Medical Use of 4-1BBL-Adjuvanted Recombinant Modified Vaccinia Virus Ankara (MVA)

Abstract

The present invention relates to a recombinant modified vaccinia virus Ankara (MVA) expressing a TAA and the costimulatory molecule 4-1BBL, for use in (i) preventing the recurrence of a solid tumor, in which the recombinant MVA is administered intratumorally, or (ii) treating, preventing, and / or preventing the recurrence of a tumor, in which the recombinant MVA is administered intratumorally, in which another solid tumor is treated with the recombinant MVA.

Description

[Technical field]

[0001] The present invention relates to the field of cancer immunotherapy, in particular to oncolytic virus therapy. In particular, the present invention relates to a recombinant modified vaccinia virus Ankara (MVA) expressing a tumor-associated antigen (TAA) and a costimulatory molecule 4-1BBL for use in the treatment, prevention and / or recurrence prevention of tumors and metastases, where the recombinant MVA is administered intratumorally to solid tumors. The present invention also relates to the respective methods of treatment. [Background technology]

[0002] Human cancers are highly heterogeneous in terms of tumor antigen expression and immune infiltration and composition. However, a common feature is that the host is unable to mount a strong immune response that effectively counteracts tumor growth. In many cases, naturally primed CD8 + T cells lack sufficient stimulation and efficient penetration of tumor tissue due to the harsh tumor microenvironment.

[0003] The lack of a strong immune response against solid tumors, due to the poor ability of immune cells to infiltrate or perform effector functions in the hostile tumor microenvironment (TME), represents a major challenge for cancer immunotherapy [1]. The concept of reprogramming the immunosuppressive TME to an inflammatory one by tumor-directed therapy has attracted considerable attention in recent years [2]. The aim is to activate immune cells already homing to the tumor and regional lymph nodes or to recruit new immune cells to the TME, while minimizing inappropriate activation of the remaining immune system [3]. To achieve this, several strategies have been explored in preclinical models and in the clinic, utilizing the local release and activation of biochemical signals derived from pathogen recognition and unprogrammed cell destruction or the local administration of immunostimulatory monoclonal antibodies and cytokines [2].

[0004] Localized oncolytic virotherapy relies on the concept of tumor-targeted therapy through specific infection and destruction of tumor cells and modulation of the TME. The great potential of oncolytic viruses (OVs) has been highlighted by the recent Food and Drug Administration (FDA) approval of the first-in-class oncolytic agent IMLYGIC, a modified herpes simplex virus 1 encoding human GM-CSF, for patients with stage III melanoma [4]. Various virus families have been investigated for their oncolytic effects, including herpesviruses, poxviruses, and adenoviruses, among others [5].

[0005] Although tumor cell-specific replication and direct killing activity of OV viruses were traditionally considered to be the main mechanism of action, it is now known that initiation or enhancement of host antitumor immune responses is essential for oncolytic virotherapy.[6] Thus, local virotherapy can be considered as an in situ vaccine that leads to immunogenic cell death accompanied by the release of damage-associated or pathogen-associated molecular patterns and tumor antigens, which ultimately triggers antitumor innate and adaptive immune responses.[7]

[0006] A striking feature of most OVs is their tumor specificity and their ability to spread through tumor tissues by viral replication and associated tumor cell lysis [8]. Despite their engineered tissue tropism for tumor cells, the use of replication-competent viruses in patients still raises safety concerns. MVA-BN is a highly attenuated vaccinia strain approved by the FDA as a non-replicating vaccine against smallpox and monkeypox (JYNNEOS®) [9]. In addition, a recombinant MVA-BN vaccine vector was recently approved by the European Medicines Agency (EMA) as part of an Ebola vaccine, and others are being used in clinical trials against various infectious agents

[10] . MVA is a potent inducer of type I interferon (IFN) [11, 12, 13] and elicits strong humoral and cellular immune responses against vector-encoded heterologous antigens [14, 16]. Importantly, MVA is unable to replicate in human cells because its replicative capacity is restricted to embryonic avian cells.

[15] Therefore, its excellent safety profile and immune-stimulating properties make MVA a prime candidate for therapeutic intervention.

[0007] MVA can accommodate large transgene inserts, facilitating the incorporation of heterologous antigens and immunostimulatory molecules to induce antigen-specific T cell responses and enhance specific immune activation pathways. CD40L-adjuvanted MVA significantly enhanced innate and adaptive immune responses upon IV injection [16, 17]. Furthermore, OVs genetically modified with costimulatory molecules or inflammatory cytokines increased therapeutic efficacy after intratumoral (IT) treatment

[18] . Thus, IT treatment using MVAs encoding tumor-associated antigens (TAAs) together with costimulatory molecules may enhance antitumor immune responses in the TME.

[0008] The tumor necrosis factor receptor (TNFR) family members 4-1BB or CD137 are defined as bona fide costimulatory molecules in T cells. 4-1BB is transiently induced upon TCR stimulation, and subsequent engagement with this costimulatory receptor leads to increased levels of cytokine secretion and upregulation of the antiapoptotic molecules Bcl-2 and Bcl-xl. This results in increased proliferation and protection against activation-induced T cell death, which is also important for the formation of immune memory [19, 20]. The expression of 4-1BB on tumor-infiltrating T cells (TILs)

[21] , coupled with its ability to promote survival, proliferation, and enhanced effector functions of activated T cells, makes it an attractive target for cancer immunotherapy. Indeed, stimulation of the costimulatory pathway 4-1BB / 4-1BBL is beneficial in many cancer treatment settings, including monotherapy or combination therapy with 4-1BB agonist antibodies or 4-1BBL-expressing viral vectors

[22] . 4-1BB costimulation is also important for therapy with third-generation chimeric antigen receptor (CAR) T cells, in which the internal domain has been engineered into a tumor-binding chimeric receptor to enhance signaling and, consequently, tumor cell killing

[23] .

[0009] Nonetheless, despite the notable progress made so far, there remains a need for further medical uses of oncolytic virus therapy. Summary of the Invention

[0010] It is an object of the present invention to provide further medical uses of localized, i.e. tumor-directed, virotherapy using oncolytic viruses.

[0011] Briefly, the object of the present invention is solved by a recombinant MVA expressing a TAA and the costimulatory molecule 4-1BBL for use in: (i) preventing recurrence of solid tumors by intratumoral administration of recombinant MVA; or (ii) For other solid tumors, the recombinant MVA is administered intratumorally to treat, prevent and / or prevent recurrence of the tumor.

[0012] In particular, the invention is defined by the appended claims and by the following aspects and embodiments thereof.

[0013] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and The present invention provides a recombinant MVA comprising:

[0014] In a further aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and The present invention provides a recombinant MVA comprising:

[0015] In another aspect, the present invention provides a method for producing a composition comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and for use in the treatment, prevention and / or prevention of recurrence of a secondary tumor in a subject, wherein the recombinant MVA is administered locally, preferably intratumorally, to an associated primary solid tumor but not to the secondary tumor; or For use in the treatment and / or prevention of recurrence of a primary solid tumor in a subject, wherein the recombinant MVA is administered locally, preferably intratumorally, to an associated secondary solid tumor but not to the primary solid tumor; or Provided is a recombinant MVA for use in the treatment, prevention and / or prevention of recurrence of a secondary tumor in a subject, wherein the recombinant MVA is administered locally, preferably intratumorally, to another related secondary solid tumor, but not to the first-mentioned secondary tumor.

[0016] In yet another aspect, the present invention provides a method for producing a composition comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and wherein the recombinant MVA is administered locally, preferably intratumorally, to another related solid tumor, but not locally, preferably intratumorally, to the first-mentioned tumor.

[0017] In yet another aspect, the present invention provides a method for producing a composition comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and The present invention provides a recombinant MVA comprising:

[0018] In yet another aspect, the present invention provides a method for producing a composition comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and The present invention provides a recombinant MVA comprising:

[0019] In yet another aspect, the present invention provides a method for producing a composition comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and The present invention provides a recombinant MVA comprising:

[0020] In yet another aspect, the present invention provides a method for producing a composition comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and The present invention provides a recombinant MVA comprising:

[0021] In yet another aspect, the present invention provides a method for producing a composition comprising: (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and The present invention provides a recombinant MVA comprising:

[0022] The present invention also provides a method of treatment for a patient having multiple tumors, comprising administering a recombinant MVA locally (preferably intratumorally) to at least one but not all of the multiple tumors, wherein the recombinant MVA comprises a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding 4-1-BB ligand (4-1-BBL).

[0023] These aspects and their embodiments are described in further detail in relation to the description of the invention. [Brief description of the drawings]

