Highly efficient immunomodulatory vaccinia virus strains

Abstract

To provide newly adapted and / or more potent viruses and / or virus vectors useful for treatment of human diseases.SOLUTION: Disclosed herein is a novel immune-modulating vaccinia virus strain (IOVA) and derivatives thereof with high replication efficiency for use in the medicine. The novel vaccinia virus strain IOVA and derivatives thereof, vectors and recombinants have an ability to replicate in a mammal cancer line and being lytic or, in the case of cancer cells, oncolytic, and elicit an increased capacity to destroy cell-cycle activated cells and / or tumor cells, compared with the gold standard Vaccinia virus WR.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a novel highly replication-efficient immunomodulatory vaccinia virus strain (IOVA) and derivatives thereof for use as a medicament. and derivatives thereof.

Background Art

[0002] In recent years, viruses have obtained market approval for use as oncolytic viruses in the treatment of human cancers for the first time. Such success has generally refocused scientific interest on viruses and also on the use of vaccinia viruses for cancer treatment. The use of vaccinia viruses has been applied, for example, not only to the vaccination approach for preventing poxvirus infections in humans, but also to many other medical treatments and clinical trials, such as for tumor vaccines. In recent years, viruses have obtained market approval for use as oncolytic viruses in the treatment of human cancers for the first time. Such success has generally refocused scientific interest on viruses and also on the use of vaccinia viruses for cancer treatment. The scientific community knows from the data obtained in the first clinical studies of oncolytic viruses that these viruses or engineered viral vectors can replicate selectively in cancer cells and actively promote the lysis of infected cancer cells. However, direct lysis of such infected cancer cells is rarely sufficient to eradicate bulky tumors and also rarely cures metastatic diseases. These results indicate an increased need not only for more potent and selective viruses to avoid disease recurrence, but also for alternative mechanisms of tumor destruction. The scientific community knows from the data obtained in the first clinical studies of oncolytic viruses that these viruses or engineered viral vectors can replicate selectively in cancer cells and actively promote the lysis of infected cancer cells. The use of vaccinia viruses has been applied, for example, not only to the vaccination approach for preventing poxvirus infections in humans, but also to many other medical treatments and clinical trials, such as for tumor vaccines. has also been applied to many other medical treatments and clinical trials, such as for tumor vaccines.

[0003] The scientific community knows from the data obtained in the first clinical studies of oncolytic viruses that these viruses or engineered viral vectors can replicate selectively in cancer cells and actively promote the lysis of infected cancer cells. However, direct lysis of such infected cancer cells is rarely sufficient to eradicate bulky tumors and also rarely cures metastatic diseases. [[ID= Thirty-two]] These results indicate an increased need not only for more potent and selective viruses to avoid disease recurrence, but also for alternative mechanisms of tumor destruction. These results indicate an increased need not only for more potent and selective viruses to avoid disease recurrence, but also for alternative mechanisms of tumor destruction. These results indicate an increased need not only for more potent and selective viruses to avoid disease recurrence, but also for alternative mechanisms of tumor destruction. These results indicate an increased need not only for more potent and selective viruses to avoid disease recurrence, but also for alternative mechanisms of tumor destruction.

[0004] More recently, vaccinia virus-based viruses and viral vectors have been tested as oncolytic agents and destroy tumors via a mechanism different from direct lysis of tumor cells. More recently, vaccinia virus-based viruses and viral vectors have been tested as oncolytic agents and destroy tumors via a mechanism different from direct lysis of tumor cells. It appears to be the most promising because it has the ability to do so (Kirn et al., 2009). On the other hand, viral replication in tumor-associated endothelial cells leads to disruption of tumor blood flow, hypoxia, and It has been demonstrated that it causes large-scale tumor necrosis (Breitbach et al. 20 13); On the other hand, replication of vaccinia virus within tumors is associated with tumor-associated antigens (TAA) after lysis. By releasing and overcoming local immunosuppression present within the tumor, an anti-tumor immune response can be induced. This is possible (Thorne et al., 2010).

[0005] These alternative mechanisms have proven important in clinical evaluations, and the immune system lyses tumors. The crucial role played in determining the activity of sex vectors was emphasized. In bed testing, the most effective viruses are those that optimize the activation of the cellular immune response. It has been proven that it is a virus that expresses immune-activating cytokines (Breitbac (h et al., 2011; Heo et al., 2013). However, These more potent cytokine-expressing viruses cause some patients to develop tumors. It does not induce an effective immune response. For the generation of an effective antitumor immune response, further immune cells are needed. The activation and collaboration of many different lineages are necessary, and the expression of a single cytokine is necessary. Russ can hardly alter the overall immunity it induces. Therefore, a strong immune response Research is still needed to generate novel strains with a higher ability to activate the response. ru. [Overview of the Initiative]

[0006] Therefore, the object of the present invention is to provide a novel, adapted, and / or a device useful for treating human diseases. The purpose is to provide a stronger virus and / or viral vector, and to provide a stronger virus and / or viral vector for infection. In the event of a virus or vector infection, additional, more potent, but for safety reasons, such To induce and / or activate a highly specific immune response in patients requiring such treatment. .

[0007] This problem relates to newly identified replication efficiencies, as specified in claim 1 of this application. This was resolved by a highly immunomodulatory vaccinia virus strain. Further embodiments of this new Identified and / or adapted immunomodulatory vaccinia viruses and vector variations Referencing the selection, including newly identified and / or suitable vaccinia virus strains. The uses and compositions are described in the dependent claims.

[0008] While studying immune responses to known vaccinia virus strains, particularly well-characterized ones... Compared to the vaccinia virus strain (Western Reserve (WR)), this The inventors were able to isolate, generate, and characterize novel vaccinia virus strains. This new strain clearly shows an improved immunological profile. Not constrained by theory. Furthermore, the novel strain strongly promotes increased immunogenic cell death during tumor cell infection, and therefore This is thought to redirect the immune response towards infected and uninfected tumor cells. .

[0009] Vaccinia virus WR strain is oncolytic due to its high replication capacity in tumor cells. It has been tested and used for the delivery of GM-CSF to increase the immune response against tumors. It is a clearly defined virus strain. To provide a tumor-selective vector, Successfully deleted different vaccinia virus genes involved in activating the metabolism of infected cells The replication of the resulting viral vector was restricted to cells with a high replication index. Genes such as thymidine kinase or viral growth factor deletions are already included in the viral candidates tested in clinical trials (Zeh et al., 2015).

[0010] WO2015027163 is an example that describes an oncolytic vaccinia virus containing a genomic deletion in a part of the immune evasion genes of a typical virus. In this particular case, the oncolytic virus contains deletions within, for example, the B8R, B18R, and A35R genes

[0011] EP2136633 is another example that describes the therapeutic use of a GM-CSF-expressing vaccinia virus in an oncolytic treatment approach

[0012] In these oncological approaches, the vaccinia virus WR strain is considered the current gold standard for the development and comparison of newly developed oncolytic vaccinia viruses

[0013] However, in addition to this claimed success, when considering vaccinia virus WR as the basis for a treatment approach in severely ill cancer patients, several concerns still exist Vaccinia virus WR was selected in vivo after multiple passages in the mouse brain, so although a virus with high replication ability in mouse cells was actually obtained, its pathogenicity is warned due to neurotropism and neurotoxicity that may prevent its safe use in the treatment of cancer patients

[0014] ​​​​​​​​​ Nevertheless, in the context of this application, vaccinia virus WR is novel to the present invention. Selected for comparison with conventionally developed vaccinia virus strains, the main objective of this invention is to A new immunological profile with improved width and high lytic and / or tumor-lytic ability. The objective is to provide immunomodulatory vaccinia virus strains with standard replication capabilities.

