Immunomodulatory replication-efficient vaccinia virus strains

Abstract

The present invention relates to new immunomodulatory, replication-efficient vaccinia virus strains (IOVA) and their derivatives for use in medicine.

Description

Field of the Invention

[0001] The present invention relates to new immunomodulatory, replication-efficient vaccinia virus strains (IOVA) and their derivatives for use in medicine. Background of the Invention

[0003] Recently, the virus has gained market acceptance for the first time as an oncolytic virus in the treatment of human cancer. Such success has refocused scientific interest on viruses in general, and also on the use of vaccinia virus for cancer treatment. The use of vaccinia virus has been shown to be effective not only in vaccination methods (e.g., to prevent human poxvirus infection), but has also been applied to many other medical treatments and clinical trials, such as tumor vaccination.

[0004] The scientific community knows from data obtained in the first clinical studies of oncolytic viruses that these viruses or designed viral vectors are able to replicate selectively in cancer cells and actively promote the lysis of such infected cancer cells. However, direct lysis of such infected cancer cells is rarely sufficient to eradicate large tumor masses and rarely cures metastatic disease. These results indicate a growing need not only for more potent and selective viruses, but also for alternative tumor-destroying mechanisms to avoid disease recurrence.

[0005] Recently, vaccinia virus-based viruses and viral vectors have been tested as oncolytic agents and are considered the most promising due to their ability to destroy tumors through mechanisms other than direct tumor cell lysis (Kirn et al., 2009). On the one hand, viral replication in tumor-associated endothelial cells has been shown to lead to disruption of tumor blood flow, hypoxia, and extensive tumor necrosis (Breitbach et al., 2013); on the other hand, vaccinia virus replication in tumors can induce antitumor immune responses due to the release of tumor-associated antigens (TAA) after lysis and overcoming the local immunosuppression present in the tumor (Thorne et al., 2010).

[0006] These alternative mechanisms have been shown to be key in clinical evaluation and highlight the key role of the immune system in determining the activity of oncolytic vectors. In randomized clinical trials, the most effective viruses have been shown to express immune-activating cytokines to optimize the activation of cellular immune responses (Breitbach et al., 2011; Heo et al., 2013). However, even with these more potent cytokine-expressing viruses, some patients do not induce an effective immune response against tumors. The generation of an effective anti-tumor immune response requires the activation and synergy of even more and different immune cell lineages, and the expression of a single cytokine can hardly change the overall immunity triggered by the virus. Therefore, it is still necessary to study the generation of new strains with higher activation robust immune response capabilities.

[0007] It is therefore an object of the present invention to provide newly adapted and / or more potent viruses and / or viral vectors for the treatment of human diseases, wherein said viruses or vectors elicit and / or activate an additional and more powerful but (for safety reasons) highly specific immune response in a patient in need of such treatment following infection.

[0008] This object has been solved by a newly identified immunomodulatory, replication efficient vaccinia virus strain as described in claim 1 of the present application. Further embodiments relate to variants of this newly identified and / or adapted immunomodulatory vaccinia virus and vectors, preferred uses and compositions comprising the newly identified and / or adapted vaccinia virus strain are described in the appended claims.

[0009] In studying the immune response to known vaccinia virus strains, and in particular in comparison with well-characterized vaccinia virus strains (Western Reserve (WR)), the inventors were able to isolate, generate and characterize new vaccinia virus strains. This new strain exhibits significantly improved immunological characteristics. Without being bound by theory, we believe that the new strain also exhibits a strong promotion of increased immunogenic cell death after infection of tumor cells and thus redirects the immune response to infected and uninfected tumor cells.

[0010] The vaccinia virus WR strain is a well-defined virus strain that has been tested as oncolytic due to its high replication capacity in tumor cells and has been used to deliver GM-CSF to increase the immune response against tumors. In order to provide a vector with tumor selectivity, different vaccinia virus genes involved in activating the metabolism of infected cells have been successfully deleted, thereby limiting the replication of the resulting viral vector to cells with a high replication index. The deletion of genes such as thymidine kinase or viral growth factors has been included in the candidate viruses tested in clinical trials (Zeh et al., 2015).

[0011] WO2015027163 is an example in which an oncolytic vaccinia virus is described that contains genomic deletions in some typical viral immune escape genes. In this particular case, the oncolytic virus contains deletions in, for example, the B8R, B18R, and A35R genes.

[0012] EP2136633 is another example, which describes the therapeutic use of GM-CSF expressing vaccinia virus in oncolytic therapy.

[0013] In these oncology approaches, the VACV WR strain is considered the current gold standard for the development and comparison of newly developed oncolytic vaccinia viruses.

[0014] However, despite this alleged success, there are still some concerns considering VACV WR as the basis for a treatment approach for critically ill cancer patients. Since VACV WR was selected in vivo after multiple passages in the mouse brain, which actually resulted in a high replication capacity of the virus in mouse cells, it also resulted in a surprising virulence due to neurotropism and neurotoxicity, which may hinder its safe use in the treatment of cancer patients.

[0015] However, in the context of the present application, vaccinia virus WR was chosen as a comparison to the newly developed vaccinia virus strain of the present invention, wherein the main object of the present invention is to provide a novel immunomodulatory, replication-competent vaccinia virus strain having significantly improved immunological characteristics and high lytic and / or oncolytic capacity.

[0016] Additional comparisons with the highly attenuated MVA (Modified Vaccinia Ankara) strain of vaccinia virus seem to be useless, since MVA is characterized by its host range restriction, or in other words, its replication defect in mammalian host cells. MVA has been widely used to deliver antigens for vaccination against pathogens (for review, see Volz and Sutter, 2017), and has been characterized for its biological and immunological characteristics (Meyer et al., 1991). In addition, MVA is a highly modified vaccinia virus that cannot replicate in mammalian cells and has been previously used for vaccination against tumors by delivering tumor-associated antigens (TAAs) (Zhang et al., 2012). Despite the fact that this virus seems to show an ideal safety profile and has been shown to be effective in generating an immune response against tumor antigens expressed in vivo, most patients do not show any verifiable anti-tumor response; it seems that the immunity elicited is hampered by immunosuppression occurring locally within the tumor, resulting in an ineffective anti-tumor response (Marigo et al., 2008).

[0017] Thus, while progress can be said to have been made toward improved immunotherapy, there is still a need to use more potent vaccinia virus strains for cancer virotherapy to establish an effective immune response capable of eradicating cancer.

[0018] Now, the present invention describes a further step towards this goal and discloses a novel vaccinia virus strain which is replication competent in human and mouse cells and, to the best of the inventors' knowledge, is even the first vaccinia virus in general that normally causes the translocation of the immune-related calreticulin (CRT) to the outer membrane and thus causes exposure of calreticulin on the outer cell membrane of infected cells, thus eliciting a potent and improved immune response.

[0019] Calreticulin (CRT) appears to be a multifunctional protein and has been described as being involved in the transcriptional regulation of hormone-responsive DNA elements and in the maturation of MHC class I proteins; it has also been described as binding and inactivating Ca in the endoplasmic reticulum. 2+ ions or misfolded proteins, and is 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, a portion of the protein can be transferred from the ER lumen to the cell surface; more specifically, it has been shown that CRT acts as an "eat-me" signal on the cell surface (Gardai et al., 2005), thereby mediating the phagocytosis of CD91-positive cells (mainly macrophages and dendritic cells (DC)).