[0024] [Figure 1] The therapeutic efficacy of intratumoral administration of MVA-TAA-4-1BBL in a model of unrelated large tumors is shown to be independent of the choice of antigen. C57BL / 6 mice (A-D) or Balb / c mice (E-F) were subcutaneously (SC) injected with 5x105 B16.OVA cells (A-B), 5x105 B16.F10 cells (C-D) or 5x105 CT26.WT cells (E-F) in the flank. 7-14 days later, when tumors exceeded 60 mm3, mice were immunized intratumorally (IT) with either PBS or the indicated MVA constructs. IT immunizations were repeated on days 4 or 5 and 8 after the first immunization (dashed lines). (A) Tumor size follow-up (n=5 mice / group) and (B) overall survival rate of B16.OVA-bearing mice injected with either PBS, 2x108 TCID50 of MVA-OVA or 2x108 TCID50 of MVA-OVA-4-1BBL (n=20 mice / group). (C) Tumor size follow-up (n=5 mice / group) and (D) overall survival rate of B16.F10-bearing mice injected with either PBS, 5x107 TCID50 of MVA-Gp70 or 5x107 TCID50 of MVA-Gp70-4-1BBL (n=15 mice / group). (E) Tumor size follow-up (n=5 mice / group) and (F) overall survival rate of B16.OVA-bearing mice injected with either PBS, 5x107 TCID50 of MVA-Gp70 or 5x107 TCID50 of MVA-Gp70-4-1BBL (n=15 mice / group). Overall survival of CT26.WT-bearing mice injected with either TCID50 of MVA-Gp70-4-1BBL (n=10 mice / group). (A, C, and E) Data are representative of at least two independent experiments. (B, D, and F) Overall survival of at least two independent experiments combined is represented. Log rank test of mouse survival was performed in panels B, D, and F. *, p<0.05; **, p<0.005; ****, p<0.0001. [Figure 2A]Figure 1 shows an increase in peripheral blood CD8+ T cell responses in tumor-bearing mice immunized with MVA-TAA-4-1BBL. Representative dot plots and frequency of peripheral blood CD44+ OVA257-264 Dex+ CD8+ T cells 3 days after the final IT immunization with PBS, MVA-OVA or MVA-OVA-4-1BBL in B16.OVA tumor-bearing mice (n=5 mice / group). Data are representative of at least two independent experiments. Data are presented as mean ± SEM. One-way ANOVA was performed. ***p<0.005, ****p<0.001. [Figure 2B] Figure 1 shows an increase in peripheral blood CD8+ T cell responses in tumor-bearing mice immunized with MVA-TAA-4-1BBL. Representative dot plots and frequency of peripheral blood CD44+ IFNγ+ CD8+ T cells restimulated with p15E604-611 peptide 3 days after the final IT immunization with PBS, MVA-Gp70 or MVA-Gp70-4-1BBL in B16.F10 tumor-bearing mice (n=5 mice / group). Data are representative of at least two independent experiments. Data are presented as mean ± SEM. One-way ANOVA was performed. ***p<0.005, ****p<0.001. [Figure 2C] Figure 1 shows an increase in peripheral blood CD8+ T cell responses in tumor-bearing mice immunized with MVA-TAA-4-1BBL. Representative dot plots and frequency of peripheral blood CD44+ IFNγ+ CD8+ T cells restimulated with AH16-14 peptide 3 days after the final IT immunization with PBS, MVA-Gp70 or MVA-Gp70-4-1BBL in CT26.WT tumor-bearing mice (n=5 mice / group). Data are representative of at least two independent experiments. Data are presented as mean ± SEM. One-way ANOVA was performed. ***p<0.005, ****p<0.001. [Figure 2D] Figure 1 shows an increase in peripheral blood CD8+ T cell responses in tumor-bearing mice immunized with MVA-TAA-4-1BBL. Representative photograph of vitiligo development in C57BL / 6 mice cured with IT MVA-TAA-4-1BBL. Data are representative of at least two independent experiments. [Figure 2E] Figure 1 shows an increase in peripheral blood CD8+ T cell responses in tumor-bearing mice immunized with MVA-TAA-4-1BBL. Pie charts showing vitiligo incidence in C57BL / 6 mice cured with IT MVA-TAA (data combining MVA-OVA and MVA-Gp70) and MVA-TAA-4-1BBL (data combining MVA-OVA-4-1BBL and MVA-Gp70-4-1BBL), respectively. Data are representative of at least two independent experiments. [Diagram 3] MVA localization upon IT MVA injection and induction of inflammatory cytokines by MVA-OVA and MVA-OVA-4-1-BBL are shown. (A) C57BL / 6 mice were subcutaneously injected with 5x105 B16.OVA cells. When tumors reached 60mm3, mice were divided into groups (n=3 mice / group) and received IT injections of PBS or 2x108 TCID50 of MVA (MVA not encoding TAA or 4-1-BBL). Six hours after IT injection, tumors, TdLNs, NdLNs, spleens and blood were snap frozen and viral DNA was extracted from tissue lysates. Gene copies (gcs) of the MVA gene MVA082L in different organs are shown. Data are presented as mean ± SEM. Two-way ANOVA was performed. *, p<0.05; ***, p<0.005; ****, p<0.0001. (B) C57BL / 6 mice were administered 5 × 105 B16.OVA cells. When tumors reached 60 mm3, mice were divided into groups (n = 3 mice / group) and administered IT with PBS or 2 × 108 TCID50 of MVA, MVA-OVA, or MVA-OVA-4-1BBL. Six hours after IT injection, tumors were excised and tumor lysates were processed. Concentrations (pg / ml) of the indicated cytokines / chemokines in tumor lysates are shown. Data are presented as mean ± SEM. One-way ANOVA was performed. *, p<0.05; **, p<0.01; ****, p<0.0001. [Figure 4]Intratumoral MVA-immunotherapy shows that it induces rejection of untreated lesions. (A-D) Bilateral tumor model. (A) Experimental layout. Balb / c mice were injected subcutaneously with 5x105 and 1x105 CT26.WT tumor cells in the right and left flank, respectively. Five days later, right flank tumors were immunized IT with either PBS or the indicated MVA constructs. IT immunizations were repeated on days 5 and 8 after the first immunization (arrow). (B) Tumor size follow-up of treated and untreated tumors after IT injection of PBS (n=10 mice / group). (C) Tumor size follow-up of treated and untreated tumors after IT injection of 5x107 TCID50 of MVA-gp70 (n=10 mice / group). (D) Tumor size follow-up of treated and untreated tumors following IT injection of 5x107 TCID50 of MVA-Gp70-4-1BBL (n=10 mice / group). [Figure 5A] Intratumoral MVA-TAA-4-1BBL-treated mice are shown to be resistant to subsequent local and systemic tumor rechallenge. Experimental layout. Naïve C57BL / 6 mice or long-term survivors (12-36 weeks after tumor disappearance) mice in Figure 1A and B were rechallenged subcutaneously in the tumor-naïve flank of cured mice with 5x105 B16.OVA cells. Peripheral blood was analyzed by flow cytometry before (day -6) and after (day 7) rechallenge. Mononuclear cells in blood, spleen, non-dLN and TdLN were analyzed 41 days after tumor cell inoculation. n=5-11 mice / group. [Figure 5B] Figure 1 shows that intratumoral MVA-TAA-4-1BBL treated mice are resistant to subsequent local and systemic tumor rechallenge. The percentage of tumor-free mice over time is shown (n=5-11 mice / group). In brackets, the number of tumor-free mice per group is shown. n=5-11 mice / group. [Figure 5C]Figure 1: Intratumoral MVA-TAA-4-1BBL treated mice are resistant to subsequent local and systemic tumor rechallenge. Frequency of peripheral blood CD44+ OVA257-264 Dex+ CD8+ in naive and long-term survivor mice after treatment with IT MVA-OVA or MVA-OVA-4-1BBL, before and after B16.OVA rechallenge. n=5-11 mice / group. Data are presented as mean ± SEM. Two-way ANOVA was performed: *, p<0.05; **, p<0.01; ***, p<0.005. [Figure 5D] Figure 1 shows that intratumoral MVA-TAA-4-1BBL treated mice are resistant to subsequent local and systemic tumor rechallenge. Frequency of CD44+ OVA257-264 Dex+ CD8+ T in blood, spleen, NdLN and TdLN. n=5-11 mice / group. Data are presented as mean ± SEM. Two-way ANOVA was performed: *, p<0.05; **, p<0.01; ***, p<0.005. [Figure 5E] Figure 1 shows that intratumoral MVA-TAA-4-1BBL-treated mice are resistant to subsequent local and systemic tumor rechallenge. Frequency of splenic CD44+ IFNγ+ TNFα+ IL2+ CD8+ T cells after restimulation with OVA257-264 peptide. n=5-11 mice / group. Data are presented as mean ± SEM. Two-way ANOVA was performed: *, p<0.05; **, p<0.01; ***, p<0.005. [Figure 5F]Intratumoral MVA-TAA-4-1BBL treated mice are resistant to subsequent local and systemic tumor rechallenge. Frequency of CD62L- CD127+ CD69- OVA257-264 Dex+ cells (TEM) in blood, spleen, NdLN and TdLN (left). Frequency of CD62L+ CD127+ OVA257-264 Dex+ cells (TCM) in blood, spleen, NdLN and TdLN (center). Frequency of CD62L- CD127+ CD69+ OVA257-264 Dex+ (TRM) in blood, spleen, NdLN and TdLN (right) 41 days after B16.OVA cell administration. n=5-11 mice / group. Data are presented as mean ± SEM. Two-way ANOVA was performed: *, p<0.05; **, p<0.01; ***, p<0.005. [Figure 5G] Figure 1 shows that intratumoral MVA-TAA-4-1BBL-treated mice are resistant to subsequent local and systemic tumor readmission. Systemic tumor readmission. Experimental layout. Naïve Balb / c mice or long-term survivors in Figure 1C were readmitted IV with 2x105 CT26.WT cells. Spleens and lungs were analyzed 19 days after tumor cell injection. n=5-11 mice / group. Data are presented as mean ± SEM. Two-way ANOVA was performed: *, p<0.05; **, p<0.01; ***, p<0.005. [Figure 5H] Figure 1 shows that intratumoral MVA-TAA-4-1BBL treated mice are resistant to subsequent local and systemic tumor readmission. Systemic tumor readmission. Representative pictures of lungs after fixation in Bouin's fluid 19 days after tumor cell introduction in naive or cured mice. The total number of macroscopic lung metastases was assessed (n = 2-9 mice / group). n = 5-11 mice / group. Data are presented as mean ± SEM. Two-way ANOVA was performed: *, p<0.05; **, p<0.01; ***, p<0.005. [Figure 5I]Figure 1 shows that intratumoral MVA-TAA-4-1BBL treated mice are resistant to subsequent local and systemic tumor rechallenge. Systemic tumor rechallenge. Frequency of splenic CD44+ IFNγ+ TNFα+ IL2+ CD8+ T cells after restimulation with AH16-14 peptide 19 days after rechallenge. Data are presented as mean ± SEM. One-way ANOVA was performed: *, p<0.05; **, p<0.005; ***, p<0.0005. [Figure 6] Figure 1C and D: Intratumoral MVA-Gp70-4-1BBL immunotherapy confers protection from local tumor rechallenge. Naive C57BL / 6 mice or long-term survivors in Figure 1C and D were rechallenged with 5x105 B16.F10 cells subcutaneously in the tumor-naive flank of cured mice. Peripheral blood was analyzed by flow cytometry before (day -6) and after (day 7) rechallenge. Blood, spleen, NdLN and TdLN were analyzed 42 days after tumor cell inoculation. (A) Percentage of tumor-free mice over time is shown (n=5-11 mice / group). In brackets, number of tumor-free mice per group is shown. (B) Frequency of CD44+ p15E604-611 Pent+ CD8+ T cells in peripheral blood before and after B16.F10 cell rechallenge. (C) Frequency of CD62L+ CD127+ p15E604-611 Pent+ CD8+ T cells (TCM) in blood, spleen, NdLN, and TdLN. (D) Frequency of CD62L- CD127+ p15E604-611 Pent+ CD8+ T cells (TEM) in blood, spleen, NdLN, and TdLN. (E) Frequency of CD62L- CD127+ CD69+ p15E604-611 Pent+ CD8+ T cells (TEM) in blood, spleen, NdLN, and TdLN. (A-E) n = 5-11 mice / group. (B-E) Data are presented as mean ± SEM. (C-E) One-way ANOVA was performed: **, p<0.005; ***, p<0.0005. [Figure 7]Replicative viruses have been shown to induce the death of infected tumor cells and immune cells [33-35]. Infection with MVA, MVA-TAA or MVA-TAA-4-1BBL enhanced the death of B16.OVA and CT26.WT tumor cells in vitro (A). Macrophages, which have been shown to be selectively infected with MVA

[36] , were effectively killed, whereby this effect was significantly augmented by the 4-1BBL adjuvant (A). Cell death leads to the release of intracellular proteins such as high mobility group box 1 (HMGB1), which are sensed by innate immune cells and contribute to the initiation of immune responses [37, 38]. A significant increase in HMGB1 was detected after MVA infection of tumor cells or macrophages, independent of 4-1BBL (B). [Figure 8A] The MVA-based vector MVA-HERV-FOLR1-PRAME-h4-1-BBL ("MVA-mBN494" or "MVA-BN-4IT") is shown. For details, see Example 7. [Figure 8B] The vector demonstrates the ability to incorporate TAAs into the HLA of infected cells. For details, see Example 7. [Figure 8C] The ability to express h4-1-BBL in a functional form, i.e., a form that binds to the h4-1-BB receptor, is demonstrated. For details, see Example 7. [Figure 9] The MVA-based vector "MVA-mBN502" (C) is shown, along with schematic maps of ERVK-env / MEL (A; for use in MVA-mBN494) and ERVK-env / MEL_03 (B; for use in MVA-mBN502). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] In this study, we combined the immune stimulatory properties of TAA-encoding MVA with the superior T cell-enhancing potential of 4-1BBL to evaluate its therapeutic efficacy against solid tumors. We found that IT injection of MVA-TAA-4-1BBL demonstrated robust objective therapeutic responses in a variety of unrelated tumor models. Treatment was mediated by robustly reactivated tumor-specific CD8+ This was due to the successful induction of multiple proinflammatory chemokines and cytokines in T cells and the TME. Furthermore, IT MVA-TAA-4-1BBL injection induced a systemic antitumor immune response and inhibited the growth of tumor deposits at distant sites. Importantly, IT MVA-TAA-4-1BBL induced diversified tumor-specific memory responses, which protected against local and metastatic recurrence.

[0026] Tumor-associated antigens (TAA) and the immune-stimulating adjuvant 4-1BBL were cloned into the genome of modified vaccinia Ankara (MVA) for intratumoral virotherapy. Local treatment with MVA-TAA-4-1BBL resulted in control of established tumors. Upon intratumoral injection, MVA was primarily localized to the tumor with minimal leakage into tumor-draining lymph nodes. In situ infection with MVA-TAA-4-1BBL induced profound changes in the tumor microenvironment, including induction of multiple proinflammatory molecules and immunogenic cell death. This led to the reactivation and proliferation of antigen-experienced, cytokine-producing tumor-specific cytotoxic T cells. Remarkably, we report here that induction of a systemic antitumor immune response by local MVA-TAA-4-1BBL treatment controlled tumor growth in distant untreated lesions and conferred protection against local and systemic tumor rechallenge. In both cases, 4-1BBL-adjuvanted MVA was superior to MVA. Thus, intratumoral 4-1BBL-adjuvanted MVA immunotherapy induced potent tumor-specific CD8 + It induced T cell proliferation and favorable pro-inflammatory changes in the tumor microenvironment, resulting in tumor elimination and protective immune memory.

[0027] definition It should be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, "a nucleic acid" includes one or more nucleic acid sequences.

[0028] As used herein, the conjunction "and / or" between multiple described elements is understood to encompass both individual and combined options. For example, when two elements are linked by "and / or", the first option refers to the first element being applicable without the second element. The second option refers to the second element being applicable without the first element. The third option refers to the first and second elements being applicable together. Any one of these options is understood to fall within the meaning and thus meet the requirements of the term "and / or" as used herein. Two or more of the options being applicable simultaneously is also understood to fall within the meaning and thus meet the requirements of the term "and / or".

[0029] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising" are understood to imply the inclusion of a stated integer or step or group of integers or steps, and not the exclusion of any other integer or step or group of integers or steps. When used in the context of a described aspect or embodiment of the invention, the term "comprising" is modifiable and thus may be replaced with the term "containing" or "including" or, as used herein, with the term "having". Similarly, any of the foregoing terms (comprising, containing, including, having), whenever used in the context of a described aspect or embodiment of the invention, include the terms "consisting of" or "consisting essentially of", each of which carries a specific legal meaning that varies from jurisdiction to jurisdiction.

[0030] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0031] The term "recombinant MVA" refers to an MVA in which an exogenous nucleic acid sequence that does not naturally occur in the parent virus has been inserted into its genome. Thus, a recombinant MVA refers to an MVA that has been created by artificial combination of an MVA nucleic acid sequence with a segment of a nucleic acid sequence of synthetic or semi-synthetic origin, which combination does not occur in nature or is arranged differently. Artificial combinations are often achieved by artificial manipulation of isolated segments of nucleic acid using well-established genetic engineering techniques. In general, "recombinant MVA" as described herein refers to an MVA that has been produced by standard genetic engineering methods, and thus, for example, a recombinant MVA is an MVA that has been genetically engineered or modified. Thus, the term "recombinant MVA" includes an MVA (e.g., MVA-BN) in which at least one recombinant nucleic acid has been integrated into its genome, preferably in the form of a transcription unit. The transcription unit may include a promoter, an enhancer, a terminator and / or a silencer. The recombinant MVA of the present invention is designed to express a heterologous antigenic determinant, polypeptide or protein (antigen) upon induction of a regulatory element, such as a promoter.

[0032] As used herein, the term "subject" refers to a recipient of recombinant MVA, typically a mammal, e.g., a non-primate or primate (e.g., monkey or human), preferably a human. The term includes human or animal "patients" and experimental animals. The terms "subject" and "patient" are used interchangeably.