[0015] MVA (Modified Vaccinia Ankara) is its host range limitation, in other words, its mammary movement Since it is characterized by its replication defects in cells derived from a physical host, it is a highly attenuated MVA strain. Further comparisons by MVA do not appear to be useful. MVA is a vaccine against pathogens. It is widely used for the delivery of antigens for species (see review by Volz & Sutter). (See 2017) Characterization of its biological and immunological profiles It is valued (Meyer et al., 1991). Furthermore, MVA is a mammalian cellular marker. It is a highly modified vaccinia virus that cannot replicate in cells, and previously it was used as a tumor-associated antigen. (TAA) was used for vaccinating tumors by delivering (Zhang et al., 2012). This virus exhibits an ideal safety profile. It appears that this effectively generates an immune response against expressed tumor antigens in vivo. Regardless of the fact that this has been proven, most patients do not show any verifiable antitumor response at all. The induced immune response is not shown, and is inhibited and ineffective by localized immunosuppression occurring within the tumor. It appears to produce an antitumor response (Marigo et al., 2008).

[0016] Therefore, progress may be described as being toward improved immunotherapy, but cancer To establish an effective immune response that can eradicate the virus, it is used in cancer viral therapy. There is still a need for more effective vaccinia virus strains than those currently being used.

[0017] This invention hereby describes further steps toward this goal, relating to human and mouse cells. It has replication ability in and, according to the best knowledge of the inventors, immunologically related Carle Translocation of ticulin (CRT) to the outer membrane causes calreticulin in the outer cell membrane of infected cells. This leads to phosphorus exposure, which initiates a strong and improved immune response, generally first We disclose a novel vaccinia virus strain, which is even a vaccinia virus.

[0018] Calreticulin (CRT) appears to be a multifunctional protein, and hormone-responsive D It is described as being involved in the transcriptional regulation of NA elements and the maturation of MHC class I proteins. Also, Ca in the endoplasmic reticulum 2+ It binds ions or incorrectly folded proteins. It is also described as being inactivated and mainly located in the storage compartment of the endoplasmic reticulum (ER). CRT represents the most abundant protein in the ER lumen, but in the case of cell death, protein The fraction may migrate from the ER lumen to the cell surface, and more specifically, CRT may migrate to the cell It functions as an "eat-me" signal on the surface (Gardai et al., 200 5) Phagocytosis by CD91-positive cells (mainly macrophages and dendritic cells (DCs)) It has been suggested that this should be done.

[0019] The novel vaccinia virus strain has been described as having the ability to replicate in mammalian cells. Without being bound by theories, this characteristic is particularly evident in the K1L inheritance in the viral skeleton. It is thought to depend on the presence and functional activity of offspring. In particular, novel strains are mammals, special Human and mouse cell lines, such as HeLa cells, 143B cells, CT26 cells, LLC It has been shown to replicate well in single cells or cancer cells such as MCF-7 cells. In comparison with these results, MVA is found to be effective in these cells or in other cells of mammalian origin. It should be noted that it is known not to duplicate within itself.

[0020] Furthermore, the new vaccinia virus strain is all and / or different from other vaccinia virus strains. They share at least some housekeeping genes. However, However, their immunological profiles differ considerably, and in the context of this application, IOVA It was named (immunotumocytic vaccinia).

[0021] Many genes in the vaccinia virus genome are immunological profiles of various virus strains. It is described as an immune evasion gene that forms and determines files (Smith et al.). (al., 2013). Over the years, research has explored the potential role of such immune evasion genes. This has been done to identify the mechanism of action. According to the results obtained, these immune evasion genes In particular, it competes with natural receptors and reduces the efficacy of cytokine-induced immune responses. It expresses cytokine soluble receptors. Other genes express various immune activation genes. It blocks the intracellular pathways for activation, and therefore interferes with the virus-specific immune response. Reduce.

[0022] Table 1 shows the currently identified immune evasion genes of vaccinia virus. And list its presence or absence in various vaccinia strains. This indicates the potential function or mechanism of action of the corresponding gene product. [Table 1] JPEG0007880583000002.jpg108115

[0023] The naming convention for open reading frames (ORFs) used in Table 1 is (Waku This refers to the established naming system for senior virus Copenhagen (COP) strains (G (Oebel et al. 1990), restricted endonuclease HindIII The alphabet assigned to the DNA fragments of the viral genome decreases in size. Identified by the letter "T". The exception is that it is not present in vaccinia virus COP strains. OR containing letters that reference DNA fragments in the cowpox virus CPXV-GRI genome F C8L and B21R. Various O23 fragments of such HindIII fragments. RF is identified by a number. The last letter indicates the direction of transcription: R is right, L is left.

[0024] Furthermore, Table 1 lists the immune evasion genes identified to date and those that have been well-characterized. Functionality with xinia virus strains COP, WR, MVA, and the MVA ancestral strain CVA This provides a comparison. In Table 1, various genes are found in various viral genomes. Functional (indicated by "+"), partially / completely absent, functionally inactive (indicated by "-") It indicates whether or not it will happen.

[0025] Novel vaccinia described herein and identified hereafter as IOVA Each virus strain has its own immunological profile. This is due to the functional expression of genes. It shares some of its offspring with CVA or WR, and some of its functionally inactive genes with MVA. This involves the publication of the full sequence of one isolate of a novel vaccinia virus IOVA. Compared to the sequences of WR, COP, CVA, and MVA, the inventors have also found that at the sequence level, This has been confirmed.

[0026] The inventors have also developed a PCR assay for easily identifying novel IOVA strains. Therefore, any vaccinia virus DNA fragment being tested is one C2 The region or sequence of L ORF (Open Reading Frame) and the region of N2L ORF PCR oligonucleotides (primers) that specifically bind to a region or sequence enhance the effect. It can be widened. PCR performed with standard parameters is effective against most vaccinia viruses. Then, an amplified fragment containing the ORF C2L-C1L-N1L-N2L sequence is generated. These PCR products are double-stranded DNA fragments, therefore, restriction endonuclei They undergo enzymatic treatment with ZeBstXI. As a result, for example, vaccinia strain WR, COP Furthermore, the specific DNA product obtained in CVA is not cleaved, and agarose gel electrophoresis During movement, it shows a single band around 3280 bp (molecular weight). In contrast, specific IOV The DNA product was cleaved by BstXI and was approximately 2360 bp and approximately on an agarose gel. It shows two bands with a molecular weight of 920 bp. The reason is that the novel IOVA virus It possesses highly specific mutations in N1L ORFs, resulting in functionally inactive N1L and novel BstXI. This results in a restriction site. In the case of MVA, the C2L gene sequence is the MVA genome. It is deleted within, and therefore, there is no amplification of this specific DNA product by PCR. It is likely.