[0020] The novel vaccinia virus strain is described as being replication-competent in mammalian cells. Without being limited by this theory, it is believed that this property depends inter alia on the presence and functional activity of the K1L gene in the viral backbone. In particular, the novel strain has been shown to replicate well in mammalian (and in particular human and mouse) cell lines, for example in cancer cells such as HeLa cells, 143B cells, CT26 cells, LLC1 cells or MCF-7 cells. In contrast to these results, it should be noted that MVA is known not to replicate in these cells or in cells of other mammalian origin.

[0021] In addition, the novel vaccinia virus strain shares several housekeeping genes with all and / or at least some other vaccinia virus strains. However, its immunological characteristics are very different and are referred to in the context of this application as IOVA (immuno-oncolytic vaccinia).

[0022] Many genes of the vaccinia virus genome have been described as immune escape genes (Smith et al., 2013), which form and determine the immunological characteristics of different virus strains. Over the years, studies have been conducted to identify the potential mechanisms of action of such immune escape genes. According to the results obtained, these immune escape genes express soluble receptors for cytokines in particular, which compete with natural receptors and reduce the efficacy of cytokine-induced immune responses. Other genes block the intracellular pathways that activate different immune activation genes, and therefore also interfere with and reduce virus-specific immune responses.

[0023] To summarize briefly, Table 1 lists the immune escape genes of vaccinia virus identified so far and their presence or absence in different vaccinia strains. It also indicates the potential function or mechanism of action of the corresponding gene product.

[0024] Table 1: Known vaccinia immunomodulators compared to IOVA sequences

[0025]

[0026]

[0027] Table 1 (continued)

[0028]

[0029] ORF: Nomenclature of vaccinia COP strain. *Nomenclature in CPXV-GRI.

[0030] +: complete and functional;

[0031] --: completely or partially missing (non-functional).

[0032] The nomenclature of the open reading frames (ORFs) used in Table 1 refers to the nomenclature system established for the Copenhagen (COP) strain of vaccinia virus (Goebel et al., 1990), and is identified by the letters assigned to the viral genomic DNA fragments of decreasing size produced by the restriction endonuclease HindIII. The exceptions are ORFs C8L and B21R with letters related to DNA fragments in the CPXV-GRI genome of the cowpox virus, because they are not present in the COP strain of vaccinia virus. Different ORFs in such HindIII fragments are identified by numbers. The letters at the end indicate the direction of transcription: R stands for right and L stands for left.

[0033] In addition, Table 1 provides a comparison of the functionality of the immune escape genes identified so far and the well-characterized vaccinia virus strains COP, WR, MVA, and the MVA ancestor strain CVA. In Table 1, it is indicated whether the different genes are functional (indicated by "+") or partially / completely deleted and functionally inactive (indicated by "-") in the different viral genomes.

[0034] The novel vaccinia virus strain, as described herein and identified below as IOVA, has its own immunological characteristics. It shares some functionally expressed genes with CVA or WR, and also shares some functionally inactivated genes with MVA. The inventors also confirmed this at the sequence level, comparing the full-length sequence of an isolate of the novel vaccinia virus IOVA with the published sequences of WR, COP, CVA and MVA.

[0035] The inventors have also developed a PCR assay for easily identifying novel IOVA strains. To this end, a DNA fragment of any vaccinia virus to be tested can be amplified with PCR oligonucleotides (primers) that specifically bind in a region or sequence of the C2L ORF (open reading frame) on the one hand and in a region or sequence of the N2L ORF on the other hand. PCR performed by standard parameters will produce an amplified fragment containing the ORF C2L-C1L-N1L-N2L sequence for most vaccinia viruses. These PCR products are double-stranded DNA fragments and as such are then enzymatically treated with the restriction endonuclease BstXI. Thus, the specific DNA products obtained in, for example, vaccinia strains WR, COP and CVA will not be cleaved after agarose gel electrophoresis and will present a single band of about 3280 bp (molecular weight). In contrast, the specific IOVA DNA product will be cleaved by BstXI and will present two bands in the agarose gel, with molecular weights of about 2360 and 920 bp, respectively. The explanation for this is that the novel IOVA virus carries a highly specific mutation in the N1L ORF, resulting in a functionally inactive N1L and a new BstXI restriction site. In the case of MVA, the C2L gene sequence in the MVA genome is deleted, and therefore, this specific PCR will not amplify a DNA product.

[0036] As can be seen in Table 1 and the data provided herein, although IOVA (itself and all its derivatives) shares many genes and / or functional deletions with CVA, WR and MVA, it is unique not only in its safety profile but also in its immunological profile.

[0037] This immunological feature, which is the essential core of the present invention, is particularly related to the presentation of calreticulin on the outer membrane of virus-infected cells.

[0038] Thus, the immunological characteristics of the novel strains are described as in particular triggering measurable calreticulin presentation on the outer membrane of virus-infected cells. Without being bound by this theory, it is believed that this property depends on the functional inactivation, partial or complete deletion of at least one of the immune escape genes in the viral backbone selected from the group consisting of: B21R*, C10L, C9L, C4L, C2L, N1L, N2L, M1L, A26R, A51R, A52R, A55R, A56R and B13R / B14R (Vaccinia Cop strain nomenclature, except for the *, which corresponds to the CPXV-GRI nomenclature).

[0039] Due to these functionally inactivated, partially or completely deleted genes, the newly generated viruses not only lose some of their abilities to evade the host's immune response, but also additionally introduce a shift in immune escape strategies.

[0040] Table 2: Differences between IOVA and WR

[0041]

[0042]

[0043] ORF: According to the nomenclature of vaccinia virus-COP strain.

[0044] +: complete and functional;

[0045] -: completely or partially missing or functionally inactive.

[0046] In this context, this effect of calreticulin is particularly interesting: calreticulin is normally a strictly internal protein and localized in the endoplasmic reticulum, but when translocated to the cell surface of cancer cells following infection with the novel vaccinia virus strain, it can elicit and enhance specific immune responses against virus-infected cells. Calreticulin is a danger-associated molecular pattern (DAMP) that has been described as acting as an "eat me" signal when present on the outer membrane and can be used as a marker of immunogenic cell death. Compared to vaccinia virus WR, the newly generated vaccinia virus strain IOVA lysed or killed cancer cells at a much lower dose and was therefore more efficient (Figure 3A).

[0047] The inventors show that WR kills only 70%-80% of cultured tumor cells in human and mouse cell lines; approximately 20%-30% of cells escape virus-mediated destruction and remain metabolically active even at greatly increased multiplicity of infection.

[0048] In contrast, the novel IOVA virus derivatives destroyed at least and even more than 95% of the cells in all tested cell lines; very surprisingly, in the human cell line HeLa, the novel IOVA derivatives showed highly improved cytotoxicity, with an efficiency at least 40 times higher than that of vaccinia virus WR, but with regard to the growth capacity of IOVA in HeLa, it was very similar to that of vaccinia virus WR. This more complete efficiency in killing virus-infected cells particularly reduces the risk of tumor remission and is therefore highly advantageous.

