Recombinant orthopoxvirus vectors encoding immunostimulatory proteins for cancer therapy

Abstract

The present invention relates to a recombinant orthopoxvirus vector comprising, in operable linkage, a first promoter comprising or consisting of (i) at least one viral early promoter element and optionally at least one viral late promoter element, or (ii) at least one viral late promoter element and at least three viral early promoter elements, and b) a first nucleic acid sequence encoding at least one immunostimulatory protein. The present invention also relates to a cell comprising the recombinant orthopoxvirus vector, as well as a composition comprising the recombinant orthopoxvirus vector or said cell; and optionally a further recombinant viral vector comprising a nucleic acid sequence encoding a checkpoint inhibitor, a nucleic acid encoding a checkpoint inhibitor, or a checkpoint inhibitor. Furthermore, the present invention provides a recombinant orthopoxvirus vector, and cells and compositions comprising same, for use in medicine, in particular for use in the treatment, amelioration or prevention of cancer.

Description

[Technical field]

[0001] The present invention relates to a recombinant orthopox viral vector comprising, in operable linkage, (i) a first promoter comprising or consisting of at least one viral early promoter element and optionally at least one viral late promoter element, or (ii) at least one viral late promoter element and at least three viral early promoter elements, and b) a first nucleic acid sequence encoding at least one immunostimulatory protein. The present invention also relates to a cell comprising the recombinant orthopox viral vector, as well as a composition comprising the recombinant orthopox viral vector or said cell; and optionally a further recombinant viral vector comprising a nucleic acid sequence encoding a checkpoint inhibitor, a nucleic acid encoding a checkpoint inhibitor, or a checkpoint inhibitor. Furthermore, the present invention provides a recombinant orthopox viral vector, and cells and compositions comprising same, for use in medicine, in particular for use in the treatment, amelioration or prevention of cancer. [Background technology]

[0002] Currently, in the field of cancer therapy, various efforts are being made to overcome the hostile tumor microenvironment (TME), which limits the function of antitumor T cells. Macrophages are mononuclear phagocytic white blood cells of the immune system that play a key role in anti-infection immunity, maintaining tissue homeostasis, and protecting the body by their function of internalizing and digesting foreign bodies, and removing harmful substances such as cellular debris and tumor cells from the body (Non-Patent Document 1, Non-Patent Document 2). Other functions of macrophages include mediating innate immunity, initiating adaptive immunity, secreting immunomodulatory substances such as various cytokines and chemokines, and activating the complement system that can cause inflammation. However, the function of macrophages can be modified and subverted in a harmful way in the presence of chemokines, cytokines, and various other factors secreted by tumor cells (e.g., CSF-1, CCL2, VEGF, TGFβ, etc.). Therefore, they are recruited to the tumor microenvironment and become so-called tumor-associated macrophages (TAMs) (Non-Patent Document 3). Tumor-associated macrophages play a major role in tumor progression at various levels, for example, promoting tumor growth and metastasis, promoting genetic instability, releasing proteases and other molecules to remodel the extracellular matrix, promoting angiogenesis, and secreting immunosuppressive mediators (Non-Patent Document 4, Non-Patent Document 5). TAMs are known to polarize to either M1, which shows antitumor activity, or M2, which leads to cancer progression, and are described as M1-like or M2-like macrophages (Non-Patent Document 6).

[0003] Therefore, among the strategies to overcome the hostile TME, several efforts have been made to develop strategies targeting TAMs, for example, inhibition of macrophage recruitment by inhibiting tumor-derived factors (TDF) via antibodies (e.g., pexidartinib, AMG820mAb, carmab, emactuzumab, etc.), depletion of TAMs by radiation therapy and / or TDF inhibitors (e.g., PLX3397, trabectedin, etc.), or targeting Toll-like receptors to reprogram TAMs from an immunosuppressive M2-like phenotype to a more inflammatory M1-like state (e.g., imiquimod, resiquimod, etc.), RNA delivery (e.g., small interfering RNA, microRNA, etc.), or antibodies (e.g., HU5F9-G4 mAb, CP-870,893, APX005M) (Non-Patent Document 7). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Zhou et al., Front. Oncol. 2020 [Non-Patent Document 2] Yona S. and Gordon S., Front Immunol. 2015 [Non-Patent Document 3] Watkins et al., J. Immunol. 2021 [Non-Patent Document 4] Mantovani et al., Nature ReviewsClinical Oncology 14, 2017 [Non-Patent Document 5] Anfrey et al., Cells 2020 [Non-Patent Document 6] Murray, PJ et al., Immunity 41, 14-20,2014 [Non-Patent Document 7] Anfrey et al., Cells 2020 Summary of the Invention [Problem to be solved by the invention]

[0005] However, despite the variety of TAM reprogramming approaches, many of them suffer from a lack of therapeutic selectivity and systemic toxicity, spurring a growing demand for safe and selective methods to shift the tumor microenvironment from an immunosuppressive to an immunostimulatory state. [Means for solving the problem]

[0006] Thus, a Vector Aided Microenvironment Programming (VAMP) strategy has been developed to deliver therapeutic proteins locally and effectively transition the TME from an immunosuppressive state to an immunostimulatory state without systemic toxicity. The strategy involves providing a recombinant orthopoxvirus vector that can effectively express an immunostimulatory therapeutic molecule in the TME under the control of a promoter that allows highly selective production of the therapeutic protein in the TME and activation of TAMs. The promoter includes one or more early element motifs to improve expression of the protein of interest in target cells, particularly TAMs. The recombinant viral vector offers the following advantages, among others: i) controlled and reproducible delivery of the protein of interest, particularly immunostimulatory proteins, via infection of normal cells infiltrating the tumor, ii) efficient reprogramming of M2-like macrophages to M1-like macrophages, iii) limited systemic toxicity, iv) high efficacy against tumors resistant to checkpoint inhibitor (CPI) treatment, and v) significant tumor shrinkage even at very low doses.

[0007] In a first aspect, the present invention provides a method for producing a composition comprising the steps of: a) a first promoter comprising or consisting of: (i) at least one viral early promoter element and optionally at least one viral late promoter element, the viral early promoter element comprising the nucleic acid sequence AAN 1 N 2AN 3 TGAAN 4 N 5 N 6 N 7 N 8 A (SEQ ID NO: 1), 1 , N 2 , N 4 , N 5 and N 6 each is independently selected from A or T; 3 is selected from C, G, or T, and N 7 is selected from C and A, and N 8 is selected from A, C and T; or (ii) at least one viral late promoter element and at least three viral early promoter elements; And, b) a first nucleic acid sequence encoding at least one immunostimulatory protein; The present invention relates to a recombinant orthopoxvirus vector comprising, in operable linkage,

[0008] In a second aspect, the present invention provides a cell comprising a recombinant orthopoxvirus vector of the present invention.

[0009] In a third aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) a recombinant orthopoxvirus vector according to the first aspect of the invention or a cell according to the second aspect of the invention, and b) a pharma- ceutically acceptable carrier, and Optionally, c) a recombinant viral vector comprising a nucleic acid sequence encoding a checkpoint inhibitor, a nucleic acid encoding a checkpoint inhibitor, or a checkpoint inhibitor; The present invention relates to a composition comprising:

[0010] In a fourth aspect, the present invention provides a recombinant orthopoxvirus vector of the first aspect of the invention, a cell of the second aspect of the invention, or a composition of the third aspect of the invention for use in medicine.

[0011] In a fifth aspect, the present invention provides a recombinant orthopoxvirus vector of the first aspect of the invention, a cell of the second aspect of the invention, or a composition of the third aspect of the invention for use in the treatment or prevention of cancer. [Brief description of the drawings]