[0027] As can be seen from Table 1 and the data presented in this application, IOVA itself and all of it The derivatives of it have many gene and / or functional deletions, CVA, WR, and MVA. While they share information, they are not unique in their understanding of its safety features, nor in its immunological profile. It is special.

[0028] This immunological profile, which is the essential core of the present invention, is, among other things, related to viral infections. It correlates with the presentation of calreticulin on the outer membrane of cells.

[0029] Therefore, the immunological profile of the novel strain is, in particular, that of the outer membrane of virus-infected cells. It is stated that this induces the presentation of the measurable calreticulin. Constrained by this theory Without any intervention, this property is functionally inactive, affecting B21R*, C10L, and C9 within the viral skeleton. L, C4L, C2L, N1L, N2L, M1L, A26R, A51R, A52R, A55 R, A56R, and B13R / B14R (except for those corresponding to the CPXV-GRI nomenclature *) At least one immune response selected from the group consisting of (vaccinia virus Cop strain nomenclature) It is thought to depend on the partial or complete deletion of the avoidance gene.

[0030] As a result of these functionally inactive, partially or completely deleted genes, new The generated virus lost some of its functions in order to evade the host's immune response. Furthermore, even changes in immune evasion strategies are being introduced. [Table 2]

[0031] What is particularly interesting in this context is the Calleti, which is usually strictly internal and located in the endoplasmic reticulum. Although it is a curin protein, after infection with a new vaccinia virus strain, it affects cancer cells. Upon migration to the surface, it initiates and enhances a specific immune response against virus-infected cells. This is the effect that allows for this. When calreticulin is presented on the outer membrane, it is "eat- Danger-associated molecular patterns (DAMPs) that are described as acting as "me" signals Yes, and it can be used as a marker of immunogenic cell death. Compared to vaccinia virus WR The newly generated vaccinia virus strain IOVA is much lower in dose, therefore It lyses or kills cancer cells far more efficiently (Figure 3A).

[0032] The inventors found that in both human and mouse cell lines, WR was found to be 70-80% of cultured tumor cells. Only % could be killed; approximately 20-30% of cells escaped destruction via the virus, and were infected. This demonstrated that metabolic activity was maintained even when the degree of chromosome density increased significantly.

[0033] In contrast, the novel IOVA virus derivatives showed at least 95% in all cell lines tested. It destroys cells by more than % and even more than 95%, and surprisingly, the human cell line HeLa In this study, the novel IOVA derivative was at least 40 times more efficient than vaccinia virus WR. While it demonstrated significantly improved cytotoxicity, it also demonstrated the proliferation capacity of IOVA in HeLa. Regarding this, it was very similar to vaccinia virus WR. It kills virus-infected cells. This more complete efficiency for achieving tumor remission, in particular, reduces the risk of tumor remission and is therefore very beneficial. It is advantageous.

[0034] Therefore, for the infection of cell cycle-activated cells and / or tumor cells, and this sentence In the pulse, particularly for use as an oncolytic virus or for use in the treatment of cancer. Not only for the purpose of vaccination and for the purpose of more general immune stimulation, therefore Using novel vaccinia virus strains is highly advantageous, even for general pharmaceutical use. That is the case. Furthermore, according to a further embodiment, the novel vaccinia virus strain IOVA is functionally inactive. It contains the A56R gene. The absence of expression of this gene product causes infected cells to fuse with adjacent cells. The ability to combine, that is, the ability to form syncytia during infection of mammalian cells and tumor cells. It correlates with Rus's abilities.

[0035] Syncytium formation has been described in many viral infections, and in most of these infections Its formation is a result of membrane destabilization caused by cytopathic effects, but other The virus in this area codes for a protein that promotes syncytial formation, and neutralizing antibodies It functions as a mechanism to spread infection without becoming a target. Interestingly, the A56R gene... The offspring are produced by vaccinia virus WR to inhibit such syncytial formation. This is shown (Figure 1).

[0036] Syncytial cell death is described as being highly immunogenic, therefore, novel vaccinia virus The discovery that IOVA induces syncytial formation in cancer cells suggests the potential of oncolytic viruses. This is considered a particularly interesting phenotype: tumor cell fusion is efficiently performed by dendritic cells. The effective use of tumor antigens, which are incorporated and therefore can enhance a highly specific immune response. This induces the release of exosome-like vesicles associated with the release of these vesicles.

[0037] Furthermore, according to a further embodiment, the novel vaccinia virus strain IOVA is functionally inactivated. It includes the sex A26R and A56R genes. The absence of expression of these gene products is in mammals. This correlates with improved syncytial formation during infection of cells and tumor cells, and therefore, infected cells are adjacent to each other. This also correlates with the virus's ability to fuse with adjacent cells.

[0038] Cincithium as a mediator with potential IOVA virus-specific antitumor effects A56R alone or in combination with A26R deletion in fusion formation and such fusions. To investigate the effect of the deletion, a single A56+ IOVA viral variant was used, mutation The modified A56R gene was created by replacing it with IOVA / A56+. (Figures 1 and 6). When A56R gene expression is restored, the IOVA / A56+ virus becomes more virulent. It maintains a certain ability to fuse tumor cells, but compared to the IOVA / A26- / A56- virus In comparison, the number of fused cells decreases (Figure 6). This is because both genes interact with each other, affecting the infected cells. This suggests that it may completely inhibit the fusion of the two.

[0039] Syncytial formation is associated with other candidate oncolytic tumors, such as those originating from measles virus or adenovirus. It is known to have a negative impact on the productive replication of detoxifying viruses. Surprisingly, IO Syncytial formation in association with VA virus has been shown to have minimal impact on replication ability. Cells infected with IOVA / A56- and IOVA / A56+ are very similar. Virus yield was obtained (Figure 2).

[0040] Surprisingly, syncytial formation has a significant positive effect on the cytotoxicity of cancer cells according to the present invention. It had an effect. In all cell lines tested, syncytosis-forming viruses were particularly prevalent in 143B and In the MCF-7 cancer cell line, more effective killing of cancer cells was achieved, and IOVA / A56 + functioned similarly to vaccinia virus WR, with reduced killing of cultured cancer cells (Figure 3). Therefore, syncytia mediated by IOVA / A56- and IOVA / A56- / A26- Body formation contributes to the destruction of enhanced cancer cells.

[0041] A large plaque phenotype was observed after infection by cancer cells. IOVA / A56- and IO Both VA / A56+ viruses are involved in the formation of plaques after infection of a single cancer cell. As indicated by the increase in diameter, it enhances the ability to kill cancer cells and spread within them. This demonstrates the ability (Figure 7).

[0042] Regarding the induction of immunogenic cell death, syncytium formation is independent of the induction of CRT migration. It is independent of the additional secretion of HMGB1 and ATP, and A56 -The virus and the A56+ virus were very similar (Figures 4a-c).