[0049] Therefore, the use of the novel vaccinia virus strains for infecting cell cycle activated cells and / or tumor cells is not only very advantageous, but also in this case particularly suitable for use as oncolytic viruses or for the treatment of cancer, but also for vaccination purposes and more general immunostimulatory purposes, and thus in medicine in general.

[0050] In addition, according to another embodiment, the novel vaccinia virus strain IOVA comprises a functionally inactivated A56R gene. The lack of expression of this gene product is associated with the ability of the virus to fuse infected cells with neighboring cells (i.e., to form syncytia when infecting mammalian cells and tumor cells).

[0051] Syncytia formation has been described in many viral infections, and in most of these infections, syncytia formation is the result of membrane destabilization due to cytopathic effects; however, some other viruses encode proteins that promote this syncytia formation as a mechanism for spreading infection rather than as a target for neutralizing antibodies. Interestingly, vaccinia virus WR expresses the A56R gene product in order to inhibit the formation of this syncytia (Figure 1).

[0052] The finding that the novel vaccinia virus strain IOVA causes syncytium formation in cancer cells is considered a potentially interesting phenotype for oncolytic viruses, as syncytial cell death has been described as highly immunogenic: fusion of tumor cells triggers the release of exosome-like vesicles associated with the efficient release of tumor antigens, which can be efficiently taken up by dendritic cells and thus improve highly specific immune responses.

[0053] In addition, according to another embodiment, the novel vaccinia virus strain IOVA comprises functionally inactivated A26R and A56R genes. The lack of expression of these gene products is associated with even improved syncytia formation after infection of mammalian cells and tumor cells, and therefore, also with the ability of the virus to fuse infected cells with neighboring cells.

[0054] To investigate syncytium formation as a possible mediator of IOVA virus-specific antitumor efficacy and the consequences of deletion of A56R alone or in combination with A26R in such fusions, a single A56+IOVA virus variant was engineered by replacing the mutant A56R gene and creating IOVA / A56+ (Figures 1 and 6). Upon restoration of A56R gene expression, the IOVA / A56+ virus retained some ability to fuse tumor cells, although the number of fused cells was reduced compared to the IOVA / A26- / A56- virus (Figures 1 and 6). Figure 6 ); this suggests that the two genes may interact to completely inhibit fusion of infected cells.

[0055] Syncytia formation is known to negatively impact the productive replication of other candidate oncolytic viruses (e.g., those derived from measles virus or adenovirus candidate oncolytic viruses). Surprisingly, syncytia formation has been shown to have minimal impact on replication capacity in the case of IOVA virus, and very similar virus yields were obtained from cells infected with IOVA / A56- and IOVA / A56+ (Figure 2).

[0056] Surprisingly, according to the present invention, the formation of syncytia has a significant positive effect on the cytotoxicity of cancer cells: in all cell lines tested, the formed syncytial viruses performed more effective killing of cancer cells, especially in the 143B and MCF-7 cancer cell lines, where IOVA / A56+ behaved similarly to vaccinia virus WR, with reduced killing of cultured cancer cells (Figure 3). Therefore, syncytium formation mediated by IOVA / A56- and IOVA / A56- / A26- can contribute to enhanced destruction of cancer cells.

[0057] Large plaque phenotype observed after cancer cell infection. Both IOVA / A56- and IOVA / A56+ viruses exhibited increased killing and ability to spread in cancer cells, as shown by the increased diameter of plaques generated after infection of single cancer cells (Figure 7).

[0058] Syncytia formation was also independent of the induction of CRT translocation and of the additional secretion of other DAMPs (such as HMGB1 and ATP) described later in inducing immunogenic cell death, which was very similar for both A56- and A56+ viruses ( Figures 4a to 4c ).

[0059] Interestingly, it was shown in the context of the present invention that the phenotype of syncytium formation, which itself already has an enhanced cytopathic effect in cancer cells, in addition to independently exposing calreticulin on the outer membrane of infected cells, also increases the antitumor effectiveness of the immunomodulatory vaccinia virus strain IOVA as described herein.

[0060] Thus, the novel vaccinia virus strains may additionally contain functionally inactivated A56R and / or A26R genes and thus cause syncytia formation and induce fusion of tumor cells. Combined with the enhanced presentation of calreticulin on the membranes of infected cells, this would lead to greater lysis and therefore to greater antitumor activity and, as a result, to enhanced destruction of cancer cells, tumor masses and, ultimately, to the generation of a stronger immune response against the tumor.

[0061] To improve the safety of the novel IOVA viruses, their derivatives and / or their viral vectors, additional modifications have been or may be introduced to restrict replication to specific cell ranges only.

[0062] Thus, according to another embodiment, the novel vaccinia virus strain IOVA is modified to have a host range in replicating cells and / or cancer cells, or in other words, to have a cell-range-restricted replication capability.

[0063] In this context, the term "replicating cells" includes cells with an activated cell cycle and replicating at a high exponential. Cells falling within this definition include continuously replicating cells, such as cancer cells, and also include cells that are part of, for example, an activated immune system and optionally have the potential to infiltrate a tumor.

[0064] In addition, the term "cancer cell" as used hereinafter includes cells that divide irreversibly and uncontrollably and have the ability to replace or dispose of normal tissue by, for example, forming a solid tumor mass or by filling the blood and / or other body cavities with abnormal cells. In addition, the term "cancer cell" also optionally refers to cells that express known tumor marker gene products and can be identified by their expression.

[0065] In order to improve the cell range specificity of the novel vaccinia virus strains, studies have shown that a cell range restricted replication capability can be obtained by inactivating one or more of the so-called housekeeping genes selected from the group consisting of F4L, J2R and C11R. For example, a virus with an inactivated or mutated J2R gene will not express viral thymidine kinase and, therefore, its efficient replication will be restricted to cells with a continuously activated cell cycle or, alternatively, to tumor cells. According to the present invention, such inactivation has been exemplarily performed by inserting a replacement expression cassette (expressing the color of the tracer) in the place of the TK gene, which is referred to as J2R in vaccinia nomenclature.

[0066] Similarly, according to the present invention, functional inactivation, deletion or mutation of C11R, F4L alone or in combination with each other or with J2R will also limit the ability of vaccinia virus strains to replicate in cells with activated cell cycle and / or tumor cells. Therefore, in order to functionally inactivate one or more genes in the group consisting of F4L, J2R and C11R with the above-mentioned result of host range restriction, such addition of mutations or deletions to the viral genome significantly improves the safety characteristics against new virus strains.

[0067] Therefore, the cell-range restricted vaccinia virus strain IOVA is particularly advantageous for use in medicine due to its improved safety profile.

[0068] The inventors have also shown that, compared to vaccinia virus WR, the novel vaccinia virus strains of the present invention are unexpectedly able to induce immunogenic cell death following infection.

[0069] In this context, the term "immunogenic cell death" should be understood as a form of cell death that is capable of initiating and activating an immune response against dying cells and is characterized by the release or exposure of damage-associated molecular pattern (DAMP) molecules that can stimulate immune responses against both microorganisms and oncogenic neoantigens (Galluzzi et al., 2017). Among the long list of DAMPs released or exposed by dying cells that may mediate an immune response against tumor cells, the exposure of calreticulin on the cell surface of cells has been described, as well as the release of high-mobility group protein 1 (HMGB1) or ATP into the extracellular space.