[0012] [Figure 1] Efficacy of intratumorally (it) injected MVA-SynE1-IL12 and MVA-7.5-IL12 in anti-PD1 resistant tumors. Tumor-bearing mice (LLC tumors) were treated with MVA encoding IL12 under the control of a synthetic early promoter (MVA-SynE1-IL12) or MVA-IL12 under the control of the 7.5 promoter (MVA-7.5-IL12) at a dose of 10e7 infectious units (ifu) in combination with anti-PD1 antibodies injected intraperitoneally (ip). Treatment started on day 0 for mice randomized according to tumor volume. Injections of MVA were repeated on days 2 and 4, and anti-PD1 treatment was performed twice a week until day 17. Time course of tumor growth of individual mice belonging to the different treatment groups is shown. Dashed lines represent non-responding mice. [Diagram 2] Efficacy of low dose MVA-SynE1-IL12 under anti-PD1 treatment. Tumor-bearing mice (LLC tumors) were treated with MVA encoding IL12 under the control of a synthetic early promoter (MVA-SynE1-IL12) at a dose of 10e7 infectious units (ifu) or 2x10e5 ifu in the presence of anti-PD1 antibodies injected intraperitoneally (ip). Treatment started on day 0 for mice randomized according to tumor volume. Injections of MVA were repeated on days 2 and 4, and anti-PD1 treatment was performed twice a week until day 17. Curves of tumor growth over time are shown. [Diagram 3]Efficacy of MVA-SynE1-IL12 as monotherapy in low dose treatment. Tumor-bearing mice (LLC tumors) were administered MVA encoding IL12 under the control of a synthetic early promoter (MVA-SynE1-IL12) at a dose of 10e7 infectious units (ifu) or 2x10e5 ifu (monotherapy). Treatment started on day 0 for mice randomized according to tumor volume. Injections of MVA were repeated on days 2 and 4, and anti-PD1 treatment was performed twice a week until day 17. Curves of tumor growth over time are shown. [Figure 4] Comparison of efficacy of MVA-SynE1-IL12 and Ad-IL12 in a peritoneal carcinomatosis model. CT26 tumor cells were injected intraperitoneally into BAlBC mice 3 days before the start of treatment. On day 0, mice were treated with MVA encoding IL12 under a synthetic early promoter (MVA-SynE1-IL12) at a dose of 10e7 infectious units (ifu) or with an equivalent dose of Adeno encoding IL12. Treatment was repeated on days 2 and 4. Overall survival of control mice (untreated), MVA-SynE1-IL12, and Ad-IL12 treated mice is shown. [Diagram 5] In vitro expression of IL12 in HeLa cells infected with Adeno or MVA encoding IL12. HeLa cells were infected with the indicated vectors encoding IL12 at 1 MOI (1 infectious unit / cell). Supernatants were harvested 24 hours post-infection and subjected to IL12 Elisa assay. For MVA infected cells, results are the mean SD of two independent experiments. [Figure 6]Intratumoral expression of IL12 following in vivo treatment in mice. Tumor-bearing mice (LLC tumors) were treated with a single injection of Adeno encoding IL12 (Ad-IL12), MVA encoding IL12 under control of the native 7.5 promoter (MVA-p7.5-IL12), or MVA encoding IL12 under control of a synthetic early promoter (MVA-SynE1-IL12) at a dose of 107 ifu. Expression of IL12 from harvested tumors was measured over time by ELISA assay and expressed as pg / ug protein lysate. [Figure 7] Expression of IL12 in muscle and tumor during in vivo treatment. Mice were injected intramuscularly or intratumorally with MVA encoding IL12 under a synthetic early promoter (MVA-SynE1-IL12) at a dose of 107 ifu. Expression of IL12 from tumor and muscle was measured by ELISA assay and expressed as pg / ug protein lysate. [Figure 8] Levels of intratumoral M1 and M2 macrophages after intratumoral treatment with MVA-IL12. Tumor-bearing mice (LLC tumors) were administered MVA encoding IL12 under a synthetic early promoter (MVA-SynE1-IL12) or MVA mock at a dose of 107 ifu by intratumoral injection on days 0, 2, and 4. Tumors were harvested on day 7 and analyzed by flow cytometry to measure levels of M1 and M2 macrophages after treatment. [Figure 9] Schematic diagram of the promoter arrangement of P7.5, SynE1, and SynE2. 7.5L: late element from the P7.5 promoter; 7.5E: early element from the P7.5 promoter; sL: synthetic late element (SEQ ID NO: 12); 7.5Emod: early element from the modified P7.5 promoter (SEQ ID NO: 4). Arrows indicate the direction of transcription. [Figure 10]Efficacy of MVA-SynE1-IL12 alone and in combination with anti-PD1 in a B16F10 tumor model resistant to anti-PD1 activity. Survival curves of C57BL / 6 mice injected subcutaneously with B16F10 cells. Mice (n=9 per group) with established tumors were randomized and treated (at a dose of 6x105 IFU for 2 cycles of 4 injections every 4 days starting on day 0) with MVA encoding IL12 under the control of a synthetic early promoter (MVA-SynE1-IL12) alone or in combination with anti-PD1 antibody, injected intraperitoneally twice weekly until day 17. Mice treated with anti-PD1 served as a control group. [Figure 11] MVA-SynE1-IL12 intratumoral injection (IT) controls tumor growth of uninjected distant tumors. (a-f) C57BL / 6 mice were inoculated subcutaneously with MC38 cells in both flanks (bilateral tumor implants). Mice with tumors were randomly divided according to whether tumor volumes were similar (n=7-10 mice per group). One of the tumors (right flank) was intratumorally injected with (a) MVA-mIL12 at 107 IFU or (b) MVA-mIL12 at 6x105 IFU in combination with anti-PD1 (two cycles of four injections every 4 days starting on day 0), and (d, e) the other tumor (left flank) did not receive IT treatment. Mice receiving only anti-PD1 were used as controls in both flanks. Tumor volumes were monitored over time. The lines in the graphs represent individual tumors (solid lines represent responding tumors, dotted lines represent non-responding tumors). The percentages on the graphs indicate the response rate (complete response rate, CR). [Figure 12-1]SynE promoters containing consensus SEQ ID NO:1 or SEQ ID NO:3 drive stronger early transgene expression compared to P7.5. (a-b) Schematic diagram of the various SynE promoters tested. The identity of the early (black box) and late (white box) promoter elements is shown in each box with the respective SEQ ID NO:5, where applicable. Consensus SEQ ID NO:3 contains early elements SEQ ID NO:4 and SEQ ID NO:5. The nucleotide sequences of early elements Early-A (SEQ ID NO:43), Early-B (SEQ ID NO:44), Early-C (SEQ ID NO:45), and Early-D (SEQ ID NO:46) are shown in b), which contain sequences contained in the more generalized consensus SEQ ID NO:1, with fixed nucleotide positions in SEQ ID NO:1 shown in bold. 7.5L: late element derived from P7.5 promoter; 7.5E: early element derived from P7.5 promoter; SynE1 corresponds to SEQ ID NO:18; SynE2 corresponds to SEQ ID NO:19; SynE5 corresponds to SEQ ID NO:22; SynE7 corresponds to SEQ ID NO:24. The arrow indicates the direction of transcription. [Figure 12-2] (c) To test early promoter activity, HeLa cells were infected with MVA-Red (MVA vector encoding the reporter HcRed protein, whose expression was not evaluated in this experiment) and 2.5 hours later, transfected with a plasmid encoding mIL12 under the control of various SynE promoters or the P7.5 promoter. AraC (40ug / ml) was added at the time of infection with MVA vectors to block intermediate / late expression. mIL12 levels were measured by ELISA in cell culture supernatants 4 hours post-transfection and are expressed as fold change relative to the levels measured in cells transfected with P7.5-mIL12. n=3 for P7.5, SynE1, SynE2, SynE7, n=2 for SynE5, SynE14, SynE15, and SynE16. FOC=fold change. [Figure 13] Description of SEQ ID NO:1 in WIPO ST.25 format. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Before describing the invention in detail below, it is to be understood that the invention is not limited to the specific methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0014] Preferably, the terms used herein are defined as set forth in "Multilingual Glossary of Biotechnology Terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Koelbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0015] Several documents are cited within the text of this specification, including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior invention.

[0016] definition

[0017] Each element of the present invention will be described below. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the present invention to only the explicitly described embodiments. The description herein should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed elements and / or preferred elements. Furthermore, any permutation and combination of all elements described herein should be considered to be disclosed by the description herein, unless otherwise indicated by context.

[0018] Throughout this specification and the claims which follow, unless the context clearly dictates otherwise, the word "comprise" and variations such as "comprises" and "comprising" are understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of other integers or steps or groups of integers or steps. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0019] In the context of the present invention, the term nucleic acid" refers to single- or double-stranded oligos or polymers of deoxyribonucleotide or ribonucleotide bases or both. Nucleotide monomers are composed of a nucleobase, a five-carbon sugar (such as ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, nucleic acids are formed by phosphodiester bonds between individual nucleotide monomers. In the context of the present invention, the term nucleic acid includes, but is not limited to, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules, and also includes synthetic forms of nucleic acids containing other bonds (e.g., peptide nucleic acid described by Nielsen et al. (Science 254:1497-1500, 1991)). Typically, nucleic acids are single- or double-stranded molecules, composed of naturally occurring nucleotides. The depiction of a single strand of a nucleic acid also defines (at least in part) the sequence of the complementary strand. Nucleic acids can be single- or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. The illustrated double-stranded nucleic acid molecules may have 3' or 5' overhangs, and thus are not required to be, nor are they assumed to be, completely double-stranded over their entire length. Nucleic acids can be obtained by any method known in the art, including, but not limited to, biological, biochemical, or chemical synthesis methods, or amplification methods, reverse transcription of RNA. The term nucleic acid includes chromosomes or chromosome segments, vectors (e.g., expression vectors), expression cassettes, naked DNA or RNA polymers, primers, probes, cDNA, genomic DNA, recombinant DNA, cRNA, mRNA, tRNA, microRNA (miRNA), or small interfering RNA (siRNA). Nucleic acids may be, for example, single-stranded, double-stranded, or triple-stranded, and are not limited to a particular length. Unless otherwise indicated, a particular nucleic acid sequence includes or encodes complementary sequences in addition to the sequence explicitly indicated.

[0020] " Isolated Nucleic AcidsA "cDNA" molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes a nucleic acid molecule that is separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an isolated nucleic acid is free of sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid). Moreover, an isolated nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized.

[0021] In the context of the present invention, the term gene " refers to a collection of nucleotides that encodes an RNA transcript or a polypeptide, and includes cDNA and genomic DNA nucleic acids. "Gene" can also refer to a nucleic acid fragment that expresses a specific protein or polypeptide, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene found in nature with its own regulatory sequences. "Chimeric gene" refers to a gene that is not a natural gene, and contains regulatory sequences and / or coding sequences that are not found together in nature. That is, a chimeric gene may contain regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences that are derived from the same source but arranged in a different way than found in nature. A chimeric gene may contain coding sequences that are derived from different sources and / or regulatory sequences that are derived from different sources. "Endogenous gene" refers to a naturally occurring gene in its natural location in the genome of an organism. "Foreign" or "heterologous" gene refers to a gene that is not normally present in the host organism, but is introduced into the host organism by gene transfer. Foreign genes may include native genes inserted into a non-native organism, or chimeric genes.

[0022] The term " genome" includes not only chromosomal DNA or RNA but also mitochondrial, chloroplast, and viral DNA or RNA.

[0023] " vector ", Vector constructs "or" Recombinant Vectors The term " vector " is used interchangeably in the context of the present invention and refers to a polynucleotide encoding a protein of interest, or a mixture containing a polynucleotide and a polypeptide encoding a protein of interest, which is introduced into a cell or whose proteins and / or nucleic acids contained therein can be introduced into a cell. Examples of vectors include, but are not limited to, plasmids, cosmids, phages, viruses, artificial chromosomes, and the like. Vectors are used to introduce a gene product of interest, e.g., foreign or heterologous DNA, into a host cell. Certain vectors are capable of directing the expression of genes to which they are operably linked.

[0024] A vector in the context of the present invention may further comprise at least one promoter suitable for driving expression of a gene in a host cell. Expression vector The term "initiator" refers to a vector, plasmid or vehicle designed to allow expression of an inserted nucleic acid sequence after transformation into a host. The cloned gene, i.e., the inserted nucleic acid sequence, is usually placed under the control of control elements such as a promoter, a minimal promoter, an enhancer, etc. Initiation control regions or promoters useful to drive expression of a nucleic acid in a desired host cell are numerous and familiar to those skilled in the art.

[0025] A vector may contain a "replicon" polynucleotide sequence that facilitates autonomous replication of the vector within a host cell. Foreign DNA is defined as heterologous DNA, which is DNA that does not naturally occur in a host cell, for example, replicating the vector molecule, encoding a selectable or screenable marker, or encoding a transgene. Once inside a host cell, the vector can replicate independently or simultaneously with the host's chromosomal DNA, resulting in multiple copies of the vector and the inserted DNA. Additionally, a vector may also contain elements necessary to transcribe the inserted DNA into an mRNA molecule or replicate the inserted DNA into multiple RNA copies. A vector may further contain an "expression control sequence" that controls the expression of a gene of interest. Typically, an expression control sequence is a polypeptide or a polynucleotide such as a promoter, enhancer, silencer, insulator, or repressor. In a vector that contains multiple polynucleotides encoding one or more gene products of interest, expression may be controlled jointly or individually by one or more expression control sequences. More specifically, each polynucleotide contained in a vector may be controlled by a separate expression control sequence, or all polynucleotides contained in a vector may be controlled by a single expression control sequence. Polynucleotides contained in a single vector controlled by a single expression control sequence may form one open reading frame. Some expression vectors further contain sequence elements adjacent to the inserted DNA, which extend the half-life of the expressed mRNA and / or allow the mRNA to be translated into a protein molecule. Many mRNA and polypeptide molecules encoded by the inserted DNA can be rapidly synthesized in this way. Such vectors may contain regulatory elements such as promoters, enhancers, terminators, etc. to cause or directly express the polypeptide when administered to a subject.

[0026] Preferably, the vector comprises an expression cassette comprising a promoter and a coding sequence, the expression of which is controlled by said promoter.

[0027] Preferably, the vector in the context of the present invention is a viral vector. Viral Vectors (viralvector)" or " Viral Vectors The term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin, has the ability to be packaged into a viral vector particle, and encodes at least one exogenous nucleic acid. The vector and / or particle can be utilized to introduce any nucleic acid into a cell in vitro or in vivo. Many forms of viral vectors are known in the art. The term "virion" is used to refer to a single infectious viral particle. Viral Vectors ", Viral Vector Particles " and " Virus particles The term also refers to the complete virus particle with a DNA or RNA core and protein coat that resides outside a cell.