[0043] Interestingly, in the context of the present invention, the individual of calreticulin on the outer membrane of infected cells In addition to its prominent exposure, it already possesses a cytopathic effect that enhances cancer cells. The phenotype of sibilitation is an antitumor of the immunomodulatory vaccinia virus strain IOVA described herein. It has been shown that the anti-inflammatory effect has been further enhanced.

[0044] Therefore, the novel vaccinia virus strain is further functionally inactive A56R and / or Alternatively, the A26R gene may be present, and therefore, it can cause syncytial formation and tumor formation. It induces cell fusion. Combined with the enhanced presentation of calreticulin on the membrane of infected cells. Furthermore, this leads to even greater lysis, and therefore antitumor activity, resulting in cancer cell cell lysis. This leads to enhanced destruction of cysts and tumor masses, ultimately resulting in a stronger immune response against the tumor. To bring about success.

[0045] Enhancing the safety of novel IOVA viruses, their derivatives, and / or their viral vectors. To achieve this, additional modifications were introduced to restrict replication to only a specific cell range, or It can be introduced.

[0046] Therefore, according to another embodiment, the novel vaccinia virus strain, IOVA, is a host characteristic In other words, the cell-limited replication ability in replicating cells and / or cancer cells. It will be modified to have it.

[0047] In this context, the term "replicating cell" refers to a cell that has an activated cell cycle and a high finger This includes cells that replicate by number. Cells that fit this definition include cancer cells and other cells that continuously replicate. Not only cells present, but also, for example, cells belonging to the activated immune system and, if necessary, tumor cells. This also includes cells that have the potential to infiltrate.

[0048] Furthermore, the term "cancer cells" used below refers to cells that divide relentlessly and uncontrollably, For example, by forming a solid tumor mass, or by abnormal cells in the blood and / or other By filling the body cavity, it replaces or discards normal tissue. Includes cells that possess. Furthermore, the term “cancer cells” is used as necessary for those known This also refers to cells that express tumor marker gene products and can be identified by their expression.

[0049] To improve the cell range specificity of novel vaccinia virus strains, F4L, J2R, and Inactivate one or more so-called housekeeping genes selected from the group consisting of C11R. By doing so, a cell-limited replication ability can be obtained. For example, inactivation Alternatively, viruses with a mutated J2R gene do not express viral thymidine kinase. Therefore, effective replication is achieved in cells whose cell cycle is continuously activated, or alternatively in cells whose cell cycle is continuously activated. This is limited to tumor cells. According to the present invention, such inactivation is limited to the nomenclature of vaccinia. Then, an alternative expression cassette (tracer color expression) is created at the location of the TK gene called J2R. This was done exemplarily by inserting (ru).

[0050] Similarly, functional inactivation, deletion, or mutation of C11R, F4L, either alone or in combination with other genes. Each of them, or in combination with J2R, enhances the replication ability of the vaccinia virus strain according to the present invention. Restrict to cell cycle activated cells and / or tumor cells. Therefore, the above host range restriction One or more genes from the group consisting of F4L, J2R, and C11R that have the result are functionally impaired. The addition of such mutations or deletions to the viral genome for activation is a novel virus. Clearly improve the safety characteristics of the stock. Therefore, the vaccinia virus strain IOVA, with its limited cell range, exhibits improved safety characteristics. Due to its characteristics, it is particularly advantageous for use in medicine.

[0051] The novel vaccinia virus strain of the present invention is remarkably different from vaccinia virus WR. In particular, the inventors have shown that it can induce immunogenic cell death during infection. These were shown.

[0052] In this context, the term "immunogenic cell death" refers to the immune response to dying cells. It can initiate and activate an epidemic response, and immunity against both microbial and carcinogenic neoantigens. The release of damage-associated molecular pattern (DAMP) molecules that can trigger an epidemic response. It should be understood as a form of cell death characterized by exposure (Galluzzi et al., 2017). Dying cells that can mediate an immune response against tumor cells. Among the long list of DAMPs released or exposed by cells, there is a significant amount of DAMPs on the cell surface of cells. Reticulin exposure, and independent high mobility group box 1 tannyl to extracellular space. The release of protein (HMGB1) or ATP is described.

[0053] Immunogenic cell death further activates dendritic cells (DCs), resulting in the death of dying cells. To activate T cells specific to antigens present in cells, different DAMPs are exposed. Apoptosis can be described as a form of cell death that involves the release of cells. Apoptosis is, for example, non-immunogenic and It is also defined as tolerogenic cell death, and cannot be defined by the same DAMP. And this cannot be triggered by external factors.

[0054] Based on the background knowledge of such markers, we have identified a virus that causes It was possible to actually describe and classify the types of cell death that occur in vaccinia. As a result, Compared to the WR virus, the novel vaccinia virus strain IOVA lyses during infection. In addition to causing increased activity and cytotoxicity, some DAMPs, namely HMGB, By measuring 1 and ATP, and the extracellular membrane of infected cells that have died due to immunogenic cell death. It has also been clearly demonstrated that this can be shown by measuring the amount of CRT presented. It was done.

[0055] This is a novel vaccine that replicates in mammalian cells, particularly tumor cells or cell cycle-activated cells. Nearvirus strains are used not only as vaccines or adjuvants for vaccination purposes, Further immunotherapy or immuno-oncology, particularly for the treatment of cancer, using oncolytic viruses and It impressively demonstrates its extreme usefulness even in such situations.

[0056] In summary, the novel vaccinia virus strain IOVA and its derivatives, vectors, and recombinants The body has the ability to replicate in mammalian cancer strains, and is the gold standard vaccinia wii. Compared to RusWR, it has an improved ability to destroy cell cycle-activated cells and / or tumor cells. Because it induces lysis, it can be described as lytic, or in the case of cancer cells, oncolytic.

[0057] Importantly, IOVA and its derivatives or recombinants are immunogenic when they infect cells. It is particularly suitable for inducing cell death, and is therefore used in immunotherapeutic approaches. They are highly anticipated to be safe and effective immunotherapies or immuno-oncology treatments. It is particularly suitable as a novel platform virus for this purpose.

[0058] According to further embodiments, the application also features isolated IOs characterized by their nucleic acid sequences. Provides VA virus. The sequence information of isolated IOVA may include base pair modifications. However, it does not affect the immunological profile of IOVA. Therefore, IOVA Wi Russ is a sequence analyzer and J2R, C11R, F4L, B21R*, C10L, C9L, C4L , C2L, N1L, N2L, M1L, A26R, A51R, A52R, A55R, A56 R and at least one function selected from the group consisting of B13R / B14R It can be identified by the presence of actively deactivated or deleted ORFs.

[0059] According to a further embodiment, IOVA and its derivatives are newly generated in N1L ORF. The resulting BstXI restriction enzyme site is introduced into C2L-C1L-N1L-N2L (sequence number) It can be identified by the presence of a unique nucleotide sequence stretch of (1). In this sequence C2L incorporates 51 nucleotide deletions distributed across three microdeletions. N1L is It incorporates two nucleotide deletions that include an early stop codon. N2L is a short N The 15 nucleotide deletions that code for the 2L version are incorporated. Therefore, the sequence The sequence shown in number 1 is a unique sequence of IOVA, incorporating a microdeletion into a part of the ORF. This represents a unique stretch of the C2L-C1L-N1L-N2L region of the OVA, but such There are no complete deletions of the gene, nor are there any functional microdeletions of C1L ORFs.