[0070] Immunogenic cell death can be further described as a form of cell death that exposes or releases different DAMPs in order to activate dendritic cells (DCs) and thus specific T cells against antigens present in dying cells. It is not triggered externally, like e.g. apoptosis, which is e.g. defined as non-immunogenic and even tolerogenic cell death, and cannot be defined by the same DAMPs.

[0071] With the background knowledge of such markers, the inventors could actually describe and classify the types of cell death caused by the virus. Thus, it was clearly demonstrated that the novel vaccinia virus strain IOVA not only caused enhanced lysis and cell killing after infection compared to vaccinia virus WR, but also that infected cells died due to immunogenic cell death by measuring certain DAMPs, namely HMGB1 and ATP, and by measuring the amount of CRT present on the outer cell membrane.

[0072] This impressively demonstrates that the novel vaccinia virus strains that replicate in mammalian cells and in particular in tumor cells or cell cycle activated cells are highly useful not only as vaccines or adjuvants for vaccination purposes, but also in other immunotherapy or immuno-oncology contexts, in particular as oncolytic viruses for the treatment of cancer.

[0073] In conclusion, the novel vaccinia virus strain IOVA and its derivatives, vectors and recombinants have the ability to replicate in mammalian cancer cell lines and can be described as lytic or, in the case of cancer cells, oncolytic, since they elicit an increased capacity to destroy cell cycle activated cells and / or tumor cells compared to the gold standard vaccinia virus WR.

[0074] Importantly, IOVA and its derivatives or recombinants are particularly suitable for inducing immunogenic cell death after cell infection and are therefore very promising for use in immunotherapy approaches. They are particularly suitable as novel platform viruses for safe and efficient immunotherapy or immuno-oncology therapy.

[0075] According to another embodiment, the present application also provides an isolated IOVA virus characterized by its nucleic acid sequence. The sequence information of the isolated IOVA may contain base pair modifications, however, it does not affect the immunological characteristics of IOVA. Therefore, the IOVA virus can be identified by sequence analysis and the presence of at least one or more functionally inactivated or deleted ORFs selected from the group consisting of: J2R, C11R, F4L, B21R*, C10L, C9L, C4L, C2L, N1L, N2L, M1L, A26R, A51R, A52R, A55R, A56R and B13R / B14R.

[0076] According to another embodiment, IOVA and its derivatives can be identified by the presence of a unique nucleotide sequence stretch of C2L-C1L-N1L-N2L (SEQ ID NO.: 1), which introduces a newly created BstXI restriction enzyme site in the N1L ORF. In this sequence, C2L incorporates a 51 nucleotide deletion distributed among 3 microdeletions; N1L incorporates a 2 nucleotide deletion that incorporates an early stop codon; and N2L incorporates a 15 nucleotide deletion that composes a shorter N2L version. Therefore, the sequence shown in SEQ ID No. 1 is a unique sequence of IOVA and represents a unique stretch of the C2L-C1L-N1L-N2L region of IOVA, thereby incorporating microdeletions in some ORFs, but not a complete deletion of such genes and without a functional microdeletion of C10 ORF.

[0077] In addition, according to one embodiment, the isolated IOVA is considered a platform technology that allows the production of related derivatives or recombinants that are identified by PCR analysis as described above or direct sequence comparison of at least one or more of the viral housekeeping genes or functionally inactivated immune escape genes selected from the group of ORFs consisting of: K1L, A56R, A26R, J2R, C11R, F4L, B21R*, C10L, C9L, C4L, C2L, N1L, N2L, M1L, A51R, A52R, A55R and B13R / B14R.

[0078] According to another embodiment, the present application also provides a viral vector derived from IOVA. This viral vector contains the same set of functionally active and functionally inactivated genes as IOVA. In the context of the present application, the term "viral vector" also includes two or more vector molecules, each of which contains or carries a portion or range of a set of functionally active and functionally inactivated genes of IOVA, and the two or more vector molecules work together to transfect cells, thereby enabling such cells to produce IOVA.

[0079] IOVA itself, isolated virus, nucleic acid sequences of IOVA, and viral vectors comprising nucleic acid sequences specific for IOVA are considered to be a platform technology.

[0080] The IOVA platform also encompasses derivatives of IOVA that can be identified by PCR and restriction enzyme digestion using the BstXI enzyme and that still possess the same functional characteristics and properties as IOVA, in particular the ability to replicate in mammalian cell cycle activated cells, the translocation of calreticulin to the outer membrane and / or the independent induction of syncytium formation in infected cells.

[0081] The IOVA platform also includes recombinants of IOVA. The backbone of IOVA contains several well-described vaccinia virus insertion sites for inserting transgenes into its genome. Due to its close relationship with the vaccinia virus, one skilled in the art knows and can use one or several sites for inserting transgenes, such as, for example, genes encoding tumor antigens, tumor-associated antigens, disease-associated antigens and / or pathogen-derived antigens.

[0082] Thus, according to another embodiment, the present invention provides recombinant or transgenic IOVA or its derivatives or viral vectors. As in each platform technology, the introduction of additional genes or genetic information at the carefully described insertion site does not affect the main properties of the claimed platform members, which are independently (i) inducing infected cell fusion or syncytia formation, (ii) inducing a significant release or exposure of DAMPs, especially calreticulin, and / or (iii) inducing immunogenic cell death of infected cells, which may contribute to even stronger immunological effects, or in the case of infected tumor cells, more effective anti-tumor activity by enhancing the destruction of tumor cells.

[0083] Without being bound by theory, it is believed that the more effective anti-tumor activity is due in particular to a stronger immune response against such infected cells, which can be triggered by the presence of calreticulin and / or increased release of other DAMPs such as HMGB1 or ATP. The released HMGB1 can bind to TLR4 and RAGE and trigger a pro-inflammatory response, while the released ATP also appears to act as a "find-me" signal for immune cells.

[0084] In conclusion, the novel vaccinia virus strain IOVA and its derivatives, vectors and recombinants have the ability to replicate in mammalian cancer cell lines and can be described as lytic or, in the case of cancer cells, oncolytic, since they elicit an increased capacity to destroy cell cycle activated cells and / or tumor cells compared to the gold standard vaccinia virus WR.

[0085] IOVA or its derivatives, due to their unique immunological and safety profiles, which are comparable to or even better than already established vaccinia virus strains, can be used in medicine, in particular in oncological approaches, or as oncolytic agents or as vaccines against cancer or other pathogens.

