=MVA-OVA-4-1BBL) or 5×10 7 TCID50 MVA-OVA was combined with 200 μg of anti-4-1BBL antibody (clone TKS-1) for intravenous immunization. Figure 5 Shown in A and 5B. Data are shown as mean ± SEM. Figure 5 A: Six hours later, the mice were bled, serum was separated from the whole blood, and the IFN-γ concentration in the serum was measured by Luminex. Figure 5 B: 3, 21, and 45 hours after immunization, mice were intravenously injected with brefeldin A to stop protein secretion. Mice were sacrificed 6, 24, and 48 hours after immunization, and splenocytes were analyzed by flow cytometry.

[0327] 4-1BB-mediated NK cell activation is consistent with increased serum levels of the NK effector cytokine IFNγ ( Figure 5 A). NK cells are known to produce large amounts of IFN-γ upon activation. To determine whether the increased IFN-γ levels in serum might be derived from NK cells, the proportion of IFN-γ-producing NK cells was measured at different time points after intravenous injection of the indicated recombinant MVA vectors. The percentage of IFN-γ+ NK cells was highest 6 hours after injection, when high serum IFN-γ levels were measured, and then slowly decreased ( Figure 5B) The highest frequency of IFN-γ-positive NK cells was observed when MVA-OVA-4-1BBL was used. Taken together, these data indicate that intravenous immunization with rMVA-4-1BBL leads to robust activation of NK cells and increased production of the NK cell effector cytokine IFN-γ.

[0328] Example 7: Intravenous rMVA-4-1BBL immunization promotes serum IFN-γ levels in B16.OVA tumor-bearing mice secretion

[0329] On day 7 after tumor inoculation, C57BL / 6 mice (n=5 / group) bearing B16.OVA tumors were divided into groups and received iv (intravenous) PBS or 5×10 7 TCID50MVA-OVA ("rMVA" in the figure) or MVA-OVA-4-1BBL ("rMVA-4-1BBL" in the figure). Six hours later, the mice were bled, serum was separated from the whole blood, and the IFN-γ concentration in the serum was measured by Luminex. The results are shown in Figure 6 Data are shown as mean ± SEM.

[0330] Figure 6 The data shown in Figure 2 indicate that similar effects on NK cells as reported in other experiments can also be obtained in a melanoma model. Six hours after immunization, serum IFN-γ levels in tumor-bearing mice immunized with MVA-OVA-4-1BBL were significantly increased, indicating strong NK cell activation ( Figure 6 ).

[0331] Example 8: Intravenous rMVA-4-1BBL priming and boosting enhances antigen- and vector-specific CD8+ T cells Amplification

[0332] Figure 7 A-7D shows antigen and vector specificity after intravenous rMVA-4-1BBL priming and boosting. C57BL / 6 mice (n=4 / group) received either saline or 5×10 7 TCID50 rMVA (=MVA-OVA), 5×10 7 TCID50rMVA-4-1BBL (=MVA-OVA-4-1BBL) or 5×10 7 TCID50 rMVA was combined with an intravenous priming of 200 μg of anti-4-1BBL antibody (clone TKS-1) and a booster immunization was received on day 41. Mice were bled on days 6, 21, 35, 48, and 64 after the priming, and peripheral blood was analyzed by flow cytometry. Mice were sacrificed on day 70 after the priming. Spleens were harvested and analyzed by flow cytometry.

[0333] The results are Figure 7 Shown in A-7D. Figure 7A shows the percentage of antigen (OVA)-specific CD8+ T cells in peripheral blood leukocytes (PBL), and Figure 7 B shows the percentage of vector (B8R)-specific CD8+ T cells in PBL. Figure 7 C shows the percentage of antigen (OVA)-specific CD8+ T cells in living cells. Figure 7 D shows the percentage of vector (B8R)-specific CD8+ T cells in living cells. Data are shown as mean ± SEM.

[0334] The results showed that B8 and OVA-specific CD8 T cells reached their maximum on day 7 after the first immunization and further expanded after the second immunization on day 41 ( Figure 7 A and B). At the 41-day time point, rMVA-4-1BBL had a clear advantage in antigen-specific T cell responses compared to rMVA for both B8 and OVA. Interestingly, co-injection of a 4-1BBL-blocking antibody showed that rMVA-induced T cell responses were completely independent of 4-1BBL but could be enhanced when rMVA-4-1BBL delivered an excessive amount of 4-1BBL signaling ( Figure 7 A and B). Consistent with these results, rMVA-4-1BBL prime / boost immunization also resulted in improved OVA- and B8-specific T cell responses in the spleen 70 days after the first immunization ( Figure 7 C and D).

[0335] Example 9: Enhanced anti-tumor effect of intravenous injection of MVA virus encoding TAA and 4-1BBL

[0336] On day 7 after tumor inoculation (black dashed line), C57BL / 6 mice (n=5 / group) bearing B16.OVA tumors were divided into groups and received iv (intravenous) PBS or 5×10 7 TCID50MVA-OVA or 5×10 7 TCID50 MVA-OVA-4-1BBL. Tumor growth was measured regularly. Figure 8 As shown in , intravenous administration of MVA virus encoding 4-1BBL resulted in a reduction in tumor volume compared to MVA or control (PBS), which was a consequence of prolonged tumor growth delay.

[0337] Example 10: Intratumoral injection of MVA viruses encoding 4-1BBL or CD40L enhances the antitumor effect

[0338] On days 7 (black dashed line), 12, and 15 (grey dashed line) after tumor inoculation, C57BL / 6 mice (n=4-5 / group) bearing B16.OVA tumors were divided into groups and received intratumoral (it) 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). Tumor growth was measured regularly. Figure 9 As shown in A-9D, enhanced antitumor effects were achieved by intratumoral injection of MVA viruses encoding TAA and 4-1BBL or CD40L. More specifically, Figure 9 As shown in Figure D, a significantly greater reduction in tumor growth was observed with the MVA virus encoding 4-1BBL. Although the present invention is not bound by any particular mechanism or mode of action, one hypothesis for the observed differences between 4-1BBL and CD40L is that 4-1BBL is intended to activate NK cells and T cells, while CD40L is intended to activate DCs. B16 melanoma tumors are more infiltrated by T cells (Mosely et al. (2016) Cancer Immunol. Res. 5(1): 29-41); therefore, in this case, MVA encoding 4-1BBL is more effective than MVA encoding CD40L.

[0339] Regardless of the exact mechanism or pathway by which 4-1BBL and CD40L exert their effects on tumor growth or diameter, Figure 9 The data in show that intratumoral injection of MVA encoding 4-1BBL resulted in prolonged tumor growth control and, in some cases, complete tumor rejection.

[0340] Example 11: Antitumor Effect of Intratumoral Injection of MVA Viruses Encoding TAA and CD40L on Established Colon Cancer Enhanced effect

[0341] On days 14 (black dashed line), 19, and 22 (black dashed line) after tumor inoculation, C57BL / 6 mice (n=5 / group) bearing MC38 tumors were divided into groups and received intratumoral (it) PBS or 5×10 7 TCID50 MVA-AH1A5-p15E-TRP2 (labeled as "rMVA" in the figure) or MVA-AH1A5-p15E-TRP2-CD40L (labeled as "rMVA-CD40L" in the figure). Tumor growth was measured regularly. Results for non-antigenic, established MC38 colon cancer are as follows Figure 10 As shown in .

[0342] These vectors encode a string of tumor-associated epitopes consisting of: a melanoma-associated TRP2-derived epitope (SVYDFFVWL, H2-K b ) and two murine leukemia virus gp70-derived CD8 + T cell epitope p15E (KSPWFTTL, H2-K b) and modified AH1, AH1A5 (SPSYAYHQF, H2-L d ), these results indicate that intratumoral injection of MVA-CD40L can significantly delay tumor growth in the MC38 colon cancer model.

[0343] Example 12: Intratumoral administration of immune checkpoint blockade and tumor antigen-specific antibodies with MVA-OVA-4-1BBL Use synergy

[0344] B16.OVA melanoma cells (5×10 5 ) were injected subcutaneously into C57BL / 6 mice. When the tumor diameter reached approximately 5 mm, the mice were divided into groups (n=5 / group) and received 200 μg IgG2a, anti-TRP-1, or anti-PD-1 intraperitoneally (ip) at the indicated time (check mark). PBS or 5×10 7 Mice were immunized intratumorally (it) with TCID50 MVA-OVA-4-1BBL. Tumor growth was measured regularly. Figure 11 When the tumor-associated antigen (TAA) Trp1-specific antibody (anti-Trp1) was combined with intratumoral administration of MVA-OVA-4-1BBL, the reduction in tumor volume was increased compared to anti-PD-1 alone ( Figure 11 , middle row). When the immune checkpoint molecule antibody PD-1 was combined with intratumoral administration of MVA-OVA-4-1BBL, the reduction in tumor volume was increased compared to anti-PD-1 alone ( Figure 11 , bottom row).

[0345] These experiments demonstrate that anti-PD-1 and anti-TRP-1 antibodies, as single agents, enhance control of tumor growth, while the combination of either antibody with MVA-OVA-4-1BBL enhances the therapeutic effect exerted by MVA-OVA-4-1BBL. Here, combination therapy with intratumoral MVA 4-1BBL and checkpoint blockade or TAA-targeting antibodies exhibits greater therapeutic activity than either monotherapy. These data also suggest that tumor-specific antibodies with the potential to induce ADCC could be combined with intratumoral administration of 4-1BBL-expressing MVA to produce a synergistic effect.

[0346] Example 13: Superior Anti-CD137 Antibody Injection in Tumors Compared to Treatment with Agonist Anti-CD137 Antibodies Tumor effects

[0347] On days 7, 12, and 15 after tumor inoculation (black dashed lines), C57BL / 6 mice bearing B16.OVA tumors (n=5 / group) were divided into groups and injected intratumorally with PBS, 5×10 7TCID50 MVA-OVA-4-1BBL or 10 μg anti-4-1BB (3H3, BioXcell). Tumor growth was measured regularly.

[0348] Figure 12 A shows the superior antitumor effect of MVA-OVA-4-1BBL compared to the agonistic anti-4-1BBL antibody (3H3). Figure 12 B shows that intratumor immunization with MVA-OVA-4-1BBL induced only OVA-specific T cell responses in the blood, whereas agonistic anti-4-1BBL antibody did not induce any OVA-specific T cells in the blood.

[0349] Thus, these data suggest that intratumoral MVA-OVA-4-1BBL treatment is more effective than agonistic anti-CD137 antibody in both tumor-specific T cell responses and tumor growth control.

[0350] Example 14: Intravenous injection of endogenous retrovirus encoding CD40L in the CT26 tumor model Enhanced antitumor activity of MVA containing the (ERV) antigen Gp70

[0351] On day 12 after tumor introduction into mice (black dashed line), Balb / c mice (n=5 / group) bearing CT26 tumors were divided into groups and received intravenous (iv) PBS or 5×10 7 TCID50 MVA-BN, MVA-Gp70 or MVA-Gp70-CD40L. Tumor growth was measured regularly. Figure 13 As shown in Figures A and 13B, intravenous administration of MVA virus encoding the endogenous retroviral antigen Gp70 reduced tumor volume compared to MVA or control (PBS). The antitumor effect was further enhanced when CD40L was additionally encoded by MVA-Gp70-CD40L.

[0352] Figure 13 C shows the induction of Gp70-specific CD8 T cells in the blood after intravenous injection of MVA-Gp70 or MVA-Gp70-CD40L.

[0353] Therefore, in these experiments, MVAs encoding a model ERV were constructed, which is the murine protein gp70 (the envelope protein of murine leukemia virus) ("MVA-gp70"). MVAs that further included the co-stimulatory molecule CD40L were also generated ("MVA-gp70-CD40L"). The anti-tumor potential of these new constructs was tested using the 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). Mice bearing CT26.wt tumors were generated and, when tumors were at least 5 mm x 5 mm, immunized intravenously as indicated above. Immunization with MVA alone induced a mild delay in tumor growth. In contrast, immunization with MVA-gp70 resulted in complete rejection of 3 / 5 tumors ( Figure 13 A and B). Immunization with MVA-Gp70-CD40L yielded even more striking results, leading to rejection of 4 / 5 tumors ( Figure 13 A and B).

[0354] To determine whether these antitumor responses were associated with the induction of gp70-specific T cells after immunization, blood was restimulated using the H-2Kd-restricted gp70 epitope AH1. Figure 13 C) shows robust induction of gp70-specific CD8 T cell responses in MVA-Gp70 and MVA-Gp70-CD40L treated mice ( Figure 13 C).

[0355] Example 15: Intravenous injection of endogenous retrovirus encoding CD40L in the B16.F10 tumor model Antitumor effects of MVA containing Gp70 antigen are enhanced

[0356] On day 7 after tumor inoculation (black dashed line), when tumors measured approximately 5×5 mm, C57BL / 6 mice (n=5 / group) bearing B16.F10 tumors were divided into groups and received intravenous (iv) PBS or 5×10 7 TCID50 of MVA-BN, MVA-Gp70, or MVA-Gp70-CD40L. Tumor growth was measured regularly. Figure 14 As shown in A, intravenous administration of MVA virus encoding the endogenous retroviral antigens Gp70 and CD40L reduced tumor volume compared to MVA or control (PBS).

[0357] Figure 14 B shows the induction of Gp70-specific CD8 T cells in the blood after intravenous injection of MVA-Gp70 or MVA-Gp70-CD40L.

[0358] Therefore, in these experiments, the efficacy of treatment with MVA-Gp70 and MVA-Gp70-CD40L was confirmed in another independent tumor model. B16.F10 is a melanoma cell line derived from C57BL / 6 and expresses high levels of Gp70 (Scrimieri (2013) Oncoimmunol 2: e26889). Treatment with MVA alone ("MVA-BN") resulted in some tumor growth delay in B16.F10 tumors, comparable to the effect of non-adjuvant MVA-Gp70 ( Figure 14 A). However, MVA-Gp70-CD40L produced a stronger antitumor effect than the MVA backbone control alone ( Figure 14 A). Additional experiments showed that both groups receiving MVA encoding the Gp70 antigen exhibited 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 CD40L was also encoded by the MVA ( Figure 14 B).