[0060] Furthermore, according to one embodiment, the isolated IOVA is used in platform technology It is considered to be G, and the aforementioned PCR analysis, or virus housekeeping, or K1 L, A56R, A26R, J2R, C11R, F4L, B21R*, C10L, C9L, C4L, C2L, N1L, N2L, M1L, A51R, A52R, A55R, and B13 Among the group of ORFs consisting of R / B14R, functionally inactive immune evasion genes selected from Related derivatives or recombinants identified by at least one direct sequence comparison It enables the generation of bait.

[0061] According to further embodiments, this application also provides a viral vector derived from IOVA. Provided. This viral vector contains functionally active and functionally inactive genes as IOVA. This includes the same set of. In the context of this application, the term “viral vector” also refers to a cell that They work in coordination to enable IOVA production from such cells. Some or a range of the set of functionally active and functionally inactive genes in IOVA It contains two or more vector molecules that contain or support each of them.

[0062] IOVA itself, isolated virus, IOVA nucleic acid sequence, and IOVA-specific nucleic acid sequence A viral vector containing columns is considered a platform technology.

[0063] The IOVA platform also includes derivatives of IOVA, which are used in PCR and B It can be identified by restriction enzyme digestion using the stXI enzyme, and has the same function as IOVA. Characteristics and properties, particularly replication ability in mammalian cell cycle activated cells, especially in infected cells. This utilizes the migration of calreticulin to the outer membrane and / or the induction of independent syncytial formation. ru.

[0064] The IOVA platform further comprises recombinant IOVA. This includes well-described vaccinia for transgenic insertion into the genome. It contains several virus insertion sites. Due to its close relationship with vaccinia virus, A skilled practitioner can identify tumor antigens, tumor-associated antigens, disease-associated antigens, and / or pathogen-derived antigens. Knowing one or more locations for inserting a transgene, such as the gene encoding the original gene. They are available and can be used.

[0065] Therefore, according to further embodiments, the present invention relates to recombinant or transgenic We provide IOVA or its derivatives or viral vectors. All platforms Similar to bleed technology, the introduction of additional genes or genetic information into well-documented insertion sites is possible. These do not affect the main characteristics of the claimed platform members, and they are independent of each other. (i) Induction of fusion or syncytial formation of infected cells, (ii) DAMP, especially Karretiki Induction of significant release or exposure of urin, and / or (iii) immunogenicity of infected cells This is an induction of death, and this can have a greater immunological effect, or in the case of infection by tumor cells. This may contribute to more effective antitumor activity through enhanced destruction of tumor cells. Without being constrained by theory, more effective antitumor activity is particularly evident in the presentation of calreticulin. Triggered by increased release of DAMP such as HMGB1 or ATP and / or other HMGB1 This is thought to be due to a stronger immune response to such infected cells that may be affected. The released HMGB1 binds to TLR4 and RAGE, triggering an inflammatory response. It can be triggered, but the released ATP is a "find-me" signal to immune cells. It seems it can also function as a lamp.

[0066] In summary, the novel vaccinia virus strain IOVA and its derivatives, vectors, and recombinants The body has the ability to replicate in mammalian cancer strains, and is the gold standard vaccinia wii. Compared to RusWR, it has an improved ability to destroy cell cycle-activated cells and / or tumor cells. Because it induces lysis, it can be described as lytic, or in the case of cancer cells, oncolytic.

[0067] IOVA or its derivatives, due to their unique immunological profile and safety characteristics, Because it is comparable to or superior to already established vaccinia virus strains, In medicine, particularly in oncological approaches, or as a tumor lytic agent or for cancer Alternatively, it can be used as a vaccine against other pathogens.

[0068] Importantly, IOVA viruses and their derivatives or recombinants are used when infecting cells. It is particularly suitable for inducing immunogenic cell death, thereby contributing to immunotherapeutic approaches. They are highly recommended for use as a safe and effective immunotherapy or immune treatment. It is particularly suitable as a novel viral platform for tumor treatment. [Brief explanation of the drawing]