[0028] Preferably, the viral vector in the context of the present invention can be used to infect cells and cell lines, particularly live animals, including humans.More preferably, the viral vector according to the present invention infects antigen-presenting cells, most preferably macrophages.Exemplary viral vectors can be selected from adenovirus, poxvirus, alphavirus, arenavirus, flavirus, rhabdovirus, retrovirus, lentivirus, herpesvirus, paramyxovirus, or picornavirus.Preferably, the viral vector is derived from poxvirus, more preferably from vaccinia virus, most preferably from Modified Vaccinia Ankara (MVA) virus.

[0029] Generally, the recombinant virus vector of the present invention can be packaged into a virus particle.For example, the recombinant modified vaccinia virus vector of the present invention can be packaged into a modified vaccinia virus particle.Preferably, the virion or virus particle is attenuated, which means that the virus can grow and replicate in avian cells, such as chicken embryo fibroblasts (CEF) and immortalized cell lines derived from avian primary cells, such as duck Cairina primary retinal cells, but cannot grow and replicate in human cell lines, such as human embryonic kidney cell line 293 cells and human cervical adenocarcinoma cell line HeLa cells.

[0030] MVA is related to vaccinia virus, which belongs to the genus Orthopoxvirus of the family Poxviridae, and has been generated by more than 570 serial passages in primary chicken embryo fibroblast (CEF) cells (Mayr, A et al., Infection 3, 6-14, 1975). As a result of these long passages, the host range of the virus has been severely restricted, rendering MVA unable to productively infect many mammalian cells. On the other hand, MVA has shown an excellent safety profile and immunogenicity in the clinic, and has been well tolerated, highlighting its potential as a safe vector for the development of vaccines and gene therapy candidates. The term "MVA" as used herein refers to any MVA strain known in the prior art. Preferred examples of MVA strains on which the recombinant orthopoxvirus vector of the invention can be based are the strain MVA-BN (the nucleic acid sequence of the genome of this strain is accessible under GenBank accession number: DQ983238.1), the strain MVA572 (the nucleic acid sequence of the genome of this strain is accessible under GenBank accession number: DQ983237.1), the strain MVA-I721 MVA-I721 (the nucleic acid sequence of the genome of this strain is accessible under GenBank accession number: DQ983236.1), Acambis 3000 (GenBank accession number: AY603355.1) or the strain MVATGN33.1 (the nucleic acid sequence of the genome of this strain is accessible under GenBank accession number: EF675191.1).

[0031] In the context of the present invention, the term " promoter " refers to a regulatory region of DNA located generally upstream (5' region of the sense strand) of a gene that allows the gene to be transcribed. Promoters contain specific DNA sequences and response elements that are recognized by proteins known as transcription factors. These factors bind to the promoter sequence and recruit RNA polymerase and enzymes that synthesize RNA from the coding region of the gene. The terms "upstream" and "downstream" are terms used to describe the relative orientation between two elements present in a nucleotide sequence or vector. An element that is "upstream" of another element is located closer to the 5' end of the sequence (i.e., closer to the end of the molecule where the phosphate group is attached to the 5' carbon of the ribose or deoxyribose backbone, if the molecule is linear) than the other element. An element is said to be "downstream" if it is located closer to the 3' end of the sequence (i.e., closer to the end of the molecule where the hydroxyl group is attached to the 3' carbon of the ribose or deoxyribose backbone, if the molecule is linear) compared to the other element.

[0032] In the context of the present invention, the term " Late promoter elements" refers to a nucleic acid sequence present in the genome of a virus that drives the expression of viral genes at a late stage of infection of a cell by the virus. The term "late promoter element" also includes mutants of naturally occurring viral late promoter elements. Preferably, such mutants have at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% nucleic acid sequence identity to a naturally occurring viral late promoter element and at least direct the transcription of genes under the control of the mutant viral late promoter element to the same level as a naturally occurring viral late promoter element. The transcription level of genes under the control of such promoters can be determined using methods known in the art, including, in particular, quantitative PCR (qPCR) of cDNA generated from RNA isolated from cells infected with a viral vector.

[0033] In the context of the present invention, Early promoter elements The term "early promoter element" refers to a nucleic acid sequence present in the genome of a virus that drives the expression of viral genes at an early stage of infection of a cell by the virus. The term "early promoter element" also includes variants of naturally occurring viral early promoter elements, such as mutant viral early promoter elements. Preferably, such variants have at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% nucleic acid sequence identity to a naturally occurring viral early promoter element, and at least direct the transcription of genes under the control of the mutant viral early promoter element to the same level as a naturally occurring viral early promoter element. The transcription level of genes under the control of such promoters can be determined using methods known in the art, including in particular quantitative PCR (qPCR) of cDNA generated from RNA isolated from cells infected with a viral vector.

[0034] Preferably, the promoter in the context of the present invention is suitable for the expression of heterologous genes in poxviruses. Poxviruses control gene expression at the transcriptional level through a cascade-like mechanism involving three major gene classes: early, middle, and late (the latter two classes are expressed after genome replication). Therefore, promoters with activity for either gene class are preferred. Promoters with both early and late activity are commonly used to direct the expression of foreign antigens in poxvirus vectors utilized as vaccine vectors, preferably injected intramuscularly, so that the appropriate expression levels are present at the appropriate time to induce a strong immune response. Such promoters include, but are not limited to, native poxvirus promoters that drive the expression of viral proteins, such as the p7.5k30k and 40k promoters. Furthermore, synthetic promoters may employ multiple early and late elements. For example, the pHyb promoter has been shown to drive antigen expression early during infection and also to induce a stronger CD8 T cell response after repeated vaccination compared to the PrS and p7.5k promoters. Thus, the term "p7.5k promoter" in the context of the present invention is a well-known promoter that is used to express antigens early during infection and to induce a stronger CD8 T cell response after repeated vaccination. Early promoter " refers to a promoter that is active in poxviruses or cells infected with poxviruses at an early stage before viral DNA replication occurs. Late promoter " refers to a promoter that is active after DNA replication has taken place. Thus, the term "early-late promoter" in the context of this specification refers to a promoter that is active in both the time frames of the early and late promoters. A promoter in the context of the present invention is preferably a synthetic promoter and comprises at least one poxvirus early element, more preferably one late poxvirus promoter element and at least three early poxvirus promoter elements.

[0035] In the context of the present invention, Operable connection "or" operably linkedThe term "operably linked" refers to the arrangement of two or more components, which are in a relationship that allows them to function in concert. By way of example, a promoter is operably linked to a coding sequence if the promoter drives transcription of the coding sequence. Aspects of the transcription process include, but are not limited to, initiation, elongation, decay, termination, etc.

[0036] The term "therapeutic protein" in the present invention refers to recombinant proteins that are widely used in the pharmaceutical field. Based on their pharmacological activity, they can be classified into five groups: (a) those that replace missing or abnormal proteins; (b) those that extend existing pathways; (c) those that provide new functions or activities; (d) those that interfere with molecules or organisms; and (e) those that deliver other compounds or proteins, such as radionuclides, cytotoxic drugs, or effector proteins. They can also be classified based on their molecular type (e.g., antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, artificial protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents, etc.) or the molecular mechanism of activity: (a) those that bind non-covalently to the target, e.g., mAbs; (b) those that affect covalent binding, e.g., enzymes; and (c) those that exert activity without specific interactions, e.g., serum albumin (Dimitrov, DS, Methods Mol. Biol. 2012).

[0037] " immunostimulatory molecules ", Immune stimulants "or" immunostimulating factors " may be used interchangeably and in the context of the present invention refers to a substance that induces the activation or increases the activity of any component of the immune system, thereby stimulating the immune system. Many molecules capable of stimulating the immune system are known to those skilled in the art. In this respect, of particular interest in the context of the present invention are inflammatory or proinflammatory molecules.

[0038] As used herein, " inflammatory molecules "or" Proinflammatory molecules The term "inflammatory molecule" refers to a molecule that can transition the tumor microenvironment (TME) from an immunosuppressive state to an immunostimulatory state. Immunostimulatory molecules can be secreted by immune cells that promote inflammation, such as, for example, helper T cells, macrophages, astrocytes, monocytes, etc. In particular, the term "inflammatory molecule" refers to cytokines.

[0039] In the context of the present invention, Cytokines "or" Inflammatory (pro)cytokines The term "macrophages" refers to polypeptides produced throughout the body, mainly by activated macrophages. These molecules play important roles in many physiological responses and have diverse effects, including autocrine (acting on the cell that secretes them), paracrine (acting on nearby cells), endocrine (acting on distant cells), and junctional secretion (transmitted via oligosaccharide, lipid, or protein components of adjacent cell membranes). A typical role of these molecules is related to the regulation of immune and inflammatory processes (Navarro-Gonzalez et al., Nat. Rev. Nephrol. 7, 327-340, 2011). Typical cytokines involved in inflammation can be classified as interleukins (e.g., IL-1, IL-6, IL-12, IL-15); interferons (e.g., IFNα, IFNβ, IFNγ); tumor necrosis factors (e.g., TNFα, TNFβ); chemokines (e.g., CC, CXC, CX3 chemokines); colony-stimulating factors (e.g., GM-CSF, G-CSF, M-CSF, IL-3); growth factors (e.g., EPO, TPO, EGF, FGF, PDGF, BDNF, VGF, TGFβ); adhesion molecules (e.g., ICAM, VCAM); enzymes (e.g., phospholipase A); complement-related molecules (e.g., C3, C5); and miscellaneous molecules (e.g., PAI-1, MIF, pentraxin, SAA, lactoferrin, procalcitonin, LCN2).

[0040] As used in the present invention, Interleukins "or" ILThe term "interleukins" refers to a type of cytokine that regulates inflammation and immune responses. The sources of interleukins are diverse, including not only white blood cells but also almost all lymphocytes and tumor cells. Interleukins are produced by various cells and act on many cells, forming a complex regulatory network. In general, the interleukin family has three main functions: i) activating and regulating immune cells, ii) transmitting information to various cells, and iii) participating in inflammatory responses. Currently, a total of 38 interleukins, named IL-1 to IL-38, have been identified, which can be further classified into IL-1, IL-6, IL-10, IL-12, and IL-17 families, interleukin members of the chemokine family, and unclassified interleukins according to differences in molecular structure and their receptors. Each interleukin family contains multiple interleukin members.

[0041] In the context of the present invention, Interleukin 12 "or" IL-12The term IL-12 refers to heterodimeric cytokines belonging to the interleukin 12 (IL-12) family, which is composed of four members: IL-12, IL-23, IL-27, and IL-35. This family plays a key role in shaping immune responses upon antigen presentation and influences the cell fate decisions of naive T cells during differentiation. In addition, the IL-12 family controls cellular pathways required for the immune system to function properly, with some members activating proinflammatory responses to provide protection against infections and others suppressing uninhibited immune responses that lead to autoimmune diseases (Sun et al., Cytokine 2015, 75(2): 249-255). IL-12, IL-23, and IL-27 are secreted by activated antigen-presenting cells (APCs) to present antigens to naive T cells, whereas IL-35 is a product of regulatory T cells and regulatory B cells. Each member consists of a helical α subunit (i.e., IL-12p35, IL-23p19, and IL27p28) and a β subunit (i.e., IL-12p40, and Ebi3) covalently linked by a disulfide bond.