[0086] Importantly, IOVA virus and its derivatives or recombinants are particularly suitable for inducing immunogenic cell death after cell infection and are therefore highly recommended for use in immunotherapy approaches. They are particularly suitable as novel viral platforms for safe and efficient immunotherapy or immuno-oncology treatment. Sequence Listing <110> Gad Souter Juan Jose Rojas Epstu <120> Immunomodulatory replication-efficient vaccinia virus strains <130> F038.002.NPECN <140> <141> 2018-12-03 <150> DE102017128538.5 <151> 2017-12-01 <160> 4 <170> BiSSAP 1.3.6 <210> 1 <211> 3234 <212> DNA <213> Vaccinia virus <220> <223> C2L-C1L-N1L-N2L has a new BstXI site <400> 1 ctattgtaga aattgttttt cacagttgct caaaaacgat ggcagtgact tatgagtttc 60 atctttagta aacatatcat atattcgat attackagtt gatatcga acaaattcca 120 agtatttgat tttggataat attcgtattt tgcatctgct atattaaga tataatcacc 180 gcaagaacac acgaacatct ttcctacatg gttaaagtac atgtacaatt ctatccattt 240 gtcttcctta actatatatt tgtatagata attacgagtc tcgtgagtaa ttccagtaat 300 tacatagatg tcgccgtcgt actctacagc ataactata ctatgatgtc taggcatggg 360 agacttttt atccaacgat ttttagtgaa acatccaca tcgtttaata ctacatattt 420 ttcatacgtg gtataaactc cacccattac atatatatca tcgtttacga ataccgacgc 480 gcctgaatat ctaggagtaa ttaagtttgg aagtcttatc cattcgaag tgccgtgttt 540 aaatattct gccacaccg ttgaaataga aaattctaat cctcctatta catataactt 600 tccatcgtta accaagtac taacttctga ttttaacgac gatatag taaccgtttt 660 ccattttttc gttcagat ctacccgcga tacggataa acatgtctat tgttaatcat 720 gccgccaata atgtatagac aattatgtaa aacatttgca ttatagaatt gtctatctgt 780 attack atcgtccaat attctgttct aggagtaa tggtttg tggatatata 840 atcagagttt ttaatgacta ctatattatg ttttacca ttcgtgtca ctggctttgt 900 agatttggat atagttaatc ccaacaatga tatagcattg cgcatagtat tagtcataaa 960 cttgggatgt aaatgttga tgatatctac atcgttttgga ttttgta tccactttaa 1020 taatatcata gctgtaacat cctcatgatt tacgttaacg tctcgtggg atagatagt 1080 tgtcagttca tcctttgata attttccaaa ttctggatcg gattcaccg cagtaatatt 1140 gttgattatt tctgacatcg acgcattata tagtttttta attccatatc ttttagaaaa 1200 gttaaacatc cttatacaat ttgtggaatt atattatga atcatagtttt ttacacatag 1260 atctacataca ggcggaacat chattatt ggcagcact agtatcattt ctacattgtt 1320 tatggtgatg tttatctttct tccagcgcat atagtctaat agcgattcaa acgcgtgata 1380 gtttatacca ttcaataa tcgctcatc ctttagatgg tgatcctga tgcgtttaaa 1440 aaaattatac ggagacccg taataatttc cttattcact tgtataattt ccccattgat 1500 agaaaatatc acgctttcca ttcttgaagt actataagta attatagtat aatgtaaagg tttatatatt caattttt tataaaaaaa tcatttcgac attaattcct ttttaaattt ccgtctatca tctatagaaa catattctat gaatttataa aatgctttta cgtgtcctat cgtaggcgat agaaccgcta aaaagcctat cgaatttcta caaaagaatc tgttatatgg 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800 taactctttc gaatacttat taatggctct tgttctgtac gaatctattt ttttgaacaa tggacctagt ggtatatctt gttctatgta tctaaaataa tgtctgacta gatccgttag tttaatatcc gcagtcatct tgtctagaat ggcaaatcta actgcgggtt taggctttag tttagtttct attacks ctatgtcttt attacks aaaaatataa tags tttattacaa tcatccggat attcttctac gatctcacta actaatgttt ctttggttat actagtatag tcacgatcag acaaataag aaaatcagat gatcgatgaa fathercattt aaattcatca tctgtaagat ttttgagatg tctcattaaa attackattag ggtcagtact cattatcatt aggcagctat tacttatttt attatttttc accatataga tcaatcatta gatcatcaaa atatgtttca atcatccaag agtatggtga atgactcttc ccatctaatt tctgaacgtt caccaatgtc tctagccact ttggcactaa tagcgatcat tcgcttagcg tcttctatat tattaactgg ttgattcaat ctatctagca atggaccgtc ggacagcgtc attctcatgt tcttaatcaa tgtacataca tcgccgtcat ctaccaattc atccaacaac onectttt taaaatcatc attachment gtttgatcgt tgtcatttct ccaaagaata tatctaata gtagagtcct catgattagt catcaactat tttttatgtt aaatcaatta gtacaccgct atgtttaata cttattcata ttttagtttt tagttgag aatcaataca aaaattaatg cats tttagaaata cttagtttcc acgtagtcaa tgaaacattt gaactcatcg tacaggacgt tctcgtacag gacgtaacta taaaccggtt tatatttgtt caagatag acaaatccga taactttttt tacgaattct acgggatcca ctttaaaagt gtcataccgg gttcttttta tttttttaaa cagattaatg gtgtgatgtt gattaggtct tttacgaatt tgatatagaa tagcgtttac atattctcca taatggtcaa tcgccatttg 3000 ttcgtatgtc ataaattctt taattatatg acactgtgta ttattagtt catccttgtt 3060 catcattagg aatctatcca atatggcaat tatactagaa ctataggtgc gttgtataca 3120 catattgatg tgtctgttta tacaatccat gctactacct tcgggtaaaa ttgtagcatc 3180 atataccatt tctagtactt taggttcatt gttatccatt gcagaggacg tcat 3234 <210> 2 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 2 atgttatcct ggacatcgta c 21 <210> 3 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 3 tcatgacgtc ctctgcaatg g 21 <210> 4 <211> 3284 <212> DNA <213> Vaccinia virus <220> <223> PCR products <400> 4 atgttatcct ggacatcgta caaataataa aaagcccata tatgttcgct attgtagaaa 60 ttgttttca cagttgctca aaaacgatgg cagtgactta tgagtttcat ctttagtaaa 120 catatcataa tattcgatat tactagttga catatcgaac aaattccaag tattttgatt 180 tggatatat tcgtatttg catctgctat aattaagata taatcaccgc agaacacac 240 gaacatcttt cctacatggt taagtacat gtacaatctt atccattgt cttccttaac 300 tatatattg tatagatatat tacgagtctc gtgagtaatt ccagtaatta catagatgtc 360 gccgtcgtac tctacagcat aaactatact atgatgtcta ggcatgggag acttttttat 420 ccaacgattt ttagtgaaac attccacatc gtttaatact acatattttt catacgtggt 480 aaactcca cccattacat atatatcatc gtttacgaat accgacgcgc ctgaatatct 540 aggagtaatt aagtttggaa gtcttatcca tttcgaagtg ccgtgtttca atattctgc 600 cacacccgtt gaaatagaaa attctaatcc tcctattaca tataactttc catcgttaac 660 acagtacta acttctgatt ttaacgacga catattagta accgttttcc attttttcgt 720 ttcagatct acccgcgata cggaataaac atgtctattg ttaatcatgc cgccaataat 780 gtatagacaa ttgtaaaa catttgcatt atagaattgt ctatctgtat taccgactat 840 cgtccaatat tctgttctag gagagtaatg ggttattgtg gatatatat cagagttttt 900 aatgactact atattatgtt ttataccatt tcgtgtcact ggctttgtag atttggatat 960 agttaatccc aacaatgata tagcattgcg catagtatta gtcataaact tgggatgtaa 1020 aatgttgatg atatctacat cgtttggatt tttagtatc cactttaata atatcatagc 1080 tgtaacatcc tcatgattta cgttaacgtc tcgtggt agatagttg tcagttcatc 1140 ctttgataat ttccaaatt ctggatcgga tgtcaccgca gtaatattgt tgattatttc 1200 tgacatcgac gcattatata gttttttaat tccatatctt ttagaaaagt taaacatcct 1260 tatacaattt gtggaattta tattatgaat catagttttt acacatagat ctactacagg 1320 cggaacatca attattatgg cagcaactag tatcatttct acatgttta tggtgatgtt 1380 tatctttcttc cagcgcatat agtctaatag cgatcaac gcgtgatt ttataccatt 1440 caataatc gcttcatcct ttagatgtg atcctgaatg cgtttaaaaa attatacgg 1500 agacgccgta ataatttcct tattcacttg tataatttcc ccattgatag aaaatatcac 1560 gctttccatt cttgaagtac tatagtaat tatagtataa tgtaaggtt tatatattca 1620 atatttttta taaaaaaatc atttcgacat taattcttt ttaatttcc gtctatcatc 1680 tatagaaaca tattctatga attataaaa tgctttacg tgtcctatcg taggcgatag 1740 aaccgctaaa aagcctatcg aatttctaca aaagaatctg ttatatggta taggagt 1800 aaaacatt aaatgtccgt acttattaaa gtattcagta gccaatccta actctttcga 1860 atacttatta atggctcttg ttctgtacga atctatttt ttgacaatg gacctagtgg 1920 tatatctgt tctatgtatc taaaataatg tctgactaga tccgttagtt taatatccgc 1980 agtcatcttg tctagaatgg caatctaac tgcggttta ggctttagtt tagttctat 2040 atctacatct atgtctttat ctaacaccaaaatataata gctatatattt tattacaatc 2100 atccggatat tcttctacga tctcactac taatgtttct tggttatac tagtatagtc 2160 acgatcagac aaataagaa atcagatga tcgatgaata atacatttaa attcatcatc 2220 tgtaagattt ttgagatgtc tcattaaaat attattaggg tcagtactca ttatcattag 2280 gcagctatta cttattttat tatttttcac catatagatc aatcattaga tcatcaaaat 2340 atgtttcaat catccaagag tatggtgaat gactcttccc atctaatttc tgaacgttca 2400 ccaatgtctc tagccacttt ggcactaata gcgatcattc gcttagcgtc ttctatatta 2460 ttaactggtt gattcaatct atctagcaat ggaccgtcgg acagcgtcat tctcatgttc 2520 ttaatcaatg tacatacatc gccgtcatct accaattcat ccaacaacat aagcttttta 2580 aaatcatcat tataataggt ttgatcgttg tcatttctcc aaagaatata tctaataagt 2640 agagtcctca tgattagtta acaactattt tttatgttaa atcaattagt acaccgctat 2700 gtttaatact tattcatatt ttagttttta ggattgagaa tcaatacaaa attaatgca 2760 tcattaattt tagaaatact tagttccac gtagtcaatg aaacatttga actcatcgta 2820 caggacgttc tcgtacagga cgtaactata aaccggttta tatttgttca agatagatac 2880 aaatccgata acttttttta cgaattctac gggatccact ttaaaagtgt cataccgggt 2940 tctttttattttttaaaca gattaatggt gtgatgttga ttaggtcttt tacgaatttg 3000 atatagaata gcgtttacat attctccata atggtcaatc gccatttgtt cgtatgtcat 3060 aaattcttta attatatgac actgtgtatt atttagttca tccttgttca tcattaggaa 3120 tctatccaat atggcaatta tactagaact ataggtgcgt tgtatacaca tattgatgtg 3180 tctgtttata caatccatgc tactaccttc gggtaaaatt gtagcatcat ataccatttc 3240 tagtacttta ggttcattgt tatccattgc agaggacgtc atga 3284 BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1. Syncytia formation in cancer cells after infection with IOVA virus. Fluorescence micrographs (40x) after infection with (a) human or (b) mouse cancer cell lines (24 hours after infection) are shown. HeLa and CT26 cells were infected with an MOI of 0.5. 143B, MCF-7 and LLC1 were infected with an MOI of 5. mCherry was expressed by all viruses under the P11 promoter. When cells were IOVA / A56-infected, a large number of fusions of cells (syncytia) could be observed.