[0069] [Figure 1A] Syncytial formation in cancer cells after IOVA virus infection. Fluorescence micrographs (40x magnification) are shown after infection (24 hours after infection) of human cancer cell lines. HeLa and CT26 cells were infected with a MOI of 0.5. 143B, MCF-7, and LLC1 were infected with a MOI of 5. mCherry is expressed from all viruses under the P11 promoter. When cells were infected with IOVA / A56-, large-scale cell fusion (synctiforms) could be observed. [Figure 1B]Syncytial formation in cancer cells after IOVA virus infection. Fluorescence micrographs (40x magnification) are shown after infection (24 hours after infection) of mouse cancer cell lines. HeLa and CT26 cells were infected with a MOI of 0.5. 143B, MCF-7, and LLC1 were infected with a MOI of 5. mCherry is expressed from all viruses under the P11 promoter. When cells were infected with IOVA / A56-, large-scale cell fusion (synctiforms) could be observed. [Figure 2A-1] Virogenesis of IOVA virus in human and mouse tumor cells. Human tumor cell lines were infected with WR / TK-, IOVA / A56-, or IOVA / A56+ at an MOI of 5, and offspring were measured by plaque assays at various time points. Viral yield was quadruplicated for each cell line by performing two independent experiments. Mean + SD is plotted. *, significant p<0.05 compared to WR / TK-. #, significant p<0.05 compared to IOVA / A56+. [Figure 2A-2] This is a continuation of Figure 2A-1. [Figure 2B] Virogenesis of IOVA virus in human and mouse tumor cells. Mouse tumor cell lines were infected with WR / TK-, IOVA / A56-, or IOVA / A56+ at an MOI of 5, and offspring were measured by plaque assays at various time points. Viral yield was quadruple-assessed for each cell line by performing two independent experiments. Mean + SD is plotted. *, significant p<0.05 compared to WR / TK-. #, significant p<0.05 compared to IOVA / A56+. [Figure 3A-1] The presence of IOVA virus exhibits increased cytotoxicity against tumor cells. Cancer cells were infected with WR / TK-, IOVA / A56-, or IOVA / A56+ at doses ranging from 100 to 0.0005 PFU / cell. Cell viability was determined 3 days post-infection. Human cancer cells were tested. Four different replicates were quantified for each cell line, and the mean ± SD of each MOI is shown. [Figure 3A-2] This is a continuation of Figure 3A-1. [Figure 3B-1]The presence of IOVA virus exhibits increased cytotoxicity against tumor cells. Cancer cells were infected with WR / TK-, IOVA / A56-, or IOVA / A56+ at doses ranging from 100 to 0.0005 PFU / cell. Cell viability was determined 3 days post-infection. Mouse cancer cells were tested. Four different replicates were quantified for each cell line, and the mean ± SD of each MOI is shown. [Figure 3B-2] This is a continuation of Figure 3B-1. [Figure 4A-1] Induction of immunogenic cell death by IOVA virus. (A) Analysis of calreticulin expression on the surface of infected cells. The indicated tumor cell lines were infected with WR / TK-, IOVA / A56-, or IOVA / A56+ at a MOI of 5, and the calreticulin+ cell population 24 hours post-infection was determined by flow cytometry. Uninfected cells (mock) and 1 μM staurosporine were used as negative and positive controls, respectively. (A) Percentage of calreticulin+ cells. Values ​​and means ± SEM of individual replicates of various treatments are plotted. ELISA assay and ENLITEN ATP assay system were used, respectively, to determine such concentrations 24 hours post-infection (MOI of 5) of the indicated tumor cell lines. Data were obtained quadruplicate and plotted as magnification change + SD relative to WR / TK-. *, significant p<0.05 compared to WR / TK-. #, significant p<0.05 compared to mock. [Figure 4A-2] This is a continuation of Figure 4A-1. [Figure 4B] Concentrations of HMGB1 in cell supernatant after infection with IOVA virus. ELISA and ENLITEN ATP assay systems were used, respectively, to determine such concentrations 24 hours after infection (MOI of 5) of the indicated tumor cell lines. Data were obtained in quadruplicate and plotted as divergence change + SD relative to WR / TK-. *, significant p<0.05 compared to WR / TK-. #, significant p<0.05 compared to mock. [Figure 4C]Concentration of ATP in the cell supernatant after infection with IOVA virus. ELISA and ENLITEN ATP assay systems were used, respectively, to determine such concentrations 24 hours after infection (MOI of 5) of the indicated tumor cell lines. Data were obtained in quadruplicate and plotted as divergence change + SD relative to WR / TK-. *, significant p<0.05 compared to WR / TK-. #, significant p<0.05 compared to mock. [Figure 5A-1] PCR assay for virus identification. Figure 5A: Sequence of PCR products used in the PCR assay. [Figure 5A-2] This is a continuation of Figure 5A-1. [Figure 5B] PCR assay for virus identification. Figure 5B: This assay clearly identifies the sequence of the IOVA strain because it produces a unique pattern of two bands (2320 and 920 bp) for IOVA, in contrast to all other vaccinia virus strains which show a single 3300 bp band for WR, COP, and CVA. The MVA strain does not produce any amplification product due to the deletion of C2L. [Figure 6] Number of nuclei in syncytia after IOVA virus infection. Human tumor cell lines were infected with WR / TK-, IOVA / A56- / A26-, or IOVA / A56+ / A26- at a MOI of 5. Sixteen hours post-infection, cultures were stained with Hoechst 33342, and the number of nuclei in a single syncytia was counted under a microscope. Individual replicate and mean ± SEM values ​​are plotted. ***, significant compared to WR / TK-, p<0.0001. [Figure 7A] Plaque size in cancer cells. A monolayer of cancer cells was infected with the indicated virus at an MOI of 0.0001 for 1 hour and cultured for 4 days covered with a 1:1 mixture of culture medium and 1% carboxymethylcellulose. After fixation and crystal violet staining, the diameter of the plaques was measured. Individual replicate values ​​and the mean ± SEM are plotted. *, significant p<0.05 compared to WR / TK-. #, significant p<0.05 compared to IOVA / A56-. [Figure 7B] This is a continuation of Figure 7A. [Figure 7C]This is a continuation of Figure 7A. [Examples]

[0070] Example 1: Deletion in the IOVA genome Novel immunotumorbic vaccinia virus (IOVA) strains contain thymidine kinase (TK It is generated by incorporating a deletion in the J2R gene and confers selective replication in cancer cells. Furthermore, the mCherry gene is used to monitor viral replication in vaccinia. It is cloned into the TK site under the control of the Russ-specific promoter P11.

[0071] Furthermore, the newly generated IOVA contains B21R* and C1, which are considered immunomodulatory factors. 0L, C9L, C4L, C2L, N1L, N2L, M1L, A26R, A51R, A52 Among the genes selected from ORFs of R, A55R, A56R, and B13R / B14R This includes several deletions or functional inactivations. The proteins that encode these genes The functions of the substance are summarized in Table 1 above.

[0072] The IOVA genome further includes mutated versions of the A26R and / or A56R genes. It is characterized by the presence of the A26 protein in virions, which directly indicates viral- It interferes with the cell fusion mechanism, and its deletion is associated with syncytial formation. A56R is found in red blood cells. It encodes a viral regulatory protein with bulb aggregation activity, and in vaccinia virus... This inactivation is thought to result in a virus with a fusion phenotype. Gene deletion or partial deletion, and all functional inactivation or gene insertions, are not recognized in sequencing. This has been confirmed by the authorities.

[0073] Example 2: Syncytium formation induced by IOVA To evaluate the advantages and disadvantages of syncytial formation in tumor destruction, the inventors of the present invention have used homologous syncytial formation. By substitution, the wild-type vaccinia virus A56R gene sequence in the novel IOVA genome It recovered. The obtained virus is named IOVA / A56-, a shortened form of A56R. It was named IOVA / A56+ in comparison to John's IOVA.

[0074] During infection, cells infected with the IOVA / A56 virus fuse with adjacent cells. (Figure 1) was observed, and the formation of giant syncytia was tracked by mCherry expression in humans. This was clearly observed in both mouse tumor cell lines. As hypothesized, in IOVA / A56+ Expression of wild-type A56 restores a phenotype very similar to that of vaccinia virus WR strains. It did not result in cell formation.

[0075] Furthermore, fusion of up to 10 cells can be observed even after infection with IOVA / A56+ / A26-. Because of the possibility that wild-type A56 expression in IOVA / A56- / A26- is syncytial It was observed that this could be explained by partially blocking body formation (Figure 6).

[0076] Example 3: IOVA replication capability A broad panel of human and mouse cancer cell lines compared with the standard vaccinia virus WR strain. The replication capability of the IOVA was tested.

[0077] To monitor newly generated IOVA replication, two IOVA virus isolates were used. The replication capabilities of (A56- and A56+) and WR / TK- as a control were compared with several H The study was conducted using both oocyte and mouse cancer cell lines.

[0078] For this purpose, human and mouse tumor cell lines were selected as WR / TK-, IOVA / A56-, and It is IOVA / A56+, infected with a MOI of 5, and its offspring plaques at various time points. Measured by Issey. Virus yield was determined by conducting two independent experiments. Each cell line was evaluated quadruple (to date, Rojas JJ et al., (As described in Cell Rep. 2016).

[0079] As shown in Figure 2, both IOVA viruses (A56- and A56+) were tested to the extent that In most cell lines, growth curves were very similar to those of the control line WR / TK-, and syncytial formation was observed. At the initial stage of mature IOVA, the yield only decreased slightly.

[0080] Example 4: Cytotoxicity of IOVA A broad panel of human and mouse cancer cell lines compared with the standard vaccinia virus WR strain. The cytotoxic effects of IOVA infection were tested.

[0081] For this reason, various cancer cell lines are classified as WR / TK-, IOVA / A56-, or IOV In A / A56+ patients, infection was administered with doses ranging from 100 to 0.0005 PFU / cell. On the third day, the cell viability was determined. Both human and mouse cancer cells were tested. Different replicates were quantified for each cell line, and the mean ± SD of each MOI is shown.