[0033] The term "solid tumor" refers to identifiable or established neoplastic tissue, as opposed to, for example, leukemia or blood cancer.

[0034] The term "primary tumor" refers to the initial or first tumor from which tumor cells may spread to other parts of the body and may form new ("secondary") tumors.

[0035] As used herein, the term "secondary tumor" refers to a tumor formed by tumor cells that have spread from a "primary tumor" or from another "secondary tumor." In particular, the term includes "metastasis."

[0036] The term "metastasis" refers to a tumor lesion originating from a cancerous primary or secondary tumor.

[0037] As used herein, the term "related" means that two tumors are believed to be responsive to the same recombinant MVA or TAA, e.g., they are of the same tumor type.

[0038] As used herein, the term "spatially distant" relates to a tumor that is clearly identifiable as an individual tumor, for example by optical means, and spaced apart from another tumor.

[0039] The term "local" administration includes, for example, intratumoral or topical administration to solid tumors. Local, therefore, means on the site. Administration can be achieved, for example, by local or topical application of recombinant MVA, or by intratumoral injection. In some embodiments, for example, in tumors close to the skin or tumors of skin cells such as melanoma, local administration can be achieved by subcutaneous injection in close proximity to the tumor. In contrast, terms such as "distal" or "systemic" mean not on the site.

[0040] As used herein, the term "systemic" means the opposite of "local." Thus, systemic can mean that the effect is not localized, i.e., rather, it pertains to the entire body.

[0041] As used herein, the term "oncotropic virotherapy" refers to localized virotherapy as opposed to systemic oncolytic virotherapy.

[0042] As used herein, the term "systemic antitumor therapy" means that recombinant MVA is administered locally to a solid tumor, but the antitumor activity extends to other tumors that are not treated locally.

[0043] As used herein, the term "systemic anti-tumor immune response" refers to an anti-tumor immune response that can occur anywhere in the body.

[0044] As used herein, the term "recurrence" refers to the relapse or return of a tumor.

[0045] As used herein, the term "prevention of recurrence" means that the relapse of a tumor in remission, i.e., a tumor in or after remission, e.g., an eradicated tumor, is prevented or inhibited. At the very least, complete recurrence of the tumor is inhibited, e.g., inhibited by at least 50%.

[0046] In contrast, the term "prevention" means that the initial appearance of a tumor is prevented.

[0047] As used herein, the term "remission" refers to, for example, a tumor that is reduced or eradicated. More generally, "remission" is the reduction or disappearance of signs and symptoms of a disease, such as a tumor. Remission can be considered partial remission or complete or full remission. For example, partial remission of a tumor can be defined as a 50% or greater decrease in a measurable parameter for tumor growth, which can be found in a physical examination, a radiological examination, or a biomarker level from a blood or urine test. On the other hand, complete remission is the complete disappearance of the tumor, regardless of the possibility of recurrence.

[0048] The term "neoadjuvant therapy" refers to treatment prior to a primary treatment, where neoadjuvant therapy using recombinant MVA is intended to reduce the size or extent of a tumor, for example, prior to surgical resection. Selected abbreviation: IT intratumoral (it) MVA Modified Vaccinia Virus Ankara (MVA) Modified Vaccinia Virus Ankara (MVA) OV oncolytic virus TAA tumor associated antigen TME tumor microenvironment

[0049] Embodiment In one embodiment, the recombinant MVA is a non-replicating or replication-deficient MVA. Preferably, the recombinant MVA is incapable of reproductive replication in human cell lines.

[0050] In one embodiment, the recombinant MVA is derived from MVA-BN deposited at the European Collection of Cell Cultures (ECACC) under number V00083008, or is derived from an MVA-BN derivative.

[0051] In one embodiment, the recombinant MVA is (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL), and (c) at least one additional nucleic acid encoding a TAA.

[0052] In one embodiment, the first nucleic acid encoding a TAA and the at least one further nucleic acid encoding a TAA are the same or different.

[0053] In one embodiment, the recombinant MVA comprises two, three, four, five, six or more nucleic acids, each encoding a different TAA.

[0054] In one embodiment, the TAA is a neoantigen or an endogenous autoantigen.

[0055] In one embodiment, the TAA is a transposable element (TE).

[0056] In one embodiment, the TAA is an endogenous retroviral protein (HERV).

[0057] In one embodiment, the TAA is a long interspersed nucleotide sequence (LINE).

[0058] In one embodiment, the TAA is a short interspersed nucleotide sequence (SINE).

[0059] In one embodiment, 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, folate receptor alpha (FOLR1), melanoma preferentially expressed antigen (PRAME), and endogenous retroviral peptide MEL, and combinations thereof. Any TAA is suitable for use in the compositions and / or methods of the invention, so long as it is an antigen associated with a known tumor or the tumor of the patient being treated, and capable of generating or stimulating an immune response when expressed.

[0060] In one embodiment, the ERV protein is from the human endogenous retrovirus K (HERV-K) family, and is preferably selected from HERV-K envelope (HERV-K-env) protein and HERV-K gag protein.

[0061] In one embodiment, 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).

[0062] In one embodiment, the recombinant MVA is (i) a nucleic acid encoding HERV-K-env / MEL; (ii) a nucleic acid encoding HERV-K gag; (iii) a nucleic acid encoding FOLR1 and PRAME, preferably expressed as a fusion protein; and (iv) a nucleic acid encoding 4-1BBL.

[0063] In one embodiment, the nucleic acid of (i) encodes HERV-K-env / MEL comprising a HERV-K env surface (SU) unit and a HERV-K transmembrane (TM) unit, wherein the HERV-K TM unit is mutated, preferably the HERV-K TM unit is mutated such that the immunosuppressive domain is inactivated. Preferably, HERV-K-MEL is inserted within the mutated HERV-K TM unit. More preferably, HERV-K-MEL replaces a portion of the immunosuppressive domain of HERV-K TM.

[0064] In one embodiment, the nucleic acid sequence of (i) encodes an amino acid sequence comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:7.

[0065] In one embodiment, the nucleic acid sequence of (i) comprises or consists of the nucleic acid sequence as set forth in SEQ ID NO:8.

[0066] In one embodiment, the nucleic acid of (i) encodes a HERV-K-env / MEL comprising a HERV-K env surface (SU) unit and a HERV-K transmembrane (TM) unit, wherein the HERV-K TM unit is truncated 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.

[0067] In one embodiment, the nucleic acid of (i) encodes HERVK-env / MEL comprising a HERV-K-env surface (SU) unit, wherein the RSKR furin cleavage site of the HERV-K-env SU unit is deleted. Preferably, the HERV-K-MEL is linked to the C-terminus of the HERV-K-env SU unit.

[0068] In one embodiment, the nucleic acid of (i) encodes a heterologous membrane anchor, preferably HERVK-env / MEL, including one derived from the human PDGF (platelet derived growth factor) receptor.

[0069] In one embodiment, the nucleic acid sequence of (i) encodes an amino acid sequence comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:11.

[0070] In one embodiment, the nucleic acid sequence of (i) comprises or consists of the nucleic acid sequence as set forth in SEQ ID NO:12.

[0071] In one embodiment, the solid tumor, primary solid tumor and / or secondary tumor is a cancerous or malignant tumor.

[0072] In one embodiment, the solid tumor, primary solid tumor and / or secondary tumor is a melanoma or a malignant breast, colon or ovarian tumor.

[0073] In one embodiment, the secondary tumor is a metastasis.

[0074] In one embodiment, the secondary tumor is a solid tumor. Alternatively, the secondary tumor is a collection of tumor cells suspended in a body fluid, such as the blood or lymph, or is present in a body cavity, such as the peritoneal cavity. However, the primary tumor or the secondary tumor to which the recombinant MVA is administered locally or intratumorally is always a solid tumor.

[0075] In one embodiment, tumors in which recombinant MVA is not administered locally or intratumorally cannot be dissected or are difficult to access by surgery.

[0076] In one embodiment, the primary tumor and the secondary tumor are spatially distal to each other.

[0077] In one embodiment, the secondary tumor and the separate secondary tumor are spatially distal to each other.

[0078] In one embodiment, the primary tumor and the secondary tumor are located within the same tissue or organ of the subject's body.

[0079] In one embodiment, the secondary tumor and the separate secondary tumor are located within the same tissue or organ of the subject.

[0080] In one embodiment, the primary tumor and the secondary tumor are located in different tissues or organs of the subject's body.

[0081] In one embodiment, the secondary tumor and the further secondary tumor are located in different tissues or organs of the subject's body. In one embodiment, the secondary tumor is naive, i.e., has not previously been treated locally, preferably intratumorally, with recombinant MVA.

[0082] In one embodiment, the tumor is in at least partial remission, eg, at least greater than 50% remission, preferably in complete remission.

[0083] In one embodiment, the recombinant MVA is not administered other than locally, preferably intratumorally, and thus is not administered to a subject, for example, systemically, intravenously, intraperitoneally, parenterally, subcutaneously, or intranasally.

[0084] In one embodiment, the anti-tumor immune memory is long-term, i.e., lasts for days, weeks, months, years, or decades.

[0085] Treatment method The present invention provides a method of treatment for a subject having two or more tumors, where less than the total number of tumors in the subject are treated by local administration of recombinant MVA. That is, the present invention provides a method of treatment for a subject having multiple tumors, comprising administering recombinant MVA locally to at least one, but not all, of the multiple tumors. In some embodiments, the step of locally administering recombinant MVA comprises intratumoral injection. In this manner, the method of treatment can be said to produce a "treated tumor" or "injected tumor" to which recombinant MVA has been administered or injected locally or intratumorally, and an "untreated tumor" or "uninjected tumor" to which recombinant MVA has not been administered locally or injected. The method of treatment can also be said to provide a method of treating an uninjected tumor in a subject by administering recombinant MVA locally or intratumorally to at least one other tumor in the subject. In some embodiments of the method of the present invention, the subject is a human patient.

[0086] In some embodiments, the method of treatment includes treating metastasis or recurrence of a first or primary tumor by local administration (in some embodiments, by intratumoral injection) of recombinant MVA to said first or primary tumor. In some embodiments, it is not possible to determine which of multiple tumors in a subject is the one that first arose (i.e., the "primary tumor"), and the method includes treating at least one of the multiple tumors by local administration or intratumoral injection of MVA. Thus, in some embodiments, by "metastasis" or "secondary tumor" it is intended that the tumor is in a different location or has different borders than the tumor designated as the "first tumor" or "primary tumor."

[0087] The methods of the invention include treating at least one of a plurality of tumors in a subject by local administration or intratumoral injection, or treating at least two, at least three, at least four, or more of the tumors, or treating all but one of the plurality of tumors in the subject. In some embodiments, the methods of the invention provide that any number of tumors in a subject may be so injected or treated by local administration or intratumoral injection of recombinant MVA, so long as fewer than the total number of tumors in the subject are so injected or treated.

[0088] The present invention is not limited to any particular mechanism or mode of action, but any method is suitable for practicing the present invention, so long as it stimulates an immune response against at least one TAA or tumor or cells thereof in said subject, or produces a decrease in the volume or size of at least one secondary tumor or uninjected tumor. By "stimulating an immune response" it is intended that indicia of a new or enhanced immune response can be identified in the subject, such as, for example, an increase in the CD8+ T cell population and / or an increase in the amount of IFN-gamma, TNF-alpha and / or IL-2 produced by the subject's CD8+ T cells, as exemplified in the Examples herein. Thus, the present invention also provides a method of stimulating an immune response against a TAA, comprising administering to a subject a recombinant MVA of the present invention intratumorally.

[0089] In some embodiments, the recombinant MVA comprises (a) a first nucleic acid encoding a tumor-associated antigen (TAA), and (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL). The recombinant MVA may further comprise an additional nucleic acid encoding an additional TAA. The methods of the invention are suitable for use with recombinant MVA encoding both at least one TAA and 4-1-BBL. In some embodiments, the recombinant MVA expresses at least one TAA and 4-1-BBL, which can be demonstrated using techniques known in the art.

[0090] Various TAAs and 4-1-BBL sequences useful in the methods and compositions of the invention are known in the art and are described, for example, in PCT / EP2019 / 081942 (published as WO2020 / 104531) and European Patent Application No. 19210369.5, both of which are incorporated herein by reference in their entirety. Exemplary 4-1-BBL sequences are described in NCBI Reference Sequence NP_003802.1 (and the amino acid sequence set forth in SEQ ID NO:3, and / or the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO:4). Recombinant MVA useful in the uses and methods of the invention are also described, for example, in European Patent Application No. 19210369.5, which are incorporated herein by reference.

[0091] In certain embodiments, the nucleic acid sequence encoding 4-1-BBL encodes 4-1-BBL that has at least 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 3. In certain embodiments, the nucleic acid sequence encodes 4-1-BBL that is the sequence set forth in SEQ ID NO: 4 or has the amino acid sequence set forth in SEQ ID NO: 3.

[0092] In certain embodiments, the nucleic acid sequence encoding a TAA encodes a TAA that has at least 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a previously known TAA, such as, for example, a sequence set forth in the sequence listing provided herein. In certain embodiments, the nucleic acid sequence encodes a TAA having an amino acid sequence set forth in the sequence listing or known in the art.

[0093] In some embodiments, at least one individual tumor is measured before and after local administration (e.g., intratumoral injection) of the recombinant MVA to determine whether the tumor continues to grow or increases or decreases in size after administration. In some methods, additional measurements may be taken at intervals after administration to track the response of at least one individual tumor. Such measurements may be by any suitable technique, including visual assessment or external measurement, as well as X-ray, ultrasound imaging, biomicroscopy, or any other suitable method known in the art. Tumor measurements used to assess tumor response to treatment and / or efficacy of treatment may include, for example, an estimation of at least one diameter of the tumor and / or tumor volume.