[0042] In the context of this specification, the term " Single chain IL-12" (sc-IL12) refers to IL-12 engineered to express an IL-12p40 polypeptide fused to an IL-12p35 polypeptide via a linker sequence, such that the p40 / p35 molecule is produced as a single polypeptide chain. The configuration can be in either order, starting with the p40 polypeptide as the amino-terminal portion (N-terminus) and linked to the p35 polypeptide as the carboxyl-terminal portion (C-terminus), producing a single polypeptide in a format called "p40-linker-p35". Conversely, in a sc-IL12 construct, the p35 portion can be the N-terminal portion linked to p40 as the C-terminal portion, in a format called "p35-linker-p40". Further possible configurations include "p40-linker-p35-linker-p40" or "p35-linker-p40-linker-p35". Secretion of sc-IL12 into the extracellular space is achieved by the presence of a signal peptide at the N-terminus of sc-IL12, preferably the signal peptide is derived from human IL-12p40 or human IL-12p35, more preferably from human IL-12p40.

[0043] In the context of the present invention, the term Linker" refers to a nucleic acid or amino acid sequence that sterically separates two parts or portions of a complex (e.g., two peptides, polypeptides or proteins, nucleic acids with a specific function (e.g., promoter elements)). The peptide linker provides flexibility between the two linked portions. In general, the smaller the amino acid, the more flexible it is. Typically, such linkers contain or consist of 1-20 amino acids. That is, flexible peptide linkers include those with an increased content of small amino acids, particularly glycine and / or alanine, and / or hydrophilic amino acids, such as, for example, serine, threonine, asparagine, and glutamine. In the context of the present invention, a linker, e.g., one or more amino acids, inserted between two domains provides sufficient mobility to the domains, e.g., in a single-chain construct. The nucleotide linker between the promoter elements is intended to provide spacing between the elements to provide sufficient space for a promoter-binding protein (e.g., a transcriptional activator) to bind to the respective promoter element, with approximately seven consecutive nucleotides constituting one turn of the double helix. When two elements are separated by seven nucleotides, they are located in the same spatial orientation (e.g., at the same site of the DNA double helix). As with the linker elements inserted between the late promoter elements, between the early promoter elements, and between the late and early promoter elements in the promoters of the present invention, the nucleic acid linkers between the nucleotide sequence elements preferably have a length of 5 to 8 nucleotides.

[0044] The " Checkpoint inhibitors "or" Immune checkpoint inhibitorsThe term "immune checkpoint inhibitors (ICIs)" refers to drugs such as monoclonal antibodies that specifically target immune checkpoints and inhibit their function, and is mainly used in the field of tumor therapy. For example, several small molecules targeting other immune checkpoints, such as LAG3, TIGIT, TIM3, B7H3, CD39, CD73, adenosine A2A receptor, and CD47, are in clinical development. Checkpoint inhibitors act by releasing the inhibitory brakes on T cells, potently activating the immune system and resulting in productive antitumor immune responses. ICIs can be classified into three FDA (U.S. Food and Drug Administration)-approved drug groups depending on the molecule they target. These drugs include antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), antibodies that block programmed cell death 1 (PD-1), an inhibitory receptor on T cells, and interact with its ligands PD-L1 and PD-L2 to disrupt active T cell responses, and antibodies against PD-L1. Examples of approved CTLA-4 inhibitors include ipilimumab. Examples of approved PD-1 or PD-L1 inhibitors include pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, and durvalumab.

[0045] The term "antigen" refers to a substance that can be recognized by an antibody, B cell, or T cell. The term "tumor antigen" or "tumor associated antigen (TAA)" used in the context of the present invention refers to a protein or polypeptide expressed by a tumor cell, or an antigenic fragment thereof. Antigenic fragments are usually presented by MHC-I or MHC-II and induce a T cell response.

[0046] In the context of the present invention, the term " Macrophages" refers to myeloid immune cells that reside in every tissue and exhibit distinct phenotypes and great functional diversity. They play important roles in development, homeostasis, tissue repair, immunity, and inflammatory processes. Roughly speaking, macrophages can be activated into two distinct subsets, classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages), based on the M1 / M2 paradigm. M1 macrophages are polarized in vitro by Th1 cytokines such as colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-α), and interferon-gamma (IFN-γ), alone or in combination with bacterial-derived lipopolysaccharide (LPS). M1 macrophages express proinflammatory cytokines such as interleukin-1β (IL-1β), IL-6, IL-12, IL-23, and TNF-α. In contrast, M2 macrophages are polarized by Th2 cytokines such as IL-4 and IL-13 and produce anti-inflammatory cytokines such as IL-10 and transforming growth factor beta (TGF-β). Macrophages also have the ability to change polarization in response to different stimuli (Zhang et al., Front. Immunol., 2021).

[0047] term" Tumor-associated macrophages (TAMs) " refers to macrophages, which are involved in shaping the tumor microenvironment. They are widely present in various tumors and can promote tumor growth, invasion, metastasis, and drug resistance.

[0048] In the context of this specification, the term " polarization " refers to the phenotypic and functional characteristics of macrophages. The phenotype can be defined through the surface markers expressed by the macrophage. The functionality can be defined, for example, based on the nature and amount of chemokines and / or cytokines expressed by the macrophage. Depending on the condition, macrophages may exhibit different phenotypic and functional characteristics, either pro-inflammatory M1-like macrophages or anti-inflammatory M2-like macrophages.

[0049] In the context of this application, the term " M1 macrophages " refers to proinflammatory or classically activated macrophages. They are highly phagocytic and generate large amounts of reactive oxygen and nitrogen species, thereby promoting Th1 responses. M1 macrophages are also defined by the expression of surface markers such as CD68 and CCR7. M1 macrophages secrete high levels of inflammatory cytokines such as IL-12 and IL-23. IL-12 induces activation and clonal expansion of Th17 cells, which in turn secrete large amounts of IL-17, which contributes to inflammation. These properties enable M1 macrophages to control metastasis, inhibit tumor growth, and control microbial infections. Furthermore, infiltration and recruitment of M1 macrophages to tumor sites correlates with improved prognosis and increased overall survival in patients with solid tumors. Once recognized, malignant cells can be destroyed by M1 macrophages through several mechanisms, including contact-dependent phagocytosis and cytotoxicity (i.e., release of cytokines such as TNF-α).

[0050] In the context of this application, M1-like macrophages "or" M1-polarized macrophages " refers to macrophages that contain a polarized state that leads to anti-tumor responses and cytotoxicity, such as that induced by GM-CSF.

[0051] As used herein, " M2-like macrophages "or" M2-polarized macrophagesThe term "M2 macrophages" refers to anti-inflammatory macrophages that aid in the processes of angiogenesis and tissue repair. They are characterized by the expression of surface markers such as CD206, PD-LI, and CD200R, express scavenger receptors, and produce large amounts of IL-10 and other anti-inflammatory cytokines. Expression of IL-10 by M2 macrophages promotes Th2 responses. As a result, Th2 cells upregulate the production of IL-4 and IL-3, which, in concert with other cytokines (e.g., erythropoietin, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-6), stimulates the proliferation of all cells of the myeloid lineage (e.g., granulocytes, monocytes, and dendritic cells). M2 macrophages exert functions that may aid in tumor progression by allowing blood vessels to nourish malignant cells and promote their growth. Furthermore, the presence of M2 macrophages is associated with the metastatic potential of breast cancer.

[0052] As used in the context of this invention, the term " amino acid" refers to any monomeric unit that includes a substituted or unsubstituted amino group, a substituted or unsubstituted carboxy group, and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, for example, thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, boric acid, boronic acid, phospho, phosphono, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. As used herein, the term "amino acid" includes the twenty naturally occurring or genetically encoded alpha amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). If an "X" residue is not defined, it is interpreted as "any amino acid." The structures of these 20 naturally occurring amino acids are shown, for example, in Stryer et al., Biochemistry, 5th ed., Freeman and Company (2002).

[0053] As referred to in this specification, Sequence identity "or" sequence homologyThe terms "sequence identity" and "sequence identity" are interchangeable and are used in relation to the comparison of polypeptide and nucleotide sequences. Comparing two sequences and calculating the percentage of sequence identity When no reference sequence is specified for comparison, the sequence identity should be calculated based on the longer of the two sequences being compared, unless otherwise specified. When a reference sequence is indicated, the sequence identity is determined based on the full length of the reference sequence, as indicated by the SEQ ID NO. For example, when comparing a 200 amino acid polypeptide sequence with a reference 300 amino acid long polypeptide sequence, the maximum percentage of sequence identity may be 66.6% (200 / 300), whereas for a 150 amino acid long sequence, the maximum percentage of sequence identity may be 50% (150 / 300). If 15 of the 150 amino acids are different from each amino acid of the 300 amino acid reference sequence, the level of sequence identity drops to 45%. The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, may be determined by sequence alignment. Such alignments can be carried out using a number of art-known algorithms, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), or hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), available, for example, at http: / / www.ebi.ac.uk / Tools / clustalw / or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html or http: / / npsa-pbil.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_clustalw.html.Preferred parameters are the default parameters set at http: / / www.ebi.ac.uk / Tools / clustalw / or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html. Grades of sequence identity (sequence matching) can be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). BLAST protein searches are performed using the BLASTP program, score=50, wordlength=3. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described by Altschulet al. (1997) Nucleic Acids Res. 25: 3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be complemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics2003b, 19 Suppl. 1: I54-I62) or Markov random fields. Structure-based alignment of multiple protein sequences and / or structures can also be used, using information from sequence database searches, available homologs with 3D structures, and user-defined constraints (Pei J, Grishin NV: PROMALS: towards accurate multiple sequence alignments of distantly related proteins. Bioinformatics 2007, 23:802-808;3DCoffee@igs: a web server for combining sequences and structures into amultiple sequence alignment. Poirot O, Suhre K, Abergel C, O'Toole E, Notredame C. Nucleic Acids Res. 2004 Jul 1; 32: W37-40).When percentages of sequence identity are referred to in this application, unless specifically indicated otherwise, these percentages are calculated over the entire length of the longer sequence.

[0054] As used herein, the term " Pharmaceutical Compositions " refers to a combination of an active agent with inert or active pharma- ceutically acceptable carriers, diluents, and excipients, resulting in a composition suitable for therapeutic use. Furthermore, the pharmaceutical compositions containing the conjugates of the present invention can be formulated for oral, parenteral, topical, inhalation, rectal, sublingual, transdermal, subcutaneous, or vaginal application routes, depending on their chemical and physical properties. The pharmaceutical compositions include solids, semisolids, liquids, or transdermal therapeutic systems (TTS). Solid compositions are selected from the group consisting of tablets, coated tablets, powders, granules, pellets, capsules, effervescent tablets, or transdermal therapeutic systems. Also included are liquid compositions selected from the group consisting of solutions, syrups, infusions, extracts, solutions for intravenous administration, solutions for injection, or solutions of the conjugates of the present invention. Semisolid compositions that can be used in the present invention include emulsions, suspensions, creams, lotions, gels, globules, buccal tablets, and suppositories. If desired, the compositions may also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained release formulations, etc. The compositions can be formulated as suppositories, with traditional binders and carriers such as triglycerides.