[0082] Interestingly, infections with IOVA virus are more common than those with vaccinia virus WR. This resulted in a clearly enhanced level of cytotoxicity in rhinocytes (Figure 3). Vaccinia Even at the highest MOI (Moment of Infection), virus-induced WR is approximately 70-80% of the cancer cells in culture. 0% were killed. Conversely, 95% of the IOVA / A56 virus cultured tumor cells were killed. It can kill up to 100%, and EC50 (the amount of virus needed to kill 50% of cells) The amount of (of) was reduced.

[0083] Surprisingly, IOVA / A56- reduces the cell culture viability of HeLa cells by 50%. The amount of virus required to cause this was compared to that of vaccinia virus WR (WR / TK-). It was reduced by more than 40 times. IOVA / A56+ also showed results in cancer cells in vitro. In contrast, it showed an enhanced cytotoxic phenotype, but the virus that recovered in A56 was HeLA. CT-26 and LLC1 cells were killed at the same rate as IOVA / A56-. Nevertheless, in 143B and MCF-7 cells, cytotoxicity was obtained with WR / TK infection. It is very similar to that, with enhanced tumor destruction due to large syncytial formation mediated by the virus. This suggests that it may be contributing to the above.

[0084] Example 5: Phenotype of large IOVA virus plaques in cancer cells Increased plaque size in cancer cells allows for better virus spread throughout the tumor. It was associated with dispersion and higher antitumor activity. Testing IOVA virus plaque size. To do this, a panel of cancer cell lines was infected with an MOI of 0.0001, and one hour after infection, Infected cells were covered with carboxymethylcellulose and cultured. After 4 hours post-infection, the culture solidified. The plaque was then identified, stained with crystal violet, and its diameter was determined. As shown in Figure 7, both IOVA / A56- and IOVA / A56+ are WR / TK-U Compared to the virus control, it induced larger plaques in all cancer cell lines tested. In eLa cells, plaques after IOVA virus infection are compared to WR / TK- They were, on average, 40% larger. Impressively, very large plaques were found to contain IOVA virus Observed in 143B and MCF-7 cells after infection by WR / TK- The plaque produced is twice the diameter of the plaque, and in the case of MCF-7, it is 2. It was a plaque six times larger. Regarding plaque size, the generation of large syncytia by IOVA / A56- is associated with MCF- Except for the case of breast cancer cell lines, there was no significant effect, and compared to IOVA / A56-, IOV Plaques generated by the A / A56+ virus were 1.4 times smaller.

[0085] Example 6: IOVA virus is involved in the presentation of calreticulin on the cell membrane of cancer cells and immunotherapy. It induces epidemic cell death. Is it possible that the IOVA virus can induce and enhance the immune response against cancer cells? To test for this, the inventors first used IOVA / A56-, or IOVA / Surface calculus of human cancer cells after infection with A56+ or control virus WR / TK- Reticulin (CRT) exposure was analyzed by flow cytometry.

[0086] For this purpose, cells were infected with a MOI of 5, and 24 hours after viral infection, non-enzymatic cells were used. Detachment was performed using a vesicular dissociation solution. Calreticulin was human anti-calreticulin-A lexaFluor405 antibody (Abcam, Ref N° ab210431) 4 Detected by incubation at °C for 1 hour. Uninfected cells and stauropoly ¹(1 μM) was used as a negative and positive control, respectively.

[0087] During HeLa cell infection, WR / TK- induces surface exposure of CRT in approximately 15% of cells. Guideline (Figure 4a); Conversely, when infected with both IOVA viruses, the rate exceeds a surprising 80%. High levels of cells expressed CRT on their surface. Similarly, CRT exposure was observed in 143B cells. It increases from approximately 35% (in the case of WR / TK-) to approximately 90% (in the case of IOVA virus), In MCF-7 cells, the percentage increased from 3% to over 72%.

[0088] Further investigation into the potential induction of significant immunogenic cell death upon infection with IOVA virus. To achieve this, the release of HMGB1 and ATP is used in ELISA assays and luciferase media. The determinations were made using each ATP assay system.

[0089] In all cell lines tested, both IOVA / A56- and IOVA / A56+ showed WR / Significantly higher concentrations of HMGB1 were detected in the supernatant of infected cells compared to cells infected with TK-. (Figure 4b), 1.23 times (143B cells, IOVA / A56-) to 1.68 times (MC The range of F-7 cells (IOVA / A56+) was increased. ATP concentration in the supernatant of infected cells. However, when infected with the IOVA virus, WR / TK levels increase compared to post-infection levels (Figure). 4c), 1.12x (143B cells, IOVA / A56+) ~ 2.27x (HeLa, I This represented an increase in the range of OVA / A56-).

[0090] These results indicate that IOVA virus, not vaccinia virus WR, is the cause of infection in humans. It demonstrates that it induces immunogenic cell death in cancer cells. Therefore, IOVA is considering it for clinical trials. As a particularly promising candidate virus that could show significant benefits in terms of antitumor activity, I can suggest it.

[0091] Example 7: PCR assay for identification of IOVA virus or its derivatives To identify the IOVA strain, viral DNA is obtained from cell extracts of infected cells. Digest with Nase K and follow the manufacturer's instructions to use the QIAamp genomic DNA kit (QIAG It is isolated using EN. C2L-C1L-N1L-N2L fragment (Figure 5a; distribution The unique sequence of IOVA covering column number 1) was obtained by PCR using the following oligonucleotides. Amplified: forward 5'-ATGTTATCCTGGACATCGTAC-3' (distribution Row number 2) and reverse 5'-TCATGACGTCCTCTGCAATGG-3'(distribution (Column number 3). PCR products using these two primers are better than the original SEQ ID NO: 1. The result is 50 bp larger, and for stability reasons, an additional 50 bp is included in the PCR reaction design. The PCR product (SEQ ID NO: 4) was purified using the QIAquick PCR purification kit. Then, digest it with BstXI restriction enzyme.

[0092] Using this assay, the IOVA strain can be clearly identified because, in contrast to all other vaccinia strains that show a single 3384 bp band, it generates a unique pattern specific to two DNA bands (2361 and 923 bp) that are visualized by electrophoresis in 1% agarose. PCR of MVA genomic DNA does not produce any PCR product due to the absence of the C2L sequence (Figure 5b). The present invention may include the following embodiments. [1] An immunomodulatory vaccinia virus wherein the virus is capable of replicating in mammalian cells, causes calreticulin transfer to the membrane of infected cells, and thereby promotes immunogenic cell death. [2] The immunomodulatory vaccinia virus according to claim 1, wherein the virus is A56R negative, or the virus is A26R negative and A56R negative. [3] The immunomodulatory vaccinia virus according to claim 1 or 2, wherein the virus and / or any derivative thereof is defined by the presence of a single BstXI restriction enzyme site on the nucleotide sequence of a mutated C2-C1-N1-N2 region defined in SEQ ID NO: 1, which is part of the viral genome. [4] The immunomodulatory vaccinia virus according to any one of claims 1 to 3, wherein the virus is K1L positive. [5] The immunomodulatory vaccinia virus according to any one of claims 1 to 4, wherein the virus further comprises one or more functionally inactivated immune evasion genes selected from the group of open reading frames consisting of B21R, C10L, C9L, C4L, C2L, N1L, N2L, M1L, A51R, A52R, A55R, and B13R / B14R. [6] The immunomodulatory vaccinia virus according to any one of claims 1 to 5, wherein the virus further comprises at least one functionally inactivated, partially deleted, or completely deleted gene selected from the group consisting of J2R, C11R, and F4L. [7] The immunomodulatory vaccinia virus according to any one of claims 1 to 6, wherein the virus is capable of replicating and is lytic in cell cycle-activated cells and / or tumor cells. [8] The immunomodulatory vaccinia virus according to any one of claims 1 to 7, wherein the virus causes syncytial formation upon infection. [9] A nucleic acid sequence or fragment thereof encoding an immunomodulatory vaccinia virus according to claims 1 to 8.