[0094] In some embodiments, a method of treating a subject having one or more tumors comprises the steps of: (a) obtaining a first measurement of a tumor in the subject; (b) administering a recombinant MVA expressing at least one, but not all, TAAs to the tumor in the subject, resulting in a treated or injected tumor and an untreated or uninjected tumor; (c) obtaining a second measurement of at least one of the untreated or uninjected tumors; and (d) comparing the second measurement to the first measurement of the untreated or uninjected tumor, whereby a decrease in the measurement or a lack of increase in the measurement indicates that the tumor has regressed or decreased in volume or that tumor growth has been slowed. In some embodiments, the recombinant MVA used in these methods expresses at least one TAA and 4-1-BBL, as further discussed elsewhere herein.

[0095] In some embodiments, the method of treating a subject with a primary tumor and one or more metastases, or potential for metastasis (i.e., malignant tumors) is intended to prevent new or additional metastases, and includes the steps of: (a) identifying one or more tumors in the subject that are at risk of metastasis; (b) administering locally (in some embodiments, by intratumoral injection) a recombinant MVA expressing a TAA to at least one, but not all, of the subject's tumors; and (c) performing tumor detection in the subject to ensure that no new tumors or metastases are detectable in the subject. In some embodiments, the recombinant MVA used in these methods expresses a TAA and 4-1-BBL, as further discussed elsewhere herein. In some embodiments, the recombinant MVA used in these methods includes a heterologous nucleic acid encoding a TAA and a heterologous nucleic acid encoding 4-1-BBL, but does not include any additional genes encoded by the heterologous nucleic acid that may or are expected to affect the subject's immune response when used as a component of the MVA or separately to treat the subject.

[0096] In some embodiments, the invention provides a method of treating an inaccessible tumor in a subject with multiple tumors, comprising locally administering a recombinant MVA to at least one accessible tumor of the multiple tumors, the recombinant MVA comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding 4-1-BB ligand (4-1-BBL). In some embodiments, the recombinant MVA comprises at least one additional nucleic acid encoding at least one additional TAA, or at least two, three, or four or more additional nucleic acids encoding at least two, three, or four or more additional TAAs. In some embodiments, locally administering the recombinant MVA comprises intratumoral injection.

[0097] By "inaccessible tumor" is intended a tumor that is difficult to treat directly by local administration of recombinant MVA, such as by intratumoral injection and / or using other techniques. An "inaccessible tumor" or unresectable tumor may be located in a sensitive area of ​​the body, such as near or around important nerves or blood vessels, or in the brain, or elsewhere where surgery and / or local administration of recombinant MVA may pose a risk of injury to the subject and / or may be difficult to administer. The present invention provides a method of treating an inaccessible tumor or an unresectable tumor in a subject having multiple tumors, comprising a step of local administration to a different tumor that is more accessible and / or where local administration is less likely to cause damage to the subject. In some embodiments, the present invention provides a method of treating an inaccessible tumor in a subject, comprising (a) identifying at least one inaccessible tumor and at least one accessible tumor in the subject, and (b) locally administering a recombinant MVA expressing a TAA to at least one accessible tumor in the subject. Optionally, the method further includes monitoring the inaccessible tumor to confirm that tumor growth has slowed or stopped and / or to determine whether the subject has an increased or new immune response following administration of the recombinant MVA.

[0098] In some embodiments, the present invention provides a method of treating a subject with multiple malignant tumors, comprising locally administering a recombinant MVA expressing a TAA to at least one of the tumors to produce a treated tumor, wherein the treatment results in a reduction in tumor volume of the treated tumor and at least one other tumor that has not been directly treated or injected with the recombinant MVA (also referred to as an "untreated tumor" or "uninjected tumor").

[0099] In some embodiments, "intratumoral injection" refers to administration within a tumor or within the borders of a tumor. In some embodiments, "local administration" refers to administration in close proximity to a tumor. Those skilled in the art will recognize that such injections may also be characterized as other types of administration, such as parenteral or subcutaneous, depending on the location of the tumor.

[0100] Detailed Description Modified vaccinia virus Ankara (MVA) In the past, MVA was generated by serial passage of the Ankara strain of vaccinia virus (CVA) in chicken embryo fibroblast cells for 516 generations (for review, see Mayr et al. 1975). The virus was renamed from CVA to MVA at passage 570, in view of its substantially altered properties. MVA underwent further passages up to passage 570. As a result of these long passages, the genome of the resulting MVA virus was deleted of approximately 31 kilobases of genomic sequence and was therefore described as being highly host cell restricted with respect to replication in avian cells (Meyer et al. 1991). The resulting MVA was shown in various animal models to be largely non-pathogenic compared to the fully replication-competent starting material (Mayr and Danner 1978).

[0101] MVAs useful in the practice of the invention include MVA-572 (deposited January 27, 1994 under ECACC V94012707), MVA-575 (deposited December 7, 2000 under ECACC V00120707), MVA-I721 (reviewed in Suter et al. 2009), NIH Clone 1 (deposited March 27, 2003 under ATCC® PTA-5095), and MVA-BN (deposited August 30, 2000 with the European Collection of Cell Cultures (ECACC) under number V00083008).

[0102] More preferably, the MVA used according to the present invention includes MVA-BN and MVA-BN derivatives. MVA-BN is described in WO02 / 042480. "MVA-BN derivatives" refers to any virus that exhibits essentially the same replication characteristics as MVA-BN as described herein, but exhibits differences in one or more parts of their genome.

[0103] MVA-BN and MVA-BN derivatives are replication-incompetent, meaning that they are incapable of reproductive replication in vivo and in vitro. More specifically, in vitro MVA-BN or MVA-BN derivatives are described as capable of reproductive replication in chicken embryo fibroblasts (CEF) but incapable of reproductive replication in the human keratinocyte cell line HaCat (Boukamp et al 1988), the human 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, MVA-BN or MVA-BN derivatives have at least two-fold, more preferably three-fold, lower viral amplification rates than MVA-575 in HeLa and HaCaT cell lines. Tests and assays for these properties of MVA-BN and MVA-BN derivatives are described in WO02 / 42480 and WO03 / 048184.

[0104] As explained above, the term "reproductively incompetent" in human cell lines in vitro is described, for example, in WO02 / 42480, which also teaches how to obtain MVA with the desired properties as described above. This term applies to viruses that have an in vitro viral amplification rate of less than 1 at 4 days after infection using the assay described in WO02 / 42480 or US6,761,893.

[0105] Exemplary Generation of Recombinant MVA Viruses Different methods may be applied to the production of the recombinant MVA disclosed herein. The DNA sequence to be inserted into the virus can be placed in an E. coli plasmid construct in which DNA homologous to a section of poxvirus DNA is inserted. Alternatively, the DNA sequence to be inserted can be linked to a promoter. This promoter-gene linkage can be placed in a plasmid construct such that it is flanked at both ends by DNA homologous to DNA sequences flanking a region of poxvirus DNA containing a non-essential locus. The resulting plasmid construct can be amplified by growth in E. coli and isolated. The isolated plasmid containing the DNA gene sequence to be inserted can be transfected into a cell culture, e.g. chicken embryo fibroblasts (CEF), at the same time that the culture is infected with MVA. Recombination between the homologous MVA viral DNA in the plasmid and the viral genome, respectively, can generate an MVA that is modified by the presence of a foreign DNA sequence, i.e. a nucleotide sequence encoding a SARS-CoV-2 antigen.

[0106] According to a preferred embodiment, cells suitable for 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, preferably under the transcriptional control of a poxvirus expression control element, that contains a foreign or heterologous gene(s), such as one or more of the nucleic acids provided herein. As explained above, the plasmid vector also contains a sequence capable of directing the insertion of the foreign sequence into a selected part of the MVA virus genome. Optionally, the plasmid vector also contains a cassette that contains a marker gene and / or a selection gene operably linked to a poxvirus promoter. The use of a selection or marker cassette simplifies the identification and isolation of the recombinant MVA produced. However, recombinant poxviruses can also be identified by PCR techniques. Further cells can then be infected with the recombinant MVA obtained as described above and transfected with a second vector that contains a second foreign or heterologous gene(s). To be sure, this gene should be introduced into a different insertion site of the poxvirus genome, and the second vector also has a different sequence homologous to the poxvirus, which induces the integration of the second foreign gene(s) into the genome of the poxvirus. After homologous recombination has taken place, a recombinant virus containing two or more foreign or heterologous genes can be isolated. If additional foreign genes are to be introduced into the recombinant virus, the infection and transfection steps can be repeated by using the recombinant virus isolated in the previous infection step and by using an additional vector containing the additional foreign gene(s) for transfection. There are many other techniques known for producing recombinant MVA.

[0107] The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, molecular biology, microbiology, cell biology, and recombinant technology, all of which are within the skill of one of ordinary skill in the art (see, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition, 1989; Current Protocols in Molecular Biology, Ausubel FM, et al., eds, 1987; Methods in Enzymology (Academic Press, Inc.) series; PCR2: A Practical Approach, MacPherson MJ, Hams BD, Taylor GR, eds, 1995; Antibodies: A Laboratory Manual, Harlow and Lane, eds, 1988). In addition, the present application relates to the invention described in the claims, but may also include the following as other aspects. 1. (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and A recombinant modified vaccinia virus Ankara (MVA) comprising: The recombinant modified vaccinia virus Ankara for use in preventing recurrence of a solid tumor in a subject during or after remission, wherein the remission is induced by local, preferably intratumoral, administration of the recombinant MVA to the solid tumor. 2. (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and A recombinant modified vaccinia virus Ankara (MVA) comprising: The recombinant modified vaccinia virus Ankara for use in inducing a systemic anti-tumor immune response in a subject, wherein the recombinant MVA is administered locally, preferably intratumorally, to a solid tumor. 3. (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and A recombinant modified vaccinia virus Ankara (MVA) comprising: For use in the treatment, prevention and / or prevention of recurrence of a secondary tumor in a subject, wherein said recombinant MVA is administered locally, preferably intratumorally, to an associated primary solid tumor but not to said secondary tumor; or For use in the treatment and / or prevention of recurrence of a primary solid tumor in a subject, wherein said recombinant MVA is administered locally, preferably intratumorally, to an associated secondary solid tumor but not to said primary solid tumor; or The recombinant modified vaccinia virus Ankara for use in the treatment, prevention and / or prevention of recurrence of a secondary tumor in a subject, wherein the recombinant MVA is administered locally, preferably intratumorally, to another related secondary solid tumor, but not to the first-mentioned secondary tumor. 4. The recombinant MVA is (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and (c) at least one further nucleic acid encoding a TAA, 5. The recombinant MVA for use according to any one of claims 1 to 4 above, wherein the TAA is a neoantigen or an endogenous autoantigen. 6. The recombinant MVA for use according to any one of claims 1 to 5 above, wherein 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, folate receptor alpha (FOLR1), melanoma preferentially expressed antigen (PRAME), and the endogenous retroviral peptide MEL, and combinations thereof. 7. The recombinant MVA for use according to claim 6, wherein said ERV protein is from the human endogenous retrovirus K (HERV-K) family, preferably selected from HERV-K envelope (HERV-K-env) protein and HERV-K gag protein. 8. The recombinant MVA for use according to claim 6, wherein said ERV peptide is from the human endogenous retrovirus K (HERV-K) family, preferably selected from the pseudogenes of the HERV-K envelope protein (HERV-K-env / MEL). 9. The recombinant MVA for use according to any one of claims 1 to 8 above, wherein said solid tumor is a malignant tumor, preferably a melanoma or a cancerous breast, colon or ovarian tumor. 10. The recombinant MVA for use according to any one of claims 3 to 9 above, wherein the secondary tumor is a metastasis. 11. A recombinant MVA for use according to any one of claims 3 to 10 above, wherein the tumor to which the recombinant MVA is not administered locally, preferably intratumorally, cannot be dissected or is difficult to access by surgery. 12. A recombinant MVA for use according to any one of claims 1 to 11 above, wherein the recombinant MVA is incompetent for reproductive replication in human cell lines. 13. A recombinant MVA for use as described in claim 12, wherein said recombinant MVA is derived from MVA-BN deposited at the European Collection of Cell Cultures (ECACC) under number V00083008. 14. A method for stimulating an immune response in a subject having multiple tumors, comprising the step of locally administering a recombinant MVA comprising at least one first nucleic acid encoding a TAA and a second nucleic acid encoding 4-1-BBL to fewer than the total number of tumors in the subject, wherein an immune response against the TAA is stimulated in the subject. 15. A method for treating a subject having at least one inaccessible tumor and at least one accessible tumor, comprising locally administering a recombinant MVA comprising at least one first nucleic acid encoding a TAA and a second nucleic acid encoding 4-1-BBL to at least one accessible tumor of the subject, thereby reducing or stopping the growth of the inaccessible tumor. EXAMPLES

[0108] The following examples serve to further illustrate the present disclosure and should not be understood as limiting the invention, the scope of which is determined by the appended claims.