[0055] In the context of the present invention, Pharmaceutically "or" Pharmaceutically acceptable The term " refers to molecular entities and compositions that do not produce adverse, allergic, or other undesirable reactions when administered to a mammal, particularly a human, as appropriate. A pharma- ceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0056] In the context of the present invention, the term " Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers may be liquid or solid. Liquid carriers include, but are not limited to, sterile liquids, such as saline solutions in water and oils, including, but not limited to, those of petroleum, animal, vegetable or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions, aqueous dextrose solutions, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Saline solutions are the preferred carrier when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0057] In the context of the present invention, Pharmaceutically acceptable carriers "teeth," Pharmaceutically acceptable diluents "or" Pharmaceutically acceptable vehicle " and may include solvents, fillers, stabilizers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0058] In the context of the present invention, the term " Excipients " refers to any substance, other than the active ingredient, present in or used in the manufacture of a drug product. Excipients function as carriers for the active ingredient and contribute to product properties such as stability, biopharmaceutical profile, appearance, and patient acceptability. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene glycol, water, ethanol, etc.

[0059] The term " Adjuvants " means a substance or combination of substances added to a vaccine, pharmaceutical composition, or drug product to enhance the effectiveness of the active ingredient and to stimulate and enhance the magnitude and duration of the immune response.

[0060] " Effective dose" and " Therapeutically Effective Dose The term "effective amount" refers to an amount that, when administered to a subject to treat a disease, may be effective to elicit a desired biological or medical response, including an amount of a compound sufficient to effect treatment of the disease. An effective amount will vary depending on the compound, the disease and its severity, the age, weight, etc., of the subject to be treated, but can be readily determined by one of ordinary skill in the art. An effective amount can include a variety of amounts. A pharma- ceutically effective amount includes an amount of an agent that is effective when combined with other agents.

[0061] As used herein, a disease or disorder is defined as Treat (treat)," Treat (treating)" or " treatment "(Treatment)" means achieving one or more of the following: (a) reducing the severity of the disease; (b) limiting or preventing the onset of symptoms characteristic of the disease being treated; (c) inhibiting the worsening of symptoms characteristic of the disease being treated; (d) limiting or preventing the recurrence of the disease in a subject who has previously had the disease; and (e) limiting or preventing the recurrence of symptoms of the disease in a subject who has previously had symptoms of the disease.

[0062] As referred to herein, a disease or disorder is defined as Prevent (prevent)," Prevent (preventing)" or " prevention The term "prevention" refers to preventing a disorder from occurring in a subject for a certain period of time. For example, when a compound described herein is administered to a subject for the purpose of preventing a disease or disorder, the disease or disorder is prevented from occurring at least on the day of administration, and one or more days from the date of administration, preferably several months or years after the date of administration. More specifically, in cancer prevention, this can be applied to the treatment of precancerous lesions to prevent progression to cancer.

[0063] As used in the context of the present invention, the terms "ameliorate", "ameliorating" or "amelioration" of a disease or disorder refer to any indication of success in treating said disease or disorder, including objective or subjective parameters, such as the alleviation, remission or reduction of symptoms, or an improvement in the physical health of a subject. Amelioration of symptoms can be based on objective or subjective parameters, including the results of a physical examination or evaluation.

[0064] According to the present invention, the term " subject " refers to animals, including humans. The term "animal" includes, but is not limited to, all animals, such as primates, including humans, gorillas, and monkeys, rodents, such as mice and rats, poultry, such as chickens, ruminants, such as goats, cows, deer, sheep, and other animals, including pigs, horses, cats, dogs, and rabbits.

[0065] Embodiment

[0066] In the research leading to the present invention, it was surprisingly found that the recombinant orthopoxvirus vector of the present invention allows for reproducible and controlled delivery of immunostimulants to tumors via infection of normal cells infiltrating the tumor. Moreover, the vector according to the present invention showed surprisingly efficient and high expression of the encoded immunostimulant while limiting systemic toxicity by controlled delivery of the immunostimulant only to the tumor. Furthermore, the vector of the present invention shows efficient reprogramming of M2-like macrophages to M1-like macrophages, shows high efficacy against tumors resistant to checkpoint inhibitor (CPI) treatment, and shows significant tumor shrinkage even at very low doses.

[0067] Based on these results, the present invention provides, in a first aspect, a) a first promoter comprising or consisting of: (i) at least one viral early promoter element, said viral early promoter element comprising the nucleic acid sequence AAN1 N 2 AN 3 TGAAN 4 N 5 N 6 N 7 N 8 A (SEQ ID NO: 1), 1 , N 2 , N 4 , N 5 and N 6 Each of N is independently selected from A or T (preferably A); 3 is selected from C, G or T (preferably T), and N 7 is selected from C and A (preferably C), and N 8 is selected from A, C and T (preferably T), or (ii) at least one viral late promoter element and at least three viral early promoter elements, and b) a first nucleic acid sequence encoding at least one immunostimulatory protein; In one aspect, a recombinant orthopoxvirus vector is provided, comprising in operative linkage:

[0068] According to some embodiments, the recombinant orthopoxvirus vector is an expression vector that can mediate the expression of inserted nucleic acid in a host cell.In this case, the expression vector of the present invention comprises an inserted nucleic acid that encodes at least one therapeutic protein under the control of an operably linked promoter.Those skilled in the art will understand that an expression vector may also comprise additional elements required for expression, such as, for example, an origin of replication, a translation initiation sequence (e.g., a ribosome binding site and a start codon), a stop codon, and a transcription termination sequence.

[0069] According to another embodiment, the recombinant orthopoxvirus vector comprises an expression cassette. In this regard, the expression cassette in the recombinant orthopoxvirus vector of the present invention comprises a promoter, a gene of interest (e.g., an immunostimulatory molecule and / or a tumor antigen). The vector may comprise sequences flanking the expression cassette, including sequences homologous to eukaryotic genomic sequences, e.g., mammalian genomic sequences or viral genomic sequences.

[0070] According to a further embodiment, the recombinant orthopoxvirus vector of the invention is an infectious virion or virus particle comprising the expression cassette. These virions or virus particles are capable of infecting various cells and cell lines, in particular live animals, including humans. Preferably, the virions or virus particles efficiently infect antigen-presenting cells (APCs), such as, for example, dendritic cells, macrophages, or B cells. More preferably, the virions or virus particles infect macrophages. Even more preferably, the virions or virus particles infect healthy macrophages. The virions or virus particles may also infect tumor-associated macrophages.

[0071] In some embodiments, the infection of cells occurs by binding of virions or viral particles to cell surface molecules, preferably receptors. More preferably, the receptor is a class A scavenger receptor. Common class A scavenger receptors include scavenger receptor type 1 (SR-A1, also called SCARA1 or MSR1), SCARA2 (also called MARCO or SR-A6), SCARA 3 (MSRL1, also called APC7 or SR-A3), SCARA4 (also called COLEC12 or SR-A4), and SCARA5 (also called TESR or SR-A5). In a preferred embodiment, the infection of cells occurs via SCARA2, also known as MARCO (macrophage receptor with collagen structure). Preferably, the virions or viral particles in the context of the present invention directly bind to SCARA2 (MARCO).

[0072] Furthermore, it is preferred that orthopoxvirus vectors in the context of the present invention are infectious whilst being impaired for viral replication within cells, thereby providing the natural limitations to viral infection.

[0073] According to one embodiment, the recombinant orthopoxvirus vector is based on a virus of a species selected from the group consisting of Orthopoxvirus variola, Orthopoxvirus vaccinia, Orthopoxvirus simiae, Orthopoxvirus bovis, Orthopoxvirus muris, Orthopoxvirus cameli, Raccoonpoxvirus or Taterapox virus. According to a more preferred embodiment, the orthopoxvirus of the Orthopoxvirus vaccinia species belongs to the subspecies Modified Vaccinia Ankara Virus (MVA).

[0074] Recombinant viral vectors based on modified vaccinia Ankara virus are of particular interest in the context of the present invention, as the inventors have described them as primarily infecting antigen-presenting cells, including macrophages, and being non-toxic as a result of deletion of genomic sequences during long-term passage. Furthermore, MVA is very promising for the expression of heterologous genes, due to its improved safety profile and high genetic plasticity, which allows the incorporation of large amounts of foreign DNA without loss of infectivity or gene expression.

[0075] The recombinant orthopoxvirus vector of the present invention also has the utility of promoting the expression of multiple exogenous nucleic acid sequences.The recombinant orthopoxvirus vector of the present invention is used in immunomodulatory therapeutic approaches.That is, the orthopoxvirus vector comprises a first nucleic acid sequence that codes for at least one immunostimulatory molecule.

[0076] In one embodiment, the recombinant orthopoxvirus vectors of the invention can be used in vitro and in vivo. Classes of genes contemplated for expression by the vectors of the invention include immunostimulatory or immunomodulatory proteins.

[0077] According to one embodiment, the immunostimulatory protein is a proinflammatory protein. According to a preferred embodiment, the proinflammatory protein is a cytokine. According to an even more preferred embodiment, the cytokine is an interleukin.

[0078] In one particularly preferred embodiment, the interleukin belongs to the interleukin 12 family. The interleukin 12 (IL-12) family is composed of four members: IL-12, IL-23, IL-27, and IL-35. According to a most preferred embodiment, the interleukin is interleukin 12.

[0079] IL-12 is secreted by various hematopoietic cells, including dendritic cells and macrophages. IL-12 is also a potent proinflammatory cytokine, inducing the secretion of other cytokines, including tumor necrosis factor alpha (TNF-α), which in combination with IFN-γ, is a prerequisite for the development of CD4+ cytotoxic T lymphocytes (CTLs). IL-12 has also been reported to induce repolarization of tumor-associated macrophages.

[0080] IL-12 is composed of IL-12p35 and IL-12p40 subunits, which must be expressed simultaneously to produce the biologically active dimeric IL-12p70. This can be achieved by expressing both subunits as one transcript, using an intervening internal ribosome entry site (IRES), or using a sequence encoding a self-cleaving amino acid sequence, or using a linker that links the subunits to produce a single chain IL-12, or by expressing two separate transgenes from the same promoter or two promoters. Suitable amino acid sequences are the murine IL-12p40 subunit having the amino acid sequence shown in SEQ ID NO:31, the murine IL-12p35 subunit having the amino acid sequence shown in SEQ ID NO:32 (without the N-terminal signal sequence), the human IL-12p40 subunit having the amino acid sequence shown in SEQ ID NO:33, and the human IL-12p35 subunit having the amino acid sequence shown in SEQ ID NO:34 (without the N-terminal signal sequence). Preferably, the IL-12 is human single chain IL-12 (sc-hIL-12), preferably having the amino acid sequence of SEQ ID NO:38-40, preferably SEQ ID NO:38.

[0081] According to some embodiments, the recombinant orthopoxvirus vector promotes IL12 production by macrophages. In some embodiments, the recombinant orthopoxvirus vector induces conversion or repolarization of macrophages to other functional phenotypes. According to another embodiment, macrophages are induced (repolarized) toward a pro-inflammatory M1 phenotype and / or away from an anti-inflammatory M2 phenotype.