[0359] Example 16: Enhanced antitumor effects of intravenous administration of MVA viruses encoding gp70 and 4-1BBL [Predictive implementation example]

[0360] On day 7 after tumor inoculation (black dashed line), C57BL / 6 mice (n=5 / group) bearing B16.OVA tumors were divided into groups and intravenously received PBS or 5×10 7 TCID50MVA-OVA or MVA-gp70-4-1BBL. Tumor growth was measured regularly. Because mouse homologs of human endogenous retrovirus (ERV) proteins are neither highly expressed in normal mouse tissues nor predominantly expressed in mouse tumor tissues, the efficacy of human ERVs cannot be effectively studied in mouse models. Gp70 is a well-studied mouse ERV protein (see, e.g., 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). Therefore, studying gp70-specific cancer vaccines in mice is likely to have strong predictive value for the efficacy of ERV-specific cancer vaccines in humans.

[0361] Example 17: Enhanced antitumor effects of intratumoral injection of MVA viruses encoding gp70 and 4-1BBL or CD40L [Predictive Example]

[0362] On days 7 (black dashed line), 12, and 15 (grey dashed line) after tumor inoculation, C57BL / 6 mice (n=4-5 / group) bearing B16.OVA tumors were divided into groups and received intratumoral (it) PBS or 5×107 TCID50 of MVA-OVA, MVA-OVA-CD40L, or MVA-OVA-4-1BBL. Tumor growth was measured regularly.

[0363] Example 18: Direct antigen presentation by infected tumor cells administered with rMVA-HERV-K-4-1BBL To influence cytokine production [Prophetic Example]

[0364] Dendritic cells (DCs) were generated after 14 days of culturing bone marrow cells from C57BL / 6 mice in the presence of recombinant Flt3L. B16.F10 cells were 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 and placed overnight. The next day, at 37°C 5% CO2, infected tumor cells were harvested and co-cultured for 4 hours at a 1:1 ratio in the presence of DC when indicated. HERV-K-specific CD8+ T cells were magnetically purified from HERV-K immune mice and added to the co-culture at a ratio of 1:5. Cells were cultured for 48 hours at 37°C 5% CO2. Culture supernatants were then collected for cytokine concentration analysis by Luminex. Cytokine level measurements included (A) IL-6, (B) GM-CSF, (C) IL-2, and (D) IFNγ. Data are expressed as mean ± SEM.

[0365] Example 19: Direct antigen presentation by infected tumor cells administered with rMVA-HERV-K-4-1BBL Directing Antigen-Specific CD8+ T Cells to Activated Effector T Cells [Prophetic Example]

[0366] Dendritic cells (DCs) were generated after culturing bone marrow cells from C57BL / 6 mice for 14 days in the presence of recombinant Flt3L. B16.F10 cells were 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 and placed overnight. The next day, at 37°C 5% CO2, infected tumor cells were harvested and co-cultured for 4 hours at a 1:1 ratio in the presence of DC when indicated. Meanwhile, HERV-K-specific CD8+ T cells were magnetically purified from HERV-K immune mice and added to the co-culture at a ratio of 1:5. The cells were cultured for 48 hours at 37°C 5% CO2. The cells were then stained and analyzed by flow cytometry. Cytokine analysis was performed on (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.

[0367] Example 20: Infection with CD40L or 4-1BBL-encoding rMVA-HERV-K in tumor cell lines and macrophages Inducing tumor cell death [Prophetic Example]

[0368] Tumor cell lines B16.OVA (A and B), MC38 (C), and B16.F10 (D) were infected at the specified MOI for 20 hours. Cell viability was then analyzed by flow cytometry. Serum HMGB1 in samples from (A) was quantified by ELISA. Bone marrow-derived macrophages (BMDM) were infected at the specified MOI for 20 hours. Cell viability was then analyzed by flow cytometry. Data are presented as mean ± SEM.

[0369] Example 21: Intratumoral administration of recombinant MVA encoding 4-1BBL leads to a decrease in Treg cells and T cells in tumors. Reduced Cell Depletion [Prophetic Example]

[0370] On day 7 after tumor inoculation (black dashed line), C57BL / 6 mice (n=5 / group) bearing B16.OVA tumors were divided into groups and received intratumoral (it) PBS or 5×10 7 TCID50 of MVA-OVA or MVA-OVA-4-1BBL. Five days later, mice were sacrificed, and spleens and tumors were harvested and stained with fluorochrome-conjugated antibodies to assess Treg infiltration and T cell exhaustion. (A) Percentage of CD4+FoxP3+ T cells among CD45+ tumor-infiltrating leukocytes; Geometric mean fluorescence intensity of PD-1 (B) and Lag-3 (C) on tumor-infiltrating CD8 T cells. Data are presented as mean ± SEM.

[0371] Example 22: Immune checkpoint blockade and tumor antigen-specific antibodies with rMVA gp-70-4-1BBL tumors Internal Administration Synergistic Effects [Prophetic Example]

[0372] C57BL / 6 mice (n=5 / group) bearing B16.OVA tumors were divided into groups and received 200 μg IgG2a, anti-TRP-1, or anti-PD-1 when indicated (checkmarks). PBS or 5×10 7 Mice were intratumorally immunized with TCID50 MVA-gp70-4-1BBL. Tumor growth was measured regularly.

[0373] Example 23: 4-1BBL adjuvant can increase cytokine / Response of the chemokine MVA-BN backbone to IT immunization answer

[0374] To evaluate the potential of recombinant MVA to induce inflammation within the tumor microenvironment (TME), cytokines and chemokines were analyzed in tissues from B16.OVA tumors. 5 B16.OVA cells were implanted subcutaneously (sc) into C57BL / 6 mice. On day 10, PBS or 2×10 8 TCID 50Mice were immunized intratumorally (it) with MVA-BN, MVA-OVA, or MVA-OVA-4-1BBL (n=5 to 6 mice / group).

[0375] Six hours after injection, the expression of cytokines and chemokines was measured ( Figure 15 Cytokine / chemokine expression in PBS-treated tissues represents the baseline inflammatory signature induced by needle insertion into the tumor and saline shear stress. Cytokines including IL-6, IFN-α, IL-15, and TNF-α, as well as chemokines such as CXCL1, CCL2, and MIP2 were upregulated ( Figure 15 IL-25 (also known as IL-17E), which is induced by NF-κβ activation and stimulates the production of IL-8 in humans, was also detected (Lee et al. (2001) J. Biol. Chem. 276:1660-64). Interestingly, tumors injected with MVA-OVA-4-1BBL showed a significant increase in proinflammatory cytokines such as IL-6, IFN-α, or IL-15 / IL15Rα compared to tumors injected with MVA-BN or tumor lesions injected with MVA-OVA.

[0376] Example 24: MVA-OVA-4-1BBL increases cytokine / chemokine promotion of intratumoral (it) immunity Inflammatory response

[0377] 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 ( Figure 16 The production of other proinflammatory cytokines, including IL-18, CCL5, CCL3, and IL-22, was enhanced by intratumoral (it) immunization with MVA-OVA or MVA-OVA-4-1BBL, rather than MVA-BN or PBS alone.

[0378] In summary, these data suggest that intratumoral (it) MVA immunization induces a shift in inflammatory cytokines and chemokines within the tumor microenvironment (TME), thereby enhancing the inflammatory response. An enhanced effect was observed with intratumoral immunization using MVA-OVA-4-1BBL compared to MVA or MVA-OVA. In this way, the addition of 4-1BBL can be said to "help" recombinant MVA.

[0379] Example 25: Quantitative and qualitative T cell analysis of the TME and draining LNs after intratumoral injection of MVA-OVA-4-1BBL

[0380] To better understand the cellular processes induced by inflammation after intratumoral (it) injection of MVA-OVA and MVA-OVA-4-1BBL, in-depth analysis of innate and adaptive immune infiltration at different time points after intratumoral (it) injection was performed. Mice bearing B16.OVA tumors were injected intratumorally (it) with PBS or 2×10 8 TCID 50 MVA-OVA or MVA-OVA-4-1BBL. Mice were sacrificed 1, 3, and 7 days after primary immunization. Tumors and tumor-draining lymph nodes (TdLN) were excised and treated with collagenase and DNase, and single cells were analyzed by flow cytometry. Immune cell populations were analyzed to determine their size, proliferation behavior, and functional status.

[0381] The results showed that 7 days after intratumoral (it) immunization, injection of MVA-OVA or MVA-OVA-4-1BBL into B16.OVA tumors induced CD45 + Leukocyte infiltration into the tumor ( Figure 17 , top row, left histogram). Interestingly, CD45 + Increased white blood cell count ( Figure 17 , top row, right histogram), especially after injection of MVA expressing 4-1BBL. This difference was further amplified in TdLN seven days after intratumoral (it) immunization, indicating that the anti-tumor effect mediated by MVA immunization begins in TdLN as early as day 3 after immunization.

[0382] One aspect of vaccination-based anti-tumor therapy is the tumor-specific CD8 + and CD4 + Expansion and rejuvenation of T cells and their enrichment in tumors. CD4 + T cells and CD8 + T cells increased ( Figure 17 , second and third rows, left histograms, respectively). CD4+ T cells increased in tumors by day 7 and in TdLNs starting on day 3 and peaked on day 7 after it immunization with MVA-OVA-4-1BBL. By day 7, CD8 + T cells contribute largely to the expression of CD45 in tumors + Compared with MVA-OVA injection in tumors (day 7) and dLN (days 3 and 7), injection of MVA-OVA-4-1BBL further expanded the CD8 + T cell population.

[0383] OVA-specific CD8 +Quantification of T cells showed an increase within the tumor microenvironment 7 days after intratumoral (it) immunization, particularly in the group treated with MVA-OVA-4-1BBL ( Figure 17 , lower left). Strikingly, OVA-specific CD8 + The expansion of T cells peaked on day 3 after immunization and was higher in the MVA-OVA-4-1BBL-treated group ( Figure 17 , lower right). Taken together, these data suggest that intratumoral immunization with MVA-OVA, and particularly MVA-OVA-4-1BBL, enhances the generation of adaptive immune responses starting 3 days after treatment of tumor-draining lymph nodes, resulting in antigen-specific CD8 + T cells increased significantly.

[0384] Example 26: Induction of antigen-specific CD8+ T cells by intratumoral injection of MVA-OVA-4-1BBL

[0385] Intratumoral injection of MVA-OVA-4-1BBL induces OVA-specific CD8 in tumor-draining lymph nodes (TdLN) + Compared with PBS, the OVA-specific CD8 T cells expressing Ki67 (a cell proliferation indicator) in TdLN after MVA-OVA treatment were significantly higher. + The percentage of T cells was higher and further increased in mice immunized with MVA-OVA-4-1BBL ( Figure 18 A). In addition, OVA-specific CD8 T cells in the tumor downregulated the exhaustion marker PD-1 7 days after immunization with MVA-OVA and MVA-OVA-4-1BBL, indicating functional recovery ( Figure 18 B).

[0386] Treg cells (also known as "regulatory T cells") are potent suppressors of anti-tumor immune responses (see, e.g., Tanaka et al. (2017) Cell Res. 27:109-118). Intratumoral injection of MVA-OVA increased the OVA-specific Teff / Treg ratio (i.e., the ratio of "Teff" cells or "effector T cells" to Treg cells) in tumors, and further increases were observed on day 7 after treatment with MVA-OVA-4-1BBL ( Figure 18 C) Thus, intratumoral treatment with MVA-OVA, and in particular with MVA-OVA-4-1BBL, reduces the frequency of intratumoral Tregs in favor of CD8+ T effector cells, which favors the antitumor immune response.

[0387] Example 27: Quantitative and qualitative NK cell analysis of the TME and draining LN after intratumoral injection of MVA-OVA-4-1BBL

[0388] Quantification of NK cells after it immunization with MVA-OVA showed a decrease in NK cells in the tumor on day 1 after intratumoral immunization ( Figure 19 , top row, left histogram). These changes were more pronounced when MVA-OVA-4-1BBL was used. Simultaneously, NK cells increased in tumor-draining lymph nodes (TdLNs) 3 and 7 days after immunization with MVA-OVA and MVA-OVA-4-1BBL ( Figure 19 , top row, right histogram), although MVA-OVA-4-1BBL induced the highest increase in NK cells in TdLN.

[0389] CD69 is a marker of early NK cell activation. Both the viral vectors MVA-OVA and MVA-OVA-4-1BBL resulted in tumors and draining lymph nodes (TdLN; Figure 19 , second row). In addition, it immunization led to the induction of granzyme B in NK cells at different time points in tumors and TdLN, suggesting enhanced cytotoxic NK cell function ( Figure 19 , third row).

[0390] Finally, we analyzed the proliferative capacity of NK cells by Ki67 expression. At day 3, Ki67 expression on NK cells was significantly increased in tumors and TdLNs of mice treated intratumorally with MVA-OVA or MVA-OVA-4-1BBL ( Figure 19 , last line).

[0391] These results indicate that 4-1BBL-assisted 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 the important role of draining lymph nodes (TdLN) in enhancing antitumor T cell and NK cell responses after intratumoral immunotherapy.

[0392] Although the present invention is not bound by any particular mechanism of operation, the expansion of T cells in TdLN at day 3 and the delayed infiltration of T cells in tumors at day 7 (see Figure 17 ) describes a scenario in which tumor-specific T cells are primed and expanded in TdLNs and subsequently migrate to the tumor to kill tumor cells. Intratumoral injection of viral vectors may also lead to the activation of NK cells directly in TdLNs, thereby inducing further DC activation.