[0088] Figure 2. Virus production of IOVA virus in human and mouse tumor cells. (a) Human and (b) mouse tumor cell lines were infected with WR / TK-, IOVA / A56-, or IOVA / A56+ at an MOI of 5, and progeny were measured by plaque assay at different time points. Virus yield was assessed in quadruplicate for each cell line by performing two independent experiments. Mean + SD are plotted. *, significant p < 0.05 compared to WR / TK-. #, significant p < 0.05 compared to IOVA / A56+.

[0089] Figure 3. Increased cytotoxicity of IOVA virus to 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 on day 3 post infection. Both human (a) and mouse (b) cancer cells were tested. Four different replicates of each cell line were quantified and the mean ± SD for each MOI is depicted.

[0090] Figure 4. Induction of immunogenic cell death by IOVA virus. (a) Analysis of the expression of calreticulin on the surface of infected cells. The indicated tumor cell lines were infected with WR / TK-, IOVA / A56- or IOVA / A56+ at an MOI of 5, and the calreticulin+ cell population was determined by flow cytometry 24 hours after infection. Uninfected cells (mock) and staurosporine 1 μm were used as negative and positive controls, respectively. (a) The percentage of calreticulin+ cells. The values ​​of individual replicates and the mean ± SEM of different treatments are plotted. (bc) The concentrations of HMGB1 (b) and ATP (c) in the cell supernatant after infection with IOVA virus. ELISA assays and ENLITEN ATP assay systems were used to determine such concentrations of the indicated tumor cell lines 24 hours after infection (MOI of 5), respectively. Data were obtained in quadruplicate and plotted as fold change relative to WR / TK-+SD. *, significant p<0.05 compared to WR / TK-. #, significant p < 0.05 compared with mock.

[0091] Figure 5. PCR assay for virus identification. Figure 5a : Sequence of PCR product used in PCR assay. Figure 5b : The results of the PCR assay clearly identified the sequence of the IOVA strain, as the assay produced two unique band patterns for IOVA (2320 and 920 bp), which was different from all other vaccinia virus strains that presented a single band of 3300 bp for WR, COP and CVA. The MVA strain did not produce an amplification product due to the deletion of C2L.

[0092] Figure 6 .Number of nuclei in syncytia after infection with IOVA virus. Human tumor cell lines were infected with WR / TK-, IOVA / A56- / A26- or IOVA / A56+ / A26- at an MOI of 5. 16 hours after infection, cultures were stained with Hoechst 33342 and the number of nuclei in one syncytia was counted under a microscope. The values ​​of individual replicates and mean ± SEM are plotted. ***, significant p<0.0001 compared to WR / TK-.