[0082] According to another embodiment, the recombinant orthopoxvirus vector further comprises a second nucleic acid sequence encoding at least one tumor antigen or an antigenic fragment thereof.

[0083] The nucleic acid sequence to be expressed is placed operably linked to a promoter.

[0084] According to one embodiment, the two transgenes are linked to one promoter and separated by an intervening internal ribosome entry site (IRES) or by a sequence encoding a self-cleaving amino acid sequence.

[0085] According to another embodiment, the two transgenes are linked to two independent promoters, which may be the same promoter or, preferably, different promoters.

[0086] Preferably, the different nucleic acid elements comprised in the orthopoxvirus vector of the invention are arranged in the 5' to 3' direction (with reference to the coding nucleotide sequence): [Late promoter element] m -[Early promoter element] n - a nucleic acid sequence encoding at least one immunostimulatory protein, wherein m is 0-5, i.e. 1, 2, 3, 4, or 5, and n is 1-10, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 2-8, more preferably 4-6. In one particular embodiment, a nucleic acid linker is present between two or all of the early promoter elements and / or between an early promoter element and a late promoter element. m is preferably 1 or 2, more preferably 1, i.e. one or two late promoter elements are located upstream (or 5' prime) of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 2-8, more preferably 4-6 early promoter elements.

[0087] In the orthopoxvirus vector of the first aspect of the invention, the viral early promoter element is one that naturally occurs in an orthopoxvirus. Preferably, the viral early promoter element is an element that naturally occurs in a virus of a species selected from the group consisting of Orthopoxvirus variola, Orthopoxvirus vaccinia, Orthopoxvirus simiae, Orthopoxvirus bovis, Orthopoxvirus muris, Orthopoxvirus cameli, raccoon poxvirus or tatterapoxvirus. Preferably, the viral early promoter element is one that is naturally found in a virus of the Orthopoxvirus vaccinia species. According to a more preferred embodiment, the viral early promoter element is an element that naturally occurs in Modified Vaccinia Ankara Virus (MVA). Alternatively, the viral early promoter element is a structural variant of such a promoter element. Preferably, such mutants have at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, even more preferably at least 95% nucleic acid sequence identity to a naturally occurring viral early promoter element and at least direct the transcription level of genes under the control of the mutant viral early promoter element to the same level as the naturally occurring viral early promoter element. The transcription level of genes under the control of such promoters can be determined using methods known in the art, including in particular quantitative PCR (qPCR) of cDNA generated from RNA isolated from cells infected with the viral vector.

[0088] In the orthopoxvirus vector of the first aspect of the invention, the viral late promoter element is one that naturally occurs in an orthopoxvirus. Preferably, the viral late promoter element is an element that naturally occurs in a virus of a species selected from the group consisting of Orthopoxvirus variola, Orthopoxvirus vaccinia, Orthopoxvirus simiae, Orthopoxvirus bovis, Orthopoxvirus muris, Orthopoxvirus cameli, raccoon poxvirus or tatterapoxvirus. Preferably, the viral late promoter element is one that is naturally found in a virus of the Orthopoxvirus vaccinia species. According to a more preferred embodiment, the viral late promoter element is an element that naturally occurs in Modified Vaccinia Ankara Virus (MVA). Alternatively, the viral late promoter element is a structural variant of such a promoter element. Preferably, such mutants have at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% nucleic acid sequence identity to a naturally occurring viral late promoter element, and at least direct the transcription level of genes under the control of the mutant viral late promoter element to the same level as the naturally occurring viral late promoter element. The transcription level of genes under the control of such promoters can be determined using methods known in the art, including, in particular, quantitative PCR (qPCR) of cDNA generated from RNA isolated from cells infected with the viral vector.

[0089] In a preferred embodiment of the recombinant orthopoxvirus vector of the first aspect, each of the viral early promoter elements comprises: (i) the vaccinia virus (VV) p7.5 early promoter element; (ii) the nucleic acid sequence AAN 1 N 2 AN 3 TGAAN4 N 5 N 6 N 7 N 8 A viral early promoter element comprising or consisting of N 1 , N 2 , N 4 , N 5 and N 6 Each of is independently selected from A or T, preferably A; 3 is selected from C, G or T, preferably T; N 7 is selected from C and A, preferably C, and N 8 is selected from A, C and T, preferably T, and therefore in a particularly preferred embodiment, N 1 , N 2 , N 4 , N 5 and N 6 is A and N 3 is T and N 7 is C and N 8 is T; Preferably, the nucleic acid sequence AAN 1 N 2 AN 3 TGAAN 4 N 5 N 6 N 7 N 8 AN 9 TCTAATTTATTGN 10 AN 11 A viral early promoter element comprising or consisting of NGG (SEQ ID NO:2), 1 , N 2 , N 4 , N 5 and N 6 Each of is independently selected from A or T, preferably A; 3 is selected from C, G or T, preferably T; N 7 is selected from C and A, preferably C; N 8 is selected from A, C and T, preferably T; N 9is selected from G and T, preferably G; N 10 is selected from C and T, preferably C, and N 11 is selected from A and C, preferably C, and therefore in a particularly preferred embodiment, N 1 , N 2 , N 4 , N 5 and N 6 is A and N 3 is T and N 7 is C and N 8 is T and N 9 is G and N 10 is C, and N 11 is C; Preferably, the nucleic acid sequence AAN 1 N 2 AN 3 TGAAN 4 N 5 N 6 N 7 N 8 A viral early promoter element comprising or consisting of AGTCTAATTTATTGCACGG (SEQ ID NO: 3), 1 , N 2 , N 4 , N 5 and N 6 Each of is independently selected from A or T, preferably A; 3 is selected from C, G or T, preferably T; N 7 is selected from C and A, preferably C, and N 8 is selected from A, C and T, preferably T, and therefore in a particularly preferred embodiment, N 1 , N 2 , N 4 , N 5 and N 6 is A and N 3 is T and N 7 is C, and N 8 is T; Preferably, it is a viral early promoter element having the nucleic acid sequence of any one of SEQ ID NOs: 4 to 11, or most preferably, SEQ ID NO: 4; and / or (i) the vaccinia virus (VV) p7.5 late promoter element; (ii) Nucleic acid sequence TTTN 1 N 2 N 3 N 4 N 5 N 6 N 7 N 8 N 9 TTTTTN 10 N 11 N 12 N 13 N 14 N 15 N 16 A viral late promoter element comprising or consisting of ATAAATA (SEQ ID NO: 41), 1 From N 9 each is independently selected from A, C, G, T or absent, preferably T; 10 From N 16 each of which is independently selected from A, C, G, T or absent, and preferably has the sequence GGCAT; Preferably, the nucleic acid sequence TTTN 1 N 2 N 3 N 4 N 5 N 6 N 7 N 8 N 9 TTTTTN 10 N 11 N 12 N 13 N 14 N 15 N 16 A viral late promoter element comprising or consisting of ATAAATA (SEQ ID NO: 42), 1 is selected from C or T, preferably T; N 2 and N 9 each is independently selected from A, G or T, preferably T; 3 , N 4 and N 8Each of is independently selected from A or T, preferably T; 5 is selected from G, T or absent, preferably T; N 6 is selected from A, T or absent, preferably T; N 7 is selected from A, G, T or absent, preferably T; N 10 is selected from C, G or T, preferably G; N 11 is selected from A, G or T, preferably G; N 12 is selected from A or C, preferably C; N 13 is selected from T or absent, preferably absent, N 14 is selected from G or absent, preferably absent, N 15 is selected from A, C or T, preferably A, and N 16 is selected from A, C or T, preferably T; Preferably, it is a viral late promoter element having the nucleic acid sequence of any one of SEQ ID NOs: 12 to 16, or most preferably, SEQ ID NO: 12; Independently selected from the group consisting of: wherein each late promoter element and / or early promoter element is optionally linked by a nucleic acid linker, preferably 5, 6 or 7 nucleotides in length.

[0090] In a preferred embodiment, all early promoter elements within the first promoter of the orthopoxvirus vector according to the first aspect of the invention are identical.

[0091] In a particularly preferred embodiment of the orthopoxvirus vector according to the first aspect of the present invention, the first promoter comprises or consists of the nucleic acid shown in SEQ ID NO:18-30.

[0092] The first promoter may comprise or consist of at least 2 and up to 10 early elements. Preferably, the first promoter comprises or consists of at least 3 and up to 7 early elements. More preferably, the first promoter comprises or consists of at least 4 and up to 6 early elements. Most preferably, the first promoter comprises or consists of at least 4 early elements.

[0093] According to one embodiment, the at least one early promoter element is selected according to the generalized consensus SEQ ID NO: 1. According to one preferred embodiment, the first promoter comprises or consists of at least one early promoter element. According to one more preferred embodiment, the early promoter element comprises or consists of any one of SEQ ID NO: 4-11 and SEQ ID NO: 43-46. According to an even more preferred embodiment, the early promoter element comprises or consists of any one of SEQ ID NO: 4, 5, 43, 44, 45 and 46. According to the most preferred embodiment, the early promoter element comprises or consists of SEQ ID NO: 4.

[0094] According to one preferred embodiment, the first promoter comprises or consists of at least three early promoter elements and at least one late promoter element, wherein at least one early promoter element comprises the nucleotide motif AAN 1 N 2 AN 3 TGAAN 4 N 5 N 6 N 7 N 8 Contains A, N 1 , N 2 , N 4 , N 5 and N 6each is independently selected from A or T (preferably A); 3 is selected from C, G or T (preferably T), and N 7 is selected from C and A (preferably C), and N 8 is selected from A, C and T (preferably T), and at least one late promoter element has the nucleotide motif TTTN 1 N 2 N 3 N 4 N 5 N 6 N 7 N 8 N 9 TTTTTN 10 N 11 N 12 N 13 N 14 N 15 N 16 ATAAATA, N 1 From N 9 each is independently selected from A, C, G, T, or absent (preferably T); 10 From N 16 Each of is independently selected from A, C, G, T, or absent (preferably has the sequence GGCAT).

[0095] According to an even more preferred embodiment, the first promoter comprises or consists of at least four early promoter elements and at least one late promoter element, each early promoter element comprising the nucleotide motif AAN. 1 N 2 AN 3 TGAAN 4 N 5 N 6 N 7 N 8 Contains A, N 1 , N 2 , N 4 , N 5 and N 6 each is independently selected from A or T (preferably A);3 is selected from C, G or T (preferably T), and N 7 is selected from C and A (preferably C), and N 8 is selected from A, C and T (preferably T); At least one late promoter element contains the nucleotide motif TTTN 1 N 2 N 3 N 4 N 5 N 6 N 7 N 8 N 9 TTTTTN 10 N 11 N 12 N 13 N 14 N 15 N 16 Contains ATAAATA, N 1 is selected from C or T (preferably T), and N 2 and N 9 each is independently selected from A, G, or T (preferably T); 3 , N 4 and N 8 each is independently selected from A or T (preferably T); N 5 is selected from G, T or absent (preferably T), and N 6 is selected from A, T or absent (preferably T), and N 7 is selected from A, G, T or absent (preferably T), and N 10 is selected from C, G or T (preferably G), and N 11 is selected from A, G or T (preferably G), and N 12 is selected from A or C (preferably C), and N 13 is selected from T or absent (preferably absent), N 14 is selected from G or absent (preferably absent), N 15 is selected from A, C or T (preferably A), and N 16is selected from A, C or T (preferably T). Preferably, the promoter element in the first promoter operably linked to the first nucleic acid sequence encoding at least one immunostimulatory protein is at the 5' prime of the first nucleic acid sequence encoding at least one immunostimulatory protein and is 5'-[late promoter element]-[early promoter element] n -[a first nucleic acid sequence encoding at least one immunostimulatory protein], where n is 4 or more, and preferably n is 4 to 6.