[0393] Example 28: Role of CD8 T cells in intratumoral MVA cancer therapy

[0394] Analysis of T cell responses in tumors and TdLN (e.g., in Figure 17Middle) shows the expansion of tumor-specific T cells at two sites after intratumoral (it) treatment. Experiments were performed to examine the contribution of T cells to the antitumor effects mediated by MVA-OVA-4-1BBL. In these experiments, C57BL / 6 mice were injected with B16.OVA melanoma cells (5×10 5 cells), and tumor growth was monitored after one of several treatments. Treatment consisted of intratumoral (it) injection 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 5 mm in diameter, MVA-OVA-4-1BBL injection (it) was performed and repeated twice within one week. One day before the first MVA-OVA-4-BBL injection, mice were injected i.p. with anti-CD8 or IgG2b antibodies, and this treatment was repeated four times over the next two weeks. Figure 20 The data presented in this study suggest that CD8 T cells are 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 are crucial events in controlling tumor growth.

[0395] Example 29: Batf3+ DC dependence of MVA-OVA and MVA-OVA-4-1BBL-mediated anti-tumor effects

[0396] To elucidate the underlying cellular and molecular entities that contribute to the anti-tumor immune response induced by MVA-OVA-4-1BBL, we investigated the roles of various immune cell players. Dendritic cells (DCs) are able to efficiently sample and present antigens and co-stimulatory signals to cells of the adaptive immune system and are considered key players in anti-tumor immunity. Multiple DC subtypes have been implicated in activating effective immune responses against tumors, including CD8α+ DCs (also known as “cDC1”). 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; Martínez-López et al. (2014) Eur. J. Immunol. 45: 119-29) and cancer (Broz et al. (2014) Cancer Cell 26: 638-52). CD8α+ DCs also effectively induce anti-tumor CD8+ T cells by cross-presenting tumor-associated antigens (Sánchez-Paulete et al. (2015) Cancer Discovery 6:71-79; Salmon et al. (2016) Immunity 44:924-38). CD8α+ DC development is critically dependent on the transcription factor Batf3 (Hildner et al. (2008) Science 322:1097-1100).

[0397] To assess the importance of this DC subset for intratumoral MVA cancer therapy, we used wild-type and Batf3-deficient (Batf3- / -) B16.OVA tumor-bearing mice. Figure 21 A shows that B16.OVA tumors grew faster in the absence of cross-presenting DCs (Batf3- / -), indicating the important role of this subset of antigen-presenting cells (APCs) in inducing tumor-directed immune responses. Consistent with previous experiments, in wild-type mice, intratumoral injection of MVA-OVA resulted in delayed tumor growth and, in one case, complete tumor clearance. This effect was ameliorated when mice were injected with MVA-OVA-4-1BBL; three of five mice treated with MVA-OVA-4-1BBL rejected the tumors ( Figure 21 Interestingly, in the absence of cross-presenting DCs (Batf3- / -), intratumoral MVA immunotherapy was not impaired at all compared to the WT group ( Figure 21 A). However, Batf3-DCs appear to be involved in the 4-1BBL-induced anti-tumor response ( Figure 21 A, bottom).

[0398] CD8 in peripheral blood 11 days after the first immunization+ Flow cytometric analysis of T lymphocyte populations ( Figure 21 B) shows that Batf3 is expressed in MVA-OVA-4-1BBL immunized cells compared to wild-type counterparts. - / - In tumor carriers, OVA-specific CD8 + T cell frequencies were only slightly reduced.While the present invention is not limited or dependent on any particular mechanism of operation, these data suggest that Batf3-dependent DCs are redundant for intratumoral cancer therapy with MVA.

[0399] Example 30: Role of NK cells in intratumoral administration of MVA-OVA-4-1BBL

[0400] NK cells are known to express 4-1BB, and ligation of 4-1BB on NK cells has been shown to lead to increased proliferation and cytotoxicity of these cells (Muntasell et al. (2017) Curr. Opin. Immunol. 45: 73-81). Figure 19 ), we found that intratumoral injection of MVA-OVA-4-1BBL strongly upregulated the activation marker CD69 and the cytotoxic marker granzyme B on NK cells, accompanied by enhanced proliferation.

[0401] To explore the role of NK cells in 4-1BBL-induced antitumor immune responses, we used IL15Rα - / - Mice. The IL-15 receptor α subunit (IL-15Rα) mediates high-affinity binding of IL-15, a pleiotropic cytokine that has been shown to be essential for the development of NK cells (Lodolce et al. (1998) Immunity 9:669-76). Wild-type and IL15Rα-deficient (IL15Rα) mice were generated. - / - ) mice bearing B16.OVA tumors were immunized intratumorally with either MVA-OVA or MVA-OVA-4-1BBL. Mice treated with MVA-OVA showed similar therapeutic efficacy regardless of the presence or absence of IL-15Rα ( Figure 22 Interestingly, the benefit observed in wild-type mice (3 out of 5 mice rejected the tumor) when treated with MVA-OVA-4-1BBL was significantly reduced in IL15Rα-deficient tumor-bearing mice treated with MVA-OVA-4-1BBL (1 out of 5 mice rejected the tumor; see Figure 22 These results were also reflected in the survival rate of mice after tumor inoculation ( Figure 22 B).

[0402] It is known that the lack of IL15Rα not only affects the development of NK cells, but also attenuates T cell homeostasis and LN migration and selectively reduces CD8 memory T cells in mice (Lodolce et al. (1998) Immunity 9:669-76). Therefore, we also investigated the T cell response to these treatments. Consistent with our previous data, we observed the induction of OVA-specific CD8 T cells after MVA-OVA intratumoral (it) immunization in wild-type animals, which was further increased in the case of MVA-OVA-4-1BBL ( Figure 22 C) However, IL15Rα - / - The OVA-specific T cell responses in the mice were similar to those found in wild-type mice.

[0403] While the present invention is not bound by any particular mechanism or mode of operation, these findings suggest that IL15Rα - / -tumor-bearing mice have enhanced tumor-specific T cell responses and thus support the notion that 4-1BBL-enhanced NK cell activation and function contributes to the efficacy of intratumoral MVA-OVA-4-1BBL therapy.

[0404] Example 31: NK cell-dependent cytokines / chemokines in response to MVA-OVA-4-1BBL intratumoral immunity spectrum

[0405] To identify cytokines selectively induced by 4-1BBL–4-1BB interactions on NK cells, NK cells from wild-type mice bearing B16.OVA tumors or treated with PBS or 5 × 10 7 TCID 50 IL15Rα in MVA-OVA or MVA-OVA-4-1BBL tumors - / - Cytokines and chemokines were analyzed in tumor tissues of mice.

[0406] Previous experiments have shown that six hours after intratumoral injection of recombinant MVA, a large number of cytokines and chemokines increase ( Figure 15 and 16 In these experiments, tumors injected with MVA-OVA-4-1BBL exhibited a significant increase in proinflammatory cytokines or chemokines (e.g., IFN-γ, CCL3, and CCL5, known to be produced by NK cells after 4-1BBL stimulation) compared to those injected with MVA-OVA ( Figure 23 This 4-1BBL-induced increase in IL15Rα - / - This finding suggests that intratumoral injection of rMVA-OVA-4-1BBL induces a unique cytokine and chemokine profile in the tumor microenvironment originating from NK cells 6 h after injection.

[0407] Example 32: Antitumor Effect of Intratumor Immunization with MVA-gp70-CD40L Compared to MVA-gp70-4-1BBL effect

[0408] Gp70 is a tumor autoantigen expressed in many syngeneic tumor models (B16.F10, CT26, MC38, 4T1, EL4, and others), all of which represent distinct tumor microenvironments (TMEs) in terms of stromal and immune cell composition. Here, we tested the efficacy of MVA encoding the tumor antigen gp70 plus CD40L or 4-1BBL for intratumoral immunization in B16.F10 tumor-bearing mice.

[0409] B16.F10 melanoma cells were injected subcutaneously into C57BL / 6 mice. When the tumors reached approximately 50 mm in size, 3 When , mice were intratumorally immunized with PBS, MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, MVA-4-1BBL, or MVA-CD40L; the results are shown in Figure 24 As shown in .

[0410] Immunization with MVA-gp70 induced transient and mild tumor growth control. This antitumor effect could be enhanced when the virus expressed CD40L. However, intratumoral immunization with MVA-gp70-4-1BBL produced the strongest therapeutic effect, leading to complete tumor clearance in 2 of 5 treated animals ( Figure 24 A).

[0411] Strikingly, mice that were tumor-free after treatment with MVA-gp70-4-1BBL exhibited a loss of pigmentation at the tumor site ( Figure 24 B). This depigmentation is a manifestation of the autoimmune condition vitiligo and is the result of melanocyte destruction by autoreactive T cells. This destruction of melanocytes suggests that recombinant MVA activation of the immune system is not limited to the MVA-encoded TAA (here, gp70). Instead, this expanded activation of the immune system against other antigens (a phenomenon known as epitope spreading) leads to a broader immune response, potentially providing better therapeutic outcomes.

[0412] To assess the antigen-specific T cell responses induced by immunization, blood was drawn 11 days after the first immunization and analyzed for the presence of antigen-specific T cells. Immunization with MVA-gp70 and MVA-gp70-CD40L, as well as with MVA-CD40L and MVA-4-1BBL, induced measurable p15E-specific T cell responses ranging from 1% to 2%. Figure 24C). Importantly, this response was dramatically increased (>5-fold) in mice receiving MVA-gp70-4-1BBL. This antigen-specific T cell response to p15E peptide restimulation correlated with the efficacy of the different treatment groups.

[0413] Example 33: Anti-tumor efficacy of MVA-gp70-4-1BBL-CD40L intratumoral immunization

[0414] Recombinant MVA expressing the tumor antigen gp70, as well as 4-1BBL and CD40L, was generated 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 3 Mice were immunized intratumorally with PBS, MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, MVA-gp70-4-1BBL-CD40L, or the corresponding MVA construct not expressing gp70.

[0415] Immunization with MVA-gp70 induced transient and significant tumor growth control ( Figure 25 A). This antitumor effect could be enhanced when the virus expressed CD40L or 4-1BBL. However, intratumoral immunization with MVA-gp70-4-1BBL-CD40L resulted in the strongest therapeutic effect—complete tumor clearance in 4 of 5 treated animals ( Figure 25 A). Strikingly, three of the four cured mice treated with MVA-gp70-4-1BBL-CD40L exhibited loss of pigmentation where the tumor had once been, suggesting the autoimmune condition vitiligo, as described above in Example 32.

[0416] In addition, gp70-specific T cell responses were measured in the blood 11 days after the first immunization. Immunization with MVA-gp70 and MVA-gp70-CD40L, as well as with MVA-CD40L and MVA-4-1BBL, induced measurable tumor-specific T cell responses ranging from 1-2%; this response was significantly increased (>5-fold) in mice receiving MVA-gp70-4-1BBL ( Figure 25 B).

[0417] In summary, in the B16.F10 melanoma model, MVA-gp70 enhanced antitumor efficacy when supplemented with CD40L or 4-1BBL, but a stronger effect was observed when 4-1BBL and CD40L were expressed together in MVA-gp70-4-1BBL-CD40L.

[0418] Example 34: Intratumoral Immunotherapy Using MVA-gp70-4-1BBL-CD40L in CT26.WT Tumors

[0419] The constructs were then tested using the CT26 colon cancer model, which is described as being rich in T cells and myeloid cells and considered 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 intratumorally immunized with PBS, MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, MVA-gp70-4-1BBL-CD40L, or MVA-4-1BBL-CD40L.

[0420] IT immunization with MVA-gp70 induced transient and significant tumor growth control. This antitumor effect was not enhanced when MVA expressed CD40L, but strikingly, immunization with MVA-gp70-4-1BBL produced the strongest therapeutic effect, resulting in complete tumor clearance in all treated animals ( Figure 26 A). However, treatment with MVA-gp70-4-1BBL-CD40L did not result in a better therapeutic effect. Notably, a virus containing only co-stimulatory molecules but without gp70 also caused a significant tumor growth delay but was unable to compete with MVA-gp70-4-1BBL. These findings were reflected in the overall survival of treated mice ( Figure 26 B).

[0421] Gp70-specific T cell responses against the H2-LdCD8+ T cell epitope AH-1 were readily detected in the blood of animals treated with MVA-gp70 and MVA-gp70-CD40L ( Figure 26 C). This response was significantly increased (>10-fold) in mice receiving MVA-gp70-4-1BBL, consistent with Figure 26 Treatment with MVA-gp70-4-1BBL-CD40L also enhanced AH-1-specific T cell responses in the blood ( Figure 26 C).

[0422] Example 35: Tumor microcirculation in mice bearing B16.F10 tumors after IT injection of MVA-gp70-4-1BBL-CD40L Comprehensive analysis of environmental and tumor-draining LNs

[0423] The data presented above show that intratumoral treatment of B16.F10 tumor-bearing mice with MVA-gp70-4-1BBL-CD40L resulted in tumor rejection in 80% of the treated mice (see Figure 26 To investigate the tumor microenvironment (TME) and TdLN in this tumor model, mice bearing B16.F10 tumors were intratumorally (it) treated with PBS or 5 × 10 7TCID50 of MVA-gp70, MVA-gp70-4-1BBL, MVA-gp70-CD40L, or MVA-gp70-4-1BBL-CD40L. Mice were sacrificed 3 days after the initial immunization. Day 3 was chosen based on previous experiments in the OVA system that showed changes in the innate and adaptive components of the immune system at that time point (see Figure 17 Tumors and TdLNs were excised and digested with collagenase / DNase to allow single-cell analysis by flow cytometry. The abundance of immune cell populations, as well as their proliferative behavior and functional status, were assessed.

[0424] Intratumoral injection of MVA assisted by 4-1BBL and CD40L did not confer an advantage in the number of CD8 T cells or p15E-specific T cells in the tumor at the day 3 time point, as determined by pentamer staining. However, in TdLN, MVA-gp70 and MVA-gp70-CD40L produced an expansion of CD8 T cells, with the addition of 4-1BBL producing a greater effect ( Figure 27 , upper right). The increase produced by the addition of 4-1BBL was more pronounced for p15E-specific CD8 T cells in TdLN, where it immunization with MVA-gp70-4-1BBL or MVA-gp70-4-1BBL-CD40L increased tumor-specific CD8 T cells ( Figure 27 , middle right). The number of p15E-specific CD8 T cells also correlated with the proliferation state of those cells; for example, the addition of 4-1BBL together with gp70 and optionally CD40L to MVA induced the greatest number of Ki67+gp70-p15E CD8 T cells in TdLN ( Figure 27 , lower right).