[0109] Example 1: Materials and Methods 1.1 Construction of MVA recombinants The production of MVA recombinants was performed as previously reported [1]. MVA-BN® was produced by Bavarian Nordic and has been deposited at the European Collection of Cell Cultures (ECACC) (V00083008). The production of recombinant MVA expressing ovalbumin (MVA-OVA) was previously reported [1, 2]. The gene encoding 4-1BBL was synthesized (Geneart, Life Technologies) and cloned into the MVA-OVA genome to generate MVA-OVA-4-1BBL. The gene encoding the murine leukemia virus-derived envelope glycoprotein Gp70 was synthesized (Geneart, Life Technologies) and cloned into the genomes of MVA and MVA-4-1BBL, respectively, to generate MVA-Gp70 and MVA-Gp70-4-1BBL. All viruses used in animal experiments were purified twice through sucrose cushions.

[0110] 1.2 Ethics Statement Animal experiments were approved by the Animal Ethics Committee of the Upper Bavaria Administration (Regierung von Oberbayern, Sachgebiet 54, Tierschutz) and were performed at Bavarian Nordic GmbH in accordance with the approved guidelines for animal experiments. Bilateral CT26.WT tumor experiments were performed at CIMA, University of Navarra (Pamplona, ​​Spain) in compliance with the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

[0111] 1.3 Mice and tumor cell lines 6-8 week-old female C57BL / 6J(H-2 b ) mice and Balb / cJ (H-2 d ) Mice were purchased from Janvier Labs. All mice were handled, fed, housed and maintained in the animal facilities of Bavarian Nordic GmbH, University of Zurich or University of Navarra in accordance with institutional guidelines.

[0112] The B16.OVA melanoma cell line was a kind gift from Roman Sporri (University of Zurich). The B16.F10 (ATCC® CRL-6475™) and CT26.WT (ATCC® CRL-2638™) cell lines were purchased from the American Type Culture Collection (ATCC). Tumor cells were cultured in DMEM Glutamax medium supplemented with 10% FCS, 1% NEAA, 1% sodium pyruvate and 1% penicillin / streptomycin (all reagents from Gibco) at 37°C in a 5% CO2 incubator. All tumor cell lines used in the experiments performed at Bavarian Nordic were routinely tested by PCR for Mycoplasma negativity.

[0113] 1.4 Tumor cell injection Mouse, 5 x 10 5 Tumor cells were injected subcutaneously into the flank. For B16.OVA and B16.F10, cells were mixed with 7 mg / ml Matrigel (Trevigen) prior to injection. For subcutaneous bilateral tumor experiments, 5 × 10 5 and 1×10 5 CT26.WT tumor cells were injected into the right and left flanks, respectively. Tumor rechallenge experiments were performed at the time of tumor disappearance, between 3 and 6 months. Subcutaneous rechallenge was performed with 5 × 10 5 The tumor cells were administered intravenously to the opposite flank using 2 × 10 5 CT26.WT cells were injected into the mice. Tumor diameters were measured periodically twice weekly using calipers.

[0114] 1.5 Immunization Intratumoral injections were administered into the solid tumor mass using a total volume of 50 μl containing each MVA recombinant. Successive intratumoral injections were performed on days 0, 5, and 8 after tumor typing and are indicated on the graph by vertical dotted lines. When required, blood was collected 3 days after the final intratumoral immunization for phenotyping of peripheral blood immune cells.

[0115] 1.6 Cell isolation Spleens and lymph nodes were harvested from mice when required. Single cell suspensions of spleens and lymph nodes were prepared by mechanical disruption through a 40 μm cell strainer (Falcon). Spleen samples were subjected to red blood cell lysis (Sigma-Aldrich).

[0116] Blood was collected in PBS containing 2% FCS, 0.1% sodium azide and 2.5U / ml heparin. Peripheral blood mononuclear cells (PBMCs) were prepared by lysing red blood cells with red blood cell lysis buffer. Mononuclear cells from the above organs were washed, resuspended in RPMI+2% FCS, counted and kept on ice until further analysis. 100 μl of TrueCount counting beads (BD Biosciences) were added to the tumor cell suspension.

[0117] 1.7 Peptide restimulation If necessary, mononuclear cells were incubated with 2.5 μg / ml of MHC class I-restricted peptide [OVA 257-264 (SIINFEKL);p15E 604-611 (KSPWFTTL);AH1 6-14 (SPSYVYHQF)] in T cell medium and 10 μg / ml BFA for 5–6 h at 37°C and 5% CO Peptides were purchased from GenScript.

[0118] 1.8 Flow cytometry Mononuclear cell suspensions, BMDMs or tumor cells were stained for 30 min at 4°C in the dark using a fixable live / dead viability kit (Life Technologies) prior to staining. Antibodies from BD Biosciences, eBioscience or Biolegend were used to stain mononuclear cells. H-2k b OVA 257-264 -Dextramer (Immudex), H-2K b p15E 604-611 -Pentamer (ProImmune) or H-2L d AH1 6-14 Cell suspensions were stained using Pentamer (ProImmune). For staining of FoxP3 transcription factor and Ki67, cells were fixed using FoxP3 Staining Kit (eBioscience). For intracellular cytokine staining, cell suspensions were stained and fixed for intracellular cytokine detection using IC Fixation & Permeabilization Staining Kit (eBioscience). Total cells were acquired using a digital flow cytometer (LSR II, BD Biosciences) and data were analyzed with FlowJo software version 10.3 (Tree Star).

[0119] 1.9 Quantitative real-time PCR for quantification of MVA-specific DNA genomic copies Genomic DNA (gDNA) was isolated from tissues using the QIAamp DNA Mini Kit according to the manufacturer's instructions (Qiagen) and quantified with a NanoVue spectrophotometer (Biochrom). Briefly, a plasmid expressing the MVA open reading frame 082L, a target for detection of MVA backbone DNA, was used to isolate 5 × 10 7 A standard curve was generated starting at 1000 genome copies (gcs). Quantitative real-time PCR was then performed with TaqMan Gene Expression Master Mix (Thermo-Fisher) using specific primers MVA082L sense 5'-acgtttagccgcctttaatagag-3', MVA082L antisense 5'-tggtcagaactatcgtcgttgg-3', and fluorescein probe 6FAM-aatcccaccgcctttctggatctc-BBQ. Calculations were performed with the 7500 software of the Real-time PCR system (Applied Biosystems). The software calculated threshold cycles (C T ), which is inversely proportional to the logarithm of the gene copy amount of the specific DNA. Based on the standard curve, the software determines the C T The values ​​were used to determine the gene copy number of each of the target genes. The quantity (gcs) of a sample was calculated by the average quantity of its replicate determinations.

[0120] 1.10 Statistical analysis Statistical analysis was performed using GraphPad Prism version 7.02 for Windows (GraphPad Software, La Jolla, CA) as described in the figure legends. For immunological data, results are presented as "mean" and "standard error of the mean" (SEM). Analysis of variance (ANOVA) with either multiple comparison tests or one-sided unpaired Student's t-test was used to determine statistical significance between treatment groups. For survival of tumor-bearing mice after treatment, Log rank test was performed to determine statistical significance between treatment groups.

[0121] Example 2: 4-1BBL enhances intratumoral MVA immunotherapy IT application of poxviruses has been shown to effectively induce antitumor responses in various tumor models [24–28]. However, the majority of these studies have been performed with replicating viruses. Here, we investigated whether local treatment of established tumors with the nonreplicating poxvirus MVA, encoding tumor-associated antigens (TAAs as defined above) and the costimulatory molecule 4-1BBL, confers a potent antitumor effect.

[0122] IT injection of MVA encoding the TAA ovalbumin (herein referred to as MVA-OVA) controlled tumor growth and prolonged survival in mice bearing established B16.OVA melanoma (Figure 1A and B). Notably, IT administration of MVA-OVA-4-1BBL increased tumor rejection in B16.OVA tumor-bearing mice by 50% (Figure 1B). Analysis of peripheral blood lymphocytes (PBL) after the final IT injection demonstrated that TAA-specific CD8 + It was found that systemic proliferation of T cells was increased by IT administration of MVA-OVA-4-1BBL (Fig. 2A).

[0123] Next, other independent, established tumor models were evaluated. IT administration of MVA encoding the endogenous retroviral antigen Gp70 (herein referred to as MVA-Gp70) [29, 30] resulted in antitumor effects in B16.F10 melanoma (Figure 1C and D). Interestingly, IT MVA-Gp70-4-1BBL significantly prolonged tumor growth control and significantly improved mouse survival (Figure 1C and D). Similar results were observed in CT26.WT tumor-bearing mice after IT immunization with MVA-Gp70 or MVA-Gp70-4-1BBL (Figure 1E and F). Local administration of MVA-Gp70-4-1BBL resulted in more than 80% CT26.WT tumor rejection. Restimulation of PBL with Gp70-derived peptides enhanced p15E(H-2K) expression during MVA-Gp70-4-1BBL IT regimen in mice bearing B16.F10 and CT26.WT tumors, respectively. b Restrictive Gp70 peptide) specific and AH1 (H-2K d Restrictive Gp70 peptide)-specific CD8 + A strong induction of interferon gamma (IFN-γ) by T cells was evident (Figures 2B and 2C, respectively). Notably, more than 70% of cured C57BL / 6 mice developed vitiligo upon IT treatment with MVA-OVA-4-1BBL or MVA-Gp70-4-1BBL (Figures 2D and 2E). Taken together, IT administration of 4-1BBL-adjuvanted MVA enhances MVA-mediated antitumor immune responses, regardless of the type of tumor antigen or tumor model used.

[0124] Example 3: IT injected MVA localizes to tumors and induces changes in the tumor microenvironment (TME) Having established that T cells rapidly proliferate in TdLNs after IT MVA injection, the question arose as to whether replication-deficient MVA resides exclusively at the injection site or migrates to other organs. To address this, we determined the presence of MVA-derived genomic DNA (gDNA) in tumors, TdLNs, NdLNs, spleen, and blood 6 hours after IT injection. Importantly, MVA gDNA was abundantly detected in B16.F10 tumors and minimally in TdLNs (Figure 3A).

[0125] After IT application, MVA infection was mainly restricted to the tumor, thus indicating that the virus-induced antitumor immune response was likely of tumor origin. This led us to hypothesize that IT injection of MVA-TAA-4-1BBL could induce changes in the TME. IT injection of MVA or MVA-OVA into B16.OVA tumors resulted in upregulation of the proinflammatory molecules IFNγ and TNFα compared to PBS. This effect was significantly augmented by MVA-OVA-4-1BBL (Figure 3B). Interestingly, cytokines such as IFNγ and GM-CSF were almost exclusively induced by 4-1BBL-adjuvanted MVA (Figure 3B).

[0126] Replicative viruses induce the death of infected tumor cells and immune cells [33-35]. Infection with MVA, MVA-TAA or MVA-TAA-4-1BBL enhanced the death of B16.OVA and CT26.WT tumor cells in vitro (Figure 7A). Macrophages, which have been shown to be selectively infected with MVA

[36] , were effectively killed, whereby this effect was significantly augmented by the 4-1BBL adjuvant (Figure 7A). Cell death leads to the release of intracellular proteins such as high mobility group box 1 (HMGB1), which are sensed by innate immune cells and contribute to the initiation of immune responses [37, 38]. A significant increase in HMGB1 was detected after MVA infection of tumor cells or macrophages, independent of 4-1BBL (Figure 7B).

[0127] Our results suggest that local injection of MVA resulted in IT expression of MVA-encoded genes, leading to the induction of inflammation in the TME, cell death of infected cells, and release of immunogenic mediators.

[0128] Example 4: Local MVA immunotherapy controls tumor growth in distant untreated lesions The goal of tumor-directed immunotherapy is to generate a systemic antitumor immune response that also eradicates distant metastases. Because local treatment with MVA-TAA-4-1BBL induced strong tumor-specific T cell responses not only in the TME but also in the blood, we next evaluated the systemic antitumor potential of IT MVA immunotherapy against distant tumor lesions. CT26.WT tumor cells were implanted subcutaneously into the left and right flanks of Balb / c mice (Figure 4A). IT injection of MVA-Gp70 delayed tumor growth compared to PBS (Figure 4B and C). IT MVA-Gp70-4-1BBL injection led to the disappearance of treated tumors in 7 out of 10 CT26.WT tumor-bearing mice (Figure 4D). Importantly, both local administration of MVA-Gp70 and MVA-Gp70-4-1BBL led to tumor growth delay and, in some cases, complete tumor disappearance of untreated tumor lesions (Figure 4C and D). The data demonstrate effective induction of antitumor immune responses against distant untreated tumor lesions by IT MVA-Gp70-4-1BBL immunotherapy.

[0129] Example 5: IT MVA-TAA-4-1BBL treatment confers protection from local and systemic tumor readmission One of the main goals of cancer vaccines is to achieve long-term protective immune memory to prevent tumor recurrence. Therefore, we first evaluated whether the antitumor response induced with IT MVA-TAA-4-1BBL would give rise to immune memory that would protect against local tumor rechallenge (Figure 5A). Tumors grew rapidly in naïve mice used as controls, whereas mice that had previously been cured after IT MVA-OVA treatment had a high prevalence of tumor regrowth upon rechallenge. In contrast, 80% of mice that had previously received IT MVA-OVA-4-1BBL were resistant to secondary tumor growth (9 of 11 mice) (Figure 5B). Similar results were obtained in mice that were cured after MVA-Gp70-4-1BBL treatment and received local rechallenge of B16.F10 cells. 60% of pretreated mice remained tumor-free after B16.F10 tumor cell implantation (Figure 6A). Thus, IT MVA-TAA-4-1BBL treatment induces strong protective immune memory against local tumor rechallenge.