[0093] Figure 7. Size of plaques in cancer cells. Cancer cell monolayers were infected with the indicated viruses at an MOI of 0.0001 for 1 hour and covered with a 1:1 mixture of culture medium and 1% carboxymethylcellulose for 4 days. After fixation and staining with crystal violet, the diameter of the plaques was determined. The values ​​of individual replicates and mean ± SEM are plotted. *, significant p < 0.05 compared to WR / TK-. #, significant p < 0.05 compared to IOVA / A56-. Example

[0094] Example 1: Deletions in the IOVA genome

[0095] A novel immuno-oncolytic vaccinia virus (IOVA) strain was generated that incorporated a deletion in the thymidine kinase (TK, J2R) gene to allow selective replication in cancer cells. In addition, the mCherry gene has been cloned into the TK locus under the control of the vaccinia virus-specific promoter P11 to monitor viral replication.

[0096] In addition, the newly generated IOVA contains several deletions or functional inactivations in genes considered to be immunomodulators and selected from the following ORFs: B21R*, C10L, C9L, C4L, C2L, N1L, N2L, M1L, A26R, A51R, A52R, A55R, A56R and B13R / B14R. The functions of the proteins encoded by these genes are summarized in Table 1 above.

[0097] The IOVA genome is also characterized by including mutant forms of the A26R and / or A56R genes. The presence of the A26 protein in the virion prevents direct virus-cell fusion mechanisms, and its absence is associated with the induction of syncytia. A56R encodes a viral regulatory protein with hemagglutination activity, and its inactivation in vaccinia virus is believed to result in a virus with a pro-fusion phenotype. All gene deletions or partial deletions and all functional inactivations or gene insertions have been confirmed by sequencing.

[0098] Example 2: Syncytia formation induced by IOVA

[0099] To evaluate the benefits and drawbacks of syncytium formation on tumor destruction, the inventors restored the wild-type vaccinia virus A56R gene sequence in the novel IOVA genome by homologous recombination. Compared with IOVA with a truncated A56R version (called IOVA / A56-), the resulting virus was named IOVA / A56+.

[0100] After infection, cells infected with IOVA / A56- virus were observed to fuse with neighboring cells (Figure 1), and giant syncytia formation was clearly observed in both human and mouse tumor cell lines tracked by mCherry expression. As hypothesized, expression of wild-type A56 in IOVA / A56+ restored a phenotype very similar to that of vaccinia virus WR strain and did not result in syncytia formation.

[0101] In addition, the observation that expression of wild-type A56 in IOVA / A56- / A26- could also be described as only partially blocking syncytia formation, since fusions of up to 10 cells could still be observed after infection with IOVA / A56+ / A26- ( Figure 6 ).

[0102] Example 3: IOVA's replication capabilities

[0103] We tested the replication capacity of IOVA compared with the standard strain vaccinia virus WR in a wide range of human and mouse cancer cell lines.

[0104] To monitor the replication of newly produced IOVA, the replication capacity of two IOVA virus isolates (A56- and A56+) and WR / TK- as a control were tested in several human and mouse cancer cell lines.

[0105] To this end, human and mouse tumor cell lines were infected with WR / TK-, IOVA / A56- or IOVA / A56+ at an MOI of 5, and progeny were measured by plaque assay at different time points. Virus yield was assessed in quadruplicate for each cell line by performing two independent experiments (previously described in: Rojas JJ et al., Cell Rep. 2016).

[0106] As shown in Figure 2 , both IOVA viruses (A56- and A56+) showed very similar growth curves to the control strain WR / TK- in most cell lines tested, with only a slight decrease in production at the early IOVA time points when syncytia were formed.

[0107] Example 4: Cytotoxicity of IOVA

[0108] We examined the cytotoxic effects of IOVA infection compared with the standard strain vaccinia virus WR in a wide range of human and mouse cancer cell lines.

[0109] To this end, various cancer cell lines were infected with WR / TK-, IOVA / A56- or IOVA / A56+ at doses ranging from 100 to 0.0005 PFU / cell. Cell viability was determined on day 3 post infection. Both human and mouse cancer cells were tested. Four different replicates of each cell line were quantified and the mean ± SD for each MOI is depicted.

[0110] Interestingly, infection with IOVA virus resulted in significantly enhanced levels of cytotoxicity in cancer cells compared to vaccinia virus WR (Figure 3). Vaccinia virus WR was able to kill approximately 70%-80% of cancer cells in culture, even at the highest multiplicity of infection (MOI). In contrast, IOVA / A56-virus was able to kill between 95%-100% of cultured tumor cells and reduced EC50 (the amount of virus required to kill 50% of the cells).

[0111] Surprisingly, IOVA / A56- reduced the amount of virus required to reduce the cell culture viability of HeLa cells by 50% by more than 40-fold compared to vaccinia virus WR (WR / TK-). IOVA / A56+ also presented a phenotype with enhanced cytotoxicity to cancer cells in vitro; A56-restored viruses killed HeLa, CT-26, and LLC1 cells at rates similar to IOVA / A56-. However, in 143B and MCF-7 cells, cytotoxicity was very similar to that obtained with WR / TK- infection, suggesting that virus-mediated macrosyncytia formation may contribute to enhanced tumor destruction.

[0112] Example 5: Large plaque phenotype of IOVA virus in cancer cells

[0113] Increased plaque size in cancer cells has been associated with better spread of the virus throughout the tumor and greater anti-tumor activity. To test the plaque size of the IOVA virus, a panel of cancer cell lines were infected at an MOI of 0.0001, and after 1 hour of infection, the infected cells were cultured with carboxymethylcellulose overlays. Four hours after infection, the cultures were fixed and stained with crystal violet, and the diameter of the plaques was determined.

[0114] As shown in Figure 7, IOVA / A56- and IOVA / A56+ induced larger plaques in all cancer cell lines tested compared to the WR / TK- virus control. In HeLa cells, plaques after infection with IOVA virus were on average 40% larger than those produced by WR / TK-. Impressively, very large plaques were observed after infection of 143B and MCF-7 with IOVA virus, where the diameter of the plaques was 2 times that of the plaques produced by WR / TK- in the case of 143B cells, and the diameter of the plaques was 2.6 times that of the plaques produced by WR / TK- in the case of MCF-7.

[0115] Regarding plaque size, there was no significant effect except in the case of the MCF-7 breast cancer cell line where large syncytia were produced by IOVA / A56-, and plaques produced by IOVA / A56+ virus were 1.4-fold smaller compared to IOVA / A56-.

[0116] Example 6: IOVA virus induces calreticulin presentation on the cell membrane and immunogenic cell death of cancer cells

[0117] To test whether IOVA virus can elicit and enhance immune responses against cancer cells, the inventors initially analyzed by flow cytometry the exposure of calreticulin (CRT) on the surface of human cancer cells after infection with IOVA / A56-A, IOVA / A56+, or the control virus WR / TK-.

[0118] To this end, cells were infected at an MOI of 5 and detached using a non-enzymatic cell dissociation solution 24 hours after viral infection. Calreticulin was detected by incubating cells with human anti-calreticulin-AlexaFluor405 antibody (Abcam, Ref N° ab210431) at 4°C for 1 hour. Uninfected cells and staurosporine 1 μM were used as negative and positive controls, respectively.