[0096] In this regard, the early and late promoter elements may be the same or different, preferably all early promoter elements within a viral vector of the invention are the same.

[0097] The first promoter may comprise or consist of multiple late promoter elements. For example, the first promoter may comprise or consist of at least two promoter late elements up to five late promoter elements. Preferably, the first promoter comprises or consists of one late promoter element.

[0098] According to another embodiment, each late promoter element and each early promoter element are linked by a short linker. According to one preferred embodiment, the linker has a length of 2 to 12 nucleotides, more preferably, the linker has a length of 3 to 8 nucleotides, and even more preferably, the linker has a length of 6 to 8 nucleotides. According to the most preferred embodiment, the linker has a length of 5 to 8 nucleotides. Preferably, the linker has a sequence selected from TCCGGT, TCCGGA, TCTGGA, TCTCGT, ATAGGA, or AGCTT. The linkers linking each late promoter element and each early promoter element in one promoter do not have to be the same.

[0099] According to one preferred embodiment, the first promoter comprises or consists of a sequence shown in any one of SEQ ID NOs: 18 to 30. According to one more preferred embodiment, the first promoter comprises or consists of a sequence shown in any one of SEQ ID NOs: 18, 19, 22, 23 or 24. According to one even more preferred embodiment, the first promoter comprises or consists of a sequence shown in any one of SEQ ID NOs: 18, 19 or 23. According to a most preferred embodiment, the first promoter comprises or consists of a sequence shown in SEQ ID NO: 18.

[0100] In a second aspect, the present invention provides a cell comprising a recombinant orthopoxvirus vector according to the first aspect of the invention. Typically, such a cell has been transformed, transduced or transfected with a viral vector of the first aspect of the invention. A cell that receives and then expresses a foreign nucleic acid or vector consisting of DNA or RNA by a process of transformation or transduction is "transformed" or "transduced". Preferably, the cell of the present invention comprises a viral vector as described above. The cell may be a eukaryotic cell, such as a mammalian cell (e.g. a human cell), yeast, plant, animal, fungus or algae, or a prokaryotic cell, such as a bacterium or a protozoan. Preferably, the cell is a mammalian cell, such as a lymphocyte or leukocyte, more preferably a macrophage. Preferably, the cell bears a scavenger receptor on its surface. More preferably, the scavenger receptor is a type 1 scavenger receptor. Most preferably, the scavenger receptor is MARCO.

[0101] According to a third aspect, the present invention provides a method for producing a composition comprising the steps of: a) a cell comprising a recombinant orthopoxvirus vector according to the first aspect of the invention or a viral vector according to the first aspect of the invention, and b) two or more orthopoxvirus vectors encoding different therapeutic proteins according to the first aspect of the invention, and c) a pharma- ceutically acceptable carrier, and Optionally, d) a recombinant viral vector comprising a nucleic acid sequence encoding a checkpoint inhibitor, a nucleic acid encoding a checkpoint inhibitor, or a checkpoint inhibitor; A composition comprising:

[0102] In one embodiment, the composition of the invention comprises a therapeutically effective amount of a recombinant orthopoxvirus vector according to the first aspect of the invention, or a cell according to the second aspect of the invention, or a therapeutically effective amount of a plurality of two or more orthopoxvirus vectors encoding different therapeutic proteins according to the first aspect of the invention, together with a suitable amount of a pharma- ceutically acceptable carrier and / or excipient to provide a form for suitable administration to a subject. The formulation of the composition should be compatible with the method of administration. For example, for intravenous administration, the carrier is preferably an aqueous carrier. According to another embodiment, the composition may further comprise a recombinant viral vector comprising a nucleic acid sequence encoding a checkpoint inhibitor, a nucleic acid encoding a checkpoint inhibitor, or optionally a checkpoint inhibitor. Preferably, the checkpoint inhibitor is selected from the group consisting of a CTLA-4 inhibitor, a PD-1 inhibitor, and a PD-L1 inhibitor. More preferably, the checkpoint inhibitor is a PD-1 inhibitor. Even more preferably, the PD-1 inhibitor is an anti-PD1 antibody selected from the group consisting of nivolumab, atezolizumab, pembrolizumab, cemiplimab, durvalumab, and avelumab.

[0103] According to one embodiment, the pharma-ceutically acceptable carrier is an aqueous carrier.Preferably, the aqueous carrier is selected from the group consisting of sterile liquids such as saline in water and oil (including but not limited to those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.).

[0104] In one embodiment, the pharmaceutical composition may further comprise therapeutic or pharmacologically active substances, such as, but not limited to, adjuvants and / or additional active ingredients, in a pharma- ceutically or physiologically acceptable formulation selected for appropriate administration according to the selected method of administration.Non-limiting examples of suitable adjuvants include alum, aluminum phosphate, aluminum salts, aluminum hydroxide, aluminum silica, calcium phosphate, incomplete Freund's adjuvant, QS21, MPL-A, RIBIDETOXTM, and / or combinations thereof.

[0105] In one embodiment, the pharmaceutical composition can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation, etc. For preparing the pharmaceutical composition of the present invention, the pharma- ceutically acceptable carrier can be either solid or liquid, preferably liquid. The liquid form of the composition includes a solution, suspension, emulsion, for example, water, saline, aqueous dextrose, glycerol solution, or water / propylene glycol solution. For parenteral injection (for example, intravenous, intraarterial, intraosseous injection, intramuscular, subcutaneous, intraperitoneal, intradermal, and intrathecal injection), the liquid preparation can be formulated, for example, by dissolving in an aqueous polyethylene glycol solution. For intravenous administration of the pharmaceutical composition, saline is the preferred carrier.

[0106] In one embodiment, the composition is in unit dosage form. In such form, the composition can be subdivided into unit doses or multiple doses containing appropriate amounts of active ingredients. The unit dosage form can be a packaged composition containing a discrete amount of the composition, such as tablets, capsules, and powders packaged in sealed vials or ampoules. The unit dosage form can also be a capsule, injection vial, tablet, cachet, or lozenge itself, or the appropriate number of any of these packaged. If necessary, the composition can also contain small amounts of wetting agents, emulsifying agents, or pH buffering agents. The composition can also be stored in a freeze-dried (lyophilized) state, with only the addition of a sterile liquid carrier, such as water for injection, immediately before use.

[0107] The form of the composition, route of administration, dosage and administration regimen will, of course, vary depending on the condition being treated, the severity of the disease, the subject's age, weight, sex, desired duration of treatment, etc. The compositions of the present invention may be in any suitable form depending on the desired method of administration to a subject.

[0108] In one embodiment, the pharmaceutical composition of the fourth aspect of the invention comprises a pharma- ceutically acceptable excipient for an injectable formulation, which may in particular be an isotonic, sterile, saline solution (such as mono- or di-sodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or a mixture of such salts), or may be a dried, in particular a lyophilized composition, which may be made up for injection, possibly by adding sterile water or saline.

[0109] To prepare a composition of the invention, an effective amount of a recombinant orthopoxvirus vector according to the first aspect of the invention, or an effective amount of a cell according to the second aspect of the invention and an effective amount of two or more orthopoxvirus vectors according to the first aspect of the invention may be dispersed in a pharma- ceutically acceptable carrier or aqueous medium. Optionally, a recombinant viral vector comprising a nucleic acid sequence encoding a checkpoint inhibitor, a nucleic acid encoding a checkpoint inhibitor, or a checkpoint inhibitor may be further added.

[0110] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions, formulations containing sesame oil, peanut oil, or aqueous propylene glycol, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the formulation must be sterile and fluid to the extent that it is easy to inject. It must be stable under the conditions of manufacture and storage and preserved against the contamination of microorganisms, such as bacteria and fungi. Solutions of the active compound free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Sterile injectable solutions are prepared by mixing the active compound in the required amount in an appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and the other required ingredients enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional ingredient from a previously sterile-filtered solution thereof.

[0111] In a fourth aspect, the present invention provides a recombinant orthopoxvirus vector according to the first aspect of the invention, a cell comprising a recombinant orthopoxvirus vector according to the first aspect of the invention, or a composition according to the third aspect of the invention, for use in medicine.

[0112] In a fifth aspect, the present invention provides a recombinant orthopoxvirus vector of the first aspect of the invention, a cell of the second aspect of the invention, or a composition of the third aspect of the invention for use in the treatment, amelioration or prevention of cancer.

[0113] According to one embodiment, the cancer is breast cancer, small intestine cancer, gastric cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, lung cancer, colon cancer, prostate cancer, B-cell lymphoma, Burkitt's lymphoma, or Hodgkin's lymphoma.

[0114] Further non-limiting examples of cancers contemplated by the present invention include: adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, neuroblastoma, basal cell carcinoma, bile duct cancer, bone cancer, brain tumor, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, bronchial adenoma, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, cutaneous T-cell lymphoma, fibroid tumor, and pulmonary tumor. Small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, glioma, head and neck cancer, cardiac cancer, hepatocellular carcinoma (liver cancer), Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, laryngeal cancer, lip and oral cavity cancer, liposarcoma, liver cancer, non-small cell and small cell lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, Medulloblastoma, melanoma, mesothelioma, metastatic squamous cell carcinoma of unknown primary, oral cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic islet cell cancer, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pituitary adenoma, breast Membrane pulmonary blastoma, plasma cell neoplasms, primary central nervous system lymphoma, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, skin cancer, Merkel cell carcinoma, soft tissue sarcoma, squamous cell carcinoma, T-cell lymphoma, pharyngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic neoplasm (gestational), cancer of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor (nephroblastoma).

[0115] In a preferred embodiment, the cancer comprises a solid tumor. In this regard, lymphomas are not generally considered to be solid tumors, although accessible solid tumors of lymphomas can form in lymph nodes.

[0116] The recombinant orthopoxvirus vector or composition described herein can be administered in a therapeutically effective amount to a subject in need of cancer treatment. Typically, the recombinant orthopoxvirus vector of the present invention is packaged into a viral particle, which is then delivered to the tumor site. For example, when the recombinant viral vector is administered as an active ingredient of a pharmaceutical composition, the dosage of the recombinant virus or viral particle is expressed in plaque-forming units (PFU) of the virus or viral particle. The term "plaque-forming unit" refers to the amount of viral particles capable of forming plaques per unit volume. A suitable amount is, for example, 10 2 ~10 14 PFU, preferably 10 5 ~10 12 PFU, more preferably 10 6 ~10 10 PFU. The recombinant orthopoxvirus vector or composition described herein may be administered multiple times. One treatment cycle corresponds to 2, 3 or 4 administrations at daily, weekly, biweekly or monthly intervals. The treatment cycle can be repeated several times to obtain a complete cure.