[0425] These data indicate that intratumoral (it) immunization with MVA-gp70 enhanced the generation of adaptive immune responses in tumors and tumor-draining lymph nodes at day 3 post-treatment, whereas adjuvant therapy with 4-1BBL or 4-1BBL plus CD40L specifically increased p15E-specific CD8 T cell responses in TdLNs.

[0426] Example 36: NK cell induction in tumors and TdLNs after intratumoral injection of MVA

[0427] Intratumoral (it) injection of MVA-OVA resulted in activation and expansion of NK cells on day 1 and day 3, respectively ( Figure 19 We then examined NK cell infiltration, activation, and expansion on day 3 after injection of the different MVA constructs. Quantification of NK cells after it immunization with recombinant MVA showed tumor infiltration ( Figure 28 , upper left) and TdLN( Figure 28, upper right) increased NK cells. When MVA encoded 4-1BBL (e.g., MVA-gp70-4-1BBL and MVA-gp70-4-1BBL-CD40L), infiltration increased. Intratumoral (it) injection of MVA-gp70 induced tumors (see Figure 28 , middle left) and TdLN( Figure 28 , middle right), and this effect was enhanced by supplementation with 4-1BBL or 4-1BBL and CD40L in TdLN.

[0428] Granzyme B is a marker of NK cell cytotoxicity (see, e.g., Ida et al. (2005) Mod. Rheumatol. 15:315-22). Following intratumoral injection of recombinant MVA, granzyme B+ NK cells were induced in tumors and TdLNs ( Figure 28 , lower left). Similarly, addition of 4-1BBL or 4-1BBL-CD40L to recombinant MVA slightly increased the number of cytotoxic NK cells in TdLN ( Figure 28 , lower right).

[0429] Together, these data highlight the important role of MVA-encoded 4-1BBL-CD40L in the expansion and function of NK cells and TAA-specific T cells after intratumoral (it) immunotherapy. Thus, intratumoral treatment with recombinant MVA encoding gp70 and 4-1BBL or gp70, 4-1BBL, and CD40L enhances T cell responses to endogenous retroviral autoantigens such as gp70.

[0430] Example 37: Intravenous Immunization with MVA-gp70-4-1BBL-CD40L in Mice Bearing CT26.WT Tumors therapy

[0431] The experiments discussed above demonstrate that novel MVA constructs encoding the tumor antigen gp70 are highly effective when administered intratumorally together with the costimulatory molecules 4-1BBL and CD40L ( Figure 25 and 26 ). In addition, Lauterbach et al. ((2013) Front. Immunol. 4:251) found that MVA-encoded CD40L enhanced innate and adaptive immune responses when administered intravenously. Here, we asked whether intravenous (iv) immunization with MVA-gp70-4-1BBL-CD40L could also provide tumor growth control.

[0432] 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 (which lacks gp70). IV immunization with MVA-gp70 resulted in tumor clearance in 2 / 5 animals ( Figure 29 A). Mice treated with gp70-expressing viruses containing either 4-1BBL or CD40L exhibited significantly improved anti-tumor responses, resulting in cure in 3 / 5 and 4 / 5 mice, respectively. Importantly, iv treatment with MVA-gp70-4-1BBL-CD40L resulted in prolonged control of tumor growth in all treated mice, with 3 / 5 mice rejecting the tumor ( Figure 29 A). Notably, recombinant MVA containing only co-stimulatory molecules but not gp70 also resulted in a significant tumor growth delay but did not induce the same tumor rejection observed with MVA-gp70-4-1BBL, MVA-gp70-CD40L, or MVA-gp70-4-1BBL-CD40L ( Figure 29 A). These findings were reflected in the overall survival of treated mice ( Figure 29 B).

[0433] Analysis of tumor-directed CD8 T-cell responses in blood by peptide restimulation of PBLs revealed a significant induction of AH1-specific CD8 T cells in all MVA-treated groups, which could be further enhanced in the presence of CD40L (i.e., MVA-gp70-CD40L and MVA-gp70-4-1BBL-CD40L). Figure 29 C).

[0434] Example 38: Recombinant MVA containing HERV-K antigen

[0435] An MVA-based vector ("MVA-mBN489," also known as "MVA-HERV-Prame-FOLR1-4-1-BBL-CD40L") was designed that includes TAAs of human endogenous retrovirus K superfamily proteins (HERV-K), specifically ERV-K-env and ERV-K-gag. The MVA was also designed to encode human FOLR1 and PRAME and to express h4-1BBL and hCD40L.

[0436] A similar MVA-based vector, termed "MVA-HERV-Prame-FOLR1-4-1-BBL," was designed to express the TAAs ERV-K-env and ERV-K-gag, as well as human FOLR1 and PRAME, and to express h4-1BBL. Specifically, the vector "MVA-BN-4IT" ("MVA-mBN494" or "MVA-HERV-FOLR1-PRAME-h4-1-BBL") was used in Figure 30 Schematic diagram in Figure A. HERV-K genes, encoding envelope (env) and group-specific antigen (gag) proteins, are normally dormant in healthy human tissue but are activated in many tumors. FOLR1 and PRAME are genes specifically upregulated in breast and ovarian cancer cells. Additional expression of the costimulatory molecule 4-1-BBL is intended to enhance immune responses against TAAs.

[0437] Another MVA-based vector, designated "MVA-HERV-Prame-FOLR-CD40L," is designed to express TAAERV-K-env and ERV-K-gag, as well as human FOLR1 and PRAME, and to express hCD40L. Each of these constructs can be used in the methods of the present invention.

[0438] Exemplary sequences are known in the art and are also listed in the provided sequence listing.Any sequence may be used in the compositions and methods of the invention so long as it provides the necessary function for the relevant MVA.

[0439] For the above-mentioned ERV-K env and gag sequences, amino acid consensus sequences were generated from at least 10 representative sequences, and the potential immunosuppressive domain was inactivated by mutation and partially replaced with the immunodominant T cell epitope HERV-K-mel, as shown below. Suitable sequences are listed in SEQ ID NO: 5 (ERV-K-gag synthetic protein consensus sequence); SEQ ID NO: 6 (ERV-K-gag synthetic nucleotide sequence); SEQ ID NO: 7 (ERV-K-env / MEL synthetic protein sequence); and SEQ ID NO: 8 (ERV-K-env / MEL nucleotide sequence).

[0440]

[0441] Modified ERVK-env consensus amino acid sequence (above):

[0442] The potential immunosuppressive domain was inactivated by mutations that replaced most of the immunosuppressive domain with the immunodominant T cell epitope HERVK-mel.

[0443] For some of these MVAs, hFOLR1 and PRAME were designed to be produced as fusion proteins. FOLR1 (folate receptor alpha) belongs to the folate receptor family. It has a high affinity for folic acid and its derivatives and is secreted or expressed on the cell surface as a membrane protein. The transmembrane protein is anchored to the plasma membrane via a GPI (glycosylphosphatidylinositol) anchor, which is likely attached to the endoplasmic reticulum (ER) via a serine (Ser) residue in the C-terminal region of the protein. To avoid modification of FOLR1 with the GPI anchor and complete processing of the hFOLR1-hPRAME fusion protein in the ER, the C-terminal region from aa 234 to 257 (including the Ser residue) was deleted.

[0444] PRAME (preferentially expressed melanoma antigen) is a transcriptional regulator. Initially described as an antigen in human melanoma, it triggers autologous cytotoxic T cell-mediated immune responses and is expressed in a variety of solid and hematological cancers. PRAME inhibits retinoic acid signaling by binding to retinoic acid receptors, potentially providing a growth advantage for cancer cells. PRAME's function requires nuclear localization, so the potential nuclear localization signal (NLS) in PRAME was modified through targeted mutagenesis in the hFOLR1-hPRAME fusion protein.

[0445] Therefore, the amino acid sequence of the hFOLR1-hPRAME fusion protein was modified by deleting the C-terminal GPI anchor signal, while in PRAME, two potential nuclear localization signals were inactivated by amino acid substitutions. In this fusion protein, the N-terminal signal sequence of hFOLR1 should result in ER targeting and incomplete processing of the fusion protein, serving as an additional safeguard against nuclear localization of PRAME.

[0446] The protein sequences of human FOLR1 and human PRAME are based on NCBI RefSeq NP_000793.1 and NP_001278644.1, respectively. In addition to the modifications described above, the nucleotide sequence of the fusion protein was optimized for human codon usage and polynucleotide stretches, repeat elements, and negative cis-acting elements were removed, and the nucleotide sequence is set forth in SEQ ID NO: 10 ("hFOLR1Δ_hPRAMEΔ fusion" nucleotide sequence), while the fusion protein sequence is set forth in SEQ ID NO: 9.

[0447]

[0448]

[0449] Sequence of hFOLR1-hPRAME fusion protein (above):

[0450] Amino acid sequence of the hFOLR1-hPRAME fusion protein, a fusion of modified human FOLR1 (N-terminal portion) and PRAME (C-terminal portion). FOLR1 was modified by deleting the C-terminal GPI anchor signal (strikethrough letters). In PRAME (underlined letters), the initial methionine was deleted, and two potential nuclear localization signals were inactivated by amino acid substitutions (bold, underlined letters).

[0451] The protein sequence of membrane-bound human 4-1BBL used in this MVA showed 100% identity to NCBI RefSeq NP_003802.1, and the protein sequence of membrane-bound human CD40L used showed 100% identity to NCBI RefSeq NP_000065.1. For both 4-1BBL and CD40L, the nucleotide sequences were optimized for human codon usage, and polynucleotide stretches, repetitive elements, and negative cis-acting elements were removed.

[0452] The amino acid sequence of hCD40L from NCBI RefSeq NP_000065.1 is set forth in SEQ ID NO: 1, and the nucleotide sequence of hCD40L is shown in SEQ ID NO: 2. The amino acid sequence of h4-1BBL from NCBI RefSeq NP_003802.1 is set forth in SEQ ID NO: 3, and the nucleotide sequence of h4-1BBL is set forth in SEQ ID NO: 4.

[0453] Each coding region is under the control of a different promoter, except for ERV-K-gag and h4-1BBL, which are both under the control of the Pr1328 promoter. The Pr1328 promoter (100 bp in length) is an exact homolog of the vaccinia virus promoter PrB2R. It drives strong immediate early expression and lower levels of late expression. In recombinant MVA-mBN489, the Pr13.5 long promoter drives expression of ERVK-env / MEL. This promoter disrupts the 124 bp intergenic region between 014L / 13.5L, drives expression of the native MVA13.5L gene, and shows very strong early expression caused by the two early promoter core sequences (see Wennier et al. (2013) PLoS One 8(8):e73511). The MVA1-40k promoter used here to drive hCD40L expression is a 161-bp fragment originally isolated in 1986 from the vaccinia virus Wyeth Hind III H region. It disrupts a 158-bp fragment of the vaccinia virus Wyeth and MVA genomes within the 094L / 095R intergenic region and drives the late gene transcription factor VLTF-4. The promoter used here to drive expression of the hFOLR1-hPRAME fusion protein, PrH5m, is a modified form of the vaccinia virus H5 gene promoter. It consists of strong early and late elements, allowing expression during both the early and late stages of recombinant MVA infection (see Wyatt et al. (1996) Vaccine 14:1451-58).

[0454] Based on MVA-mBN494 (see above), another vector was designed to include the modifications in ERVK-env / MEL. The resulting vector was called "MVA-mBN502" and is available in Figure 31 Schematically shown in C. In addition to the modified ERVK-env / MEL, MVA-mBN502 also encodes ERVK-gag, hFOLR1-hPRAME fusion protein and h4-1BBL

[0455] Naturally, HERVK-env consists of a signal peptide that is cleaved after translation, a surface (SU) and a transmembrane unit (TM). Cleavage into two domains is achieved by cellular proteases. The RSKR cleavage motif is necessary and sufficient for cleaving the full-length 90kDa protein into SU (about 60kDa) and TM (about 40kDa) domains. As described above for the preparation of MVA-mBN494, an env amino acid consensus sequence derived from at least ten representative sequences was generated, and the potential immunosuppressive domain in TM was inactivated by mutation. The introduced mutation replaced most of the immunosuppressive domain with the immunodominant T cell epitope HERV-K-mel. This transgenic (for MVA-mBN494) is called ERVK-env / MEL ( Figure 31 A).

[0456] Compared to MVA-mBN494, the TM domain in ERVK-env / MEL is missing in MVA-mBN502. This ERVK-env / MEL variant was named "ERVK-env / MEL_03" and consists of the entire SU domain, except for the missing RSKR furin protease cleavage site. The MEL peptide is inserted at the C-terminus, followed by 6 amino acids of the TM domain (excluding the fusion peptide sequence with strong hydrophobicity). In addition, this modified ERVK-env / MEL is targeted to the plasma membrane by adding a membrane anchor derived from the human PDGF (platelet-derived growth factor) receptor. This membrane anchor is connected to the SU domain ( Figure 31 B). The resulting ERVK-env / MEL variant, ERVK-env / MEL_03, was included in MVA-mBN502 ( Figure 31 C). Suitable sequences of variants are set forth in SEQ ID NO: 11 (ERV-K-env / MEL_03 synthetic protein sequence) and SEQ ID NO: 12 (ERV-K-env / MEL_03 nucleotide sequence).

[0457] Example 39: Biological activity of MVA-HERV-FOLR1-PRAME-h4-1-BBL (MVA-BN-4IT)

[0458] We investigated whether infection with MVA-BN-4IT (i.e., MVA-HERV-FOLR1-PRAME-h4-1-BBL; see also Example 38 above) would result in HLA molecules presenting vaccine-derived tumor antigens on human cells. To this end, we immunoprecipitated HLA-ABC peptide complexes on antigen-presenting cells and analyzed which HLA-bound peptides could be identified by mass spectrometry.