[0119] After infection of HeLa cells, WR / TK-induced surface exposure of CRT in approximately 15% of cells ( Figure 4a ); in contrast, after infection with both IOVA viruses, surprisingly high levels of over 80% to 72% of cells expressed CRT on the surface. Similarly, in 143B cells, CRT exposure increased from about 35% (with WR / TK-) to almost 90% (with IOVA viruses), and in MCF-7 cells from 3% to over 72%.

[0120] To further investigate the possible induction of overt immunogenic cell death after infection with IOVA virus, the release of HMGB1 and ATP was measured using an ELISA assay and a luciferase-mediated ATP assay system, respectively.

[0121] In all cell lines tested and infected with both IOVA / A56- and IOVA / A56+, significantly higher concentrations of HMGB1 could be detected in the supernatants of infected cells compared to cells infected with WR / TK-. Figure 4b ), with increases ranging from 1.23-fold (143B cells, IOVA / A56-) to 1.68-fold (MCF-7 cells, IOVA / A56+). ATP concentrations in the supernatant of infected cells also increased when infected with IOVA virus compared to levels after infection with WR / TK- ( Figure 4c ), with the increase ranging from 1.12-fold (143B cells, IOVA / A56+) to 2.27-fold (HeLa, IOVA / A56-).

[0122] These results suggest that IOVA virus, but not vaccinia virus WR, induces immunogenic cell death in infected human cancer cells. Therefore, IOVA can be suggested as a particularly promising candidate virus that could have great benefits in terms of antitumor activity in clinical trials.

[0123] Example 7: PCR assay for identification of IOVA virus or its derivatives

[0124] To identify the IOVA strain, cell extracts from infected cells were digested with proteinase K and viral DNA was isolated using the QIAamp genomic DNA kit (QIAGEN) according to the manufacturer's instructions. The IOVA unique sequence covering the C2L-C1L-N1L-N2L segment was amplified by PCR using the following oligonucleotides: Figure 5a ; SEQ ID No.1): forward 5'-ATGTTATCCTGGACATCGTAC-3' (SEQ ID No.2) and reverse 5'-TCATGACGTCCTCTGCAATGG-3' (SEQ ID No.3). The PCR product using these two primers is 50 bp larger than the unique SEQ ID No.1; for stability reasons, 50 bp was also included in the design of the PCR reaction. The PCR product (SEQ ID No.4) was purified by using the QIAquick PCR purification kit and digested with the BstXI restriction enzyme.

[0125] By using this assay, the IOVA strain can be clearly identified as it produces a specific and unique pattern of two DNA bands (2361 and 923 bp) visible on electrophoresis in 1% agarose, which is different from all other vaccinia strains that present a single band of 3384 bp. PCR of MVA genomic DNA does not produce a PCR product due to the absence of the C2L sequence ( Figure 5b ).

[0126] References

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[0129] S.H.Thorne et al.,Targetinglocalized immune suppression within thetumour through repeat cycles of immune cell-oncolytic virus combinationtherapy.Molecular therapy:thejournal of the American Society of Gene Therapy18,1698(Sep,2010).

[0130] C.J.Breitbach et al.,Intravenous delivery of a multi-mechanisticcancer-targeted oncolytic poxvirus in humans.Nature 477,99(sep 1,2011).

[0131] J.Heo et al.,Randomized dose-finding clinical trial of oncolyticimmunotherapeutic vaccinia JX-594 in liver cancer.Nature medicine 19,329(Mar,2013).

[0132] H.J.Zeh et al.,First-in-man study of western reserve strain oncolyticvaccinia virus:safety,systemic spread,and antitumouractivity.Moleculartherapy:thejournal of the American Society of Gene Therapy 23,202(Jan,2015).

[0133] A.Volz,G.Sutter,Protective efficacy of Modified Vaccinia virus Ankara in preclinical studies.Vaccine 31,4235(Sep 6,2013).

[0134] H.Meyer,G.Sutter,A.Mayr,Mapping of deletions in the genome of thehighly attenuated vaccinia virus MVA and their influence on virulence.TheJournal of general virology 72(Pt 5),1031(May,1991).

[0135] R.T.Zhang,s.D.Bines,C.Ruby,H.L.Kaufman,TroVax((R))vaccine therapy forrenal cell carcinoma.Immunotherapy 4,27(Jan,2012).

[0136] I.Marigo,L.Dolcetti,P.Serafini,P.Zanovello,V.Bronte,Tumour-inducedtolerance and immune suppression by myeloid derived suppressor cells.ImmunolRev 222,162(Apr,2008).

[0137] S.J.Gardai et al.,Cell-surface calreticulin initiates clearance ofviable or apoptotic cells through trans-activation of LRP on thephagocyte.Cell 123,321(Oct 21,2005).

[0138] G.L.Smith et al.,Vaccinia virus immune evasion:mechanisms,virulenceand immunogenicity.The Journal of generol virology 94,2367(Nov,2013).

[0139] Goebel et al.The complete DNA sequence of vaccinia virus.Virology,1990 Nov;179(1):247-66,517-63.

[0140] L.Galluzzi,A.Buque,O.Kepp,L.Zitvogel,G.Kroemer,Immunogenic cell deathin cancer and infectious disease.Nat Rev Immunol 17,97(Feb,2017).

[0141] Rojas JJ et al.,Manipulating TLR signaling increases the anti-tumourT cell response induced by viral cancer therapies.Cell Rep.,2016 Apr 12;15(2):264-73.

Claims

1. An immunomodulatory vaccinia virus, wherein the virus is identified by the presence of SEQ ID NO: 1, has a functionally active K1L, is replication competent in mammalian cells, and induces translocation of calreticulin to the membrane of infected HeLa cells, wherein A56R and A26R of the virus are functionally inactivated; wherein one or more immune escape genes of the virus comprising an open reading frame selected from the group consisting of B21R, C10L, C9L, C4L, M1L, A51R, A52R, A55R and B13R / B14R are functionally inactivated.

2. The immunomodulatory vaccinia virus according to claim 1, 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.

3. The immunomodulatory vaccinia virus according to claim 1, wherein the virus is replication competent and lytic in cell cycle activated cells and / or tumor cells.

4. The immunomodulatory vaccinia virus of claim 1, wherein the virus causes syncytia formation upon infection.

5. A nucleic acid sequence encoding the immunomodulatory vaccinia virus according to any one of claims 1 to 4.

6. An immunomodulatory vaccinia virus vector comprising the nucleic acid sequence according to claim 5.

7. The immunomodulatory vaccinia virus according to any one of claims 1 to 4, or the immunomodulatory vaccinia virus vector according to claim 6, characterized in that Carrying one or more insertion sites, at least one of which has one or more transgenes inserted therein.

8. The immunomodulatory vaccinia virus or vector of claim 7, wherein the transgene is a gene encoding a disease-associated antigen.

9. The immunomodulatory vaccinia virus or vector of claim 7, wherein the transgene is selected from the group consisting of genes encoding tumor antigens, tumor-associated antigens, and pathogen-derived antigens.

10. A pharmaceutical composition comprising the immunomodulatory vaccinia virus according to any one of claims 1 to 4, the vector according to claim 6 or the immunomodulatory vaccinia virus according to any one of claims 8 to 9 and a pharmaceutically acceptable carrier, diluent or excipient.

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