[0130] OVA-specific CD8 + T cells could be detected immediately before rechallenge in mice that rejected tumors after IT treatment with MVA-OVA-4-1BBL, but not MVA-OVA (Figure 5C). Seven days after tumor cell injection, the OVA-specific T cell population had significantly expanded, indicating effective tumor recognition (Figure 5C). Splenocyte OVA 257-264 With peptide restimulation, IT MVA-OVA-4-1BBL therapy induced antigen-specific CD8 + Analysis of the spleen, blood, TdLN, and NdLN on day 41 after tumor rechallenge demonstrated that OVA-specific CD8 T cells were induced in all organs analyzed (Figure 5E). + Accumulation of T cells was evident (Figure 5D). Memory subset analysis

[31] revealed that OVA-specific T CM While the cells were distributed equally across all organs, T EMWe found that T cells were predominantly found in the blood, spleen, and TdLNs, but not in NdLNs (Figure 5F). Next, we investigated the role of tissue-resident memory T cells (T RM ) [32, 33]. Remarkably, resident memory T cells (T RM ) could also be detected in blood (Figure 5F)

[34] . + T cells were detected before and after rechallenge in mice in which primary B16.F10 tumors had been removed during IT MVA-Gp70-4-1BBL treatment (Fig. 6B). CM , T EM and T RM We were able to identify 2D and 3D TdLNs 42 days after B16.F10 rechallenge (Fig. 6C–E), where the latter were found exclusively in TdLNs (Fig. 6E). Our results suggest that IT MVA-TAA-4-1BBL immunotherapy enhances T CM cell, T EM Cells and T RM Tumor-specific memory CD8 containing cells + These results indicate that the antibody produced a diverse population of T cells.

[0131] IT MVA-TAA-4-1BBL injection induced a systemic immune response that mediated control of local recurrent tumors and naive lesions. We speculated that tumor-specific T cell memory generated by IT MVA injection might protect against metastatic recurrence (Figure 5G). Macroscopic quantification of tumor nodules in the lungs after IV injection of CT26.WT tumor cells showed the development of multiple lesions in naive mice. No macroscopic metastatic lesions were found in the lungs of mice previously cured by IT MVA-Gp70 or MVA-Gp70-4-1BBL (Figure 5H). T cell analysis revealed polyfunctional AH1-specific CD8 T cells in MVA-Gp70-4-1BBL-cured mice. + A population of T cells was evident ( Fig. 5I ).

[0132] In summary, local immunotherapy using MVA genetically engineered to express tumor antigens together with the costimulatory molecule 4-1BBL confers potent antitumor activity by combining activation of innate and adaptive immunity, which not only induced systemic antitumor effects but also generated potent tumor-specific memory responses that protected against local and systemic tumor rechallenge.

[0133] Example 6: Further discussion of the results In the present study, we used a novel approach to tumor-directed virotherapy by using non-replicating MVA genetically modified to express TAA and the costimulatory molecule 4-1BBL, which combines the excellent immunostimulatory properties of MVA

[12] and its favorable safety profile

[35] with the immune-activating potential of 4-1BBL

[22] . IT MVA-TAA-4-1BBL injection activates a series of immediate and long-term immune events, ultimately resulting in tumor eradication. The induction of multiple proinflammatory mediators by IT MVA treatment represents a fundamental change in the previously immunosuppressive TME, promoting the reactivation and proliferation of tumor-specific T cells. Marked qualitative and quantitative changes in cytotoxic antitumor immune responses, essential for both therapeutic efficacy and the formation of local and systemic long-term immune memory against the primary tumor, were induced by MVA-encoded 4-1BBL.

[0134] It is now shown for the first time that IT injection of nonreplicating MVA confers a strong therapeutic antitumor effect. Interestingly, IT delivery of heat-inactivated MVA, but not MVA, has been reported to induce a strong antitumor effect that was mainly dependent on the activation of cytotoxic T cells

[25] . In contrast to this study, here we utilized active MVA encoding TAA with and without additionally expressed 4-1BBL. Importantly, while MVA-TAA alone was already effective in delaying tumor growth, adjuvant action with 4-1BBL markedly improved the therapeutic effect, resulting in the rejection of established tumors in multiple models. Moreover, the antitumor effect was independent of the choice of tumor antigen. Apart from the model antigen OVA, the endogenous retroviral protein Gp70 was examined for its immunogenicity as a TAA. Endogenous retroviral elements are epigenetically silenced in healthy tissues but are reactivated and expressed in various cancers

[36] . Similarly, Gp70 is highly expressed in several mouse tumor cell lines

[37] . In humans, there is growing evidence that these non-coding regions have strong immunogenic properties and therefore represent excellent TAA targets for cancer immunotherapy [38-40]. Given the properties of Gp70 itself

[41] , the strong therapeutic effect obtained by IT MVA-Gp70-4-1BBL therapy in Gp70-expressing tumor models suggests that local MVA therapy can not only induce rejection of tumors expressing neoantigens, but also break tolerance to endogenous self-antigens.

[0135] IT virotherapy uses virus-induced inflammation and cell death with the goal of altering the immunosuppressive TME

[50] . This sequence of events would enhance antitumor-specific immunity. Similarly, our data show that MVA infection promotes tumor cell death and thus HMGB1 release (Figures 7A and 7B), similar to oncolytic vaccinia virus

[51] . Furthermore, IT injection of MVA induced a strong inflammatory response in the TME, accompanied by the induction of multiple MVA-associated cytokines and chemokines

[52] . IT application of 4-1BBL-adjuvanted MVA significantly increased the concentrations of IFNγ and GM-CSF in B16.OVA tumors (Figure 3B), indicating that the induction of these molecules is downstream of 4-1BB signaling. Indeed, the OVA-specific CD8 + In vitro activation of T cells resulted in large amounts of IFNγ and GM-CSF production only in the presence of MVA-encoded 4-1BBL (Figure 3B). We investigated the distribution and T cell priming potential of MVA-encoded antigens in TdLNs and other organs upon IT rMVA administration. We addressed this by performing a comprehensive analysis of rMVA localization in different organs after IT injection. MVA gDNA was mostly restricted to the tumor site. However, MVA gDNA was also detected in TdLNs, albeit in much lower amounts compared to tumors. Thus, TdLNs may also serve as a priming site for tumor-specific T cells. Our results are consistent with a previous study showing that MVA localizes to the paracortical region of draining LNs after footpad injection

[42] . Consistent with this, no protein or gDNA was found in NdLNs.

[0136] An important aspect of tumor-directed immunotherapy is to generate a systemic antitumor immune response that eradicates distant metastases and induces long-term tumor immunity. Here, we show that local MVA-TAA-4-1BBL treatment not only induced an immune response within the TME, but also increased the number of systemic antigen-specific CD8 +We showed that T cell responses were also generated. Moreover, across many individual experiments and different TAAs used, mice that rejected melanoma upon IT MVA-TAA-4-1BBL treatment developed vitiligo. Vitiligo is caused by autoreactive CD8 T cells that target melanocytes in the skin, which pigment hair. These cells were activated by IT MVA-TAA-4-1BBL virotherapy, likely via antigen spreading, a phenomenon that explains the diversification of epitope specificity from the primary epitope-specific immune response initially focused on, e.g., Gp70 or OVA. Tumor antigen spreading is a desirable feature of cancer immunotherapy, as it broadens antitumor responses and prevents potential tumor escape due to TAA loss.

[0137] Furthermore, our data from a bilateral tumor model clearly demonstrated that IT MVA-TAA-4-1BBL injection produced a significant antitumor effect in untreated lesions. Thus, these results strongly suggest that the antitumor response induced by local MVA-TAA-4-1BBL administration was associated with system-wide immunity against the primary tumor.

[0138] The ability of the immune system to maintain memory of previous antigen encounters is fundamental for long-term immunity. Here, we defined the components of immune memory induced upon IT MVA administration. Regardless of the tumor model or mouse strain used, blood TAA-specific CD8 + T cells were detected in mice. CD8 + The frequency of T cells was significantly increased when 4-1BBL-adjuvanted MVA was used. We found that mice previously cured with IT MVA-TAA-4-1BBL were more resistant to subcutaneous tumor rechallenge with B16.OVA or B16.F10 than cured mice treated with MVA-TAA. Analysis of tissues from these mice showed that T cell memory subsets were found not only in the blood but also at multiple anatomical sites, suggesting immune surveillance. Antigen-specific CD8 + T CM and TEM Increased frequencies of the subsets were detected in the spleen and blood after local tumor rechallenge of cured mice previously administered MVA encoding 4-1BBL. It is well established that 4-1BBL / 4-1BB signals are particularly potent in enhancing the proliferation and maintenance of CD8 effector and memory T cells [43-45]. Similarly, MVA-encoded 4-1BBL costimulation enhanced the activation and effector function of tumor-specific cytotoxic T cells, thereby leading to the formation of a potent and diverse memory compartment.

[0139] Blood CD8 + T CM and T EM In addition to subsets, resident CD8 + T RM These cells have been shown to cooperate in antitumor immunity [32, 33, 46]. Interestingly, in mice cured after IT 4-1BBL-adjuvanted MVA, tumor-specific T cells were only expressed in the TdLNs after local re-administration of B16.OVA or B16.F10. RM The frequency of CD8 cells was increased. + T RM Although the cells were first identified in tissues, they can also migrate from mouse tissues and accumulate in draining LNs upon re-encounter with antigen [34, 46, 47]. Consistent with our results, 4-1BB inhibits influenza-specific CD8 + T RM It has been shown that IL-1BBL promotes the establishment of a pool of tumor-specific CD8 + T RM We hypothesize a relationship between cell proliferation and favorable response by cured mice to local secondary tumor rechallenge.

[0140] In cancer, memory CD8 +T cells are often dysfunctional due to suboptimal differentiation or maintenance states and chronic exposure to antigens

[49] . This phenomenon is associated with an inability to secrete IL-2 and TNFα [50, 51]. Importantly, IT 4-1BBL-adjuvanted MVA induces highly potent CD8 + It generated a T cell memory pool that expanded upon re-encounter with tumor antigens and produced greater amounts of IFNγ, TNFα and IL-2 compared to IT MVA in all rechallenge models tested.

[0141] In summary, we describe here a novel therapeutic platform based on the local injection of non-replicating MVA expressing tumor antigens in conjunction with 4-1BBL. IT viral injection induces severe pro-inflammatory changes in the TME and upregulates tumor-specific CD8 + Furthermore, it resulted in the reactivation and proliferation of diverse CD8 T cells that protected against local and systemic tumor rechallenge. + The generation of T cell memory populations was demonstrated. Combined with the favorable safety profile of MVA, these preclinical data provide a strong rationale for exploring this approach in the clinic.

[0142] Final Remarks: Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) is incorporated herein by reference in its entirety. To the extent that the material incorporated by reference conflicts or is inconsistent with this specification, this specification takes precedence over any such material. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0143] Example 7: MVA Recombinant for Medical Use 7.1 Construction of MVA recombinants Recombinant MVA viruses embodying elements of the present disclosure were generated by inserting the indicated transgenes together with the promoters of such transgenes into the vector MVA-BN. The transgenes were inserted using a recombination plasmid containing the transgene and a selection cassette, as well as sequences homologous to the target locus in MVA-BN. Homologous recombination between the viral genome and the recombination plasmid was achieved by transfection of the recombination plasmid into CEF cells infected with MVA-BN. The selection cassette was then removed in a second step, utilizing a plasmid expressing CRE recombinase that specifically targets the loxP sites flanking the selection cassette and excises the intervening sequences. Alternatively, removal of the selection cassette was achieved by MVA-mediated recombination using internal repeat sequences from MVA.

[0144] For the generation of recombinant MVA-mBN viruses, CEF cell cultures were inoculated with MVA-BN, respectively, and transfected with the corresponding recombinant plasmid. Samples from these cell cultures were then inoculated into CEF cultures in medium containing a drug that exerts selective pressure, and fluorescence-expressing virus clones were isolated by plaque purification. Deletion of the fluorescent protein-containing selection cassette from these virus clones was mediated in a second step by CRE-mediated recombination, which contains two loxP sites flanking the selection cassette in each construct, or by MVA-mediated internal recombination. After the second recombination step, only the transgene sequence (e.g., 4-1BBL) and its promoter inserted into the target locus of MVA-BN were retained. Stocks of plaque-purified viruses lacking the selection cassette were prepared. Expression of the identified transgenes was demonstrated in cells inoculated with the constructs described.

[0145] 7.2 Recombinant MVA containing HERV-K antigen An MVA-based vector ("MVA-mBN489," also referred to as "MVA-HERV-Prame-FOLR1-4-1-BBL-CD40L") was designed to include TAAs that are human endogenous retroviral K superfamily proteins (HERV-K), specifically, ERV-K-env and ERV-K-gag. MVA was also designed to encode human FOLR1 and PRAME, and to express h4-1BBL and hCD40L.

[0146] A similar MVA-based vector, called "MVA-HERV-Prame-FOLR1-4-1-BBL", was designed to express TAAERV-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") is illustrated diagrammatically in FIG. 8A. The HERV-K genes, which code for the envelope (env) and group-specific antigen (gag) proteins, are normally dormant in healthy human tissues, but are activated in many tumors. FOLR1 and PRAME are genes that are specifically upregulated in breast and ovarian cancer cells. The additional expression of the costimulatory molecule 4-1-BBL is intended to enhance the immune response to the TAAs.