[0459] First, the human monocytic cell line THP-1 differentiates into macrophages (Daigneault et al. PLoS One, 2010), which exert antigen presenting capacity because antigens can be loaded onto HLA class I (Nyambura L. et al. J. Immunol 2016). In fact, THP-1 cells express HLA-A*0201 + , which is one of the most common haplotypes in the United States and Europe (approximately 30% of the population). In addition to HLA-A*02:01:01G, THP-1 cells have been reported to express HLA-B*15 and HLA-C*03 (Battle R. et al., Int. J. of Cancer). Here, 8×10 5 / ml THP-1 cells were cultured in the presence of 200ng / ml PMA (phorbol-12-myristate-13-acetate) for 3 days, then the medium was changed and the cells were cultured for another 2 days in the absence of PMA. On day 5, the cells were infected with MVA-BN-4IT at an InfU (infectious unit) of 4 for 12 hours. Figure 30 As shown in B, in the presence of MVA-BN-4IT ( Figure 30 After infection of THP-1 cells with mBN494 ("mBN494" in B), HERVK-env / MEL, HERVK-gag, and the fusion protein FOLR1-PRAME were expressed. In contrast, the antigen was not expressed in uninfected THP-1 cells ( Figure 30 endogenously expressed in the 'ctr' in B).

[0460] Next, a "ProPresent" HLA-ABC ligand analysis (ProImmune) was performed. In cells infected with MVA-BN-4IT, four tumor antigen-derived peptides were identified: the HERV-K env peptide ILTEVLKGV, the HERV-K gag peptide YLSFIKILL, and the PRAME peptides ALQSLLQHL and SLLQHLIGL. The two identified PRAME peptides overlap to a large extent and are likely to share a common core epitope. It is expected that both peptides bind very strongly to HLA-A*02:01, whereby ALQSLLQHL has a binding rank almost similar to that of HLA-B*15. It is noteworthy that the PRAME peptide SLLQHLIGL has been described as a cytotoxic T lymphocyte epitope presented by the human immunogenic HLA-A*0201 (Kessler JH. et al., J Exp Med., 2001). In summary, the data show that the antigens expressed by MVA-BN-4IT can be loaded into the HLA of infected cells.

[0461] In addition, the ability of MVA-BN-4IT to express 4-1-BBL in a functional form bound to its receptor 4-1-BB was tested. To this end, a commercial kit ("4-1BB Bioassay", Promega) was used. The analysis consists of a genetically engineered Jurkat T cell line expressing h4-1-BB and a luciferase reporter gene driven by a response element (RE) that can respond to stimulation by the 4-1-BB ligand. When h4-1-BB is stimulated by h4-1-BBL, RE activates cellular luciferase production in the cells. After cell lysis and addition of "Bio-Glo" reagent (Promega), luminescence was measured and quantified using a luminometer. Briefly, HeLa cells (1×10 6 ) and use Figure 30 Infection with the MVA-based constructs shown in C (TCID 50 =2), cultured overnight (37°C, 5% CO2), and then co-cultured with Jurkat-h4-1-BB cells (HeLa:Jurkat ratio = 4:1) for 6 hours. His-tagged h4-1BBL cross-linked to Fc was used as a reference (positive control), and the luciferase expression of Jurkat-h4-1BB cells cultured with 1 μg / ml cross-linked h4-1BBl was set to 1 ( Figure 30 C, dotted line). MVA-BN (i.e., not encoding h4-1-BBL) was used as a backbone control. Figure 30 As shown in Figure C, HeLa cells infected with an MVA-based vector expressing h4-1-BBL induced luciferase production (by co-cultured Jurkat-h4-1-BB cells) that was more than 6-fold higher than the reference. Notably, luciferase production mediated by MVA-BN-4IT was even higher than that mediated by the other two MVA vectors expressing h4-1-BBL. Thus, MVA-mBN494 expresses functional h4-1-BBL that effectively binds to its 4-1BB receptor.

[0462] Example 40: Intratumoral immunization with MVA encoding brachyury antigen

[0463] The highly attenuated, non-replicating vaccinia virus MVA-BN-Brachyury is designed to consist of four human transgenes to elicit specific and potent immune responses against a variety of cancers. The vector co-expresses the brachyury human TAA and three human co-stimulatory molecules: 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 co-stimulatory molecules (or triad of co-stimulatory molecules, TRICOM) TM) were included to maximize the immune response to the brachyury human TAA.

[0464] Brachyury is a transcription factor in the T-box family and a driver of EMT, a process associated with cancer progression. It is overexpressed in cancer cells compared to normal tissues and has been linked to resistance to several therapeutic modalities and metastatic potential. Cancers known to express brachyury include lung, breast, ovarian, chordoma, prostate, colorectal, and pancreatic cancers.

[0465] In vitro and clinical studies were conducted to demonstrate the safety and potential 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”); 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”).

[0466] A GLP-compliant repeated-dose toxicity study was conducted to evaluate any potential toxicity of MVA-BN-Brachyury (MVA-mBN240B) in NHPs (cynomolgus macaques) to support the use of the intravenous route in Phase 1 clinical development. The toxicity study included a biodistribution component that evaluated the spatial and temporal distribution of MVA-BN-Brachyury in NHPs.

[0467] MVA-BN-Brachyury is being used in a Phase III trial in which cancer patients receive intratumoral injections of MVA, optionally in combination with another treatment, such as radiation and / or a checkpoint inhibitor.

[0468] Obviously, the precise details of the methods or compositions described herein may be changed or modified without departing from the spirit of the invention described. We claim all such modifications and variations that come within the scope and spirit of the following claims.

[0469] Sequence Listing

[0470] The nucleic acid and amino acid sequences listed in the accompanying sequence listing use standard letter abbreviations to indicate nucleotide bases and either the single-letter code or the three-letter code to indicate amino acids, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included in any reference to a displayed strand.

[0471] Sequences in the sequence listing:

[0472] SEQ ID NO: 1: hCD40L amino acid sequence from NCBI RefSeq NP_000065.1 (261 amino acids)

[0473] SEQ ID NO: 2: hCD40L from NCBI RefSeq NP_000065.1 (792 nucleotides)

[0474] SEQ ID NO: 3: h4-1BBL from NCBI RefSeq NP_003802.1 (254 amino acids)

[0475] SEQ ID NO: 4: h4-1BBL from NCBI RefSeq NP_003802.1

[0476] SEQ ID NO: 5: ERV-K-gag (666 amino acids) synthetic consensus sequence

[0477] SEQ ID NO:6: ERV-K-gag; nt sequence

[0478] SEQ ID NO: 7: ERV-K-env / MEL (699 amino acids) synthetic sequence

[0479] SEQ ID NO:8: ERV-K-env / MEL nt sequence

[0480] SEQ ID NO: 9: hFOLR1Δ_hPRAMEΔ fusion (741 amino acids)

[0481] SEQ ID NO: 10: hFOLR1Δ_hPRAMEΔ fusion (741 amino acids) nt sequence

[0482] SEQ ID NO: 11: ERV-K-env / MEL_03 (517 amino acids) synthetic sequence

[0483] SEQ ID NO: 12: ERV-K-env / MEL_03nt sequence

[0484] SEQ ID NO: 1

[0485] From NCBI RefSeq NP_000065.1 hCD40L (261 amino acids)

[0486] MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEA SSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

[0487] SEQ ID NO:2

[0488] From NCBI RefSeq NP_000065.1 hCD40L (792 nucleotides)

[0489] nt sequence:

[0490] atgatcgagacatacaaccagacaagccctagaagcgccgccacaggactgcctatcagcatgaagatcttcatgtacctgctgaccgtgttcctgatcacccagatgatcggcagcgccctgtttgccgtgtacctgcacagacggctggacaagatcgaggacgagagaaacctgcacgaggacttcgtgttcatgaagaccatccagcggtgcaacaccggcgagagaagtctgagcctgctgaactgcgaggaaatcaagagccagttcgagggcttcgtgaaggacatcatgctgaacaaagaggaaacgaagaaagagaactccttcgagatgcagaagggcgaccagaatcctcagatcgccgctcacgtgatcagcgaggccagcagcaagacaacaagcgtgctgcagtgggccgagaagggctactacaccatgagcaacaacctggtcaccctggagaacggcaagcagctgacagtgaagcggcagggcctgtactacatctacgcccaagtgaccttctgcagcaacagagaggccagctctcaggctcctttcatcgccagcctgtgcctgaagtctcctggcagattcgagcggattctgctgagagccgccaacacacacagcagcgccaaaccttgtggccagcagtctattcacctcggcggagtgtttgagctgcagcctggcgcaagcgtgttcgtgaatgtgacagaccctagccaggtgtcccacggcaccggctttacatctttcggactgctgaagctgtgatgatag

[0491] SEQ ID NO: 3

[0492] From of NCBI RefSeq NP_003802.1 h4-1BBL . (254 amino acids)

[0493] MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

[0494] SEQ ID NO:4

[0495] From of NCBI RefSeq NP_003802.1 h4-1BBL 。

[0496] nt sequence:

[0497] atggaatacgccagcgacgcctctctggaccctgaagctccttggcctccagctcctagagccagggcttgtagagtgctgccttgggctcttgtggctggacttctgcttctgttgctcctggctgctgcctgcgcagtgtttcttgcttgtccatgggctgtgtcaggagccagagcatctcctggatctgccgcttctcccagactgagagagggacctgaactgagccctgatgatcctgctggactgctcgacctgagacagggcatgtttgcccagctggtggcccagaatgtgctgctgattgatggccctctgagctggtacagcgatcctggacttgctggcgttagcctgactggaggcctgagctacaaggaggacaccaaagaactggtggtggccaaggctggcgtgtactacgtgttctttcagctggaactgcggagagtggtggcaggcgaaggatctggatccgtgtctctggcactgcatctgcagcctctgagatctgctgctggtgcagctgccctggctctgacagttgatctgcctcctgcctccagcgaagccagaaacagcgcctttggcttccaaggcagactgctgcacctgtctgctggccagagactgggagtgcacctccacacagaagcaagagcaagacacgcctggcagcttacacaaggcgctacagtgctgggcctgttcagagtgacacctgagattccagctggcttgccatctcctcgcagcgagtaatga

[0498] SEQ ID NO:5

[0499] ERV-K-env / MEL (699 amino acids)

[0500] MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPTWAQLKKLTQLATKYLENTKVTQTPESMLLAALMIVSMVVSLPMPAGAAAANYTYWAYVPFPPMIRAVTWMDNPIEVYVNDSVWVPGPIDDRCPAKPEEEGMMINISIGYRYPPICLGRAPGCLMPAVQNWLVEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPKEIPKESKNTEVLVWEECVANSAVILQNNEFGTIIDWAPRGQFYHNCSGQTQSCPSAQVSPAVDSDLTESLDKHKHKKLQSFYPWEWGEKGISTPRPKIISPVSGPEHPELWRLTVASHHIRIWSGNQTLETRDRKPFYTVDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLLTCIDSTFNWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNRSKRFIFTLIAVIMGLIAVTATAAVAGVALHSSVQSVNFVNDWQKNSTRLWNSQSSIDQKMLAVISCAVQTVIWMGDRLMSLEHRFQLQCDWNTSDFCITPQIYNESEHHWDMVRRHLQGREDNLTLDISKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV

[0501] SEQ ID NO:6

[0502] ERV-K-env / MEL

[0503] nt sequence

[0504]

[0505] SEQ ID NO:7

[0506] ERV-K-gag (666 amino acids)

[0507] MGQTKSKIKSKYASYLSFIKILLKRGGVKVSTKNLIKLFQIIEQFCPWFPEQGTLDLKDWKRIGKELKQAGRKGNIIPLTVWNDWAIIKAALEPFQTEEDSVSVSDAPGSCIIDCNENTRKKSQKETESLHCEYVAEPVMAQSTQNVDYNQLQEVIYPETLKLEGKGPELVGPSESKPRGTSPLPAGQVPVTLQPQKQVKENKTQPPVAYQYWPPAELQYRPPPESQYGYPGMPPAPQGRAPYPQPPTRRLNPTAPPSRQGSELHEIIDKSRKEGDTEAWQFPVTLEPMPPGEGAQEGEPPTVEARYKSFSIKMLKDMKEGVKQYGPNSPYMRTLLDSIAHGHRLIPYDWEILAKSSLSPSQFLQFKTWWIDGVQEQVRRNRAANPPVNIDADQLLGIGQNWSTISQQALMQNEAIEQVRAICLRAWEKIQDPGSTCPSFNTVRQGSKEPYPDFVARLQDVAQKSIADEKARKVIVELMAYENANPECQSAIKPLKGKVPAGSDVISEYVKACDGIGGAMHKAMLMAQAITGVVLGGQVRTFGGKCYNCGQIGHLKKNCPVLNKQNITIQATTTGREPPDLCPRCKKGKHWASQCRSKFDKNGQPLSGNEQRGQPQAPQQTGAFPIQPFVPQGFQGQQPPLSQVFQGISQLPQYNNCPPPQAAVQQ

[0508] SEQ ID NO:8

[0509] ERV-K-gag

[0510] nt sequence

[0511]

[0512] SEQ ID NO:9

[0513] hFOLR1Δ_hPRAMEΔ fusion (741 amino acids)

[0514] MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTTKAKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVAAKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPN

[0515] SEQ ID NO: 10

[0516] hFOLR1Δ_hPRAMEΔ fusion (741 amino acids)

[0517] nt sequence

[0518]

[0519] SEQ ID NO:11

[0520] ERV-K-env / MEL_03 (517 amino acids)