[10] A viral vector or a derivative of immunomodulatory vaccinia virus comprising the nucleic acid sequence or a substantial portion thereof as described in claim 9.

[11] An immunomodulatory vaccinia virus according to any one of claims 1 to 8, characterized by carrying one or more insertion sites having at least one insertion of one or more recombinant transgenes, a vector according to claim 10, and a derivative of an immunomodulatory vaccinia virus.

[12] The recombinant immunomodulatory vaccinia virus or vector according to claim 11, wherein the introduced gene is selected from the group comprising genes encoding tumor antigens, tumor-associated antigens, disease-associated antigens, and pathogen-derived antigens.

[13] An immunomodulatory vaccinia virus according to any one of claims 1 to 8, a vector according to claim 10, or a recombinant immunomodulatory vaccinia virus according to claim 12, for use in pharmaceuticals.

[14] [[ID=4�]] For use in the treatment of cancer and / or as a cancer vaccine, an immunomodulatory vaccinia virus according to any one of claims 1 to 8, a vector according to claim 10, or a recombinant immunomodulatory vaccinia virus according to claim 12.

[15] A pharmaceutical composition comprising an immunomodulatory vaccinia virus according to any one of claims 1 to 8, a vector according to claim 10, or recombinant immunomodulatory vaccinia according to claim 12, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0093] References D. Kirn, T. Hermiston, F. McCormick, ONY X-015: clinical data are encouraging. Na ture medicine 4, 1341 (Dec, 1998). C. J. Breitbach et al., Oncolytic vaccin ia virus disrupts tumour-associated vasc ulature in humans. Cancer research 73, 1 265 (Feb 15, 2013). S. H. Thorne et al., Targeting localized immune suppression within the tumour th rough repeat cycles of immune cell-oncol ytic virus combination therapy. Molecula r therapy : the journal of the American Society of Gene Therapy 18, 1698 (Sep, 2 010). C. J. Breitbach et al., Intravenous deli very of a multi-mechanistic cancer-targe ted oncolytic poxvirus in humans. Nature 477, 99 (Sep 1, 2011). J. Heo et al., Randomized dose-finding c linical trial of oncolytic immunotherape utic vaccinia JX-594 in liver cancer. Na ture medicine 19, 329 (Mar, 2013). H. J. Zeh et al., First-in-man study of western reserve strain oncolytic vaccini a virus: safety, systemic spread, and an titumour activity.Molecular therapy : th e journal of the American Society of Gen e Therapy 23, 202 (Jan, 2015). A. Volz, G. Sutter, Protective efficacy of Modified Vaccinia virus Ankara in pre clinical studies. Vaccine 31, 4235 (Sep 6, 2013). H. Meyer, G. Sutter, A. Mayr, Mapping of deletions in the genome of the highly a ttenuated vaccinia virus MVA and their i nfluence on virulence.The Journal of gen eral virology 72 ( Pt 5), 1031 (May, 199 1). R. T. Zhang, S. D. Bines, C. Ruby, H. L. Kaufman, TroVax((R)) vaccine therapy fo r renal cell carcinoma. Immunotherapy 4, 27 (Jan, 2012). I. Marigo, L. Dolcetti, P. Serafini, P. Zanovello, V. Bronte, Tumour-induced tol erance and immune suppression by myeloid derived suppressor cells. Immunol Rev 2 22, 162 (Apr, 2008). S. J. Gardai et al., Cell-surface calret iculin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte.Cell 123, 321 ( Oct 21, 2005). G. L. Smith et al., Vaccinia virus immun e evasion: mechanisms, virulence and imm unogenicity.The Journal of general virol ogy 94, 2367 (Nov, 2013). Goebel et al.The complete DNA sequence o f vaccinia virus. Virology, 1990 Nov;179 (1): 247-66, 517-63. L. Galluzzi, A. Buque, O. Kepp, L. Zitvo gel, G. Kroemer, Immunogenic cell death in cancer and infectious disease. Nat Re v Immunol 17, 97 (Feb, 2017). Rojas JJ et al., Manipulating TLR signal ing increases the anti-tumour T cell res ponse induced by viral cancer therapies. Cell Rep., 2016 Apr 12;15(2): 264-73.

Claims

1. A genetically modified immunomodulatory vaccinia virus wherein the virus is capable of replicating in mammalian cells and has a functionally active K1L open reading frame and functionally inactive A56R, A26R, and A52R open reading frames, causing calreticulin transfer to the membrane of infected HeLa cells, thereby promoting immunogenic cell death.

2. The immunomodulatory vaccinia virus according to claim 1, wherein the virus further comprises one or more functionally inactive immune evasion genes selected from the group of open reading frames consisting of B21R, C10L, C9L, C4L, C2L, N1L, N2L, M1L, A51R, A55R, and B13R / B14R.

3. The immunomodulatory vaccinia virus according to claim 1 or 2, wherein the virus further comprises at least one functionally inactivated, partially deleted, or completely deleted gene selected from the group consisting of J2R, C11R, and F4L.

4. The immunomodulatory vaccinia virus according to any one of claims 1 to 3, wherein the virus is capable of replicating and is lytic in cell cycle-activated cells and / or tumor cells.

5. The immunomodulatory vaccinia virus according to any one of claims 1 to 4, wherein the virus causes syncytial formation upon infection.

6. A nucleic acid encoding an immunomodulatory vaccinia virus according to any one of claims 1 to 5.

7. A viral vector or immunomodulatory vaccinia virus comprising the nucleic acid described in claim 6.

8. An immunomodulatory vaccinia virus according to any one of claims 1 to 5, characterized by carrying one or more insertion sites having at least one insertion of one or more recombinant transgenes.

9. The recombinant immunomodulatory vaccinia virus according to claim 8, wherein the introduced gene is selected from the group including genes encoding tumor antigens, tumor-associated antigens, disease-associated antigens, and pathogen-derived antigens.

10. A pharmaceutical composition comprising the immunomodulatory vaccinia virus described in any one of claims 1 to 5.

11. A pharmaceutical composition for the treatment or prevention of cancer, comprising the immunomodulatory vaccinia virus described in any one of claims 1 to 5.

12. A pharmaceutical composition comprising an immunomodulatory vaccinia virus according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier, diluent, or excipient.

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