[0147] "Another MVA-based vector, designated MVA-HERV-Prame-FOLR-CD40L, was 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 is useful in the methods of the invention.

[0148] Exemplary sequences are known in the art and are also set forth in the sequence listing provided, and any sequence can be used in the compositions and methods of the invention, so long as it provides the required functionality for the associated MVA.

[0149] For the ERV-K env and gag sequences above, amino acid consensus sequences were generated from at least 10 representative sequences, and potential immunosuppressive domains were mutated to be inactivated and partially replaced with the immunodominant T cell epitope HERV-K-mel, as shown below: Suitable sequences are set forth 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).

[0150] [ka]

[0151] The modified consensus amino acid sequence of ERVK-env (above): The potential immunosuppressive domain was inactivated by mutations that replaced a significant portion of the immunosuppressive domain with the immunodominant T cell epitope HERV-K-mel.

[0152] In some of these MVAs, hFOLR1 and PRAME were designed to be produced as fusion proteins. FOLR1 (folate receptor alpha) belongs to the family of folate receptors. It has a high affinity for folate 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 and is likely bound in the endoplasmic reticulum (ER) via a serine (Ser) residue in the C-terminal region of the protein. To avoid modification of FOLR1 by the GPI-anchor and complete processing of the hFOLR1-hPRAME fusion protein in the ER, the C-terminal region from amino acids 234 to 257 (including the Ser residue) was deleted.

[0153] PRAME (Preferentially Expressed Antigen in Melanoma) is a transcriptional regulator protein. It was first reported as an antigen in human melanoma, triggers autologous cytotoxic T cell-mediated immune responses, and is expressed in a variety of solid and hematological cancers. PRAME inhibits retinoic acid signaling through binding to retinoic acid receptors, which may provide a growth advantage to cancer cells. Because PRAME functionality requires nuclear localization, we modified the potential nuclear localization signal (NLS) of PRAME by targeted mutation of the hFOLR1-hPRAME fusion protein.

[0154] Therefore, in the amino acid sequence of the hFOLR1-hPRAME fusion protein, FOLR1 was modified by deleting the C-terminal GPI anchor signal, and 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, acting as an additional protective mechanism to avoid nuclear localization of PRAME.

[0155] The protein sequences of human FOLR1 and human PRAME were based on NCBI reference sequences NP_000793.1 and NP_001278644.1, respectively. In addition to the above modifications, the nucleotide sequences of the fusion proteins were optimized for human codon usage and polynucleotide stretches, repetitive elements, and negative cis-acting elements were removed. The nucleotide sequence is set forth in SEQ ID NO: 10 ("hFOLR1Δ_hPRAMEΔ fusion" nucleotide sequence) and the fusion protein sequence is set forth in SEQ ID NO: 9.

[0156] [ka]

[0157] Sequence of the hFOLR1-hPRAME fusion protein (above): Amino acid sequence of the hFOLR1-hPRAME fusion protein, a fusion of modified human FOLR1 (N-terminal part) and PRAME (C-terminal part). FOLR1 has been modified by deleting the GPI anchor signal at the C-terminus (strikethrough letters). In PRAME (underlined letters), the initiation methionine has been deleted and two potential nuclear localization signals have been inactivated by amino acid substitutions (bold, underlined letters).

[0158] The protein sequence of membrane-bound human 4-1BBL used in this MVA shows 100% identity with the NCBI reference sequence NP_003802.1, and the protein sequence of membrane-bound human CD40L used shows 100% identity with the NCBI reference sequence NP_000065.1. In both cases 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.

[0159] The amino acid sequence of hCD40L from NCBI Reference Sequence NP_000065.1. is set forth in SEQ ID NO: 1, and the nucleotide sequence of hCD40L is set forth in SEQ ID NO: 2. The amino acid sequence of h4-1BBL from NCBI Reference Sequence 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.

[0160] Each coding region was placed under the control of a different promoter, except for ERV-K-gag and h4-1BBL, both of which were placed under the control of the Pr1328 promoter. The Pr1328 promoter (100 bp long) is an exact homologue of the vaccinia virus promoter PrB2R. It drives strong immediate early expression and low levels of late expression. In recombinant MVA-mBN489, the Pr13.5 long promoter drives the expression of ERVK-env / MEL. This promoter contains a 124 bp intergenic region between 014L / 13.5L that drives the expression of the native MVA13.5L gene, and shows very strong early expression caused by two early promoter core sequences (see Wennier et al. (2013) PLoS One 8(8):e73511). The MVA1-40k promoter used here to drive the expression of hCD40L was originally isolated in 1986 as a 161 bp fragment from the Hind III H region of vaccinia virus Wyeth. It contains 158 bp of the vaccinia virus Wyeth and MVA genomes within the 094L / 095R intergenic region, which drives the late gene transcription factor VLTF-4. The promoter PrH5m used here to drive the expression of the hFOLR1-hPRAME fusion protein is a modified version of the vaccinia virus H5 gene promoter. It consists of strong early and late elements, resulting in expression of recombinant MVA at both early and late stages of infection (see Wyatt et al. (1996) Vaccine 14:1451-58).

[0161] Based on MVA-mBN494 (see above), yet another vector was designed to contain modifications to ERVK-env / MEL. The resulting vector is called "MVA-mBN502" and is illustrated diagrammatically in FIG. 9C. In addition to the modified ERVK-env / MEL, MVA-mBN502 also encodes ERVK-gag, hFOLR1-hPRAME fusion protein, and h4-1BBL.

[0162] Natively, HERV-K-env consists of a signal peptide, a surface (SU) and a transmembrane unit (TM) that are cleaved post-translationally. Cleavage into the two domains is achieved by cellular proteases. The RSKR cleavage motif is necessary and sufficient to cleave the full-length 90 kDa protein into the SU (approximately 60 kDa) and TM (approximately 40 kDa) domains. As described above for the preparation of MVA-mBN494, an amino acid consensus sequence of env derived from at least 10 representative sequences was generated, and the potential immunosuppressive domain of the TM was inactivated by mutation. The introduced mutations replaced a significant portion of the immunosuppressive domain with the immunodominant T cell epitope HERV-K-mel. This transgene (used for MVA-mBN494) was designated ERVK-env / MEL (Figure 9A).

[0163] Compared to MVA-mBN494, the TM domain of ERVK-env / MEL is deleted in MVA-mBN502. This ERVK-env / MEL variant, designated “ERVK-env / MEL_03”, is composed of the entire SU domain, excluding the deleted RSKR furin cleavage site. The MEL peptide was inserted at the C-terminus, followed by six amino acids of the TM domain (excluding the highly hydrophobic fusion peptide sequence). Furthermore, this modified ERVK-env / MEL was targeted to the plasma membrane by adding a membrane anchor derived from the human PDGF (platelet-derived growth factor) receptor. This membrane anchor was linked to the SU domain via a flexible glycine-containing linker (Figure 9B). The resulting ERVK-env / MEL variant, ERVK-env / MEL_03, is contained in MVA-mBN502 (Figure 9C). Suitable sequences for the variants are set out 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).

[0164] 7.3 Biological activity of MVA-HERV-FOLR1-PRAME-h4-1-BBL (MVA-BN-4IT) We investigated whether infection with MVA-BN-4IT (i.e., MVA-HERV-FOLR1-PRAME-h4-1-BBL; see also above) results in the presentation of vaccine-derived tumor antigens by HLA molecules on human cells. To this end, we immunoprecipitated HLA-ABC peptide complexes on antigen-presenting cells and analyzed which HLA-binding peptides could be identified by mass spectrometry.

[0165] Since antigens can be loaded onto HLA class I (Nyambura L. et al. J. Immunol 2016), we first differentiated the human mononuclear cell line THP-1 into macrophages that exhibit antigen-presenting ability (Daigneault et al. PLoS One, 2010). In fact, THP-1 cells harbored HLA-A, one of the most frequent haplotypes in the United States and Europe (approximately 30% of the population). * 0201 + Expresses HLA-A. * Apart from 02:01:01G, THP-1 cells also express HLA-B * 15 and HLA-C * It has been reported that 03 is expressed (Battle R. et al., Int. J. of Cancer). Here, 8 × 10 5 THP-1 cells were cultured for 3 days in the presence of 200 ng / ml PMA (phorbol-12-myristate-13-acetate), then the medium was changed and the cells were cultured for another 2 days in the absence of PMA. On the fifth day, the cells were infected with 4 InfU (infectious units) of MVA-BN-4IT for 12 hours. As shown in FIG. 8B, HERVK-env / MEL, HERVK-gag and the fusion protein FOLR1-PRAME were expressed after MVA-BN-4IT infection of THP-1 cells ("mBN494" in FIG. 8B). In contrast, the antigen was not endogenously expressed in uninfected THP-1 cells ("ctr" in FIG. 8B).

[0166] Next, a "ProPresent" HLA-ABC ligandome analysis (ProImmune) was performed. Four tumor antigen-derived peptides were identified in MVA-BN-4IT-infected cells, namely, the HERV-Kenv peptide ILTEVLKGV, the HERV-Kgag peptide YLSFIKILL, and the PRAME peptides ALQSLLQHL and SLLQHLIGL. The two PRAME peptides identified are largely overlapping and likely share a common core epitope. Both peptides bind to HLA-A * Since it is predicted to bind very strongly to 02:01, ALQSLLQHL has a binding order of HLA-B * 15. Of note, the PRAME peptide SLLQHLIGL does not bind to the immunogenic HLA-A * It has already been reported as a cytotoxic T lymphocyte epitope presented by 0201 (Kessler JH. et al., J Exp Med., 2001). Taken together, the data indicate that the antigen expressed by MVA-BN-4IT can be incorporated into the HLA of infected cells.

[0167] Furthermore, we tested the ability of MVA-BN-4IT to express 4-1-BBL in a functional form that binds to its receptor, 4-1-BB. For that purpose, a commercial kit ("4-1BB Bioassay", Promega) was used. The assay consists of a genetically engineered JurkatT cell line expressing h4-1-BB and a luciferase reporter driven by a response element (RE) that can respond to 4-1-BB ligand stimulation. When h4-1-BB is stimulated by h4-1-BBL, the RE activates cellular luciferase production inside the cells. After lysing the cells and adding "Bio-Glo" reagent (Promega), the luminescence is measured and quantified using a luminometer. Briefly, HeLa cells are seeded (1 × 10 6 ) and infected with the MVA-based constructs shown in Figure 8C (TCID 50=2) overnight (37°C, 5% CO2) and then co-cultured with Jurkat-h4-1-BB cells (HeLa:Jurkat = 4:1 ratio) for 6 h. Fc-crosslinked His-tagged h4-1BBL was used as a reference (positive control), and luciferase expression by Jurkat-h4-1BB cells cultured with 1 μg / ml crosslinked h4-1BBl was set to 1 (Figure 8C, dotted line). MVA-BN (i.e., not encoding h4-1-BBL) was used as a backbone control. As shown in Figure 8C, six-fold higher luciferase production was induced in HeLa cells infected with MVA-based vectors expressing h4-1-BBL compared to the reference (by co-cultured Jurkat-h4-1-BB cells). Notably, luciferase production mediated by MVA-BN-4IT was even higher than that mediated by the other two h4-1-BBL expressing MVA vectors. Thus, MVA-mBN494 expresses functional h4-1-BBL that effectively binds to its own 4-1BB receptor.

[0168] Sequence Listing The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard abbreviations for nucleotide bases and either one-letter or three-letter codes for amino acids, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but any reference to the displayed strand is understood to include the complementary strand. Sequences in the sequence listing: SEQ ID NO: 1: Amino acid sequence of hCD40L from the NCBI reference sequence NP_000065.1. (261 amino acids) SEQ ID NO:2: hCD40L (792 nucleotides) from NCBI reference sequence NP_000065.1 SEQ ID NO: 3: h4-1BBL (254 amino acids) from NCBI reference sequence NP_003802.1 SEQ ID NO: 4: h4-1BBL from NCBI reference sequence NP_003802.1 SEQ ID NO: 5: Synthetic consensus sequence of ERV-K-gag (666 amino acids) SEQ ID NO:6: ERV-K-gag; nucleotide sequence SEQ ID NO: 7: Synthetic sequence of ERV-K-env / MEL (699 amino acids) SEQ ID NO: 8: Nucleotide sequence of ERV-K-env / MEL SEQ ID NO: 9: hFOLR1Δ_hPRAMEΔ fusion (741 amino acids) SEQ ID NO: 10: Nucleotide sequence of hFOLR1Δ_hPRAMEΔ fusion (741 amino acids) SEQ ID NO: 11: Synthetic sequence of ERV-K-env / MEL_03 (517 amino acids) SEQ ID NO: 12: Nucleotide sequence of ERV-K-env / MEL_03

[0169] SEQ ID NO:1 hCD40L from NCBI reference sequence NP_000065.1. (261 amino acids) MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEA SSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