[0521] MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPTWAQLKKLTQLATKYLENTKVTQTPESMLLAALMIVSMVVSLPMPAGAAAANYTYWAYVPFPPMIRAVTWMDNPIEVYVNDSVWVPGPIDDRCPAKPEEEGMMINISIGYRYPPICLGRAPGCLMPAVQNWLVEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPKEIPKESKNTEVLVWEECVANSAVILQNNEFGTIIDWAPRGQFYHNCSGQTQSCPSAQVSPAVDSDLTESLDKHKHKKLQSFYPWEWGEKGISTPRPKIISPVSGPEHPELWRLTVASHHIRIWSGNQTLETRDRKPFYTVDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLLTCIDSTFNWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNMLAVISCAVAGVALHGSAGSAAGSGEFVVISAILALVVLTIISLIILIMLWQKKPR

[0522] SEQ ID NO:12

[0523] ERV-K-env / MEL_03

[0524] nt sequence

[0525] Sequence Listing <110> Bavarian Nordic A / S <120> Recombinant MVA viruses for intratumoral and / or intravenous administration to treat cancer <130> BNIT0015PCT <150> EP 19210369.5 <151> 2019-11-20 <150> EP 20193706.7 <151> 2020-08-31 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 261 <212> PRT <213> Homo sapiens <400> 1 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 115 120 125 Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140 Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145 150 155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250 255 Gly Leu Leu Lys Leu 260 <210> 2 <211> 792 <212> DNA <213> Homo sapiens <400> 2 atgatcgaga catacaacca gacaagccct agaagcgccg ccacaggact gcctatcagc 60 atgaagatct tcatgtacct gctgaccgtg ttcctgatca cccagatgat cggcagcgcc 120 ctgtttgccg tgtacctgca cagacggctg gacaagatcg aggacgagag aaacctgcac 180 gaggacttcg tgttcatgaa gaccatccag cggtgcaaca ccggcgagag aagtctgagc 240 ctgctgaact gcgaggaaat caagagccag ttcgagggct tcgtgaagga catcatgctg 300 aacaaagagg aaacgaagaa agagaactcc ttcgagatgc agaagggcga ccagaatcct 360 cagatcgccg ctcacgtgat cagcgaggcc agcagcaaga caacaagcgt gctgcagtgg 420 gccgagaagg gctactacac catgagcaac aacctggtca ccctggagaa cggcaagcag 480 ctgacagtga agcggcaggg cctgtactac atctacgccc aagtgacctt ctgcagcaac 540 agagaggcca gctctcaggc tcctttcatc gccagcctgt gcctgaagtc tcctggcaga 600 ttcgagcgga ttctgctgag agccgccaac acacacagca gcgccaaacc ttgtggccag 660 cagtctattc acctcggcgg agtgtttgag ctgcagcctg gcgcaagcgt gttcgtgaat 720 gtgacagacc ctagccaggt gtcccacggc accggcttta catctttcgg actgctgaag 780 ctgtgatgat ag 792 <210> 3 <211> 254 <212> PRT <213> Homo sapiens <400> 3 Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro 1 5 10 15 Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val 20 25 30 Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe 35 40 45 Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser 50 55 60 Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp 65 70 75 80 Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val 85 90 95 Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp 100 105 110 Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu 115 120 125 Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe 130 135 140 Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser 145 150 155 160 Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala 165 170 175 Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala 180 185 190 Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala 195 200 205 Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His 210 215 220 Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val 225 230 235 240 Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 245 250 <210> 4 <211> 768 <212> DNA <213> Homo sapiens <400> 4 atggaatacg ccagcgacgc ctctctggac cctgaagctc cttggcctcc agctcctaga 60 gccagggctt gtagagtgct gccttgggct cttgtggctg gacttctgct tctgttgctc 120 ctggctgctg cctgcgcagt gtttcttgct tgtccatggg ctgtgtcagg agccagagca 180 tctcctggat ctgccgcttc tcccagactg agagagggac ctgaactgag ccctgatgat 240 cctgctggac tgctcgacct gagacagggc atgtttgccc agctggtggc ccagaatgtg 300 ctgctgattg atggccctct gagctggtac agcgatcctg gacttgctgg cgttagcctg 360 actggaggcc tgagctacaa ggaggacacc aaagaactgg tggtggccaa ggctggcgtg 420 tactacgtgt tctttcagct ggaactgcgg agagtggtgg caggcgaagg atctggatcc 480 gtgtctctgg cactgcatct gcagcctctg agatctgctg ctggtgcagc tgccctggct 540 ctgacagttg atctgcctcc tgcctccagc gaagccagaa acagcgcctt tggcttccaa 600 ggcagactgc tgcacctgtc tgctggccag agactgggag tgcacctcca cacagaagca 660 agagcaagac acgcctggca gcttacacaa ggcgctacag tgctgggcct gttcagagtg 720 acacctgaga ttccagctgg cttgccatct cctcgcagcg agtaatga 768 <210> 5 <211> 666 <212> PRT <213> Artificial sequence <220> <223> Synthetic ERV-K-gag sequence <400> 5 Met Gly Gln Thr Lys Ser Lys Ile Lys Ser Lys Tyr Ala Ser Tyr Leu 1 5 10 15 Ser Phe Ile Lys Ile Leu Leu Lys Arg Gly Gly Val Lys Val Ser Thr 20 25 30 Lys Asn Leu Ile Lys Leu Phe Gln Ile Ile Glu Gln Phe Cys Pro Trp 35 40 45 Phe Pro Glu Gln Gly Thr Leu Asp Leu Lys Asp Trp Lys Arg Ile Gly 50 55 60 Lys Glu Leu Lys Gln Ala Gly Arg Lys Gly Asn Ile Ile Pro Leu Thr 65 70 75 80 Val Trp Asn Asp Trp Ala Ile Ile Lys Ala Ala Leu Glu Pro Phe Gln 85 90 95 Thr Glu Glu Asp Ser Val Ser Val Ser Asp Ala Pro Gly Ser Cys Ile 100 105 110 Ile Asp Cys Asn Glu Asn Thr Arg Lys Lys Ser Gln Lys Glu Thr Glu 115 120 125 Ser Leu His Cys Glu Tyr Val Ala Glu Pro Val Met Ala Gln Ser Thr 130 135 140 Gln Asn Val Asp Tyr Asn Gln Leu Gln Glu Val Ile Tyr Pro Glu Thr 145 150 155 160 Leu Lys Leu Glu Gly Lys Gly Pro Glu Leu Val Gly Pro Ser Glu Ser 165 170 175 Lys Pro Arg Gly Thr Ser Pro Leu Pro Ala Gly Gln Val Pro Val Thr 180 185 190 Leu Gln Pro Gln Lys Gln Val Lys Glu Asn Lys Thr Gln Pro Pro Val 195 200 205 Ala Tyr Gln Tyr Trp Pro Pro Ala Glu Leu Gln Tyr Arg Pro Pro Pro 210 215 220 Glu Ser Gln Tyr Gly Tyr Pro Gly Met Pro Pro Ala Pro Gln Gly Arg 225 230 235 240 Ala Pro Tyr Pro Gln Pro Pro Thr Arg Arg Leu Asn Pro Thr Ala Pro 245 250 255 Pro Ser Arg Gln Gly Ser Glu Leu His Glu Ile Ile Asp Lys Ser Arg 260 265 270 Lys Glu Gly Asp Thr Glu Ala Trp Gln Phe Pro Val Thr Leu Glu Pro 275 280 285 Met Pro Pro Gly Glu Gly Ala Gln Glu Gly Glu Pro Pro Thr Val Glu 290 295 300 Ala Arg Tyr Lys Ser Phe Ser Ile Lys Met Leu Lys Asp Met Lys Glu 305 310 315 320 Gly Val Lys Gln Tyr Gly Pro Asn Ser Pro Tyr Met Arg Thr Leu Leu 325 330 335 Asp Ser Ile Ala His Gly His Arg Leu Ile Pro Tyr Asp Trp Glu Ile 340 345 350 Leu Ala Lys Ser Ser Leu Ser Pro Ser Gln Phe Leu Gln Phe Lys Thr 355 360 365 Trp Trp Ile Asp Gly Val Gln Glu Gln Val Arg Arg Asn Arg Ala Ala 370 375 380 Asn Pro Pro Val Asn Ile Asp Ala Asp Gln Leu Leu Gly Ile Gly Gln 385 390 395 400 Asn Trp Ser Thr Ile Ser Gln Gln Ala Leu Met Gln Asn Glu Ala Ile 405 410 415 Glu Gln Val Arg Ala Ile Cys Leu Arg Ala Trp Glu Lys Ile Gln Asp 420 425 430 Pro Gly Ser Thr Cys Pro Ser Phe Asn Thr Val Arg Gln Gly Ser Lys 435 440 445 Glu Pro Tyr Pro Asp Phe Val Ala Arg Leu Gln Asp Val Ala Gln Lys 450 455 460 Ser Ile Ala Asp Glu Lys Ala Arg Lys Val Ile Val Glu Leu Met Ala 465 470 475 480 Tyr Glu Asn Ala Asn Pro Glu Cys Gln Ser Ala Ile Lys Pro Leu Lys 485 490 495 Gly Lys Val Pro Ala Gly Ser Asp Val Ile Ser Glu Tyr Val Lys Ala 500 505 510 Cys Asp Gly Ile Gly Gly Ala Met His Lys Ala Met Leu Met Ala Gln 515 520 525 Ala Ile Thr Gly Val Val Leu Gly Gly Gln Val Arg Thr Phe Gly Gly 530 535 540 Lys Cys Tyr Asn Cys Gly Gln Ile Gly His Leu Lys Lys Asn Cys Pro 545 550 555 560 Val Leu Asn Lys Gln Asn Ile Thr Ile Gln Ala Thr Thr Thr Gly Arg 565 570 575 Glu Pro Pro Asp Leu Cys Pro Arg Cys Lys Lys Gly Lys His Trp Ala 580 585 590 Ser Gln Cys Arg Ser Lys Phe Asp Lys Asn Gly Gln Pro Leu Ser Gly 595 600 605 Asn Glu Gln Arg Gly Gln Pro Gln Ala Pro Gln Gln Thr Gly Ala Phe 610 615 620 Pro Ile Gln Pro Phe Val Pro Gln Gly Phe Gln Gly Gln Gln Pro Pro 625 630 635 640 Leu Ser Gln Val Phe Gln Gly Ile Ser Gln Leu Pro Gln Tyr Asn Asn 645 650 655 Cys Pro Pro Pro Gln Ala Ala Val Gln Gln 660 665 <210> 6 <211> 2004 <212> DNA <213> Artificial sequence <220> <223> Synthetic ERV-K-gag sequence <400> 6 atgggacaga ccaagagtaa gatcaagtct aagtacgcca gctacctcag cttcatcaag 60 atcctgctga agagaggagg cgtgaaagtg tccaccaaga acctgatcaa gctgttccag 120 atcatcgagc agttctgtcc ctggtttcct gagcagggca ccctggatct gaaggactgg 180 aagcggatcg gcaaagagct gaagcaggct ggcagaaagg gcaacatcat ccctctgacc 240 gtgtggaacg actgggccat catcaaagca gctctggaac ccttccagac cgaagaggat 300 agcgtgtccg tgtctgatgc tcctggcagc tgcatcatcg actgcaacga gaacacccgg 360 aagaagtccc agaaagagac agagagcctg cactgcgagt acgtggccga acctgtgatg 420 gctcagagca cccagaacgt ggactacaac cagctccaag aagtgatcta tcccgaaaca 480 ctgaagctgg aaggcaaggg acctgaactc gtgggtcctt ctgagctaa gcccagaggc 540 acatctcctc tgcctgcagg acaggtgcca gtgacactgc agcctcagaa acaagtgaaa 600 gagaacaaga cccagcctcc tgtggcctac cagtattggc ctccagccga gctgcagtac 660 agacctctc cagagagcca gtacggctac cctggaatgc ctcctgctcc tcaaggcaga 720 gctccttatc ctcagcctcc taccagacgg ctgaacccta cagctcctcc tagcagacag 780 ggctctgagc tgcacgagat cattgacaag agccggaaag agggcgacac cgaggcttgg 840 cagtttcccg ttacactgga acccatgcct ccaggcgaag gcgctcaaga aggcgaacct 900 cctacagtgg aagccaggta caagagcttc agcatcaaga tgctgaagga catgaaggaa 960 ggcgtcaagc agtacggacc taacagccca tacatgcgga ccctgctgga ttctattgcc 1020 cacggccacc ggctgatccc ttacgattgg gagatcctgg ctaagtcctc tctgagccct 1080 agccagttcc tgcagttcaa gacctggtgg atcgacggcg tgcaagaaca agtgagacgg 1140 aacagagctg ccaatcctcc tgtgaacatc gacgccgacc agctcctcgg aatcggccag 1200 aattggagca ccatctctca gcaggctctg atgcagaacg aggccattga aacagtcaga 1260 gccatctgcc tgagagcttg ggagaagaatt caggacccag gcagcacatg tcccagcttc 1320 aataccgttc ggcagggcag caaagagccc tatcctgact ttgtggctag actgcaggat 1380 gtggcccaga agtctattgc cgacgagaag gctcgggaaag tgatcgtgga actgatggcc 1440 tacgagaacg ctaatccaga gtgccagagc gccatcaagc ccttgaaggg caaagtgcct 1500 gccggatccg atgtgatcag cgagtatgtg aaggcctgcg acggaatcgg aggtgccatg 1560 cacaaagcca tgctgatggc acaggccatc actggcgttg tgctcggagg acaagttcgg 1620 acctttggag gcaagtgcta caactgtggc cagatcggac acctgaagaa gaactgccct 1680 gtgctgaaca agcagaacat caccatccag gccaccacca ccggcagaga acctccagat 1740 ctgtgcccta gatgcaagaa gggcaagcac tgggccagcc agtgcagaag caagttcgac 1800 aagaacggcc agcctctgag cggcaacgaa caaagaggac agcctcaggc tcctcagcag 1860 actggcgcat ttccaatcca gcccttcgtg cctcaaggct tccagggaca acagcctcca 1920 ctgtctcagg tgttccaggg cattagccag ctccctcagt acaacaactg ccctccacct 1980 caggctgctg tgcagcagtg atga 2004 <210> 7 <211> 699 <212> PRT <213> Artificial sequence <220> <223> Synthetic ERV-K-env / MEL sequence <400> 7 Met Asn Pro Ser Glu Met Gln Arg Lys Ala Pro Pro Arg Arg Arg Arg 1 5 10 15 His Arg Asn Arg Ala Pro Leu Thr His Lys Met Asn Lys Met Val Thr 20 25 30 Ser Glu Glu Gln Met Lys Leu Pro Ser Thr Lys Lys Ala Glu Pro Pro 35 40 45 Thr Trp Ala Gln Leu Lys Lys Leu Thr Gln Leu Ala Thr Lys Tyr Leu 50 55 60 Glu Asn Thr Lys Val Thr Gln Thr Pro Glu Ser Met Leu Leu Ala Ala 65 70 75 80 Leu Met Ile Val Ser Met Val Val Ser Leu Pro Met Pro Ala Gly Ala 85 90 95 Ala Ala Ala Asn Tyr Thr Tyr Trp Ala Tyr Val Pro Phe Pro Pro Met 100 105 110 Ile Arg Ala Val Thr Trp Met Asp Asn Pro Ile Glu Val Tyr Val Asn 115 120 125 Asp Ser Val Trp Val Pro Gly Pro Ile Asp Asp Arg Cys Pro Ala Lys 130 135 140 Pro Glu Glu Glu Gly Met Met Ile Asn Ile Ser Ile Gly Tyr Arg Tyr 145 150 155 160 Pro Pro Ile Cys Leu Gly Arg Ala Pro Gly Cys Leu Met Pro Ala Val 165 170 175 Gln Asn Trp Leu Val Glu Val Pro Thr Val Ser Pro Ile Ser Arg Phe 180 185 190 Thr Tyr His Met Val Ser Gly Met Ser Leu Arg Pro Arg Val Asn Tyr 195 200 205 Leu Gln Asp Phe Ser Tyr Gln Arg Ser Leu Lys Phe Arg Pro Lys Gly 210 215 220 Lys Pro Cys Pro Lys Glu Ile Pro Lys Glu Ser Lys Asn Thr Glu Val 225 230 235 240 Leu Val Trp Glu Glu Cys Val Ala Asn Ser Ala Val Ile Leu Gln Asn 245 250 255 Asn Glu Phe Gly Thr Ile Ile Asp Trp Ala Pro Arg Gly Gln Phe Tyr 260 265 270 His Asn Cys Ser Gly Gln Thr Gln Ser Cys Pro Ser Ala Gln Val Ser 275 280 285 Pro Ala Val Asp Ser Asp Leu Thr Glu Ser Leu Asp Lys His Lys His 290 295 300 Lys Lys Leu Gln Ser Phe Tyr Pro Trp Glu Trp Gly Glu Lys Gly Ile 305 310 315 320 Ser Thr Pro Arg Pro Lys Ile Ile Ser Pro Val Ser Gly Pro Glu His 325 330 335 Pro Glu Leu Trp Arg Leu Thr Val Ala Ser His His Ile Arg Ile Trp 340 345 350 Ser Gly Asn Gln Thr Leu Glu Thr Arg Asp Arg Lys Pro Phe Tyr Thr 355 360 365 Val Asp Leu Asn Ser Ser Leu Thr Val Pro Leu Gln Ser Cys Val Lys 370 375 380 Pro Pro Tyr Met Leu Val Val Gly Asn Ile Val Ile Lys Pro Asp Ser 385 390 395 400 Gln Thr Ile Thr Cys Glu Asn Cys Arg Leu Leu Thr Cys Ile Asp Ser 405 410 415 Thr Phe Asn Trp Gln His Arg Ile Leu Leu Val Arg Ala Arg Glu Gly 420 425 430 Val Trp Ile Pro Val Ser Met Asp Arg Pro Trp Glu Ala Ser Pro Ser 435 440 445 Val His Ile Leu Thr Glu Val Leu Lys Gly Val Leu Asn Arg Ser Lys 450 455 460 Arg Phe Ile Phe Thr Leu Ile Ala Val Ile Met Gly Leu Ile Ala Val 465 470 475 480 Thr Ala Thr Ala Ala Val Ala Gly Val Ala Leu His Ser Ser Val Gln 485 490 495 Ser Val Asn Phe Val Asn Asp Trp Gln Lys Asn Ser Thr Arg Leu Trp 500 505 510 Asn Ser Gln Ser Ser Ile Asp Gln Lys Met Leu Ala Val Ile Ser Cys 515 520 525 Ala Val Gln Thr Val Ile Trp Met Gly Asp Arg Leu Met Ser Leu Glu 530 535 540 His Arg Phe Gln Leu Gln Cys Asp Trp Asn Thr Ser Asp Phe Cys Ile 545 550 555 560 Thr Pro Gln Ile Tyr Asn Glu Ser Glu His His Trp Asp Met Val Arg 565 570 575 Arg His Leu Gln Gly Arg Glu Asp Asn Leu Thr Leu Asp Ile Ser Lys 580 585 590 Leu Lys Glu Gln Ile Phe Glu Ala Ser Lys Ala His Leu Asn Leu Val 595 600 605 Pro Gly Thr Glu Ala Ile Ala Gly Val Ala Asp Gly Leu Ala Asn Leu 610 615 620 Asn Pro Val Thr Trp Val Lys Thr Ile Gly Ser Thr Thr Ile Ile Asn 625 630 635 640 Leu Ile Leu Ile Leu Val Cys Leu Phe Cys Leu Leu Leu Val Cys Arg 645 650 655 Cys Thr Gln Gln Leu Arg Arg Asp Ser Asp His Arg Glu Arg Ala Met 660 665 670 Met Thr Met Ala Val Leu Ser Lys Arg Lys Gly Gly Asn Val Gly Lys 675 680 685 Ser Lys Arg Asp Gln Ile Val Thr Val Ser Val 690 695 <210> 8 <211> 2103 <212> DNA <213> Artificial sequence <220> <223> Synthetic ERV-K-env / MEL sequence <400> 8 atgaacccta gcgagatgca gagaaaggct ccacctagac ggagaagaca cagaaacagg 60 gctcctctga cacacaagat gaacaagatg gtcaccagcg aggaacagat gaaactgccc 120 agcaccaaga aggccgagcc tccaacatgg gctcagctga agaaactgac ccagctggcc 180 accaagtacc tggagaacac caaagtgacc cagacacctg agagcatgct gctggcagct 240 ctgatgatcg tgtccatggt ggtgtccctg cctatgcctg ctggtgctgc cgctgccaac 300 tacacatact gggcctacgt gccctttcct cctatgatca gagccgtgac ctggatggac 360 aaccctattg aggtgtacgt gaacgacagc gtgtgggtgc caggacctat cgacgataga 420 tgtcctgcca aacctgagga agagggcatg atgatcaaca tcagcatcgg ctaccggtat 480 cctccaatct gcctgggcag agcacctggc tgtcttatgc cagctgtgca gaattggctg 540 gtggaagtgc ctaccgtgtc tcccatcagc cggttcacct accacatggt gtccggcatg 600 agcctcagac ctagagtgaa ctacttgcag gacttcagct atcagcggag cctgaagttc 660 agacccaagg gaaagccctg tcctaaagag attcccaaag agtccaagaa caccgaggtg 720 ctcgtgtggg aagagtgcgt ggccaattct gccgtgatcc tgcagaacaa cgagttcggc 780 accatcattg actgggctcc tagaggccag ttctaccaca attgcagcgg acagacacag 840 agctgtccta gcgcacaagt gtcaccagcc gtggatagcg atctgaccga gagcctggac 900 aagcacaaac acaagaaact tcagagcttc tatccctggg agtggggaga gaagggcatc 960 tctacaccaa ggcctaagat cattagccct gtgtctggac cagaacatcc cgaactttgg 1020 agactgacag tggccagcca ccacatcaga atctggagcg gcaatcagac cctggaaaca 1080 cgggacagaa agcccttcta caccgtcgat ctgaacagca gcctgaccgt gcctctccag 1140 agctgtgtga agcctcctta catgctggtc gtgggcaaca ttgtgatcaa gcccgactcc 1200 cagaccatca catgcgagaa ctgcagactg ctgacctgca tcgacagcac cttcaactgg 1260 cagcaccgga tcctgctcgt gcgagctaga gaaggcgtgt ggatccctgt ctctatggac 1320 aggccttggg aagccagccc tagcgtgcac attctgacag aggtgctgaa gggcgtgctc 1380 aacagatcca agcggttcat cttcaccctg atcgccgtca tcatgggcct gattgctgtg 1440 acagccacag ctgctgttgc tggcgtggcc ctgcatagct ctgtgcagag cgtgaacttc 1500 gtgaacgatt ggcagaagaa cagcacacgg ctgtggaaca gccagagcag catcgaccag 1560 aagatgctgg ccgtgatctc ctgtgccgtg cagacagtta tctggatggg cgacagactg 1620 atgagcctgg aacaccggtt ccagctgcag tgcgactgga ataccagcga cttctgcatc 1680 acacctcaga tctacaacga gagcgagcac cactgggata tggtccgaag gcatctgcag 1740 ggcagagagg acaacctgac actggacatc agcaagctga aagagcagat cttcgaggcc 1800 agcaaggctc acctgaatct ggtgcctgga accgaagcta ttgctggagt tgcagatggc 1860 ctggccaatc tgaatcctgt gacctgggtc aagaccatcg gcagcaccac aatcatcaac 1920 ctgatcctga tcctcgtgtg cctgttttgc ctgctgcttg tgtgcagatg cacccagcag 1980 ctgagaagag acagcgacca tagagaaaga gccatgatga ccatggccgt cctgagcaag 2040 agaaagggag gcaacgtggg caagagcaag cgggatcaga tcgtgaccgt gtccgtttga 2100 taa 2103 <210> 9 <211> 741 <212> PRT <213> Artificial sequence <220> <223> Synthetic hFOLR1 - hPRAME fusion sequence <400> 9 Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val 1 5 10 15 Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu 20 25 30 Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35 40 45 Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala 50 55 60 Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr 65 70 75 80 Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85 90 95 Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100 105 110 Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115 120 125 Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu 130 135 140 Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp 145 150 155 160 Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln 165 170 175 Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile 180 185 190 Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg 195 200 205 Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210 215 220 Val Ala Arg Phe Tyr Ala Ala Ala Met Glu Arg Arg Arg Leu Trp Gly 225 230 235 240 Ser Ile Gln Ser Arg Tyr Ile Ser Met Ser Val Trp Thr Ser Pro Arg 245 250 255 Arg Leu Val Glu Leu Ala Gly Gln Ser Leu Leu Lys Asp Glu Ala Leu 260 265 270 Ala Ile Ala Ala Leu Glu Leu Leu Pro Arg Glu Leu Phe Pro Pro Leu 275 280 285 Phe Met Ala Ala Phe Asp Gly Arg His Ser Gln Thr Leu Lys Ala Met 290 295 300 Val Gln Ala Trp Pro Phe Thr Cys Leu Pro Leu Gly Val Leu Met Lys 305 310 315 320 Gly Gln His Leu His Leu Glu Thr Phe Lys Ala Val Leu Asp Gly Leu 325 330 335 Asp Val Leu Leu Ala Gln Glu Val Arg Pro Arg Arg Trp Lys Leu Gln 340 345 350 Val Leu Asp Leu Arg Lys Asn Ser His Gln Asp Phe Trp Thr Val Trp 355 360 365 Ser Gly Asn Arg Ala Ser Leu Tyr Ser Phe Pro Glu Pro Glu Ala Ala 370 375 380 Gln Pro Met Thr Thr Lys Ala Lys Val Asp Gly Leu Ser Thr Glu Ala 385 390 395 400 Glu Gln Pro Phe Ile Pro Val Glu Val Leu Val Asp Leu Phe Leu Lys 405 410 415 Glu Gly Ala Cys Asp Glu Leu Phe Ser Tyr Leu Ile Glu Lys Val Ala 420 425 430 Ala Lys Lys Asn Val Leu Arg Leu Cys Cys Lys Lys Leu Lys Ile Phe 435 440 445 Ala Met Pro Met Gln Asp Ile Lys Met Ile Leu Lys Met Val Gln Leu 450 455 460 Asp Ser Ile Glu Asp Leu Glu Val Thr Cys Thr Trp Lys Leu Pro Thr 465 470 475 480 Leu Ala Lys Phe Ser Pro Tyr Leu Gly Gln Met Ile Asn Leu Arg Arg 485 490 495 Leu Leu Leu Ser His Ile His Ala Ser Ser Tyr Ile Ser Pro Glu Lys 500 505 510 Glu Glu Gln Tyr Ile Ala Gln Phe Thr Ser Gln Phe Leu Ser Leu Gln 515 520 525 Cys Leu Gln Ala Leu Tyr Val Asp Ser Leu Phe Phe Leu Arg Gly Arg 530 535 540 Leu Asp Gln Leu Leu Arg His Val Met Asn Pro Leu Glu Thr Leu Ser 545 550 555 560 Ile Thr Asn Cys Arg Leu Ser Glu Gly Asp Val Met His Leu Ser Gln 565 570 575 Ser Pro Ser Val Ser Gln Leu Ser Val Leu Ser Leu Ser Gly Val Met 580 585 590 Leu Thr Asp Val Ser Pro Glu Pro Leu Gln Ala Leu Leu Glu Arg Ala 595 600 605 Ser Ala Thr Leu Gln Asp Leu Val Phe Asp Glu Cys Gly Ile Thr Asp 610 615 620 Asp Gln Leu Leu Ala Leu Leu Pro Ser Leu Ser His Cys Ser Gln Leu 625 630 635 640 Thr Thr Leu Ser Phe Tyr Gly Asn Ser Ile Ser Ile Ser Ala Leu Gln 645 650 655 Ser Leu Leu Gln His Leu Ile Gly Leu Ser Asn Leu Thr His Val Leu 660 665 670 Tyr Pro Val Pro Leu Glu Ser Tyr Glu Asp Ile His Gly Thr Leu His 675 680 685 Leu Glu Arg Leu Ala Tyr Leu His Ala Arg Leu Arg Glu Leu Leu Cys 690 695 700 Glu Leu Gly Arg Pro Ser Met Val Trp Leu Ser Ala Asn Pro Cys Pro 705 710 715 720 His Cys Gly Asp Arg Thr Phe Tyr Asp Pro Glu Pro Ile Leu Cys Pro 725 730 735 Cys Phe Met Pro Asn 740 <210> 10 <211> 2228 <212> DNA <213> Artificial sequence <220> <#223> Synthetic hFOLR1-hPRAME fusion nucleotide sequence <400> 10 tggcccagag aatgaccaca caactgctgc tgctcctggt gtgggttgcc gttgttggag 60 aggcccagac cagaattgcc tgggccagaa ccgagctgct gaacgtgtgc atgaacgcca 120 agcatcacaa agagaagcct ggacctgaag acaagctgca tgaacagtgt cggccttgga 180 gaaagaatgc ttgctgtagc accaacacca gccaagaggc ccacaaggac gtgtcctacc 240 tgtaccggtt caactggaac cactgcggag aaatggctcc tgcctgcaag agacacttca 300 tccaggatac ctgcctgtac gagtgctctc ccaatctcgg accttggatc cagcaagtgg 360 accagagctg gcggaaagaa cgggtgctga atgtgccctt gtgcaaagag gattgcgagc 420 agtggtggga agattgccgg accagctaca catgtaagag caactggcac aaaggctgga 480 actggaccag cggcttcaac aagtgtgccg tgggagctgc ctgccagcct ttccacttct 540 acttcccaac acctaccgtg ctgtgcaacg aaatctggac ccacagctac aaggtgtcca 600 actacagcag aggcagcggc aggtgtatcc agatgtggtt cgatcccgct cagggcaatc 660 ccaatgagga agtggctaga ttctacgctg ctgccatgga aagaagaagg ctctggggca 720 gcatccagag ccggtacatt agcatgagcg tgtggacaag ccctagacgg ctggttgaac 780 tggctggaca gagcctgctc aaggatgagg ccctggccat tgctgctctg gagctgctgc 840 ctagagagct gttccctcct ctgttcatgg ctgccttcga cggcagacac agccagacac 900 tgaaagccat ggtgcaggcc tggcctttca cctgtctgcc tctgggagtg ctgatgaagg 960 gccagcatct gcacctggaa accttcaagg ccgtgctgga cggcctggat gttctcctgg 1020 ctcaagaggt gaggcctcgg cgttggaaac tgcaggttct ggatctgcgg aagaactctc 1080 accaggattt ctggaccgtt tggtccggca acagagccag cctgtacagc tttcctgaac 1140 ctgaggctgc ccagcccatg accacaaagg ccaaagtgga tggcctgagc acagaggccg 1200 agcagccttt cattcccgtc gaagtgctgg tggacctgtt cctgaaagaa ggagcctgcg 1260 atgagctgtt cagctacctg attgagaagg tggcagccaa gaagaacgtg ctgcggctgt 1320 gctgcaagaa gctgaagaatc tttgccatgc ctatgcagga tatcaagatg atcctgaaga 1380 tggtgcagct ggacagcatc gaggacctgg aagtgacctg tacctggaag ctgcccacac 1440 tggccaagtt cagcccttac ctgggacaga tgattaacct gcggaggctg ctgctgtctc 1500 acatccacgc cagctcctac atcagccctg agaagaga aagttatc gcccagttca 1560 caagccagtt tctgagcctg cagtgtctgc aggccctgta cgtggacagc ctgttctttc 1620 tgagaggcag gctggatcag ctgctgcggc acgtgatgaa ccctctggaa accctgagca 1680 tcaccaactg tagactgagc gagggcgacg tgatgcacct gtctcagagc ccatctgtgt 1740 ctcagctgag cgtgctgtct ctgtctggcg tgatgctgac cgatgtgagc cctgaacctc 1800 tgcaggcact gctggaaaga gcctccgcta ctctgcagga cctggtgttc gatgagtgcg 1860 gcatcaccga tgaccagctg cttgctctgc tgccaagcct gagccactgt agccagctga 1920 caaccctgtc cttctacggc aacagcatct ccatctctgc cctgcagtct ctcctgcagc 1980 atctgatcgg cctgtccaat ctgacccacg tgctgtaccc tgtgccactg gaaagctacg 2040 aggacatcca cggaaccctg cacctcgaga gactggccta tctgcatgct cggctgagag 2100 aactgctgtg cgaactgggc agacccagca tggtttggct gagcgccaat ccatgtcctc 2160 actgtggcga ccggaccttc tacgaccctg agcctatcct gtgtccttgc ttcatgccca 2220 actaatag 2228 <210> 11 <211> 517 <212> PRT <213> Artificial sequence <220> <223> Synthetic ERV-K-env / MEL_03 sequence <400> 11 Met Asn Pro Ser Glu Met Gln Arg Lys Ala Pro Pro Arg Arg Arg Arg 1 5 10 15 His Arg Asn Arg Ala Pro Leu Thr His Lys Met Asn Lys Met Val Thr 20 25 30 Ser Glu Glu Gln Met Lys Leu Pro Ser Thr Lys Lys Ala Glu Pro Pro 35 40 45 Thr Trp Ala Gln Leu Lys Lys Leu Thr Gln Leu Ala Thr Lys Tyr Leu 50 55 60 Glu Asn Thr Lys Val Thr Gln Thr Pro Glu Ser Met Leu Leu Ala Ala 65 70 75 80 Leu Met Ile Val Ser Met Val Val Ser Leu Pro Met Pro Ala Gly Ala 85 90 95 Ala Ala Ala Asn Tyr Thr Tyr Trp Ala Tyr Val Pro Phe Pro Pro Met 100 105 110 Ile Arg Ala Val Thr Trp Met Asp Asn Pro Ile Glu Val Tyr Val Asn 115 120 125 Asp Ser Val Trp Val Pro Gly Pro Ile Asp Asp Arg Cys Pro Ala Lys 130 135 140 Pro Glu Glu Glu Gly Met Met Ile Asn Ile Ser Ile Gly Tyr Arg Tyr 145 150 155 160 Pro Pro Ile Cys Leu Gly Arg Ala Pro Gly Cys Leu Met Pro Ala Val 165 170 175 Gln Asn Trp Leu Val Glu Val Pro Thr Val Ser Pro Ile Ser Arg Phe 180 185 190 Thr Tyr His Met Val Ser Gly Met Ser Leu Arg Pro Arg Val Asn Tyr 195 200 205 Leu Gln Asp Phe Ser Tyr Gln Arg Ser Leu Lys Phe Arg Pro Lys Gly 210 215 220 Lys Pro Cys Pro Lys Glu Ile Pro Lys Glu Ser Lys Asn Thr Glu Val 225 230 235 240 Leu Val Trp Glu Glu Cys Val Ala Asn Ser Ala Val Ile Leu Gln Asn 245 250 255 Asn Glu Phe Gly Thr Ile Ile Asp Trp Ala Pro Arg Gly Gln Phe Tyr 260 265 270 His Asn Cys Ser Gly Gln Thr Gln Ser Cys Pro Ser Ala Gln Val Ser 275 280 285 Pro Ala Val Asp Ser Asp Leu Thr Glu Ser Leu Asp Lys His Lys His 290 295 300 Lys Lys Leu Gln Ser Phe Tyr Pro Trp Glu Trp Gly Glu Lys Gly Ile 305 310 315 320 Ser Thr Pro Arg Pro Lys Ile Ile Ser Pro Val Ser Gly Pro Glu His 325 330 335 Pro Glu Leu Trp Arg Leu Thr Val Ala Ser His His Ile Arg Ile Trp 340 345 350 Ser Gly Asn Gln Thr Leu Glu Thr Arg Asp Arg Lys Pro Phe Tyr Thr 355 360 365 Val Asp Leu Asn Ser Ser Leu Thr Val Pro Leu Gln Ser Cys Val Lys 370 375 380 Pro Pro Tyr Met Leu Val Val Gly Asn Ile Val Ile Lys Pro Asp Ser 385 390 395 400 Gln Thr Ile Thr Cys Glu Asn Cys Arg Leu Leu Thr Cys Ile Asp Ser 405 410 415 Thr Phe Asn Trp Gln His Arg Ile Leu Leu Val Arg Ala Arg Glu Gly 420 425 430 Val Trp Ile Pro Val Ser Met Asp Arg Pro Trp Glu Ala Ser Pro Ser 435 440 445 Val His Ile Leu Thr Glu Val Leu Lys Gly Val Leu Asn Met Leu Ala 450 455 460 Val Ile Ser Cys Ala Val Ala Gly Val Ala Leu His Gly Ser Ala Gly 465 470 475 480 Ser Ala Ala Gly Ser Gly Glu Phe Val Val Ile Ser Ala Ile Leu Ala 485 490 495 Leu Val Val Leu Thr Ile Ile Ser Leu Ile Ile Leu Ile Met Leu Trp 500 505 510 Gln Lys Lys Pro Arg 515 <210> 12 <211> 1557 <212> DNA <213> Artificial sequence <220> <223> ERV-K-env / MEL_03 nucleotide sequence <400> 12 atgaacccta gcgagatgca gagaaaggct ccacctagac ggagaagaca cagaaacagg 60 gctcctctga cacacaagat gaacaagatg gtcaccagcg aggaacagat gaaactgccc 120 agcaccaaga aggccgagcc tccaacatgg gctcagctga agaaactgac ccagctggcc 180 accaagtacc tggagaacac caaagtgacc cagacacctg agagcatgct gctggcagct 240 ctgatgatcg tgtccatggt ggtgtccctg cctatgcctg ctggtgctgc cgctgccaac 300 tacacatact gggcctacgt gccctttcct cctatgatca gagccgtgac ctggatggac 360 aaccctattg aggtgtacgt gaacgacagc gtgtgggtgc caggacctat cgacgataga 420 tgtcctgcca aacctgagga agagggcatg atgatcaaca tcagcatcgg ctaccggtat 480 cctccaatct gcctgggcag agcacctggc tgtcttatgc cagctgtgca gaattggctg 540 gtggaagtgc ctaccgtgtc tcccatcagc cggttcacct accacatggt gtccggcatg 600 agcctcagac ctagagtgaa ctacttgcag gacttcagct atcagcggag cctgaagttc 660 agacccaagg gaaagccctg tcctaaagag attcccaaag agtccaagaa caccgaggtg 720 ctcgtgtggg aagagtgcgt ggccaattct gccgtgatcc tgcagaacaa cgagttcggc 780 accatcattg actgggctcc tagaggccag ttctaccaca attgcagcgg acagacacag 840 agctgtccta gcgcacaagt gtcaccagcc gtggatagcg atctgaccga gagcctggac 900 aagcacaaac acaagaaact tcagagcttc tatccctggg agtggggaga gaagggcatc 960 tctacaccaa ggcctaagat cattagccct gtgtctggac cagaacatcc cgaactttgg 1020 agactgacag tggccagcca ccacatcaga atctggagcg gcaatcagac cctggaaaca 1080 cgggacagaa agcccttcta caccgtcgat ctgaacagca gcctgaccgt gcctctccag 1140 agctgtgtga agcctcctta catgctggtc gtgggcaaca ttgtgatcaa gcccgactcc 1200 cagaccatca catgcgagaa ctgcagactg ctgacctgca tcgacagcac cttcaactgg 1260 cagcaccgga tcctgctcgt gcgagctaga gaaggcgtgt ggatccctgt ctctatggac 1320 aggccttggg aagccagccc tagcgtgcac attctgacag aggtgctgaa gggcgtgctc 1380 aacatgctgg ccgtgatctc ctgtgccgtg gctggcgtgg ccctgcatgg ctctgctgga 1440 tctgctgctg gaagcggcga gttcgtggtc atctctgcca ttctggctct ggtggtgctg 1500 accatcatca gcctgatcat cctgattatg ctgtggcaga agaagccccg gtgataa 1557