[0170] SEQ ID NO:2 hCD40L (792 nucleotides) from the NCBI reference sequence NP_000065.1. Nucleotide sequence: atgatcgagacatacaaccagacaagccctagaagcgccgccacaggactgcctatcagcatgaagatcttcatgtacctgctgaccgtgttcctgatcacccagatgatcggcagcgccctgtttgccgtgtacctgcacagacggctggacaagatcgaggacgagagaaacctgcacgaggacttcgtgttcatgaagaccatccagcggtgcaacaccggcgagagaagtctgagcctgctgaactgcgaggaaatcaagagccagttcgagggcttcgtgaaggacatcatgctgaacaaagaggaaacgaagaaagagaactccttcgagatgcagaagggcgaccagaatcctcagatcgccgctcacgtgatcagcgaggccagcagcaagacaacaagcgtgctgcagtgggccgagaagggctactacaccatgagcaacaacctggtcaccctggagaacggcaagcagctgacagtgaagcggcagggcctgtactacatctacgcccaagtgaccttctgcagcaacagagaggccagctctcaggctcctttcatcgccagcctgtgcctgaagtctcctggcagattcgagcggattctgctgagagccgccaacacacacagcagcgccaaaccttgtggccagcagtctattcacctcggcggagtgtttgagctgcagcctggcgcaagcgtgttcgtgaatgtgacagaccctagccaggtgtcccacggcaccggctttacatctttcggactgctgaagctgtgatgatag

[0171] Sequence number 3 h4-1BBL (254 amino acids) from NCBI reference sequence NP_003802.1 MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

[0172] SEQ ID NO:4 h4-1BBL from NCBI reference sequence NP_003802.1. Nucleotide sequence: atggaatacgccagcgacgcctctctggaccctgaagctccttggcctccagctcctagagccagggcttgtagatgctgccttgggctcttgtggctggacttctgcttctgttgctcctggctgctgcctgcgcagtgtttcttgcttgtccatgggctgtgtcaggagccagagcatctcctggatct gccgcttctcccagactgagagagggacctgaactgagccctgatgatcctgctggactgctcgacctgagacagggcatgtttgcccagctggtggcccagaatgtgctgctgattgatggccctctgagctggtacagcgatcctggacttgctggcgttagcctgactggaggcctgagctacaaggag gacaccaaagaactggtggtggccaaggctggcgtgtactacgtgttctttcagctgggaactgcggagagtggtggcaggcgaaggatctggatccgtgtctctggcactgcatctgcagcctctgagatctgctgctggtgcagctgccctggctctgacagttgatctgcctcctgcctccagcgaagcc agaaacagcgcctttggcttccaaggcagactgctgcacctgtctgctggccagagactgggagtgcacctccacacagaagcaagagcaagacacgcctggcagcttacacaaggcgctacagtgctgggcctgttcagagtgacacctgagattccagctggcttgccatctctcgcagcgagtaatga

[0173] sequence number 5 ERV-K-env / MEL (699 amino acids) MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPTWAQLKKLTQLATKYLENTKVTQTPESMLLAALMIVSMVVSLPPMPAGAAAANYTYWAYVPFPPMIRAVTWMDNPIEVYVNDSVWVPGPIDDRCPAKPEEEGMMINISIGYRYPPICLGRAPGCLMP AVQNWLVEVVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPKEIPKESKNTEVLVWEECVANSAVILQNNEFGTIIDWAPRGQFYHNCSGQTQSCPSAQVSPAVDSDLTESLDKHKHKKLQSFYPWEWGEKGISTPRPKIISPVSGPEHPELWRLTVASHHI RIWSGNQTLETRDRKPFYTVDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLLTCIDSTFNWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNRSKRFIFTLIAVIMGLIAVTATAAVAGVALHSSVQSVNFVNDWQKNSTRLWNSQSSIDQKMLA VISCAVQTVIWMGDRLMSLEHRFQLQCDWNTSDFCITPQIYNESEHHWDMVRRHLQGREDNLTLDISKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV

[0174] SEQ ID NO:6 ERV-K-env / MEL Nucleotide sequence

[0175] SEQ ID NO:7 ERV-K-gag (666 amino acids) MGQTKSKIKSKYASYLSFIKILLKRGGVKVSTKNLIKLFQIIEQFCPWFPEQGTLDLKDWKRIGKELKQAGRKGNIIPLTVWNDWAIIKAALEPFQTEEDSVSVSDAPGSCIIDCNENTRKKSQKETESLHCEYVAEPVMAQSTQNVDYNQLQEVIYPETLKLEGK GPELVGPSESKPRGTSPLPAGQVPVTLQPQKQVKENKTQPPVAYQYWPPAELQYRPPPESQYGYPGMPPAPQGRAPYPQPPTRRLNPTAPPSRQGSELHEIIDKSRKEGDTEAWQFPVTLEPMPPGEGAQEGEPPTVEARYKSFSIKMLKDMKEGVKQYGPNSPYMR TLLDSIAHGHRLIPYDWEILAKSSLSPSQFLQFKTWWIDGVQEQVRRNRAANNPPVNIDADQLLGIGQNWSTISQQALMQNEAIEQVRAICLRAWEKIQDPGSTCPSFNTVRQGSKEPYPDFVARLQDVAQKSIADEKARKVIVELMAYENANPECQSAIKPLKGKV PAGSDVISEYVKACDGIGGAMHKAMLMAQAITGVVLGGQVRTFGGKCYNCGQIGHLKKNCPVLNKQNITIQATTTGREPPDLCPRCKKGKHWASQCRSKFDKNGQPLSGNEQRGQPQAPQQTGAFPIQPFVPQGFQGQQPPLSQVFQGISQLPQYNNCPPPQAAVQQ

[0176] SEQ ID NO:8 ERV-K-gag Nucleotide sequence

[0177] SEQ ID NO:9 hFOLR1Δ_hPRAMEΔ fusion (741 amino acids) MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTP TVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEVARFYAAAMERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSG NRASLYSFPEPEAAQPMTTKAKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVAAKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNP LETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPN

[0178] SEQ ID NO:10 hFOLR1Δ_hPRAMEΔ fusion (741 amino acids) Nucleotide sequence

[0179] SEQ ID NO:11 ERV-K-env / MEL_03 (517 amino acids) MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPTWAQLKKLTQLATKYLENTKVTQTPESMLLAALMIVSMVVSLPPMPAGAAAANYTYWAYVPFPPMIRAVTWMDNPIEVYVND SVWVPGPIDDRCPAKPEEEGMMINISIGYRYPPICLGRAPGCLMPAVQNWLVEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPKEIPKESKNTEVLVWEECVANSAVILQNNE FGTIIDWAPRGQFYHNCSGQTQSCPSAQVSPAVDSDLTESLDKHKHKKLQSFYPWEWGEKGISTPRPKIISPVSGPEHPELWRLTVASHHIRIWSGNQTLETRDRKPFYTVDLNSSLTVPLQSCVKPPY MLVVGNIVIKPDSQTITCENCRLLTCIDSTFNWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNMLAVISCAVAGVALHGSAGSAAGSGEFVVISAILALVVLTIISLIILIMLWQKKPR

[0180] SEQ ID NO:12 ERV-K-env / MEL_03 Nucleotide sequence

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Claims

1. (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and 1. Use of a recombinant modified vaccinia virus Ankara (MVA) comprising:

2. (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and 1. Use of a recombinant modified vaccinia virus Ankara (MVA) in the manufacture of a medicament for inducing a systemic anti-tumor immune response in a subject, wherein the recombinant MVA is administered intratumorally to a solid tumor.

3. (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and A recombinant modified vaccinia virus Ankara (MVA) comprising: Use in the manufacture of a medicament for the treatment, prevention and / or prevention of recurrence of a secondary tumor in a subject, wherein said recombinant MVA is administered intratumorally to an associated primary solid tumor but not to said secondary tumor; or In the manufacture of a medicament for use in the treatment and / or prevention of recurrence of a primary solid tumor in a subject, wherein the recombinant MVA is administered intratumorally to an associated secondary solid tumor but not to the primary solid tumor; or Use in the manufacture of a medicament for use in the treatment, prevention and / or prevention of recurrence of a secondary tumor in a subject, wherein the recombinant MVA is administered intratumorally to another related secondary solid tumor, but not to the first-mentioned secondary tumor.

4. The recombinant MVA is (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and (c) at least one further nucleic acid encoding a TAA.

5. The use according to any one of claims 1 to 3, wherein the TAA is a neoantigen or an endogenous autoantigen.

6. 4. The use according to any one of claims 1 to 3, wherein 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, folate receptor alpha (FOLR1), melanoma preferentially expressed antigen (PRAME), and the endogenous retroviral peptide MEL, and combinations thereof.

7. The use according to claim 6, wherein the ERV protein is from the Human Endogenous Retrovirus K (HERV-K) family.

8. The use according to claim 6, wherein the ERV protein is selected from HERV-K envelope (HERV-K-env) protein and HERV-K gag protein.

9. The use according to claim 6, wherein the ERV peptide is from the Human Endogenous Retrovirus K (HERV-K) family.

10. The use according to claim 6, wherein the ERV peptide is a pseudogene of the HERV-K envelope protein (HERV-K-env / MEL).

11. The use according to any one of claims 1 to 3 and 8 to 10, wherein the solid tumor is a malignant tumor.

12. 12. The use according to claim 11, wherein the solid tumor is a melanoma or a cancerous breast, colon or ovarian tumor.

13. The use according to any one of claims 3, 7 to 10 and 12, wherein the secondary tumor is a metastasis.

14. The use according to any one of claims 3, 7 to 10 and 12, wherein the tumor to which the recombinant MVA is not administered cannot be dissected or is difficult to access by surgery.

15. The use according to any one of claims 1 to 3, 7 to 10 and 12, wherein the recombinant MVA is reproductively replication incompetent in human cell lines.

16. The use according to claim 15, wherein said recombinant MVA is derived from MVA-BN deposited at the European Collection of Cell Cultures (ECACC) under number V00083008.

17. Use of a recombinant MVA in the manufacture of a medicament for stimulating an immune response in a subject having multiple tumors, said recombinant MVA comprising at least one first nucleic acid encoding a TAA and a second nucleic acid encoding 4-1-BBL, said stimulating comprising administering said MVA to fewer than the total number of tumors in said subject, wherein an immune response against said TAA is stimulated in said subject.

18. Use of a recombinant MVA comprising at least one first nucleic acid encoding a TAA and a second nucleic acid encoding 4-1-BBL in the manufacture of a medicament for treating a subject having at least one inaccessible tumor and at least one accessible tumor, said treatment comprising administering said MVA to at least one accessible tumor of said subject, thereby reducing or stopping the growth of said inaccessible tumor.

19. (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and A pharmaceutical for preventing recurrence of a solid tumor in a subject during or after remission thereof, comprising a recombinant modified vaccinia virus Ankara (MVA) comprising:

20. (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and A pharmaceutical for inducing a systemic anti-tumor immune response in a subject, comprising a recombinant Modified Vaccinia Virus Ankara (MVA) comprising:

21. (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and or a medicament for the treatment, prevention and / or prophylaxis of recurrence of a secondary tumor in a subject, comprising a recombinant Modified Vaccinia Virus Ankara (MVA) comprising: A medicament for the treatment and / or prevention of recurrence of a primary solid tumor in a subject, comprising the recombinant Modified Vaccinia Virus Ankara (MVA), wherein the recombinant MVA is administered intratumorally to an associated secondary solid tumor, but not to the primary solid tumor; or A pharmaceutical for the treatment, prevention and / or prevention of recurrence of a secondary tumor in a subject, comprising the recombinant modified vaccinia virus Ankara (MVA), wherein the recombinant MVA is administered intratumorally to another related secondary solid tumor, but not to the first-mentioned secondary tumor.

22. The recombinant MVA is (a) a first nucleic acid encoding a tumor-associated antigen (TAA); (b) a second nucleic acid encoding a 4-1BB ligand (4-1BBL); and The pharmaceutical according to any one of claims 19 to 21, comprising: (c) at least one further nucleic acid encoding a TAA.

23. The pharmaceutical according to any one of claims 19 to 21, wherein the TAA is a neoantigen or an endogenous autoantigen.

24. The pharmaceutical of any one of claims 19 to 21, wherein 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, folate receptor alpha (FOLR1), melanoma preferentially expressed antigen (PRAME), and the endogenous retroviral peptide MEL, and combinations thereof.

25. The pharmaceutical according to claim 24, wherein the ERV protein is derived from the human endogenous retrovirus K (HERV-K) family.

26. The pharmaceutical according to claim 24, wherein the ERV peptide is derived from the human endogenous retrovirus K (HERV-K) family.

27. The pharmaceutical composition according to any one of claims 19 to 21 and 26, wherein the solid tumor is a malignant tumor.

28. The method of any one of claims 21, 25 and 26, wherein the secondary tumor is a metastasis.

29. The pharmaceutical agent of any one of claims 21, 25 and 26, wherein the tumor to which the recombinant MVA is not administered cannot be dissected or is difficult to access by surgery.

30. The method according to any one of claims 19 to 21, 25 and 26, wherein the recombinant MVA is reproductively replication incompetent in human cell lines.

31. The method of claim 30, wherein the recombinant MVA is derived from MVA-BN deposited at the European Collection of Cell Cultures (ECACC) under number V00083008.

32. A pharmaceutical for stimulating an immune response in a subject having multiple tumors, comprising a recombinant MVA comprising at least one first nucleic acid encoding a TAA and a second nucleic acid encoding 4-1-BBL, wherein the stimulating comprises administering the MVA to fewer than all of the subject's tumors, and wherein an immune response to the TAA is stimulated in the subject.

33. A pharmaceutical for treating a subject having at least one inaccessible tumor and at least one accessible tumor, comprising a recombinant MVA comprising at least one first nucleic acid encoding a TAA and a second nucleic acid encoding 4-1-BBL, said treatment comprising administering said MVA to at least one accessible tumor of said subject, thereby reducing or stopping the growth of said inaccessible tumor.

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