Claims

1. A recombinant modified vaccinia virus Ankara (MVA), comprising: (i) a nucleic acid encoding HERV-K-env / MEL; (ii) a nucleic acid encoding HERV-K gag; (iii) a nucleic acid encoding a fusion protein comprising FOLR1 and PRAME; and (iv) a nucleic acid encoding 4-1BBL.

2. The recombinant MVA according to claim 1, further comprising: (v) Nucleic acid encoding CD40L.

3. The recombinant MVA according to claim 1, wherein the fusion protein comprising FOLR1 and PRAME has the sequence shown in SEQ ID NO:

9. The recombinant MVA according to claim 1 , wherein the recombinant MVA is derived from MVA-BN.

5. A pharmaceutical preparation or composition comprising the recombinant MVA according to any one of claims 1 to 4.

6. The pharmaceutical formulation or composition according to claim 5, which is suitable for intratumoral and / or intravenous administration.

7. A vaccine comprising the recombinant MVA according to any one of claims 1 to 4.

8. Use of the recombinant MVA according to any one of claims 1 to 4 in the preparation of a medicament for treating melanoma or colon cancer in a subject.

9. Use of the recombinant MVA according to any one of claims 1 to 4 in the preparation of a medicament for enhancing the inflammatory response of a melanoma or colon cancer, reducing the size of a melanoma or colon cancer, delaying or preventing the growth of a melanoma or colon cancer and / or increasing the overall survival rate in a subject.

10. Use according to any one of claims 8 to 9, wherein the recombinant MVA is administered intratumorally and / or intravenously.

11. Use according to any one of claims 8 to 9, wherein the recombinant MVA is used in combination with a TAA-specific antibody.

12. The use according to any one of claims 8 to 9, wherein the recombinant MVA is used in combination with an immune checkpoint molecule antagonist or agonist.

13. The use according to any one of claims 8 to 9, wherein the subject is a human.

14. The recombinant MVA according to any one of claims 1 to 4, wherein the nucleic acid encoding HERV-K-env / MEL has the sequence shown in SEQ ID NO: 12 or encodes the protein sequence shown in SEQ ID NO: 11.

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