[0117] It should be understood that the amount of the recombinant orthopoxvirus vector of the first aspect of the invention, the cell of the second aspect of the invention, or the composition of the third and fourth aspects of the invention may vary depending on the particular compound used, the particular composition formulated, the method of application, the size and type of the tumor, and the recipient of the compound. The recombinant orthopoxvirus vector of the first aspect of the invention, the cell of the second aspect of the invention, or the composition of the third and fourth aspects of the invention may be administered once or repeatedly.

[0118] One suitable route of administration is by injecting the viral particles in a sterile solution. The particles may be administered alone. It is preferred to provide the viral particles as a pharmaceutical composition or formulation. Thus, the composition preferably comprises the viral particles, one or more acceptable carriers, and optionally other therapeutic ingredients as described above. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition or formulation and not harmful to the recipient thereof.

[0119] According to one embodiment, the recombinant orthopoxvirus vector of the first aspect of the invention, the cell of the second aspect of the invention, or the composition of the third and fourth aspects of the invention can be administered directly to the tumor tissue. According to one preferred embodiment, the recombinant orthopoxvirus vector of the first aspect of the invention, the cell of the second aspect of the invention, or the composition of the third and fourth aspects of the invention is directly injected or administered by catheter before or during surgery. The recombinant orthopoxvirus vector of the first aspect of the invention, the cell of the second aspect of the invention, or the composition of the third and fourth aspects of the invention can also be administered by local perfusion, direct administration into the tumor, direct administration into a body cavity (intracavitary administration), for example by intraperitoneal injection. Preferably, the route of administration is parenteral injection, such as intradermal or intramuscular injection.

[0120] Other routes of administration include, but are not limited to, topical, oral, enteral, nasal (i.e., intranasal), inhalation, intrathecal, rectal, intravaginal, intraocular, subconjunctival, sublingual, intradermal, transdermal, or parenteral administration (including subcutaneous, transdermal, intravenous, intramuscular, intratumor, intralymph node, intrasternal, intracavernosal, intravesical, or intraurethral injection or infusion, etc.).

[0121] According to one embodiment, recombinant orthopoxvirus vector and recombinant viral vector comprising nucleic acid sequence encoding checkpoint inhibitor, nucleic acid encoding checkpoint inhibitor, or checkpoint inhibitor are administered simultaneously or consecutively.For example, recombinant orthopoxvirus vector and recombinant viral vector comprising nucleic acid sequence encoding checkpoint inhibitor, nucleic acid encoding checkpoint inhibitor, or checkpoint inhibitor can be administered consecutively, for example, at intervals of several hours, daily, weekly, monthly, or according to the specific needs of the subject.In a specific embodiment, recombinant orthopoxvirus vector is administered at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, or at least 12 hours, or at least 18 hours before administration of recombinant viral vector comprising nucleic acid sequence encoding checkpoint inhibitor, nucleic acid encoding checkpoint inhibitor, or checkpoint inhibitor. In other specific embodiments, the recombinant orthopoxvirus vector is administered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days prior to administration of the recombinant viral vector comprising a nucleic acid sequence encoding a checkpoint inhibitor, a nucleic acid encoding a checkpoint inhibitor, or a checkpoint inhibitor. In other embodiments, the recombinant orthopoxvirus vector is administered 1-36 days prior to administration of the recombinant viral vector comprising a nucleic acid sequence encoding a checkpoint inhibitor, a nucleic acid encoding a checkpoint inhibitor, or a checkpoint inhibitor.The recombinant orthopoxvirus vector can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 days prior to administration of the recombinant viral vector comprising a nucleic acid sequence encoding a checkpoint inhibitor, the nucleic acid encoding a checkpoint inhibitor, or the checkpoint inhibitor. In yet another embodiment, the recombinant orthopoxvirus vector and the recombinant viral vector comprising a nucleic acid sequence encoding a checkpoint inhibitor, the nucleic acid encoding a checkpoint inhibitor, or the checkpoint inhibitor, respectively, are administered one or more (e.g., two, three, or four) consecutive times to a subject in need thereof.

[0122] The present invention is illustrated by the following examples, which should be construed as merely illustrative and not limiting the scope of the invention. EXAMPLES

[0123] Preparation of recombinant orthopoxvirus vectors

[0124] Methods for inserting an expression cassette or promoter according to the invention into a viral genome, in particular the genome of a vaccinia virus, most preferably the genome of MVA, are known to those skilled in the art. As an example, an expression cassette or promoter or derivative thereof according to the invention can be inserted into the genome of MVA by homologous recombination. For this purpose, a nucleic acid is transfected into a permissive cell line, such as a primary avian cell line or an avian-derived cell line, said nucleic acid comprising an expression cassette or promoter or derivative thereof according to the invention flanked by nucleotide sequences that are homologous to the region of the MVA genome into which the expression cassette or promoter or derivative thereof according to the invention is inserted. The cell is infected with MVA, and homologous recombination occurs between the nucleic acid and the viral genome in the infected cell. Alternatively, it is possible to first infect a cell with MVA and then introduce the nucleic acid into the infected cell. The recombinant MVA is then selected by methods known in the art. The construction of the recombinant MVA is not limited to this particular method. Instead, any suitable method known to those skilled in the art can be used for this purpose.

[0125] Example 1: Intratumoral treatment combining MVA-IL12 and anti-PD1 therapy is highly effective in tumors resistant to checkpoint inhibitors (CPIs) and uses MVA encoding single-chain murine IL-12 (sc-mIL12) (according to SEQ ID NO: 37) under the control of a synthetic promoter (SynE1) (according to SEQ ID NO: 18) that exerts a stronger effect compared to the native p7.5 promoter (SEQ ID NO: 17) (Figure 1).

[0126] Tumor-bearing mice (Lewis Lung Carcinoma model, LLC) were treated intratumorally (it) with a dose of 10e7 infectious units (ifu) of MVA encoding IL12 under the control of a synthetic early promoter (MVA-SynE1-IL12) or MVA-IL12 under the control of the 7.5 promoter (MVA-7.5-IL12) in combination with an anti-PD1 antibody injected intraperitoneally (ip). Treatment began on day 0 for mice randomized according to tumor volume. Treatment with MVA was performed on days 0, 2, and 4, while anti-PD1 treatment was performed twice weekly until day 17. Tumor growth was measured over time using digital calipers every 3–4 days. Tumor volume was calculated using the formula 0.5 x length x width 2, where length is the longer dimension. The results showed that the therapeutic effect of combining MVA encoding IL-12 with anti-PD1 was very strong, with tumor regression and cure observed in 90% and 62.5% of mice treated with MVA-SynE1-IL12 and MVA-7.5-IL12, respectively (Figure 1), highlighting the more potent activity of MVA-SynE1-IL12. Anti-PD1 treatment alone was ineffective in this model.

[0127] Example 2: Efficacy of intratumoral MVA-SynE1-IL12 is maintained even at low doses (Figure 2).

[0128] Tumor-bearing mice (Lewis Lung Carcinoma model, LLC) were treated intratumorally (it) with MVA encoding IL12 under the control of the SynE1 synthetic early promoter (MVA-SynE1-IL12) at a dose of 10e7 infectious units (ifu) or 2x10e5 ifu in the presence of anti-PD1 antibodies injected intraperitoneally (ip). For both groups of mice, treatment was started on day 0 with mice randomized according to tumor volume. Injections of MVA were repeated on days 2 and 4, and anti-PD1 treatment was performed twice a week until day 17. The results show that even reduced doses of MVA-SynE1-IL12 have an effective and potent antitumor effect.

[0129] Example 3: MVA-SynE1-IL12 is effective as a monotherapy (Figure 3)

[0130] The antitumor efficacy of MVA-SynE1-IL12 monotherapy was investigated in tumor-bearing mice (same tumor model and treatment regimen as reported in Examples 1 and 2) at a dose of 10e7 infectious units (ifu) or 2x10e5 ifu. Tumor volume (mm 3 ) over time demonstrated activity of MVA-SynE1-IL12 at both doses, even in the absence of anti-PD1 treatment.

[0131] Example 4: Adenoviral vector encoding IL12 is ineffective in a peritoneal carcinomatosis model (Figure 4)

[0132] The activity of MVA-SynE1-IL12 was compared with that of different viral vectors, more specifically sc-mIL-12 encoding Ad5 under the control of the CMV promoter. CT26 (murine colon carcinoma cells) were injected into the peritoneum of BAlBC mice. Three days later (day 0), mice were treated with MVA encoding IL12 under a synthetic early promoter (MVA-SynE1-IL12) at a dose of 10e7 infectious units (ifu) or with an equivalent dose of Ad5 encoding IL12. Treatment was repeated on days 2 and 4. Overall survival was monitored over time and compared with that of untreated control mice, and the results showed efficient inhibition of peritoneal carcinomatosis by MVA-SynE1-IL12, but not Ad-IL12, with survival rates of 100% and 10%, respectively, at day 30.

[0133] Example 5: Adenoviral vectors and MVA vectors encoding IL12 express similar cargo levels in vitro (Figure 5).

[0134] Expression of IL12 produced by Adeno or MVA encoding IL12 was measured in vitro. HeLa cells were infected with MVA-SynE1-IL12 or MVA-p7.5-IL12, or Ad5-IL12 at 1 MOI (1 infectious unit / cell). Supernatants were harvested 24 hours post-infection and subjected to IL12 Elisa assay, which showed similar expression levels for all three vectors.

[0135] Example 6: The MVA-SynE1 promoter drives extremely high IL12 expression when injected into tumors (Figure 6)

[0136] The levels of intratumoral IL12 produced by Adeno or MVA encoding IL12 were measured upon in vivo treatment in mice. Tumor-bearing mice (LLC tumors) were treated with a single intratumoral injection of Adeno encoding IL12 (Ad-IL12), MVA encoding IL12 under the native 7.5 promoter (MVA-p7.5-IL12), or MVA encoding IL12 under a synthetic early promoter (MVA-SynE1-IL12) at a dose of 10^7ifu. Expression of IL12 from recovered tumors was measured over time by ELISA assay, showing that the MVA-SynE1 promoter drives very high IL12 expression with approximately 50-fold higher expression than the MVA-p7.5 promoter. IL12 levels from tumors treated with Ad-IL12 were very low (Figure 6).

[0137] Example 7: The MVA-SynE1 promoter drives very high IL12 expression in tumors but not in muscle (Figure 7).

[0138] Upon in vivo treatment with MVA-SynE1-IL12, we measured the expression of IL12 in muscle and tumor. Mice were injected with a single dose of 10^7 ifu of MVA-SynE1-IL12 in a given muscle or tumor. The levels of IL12 from tumor and muscle were measured by ELISA assay, demonstrating that the MVA-SynE1 promoter drives very high IL12 expression in tumor but not in muscle (Figure 7).

[0139] Example 8: Intratumoral treatment with MVA-SynE1-IL12 efficiently reprograms the tumor microenvironment by decreasing the levels of suppressive M2 macrophages and increasing the amount of proinflammatory M1 macrophages (Figure 8).

[0140] In this example, we measured the frequency of M1 and M2 macrophages after in vivo treatment with MVA-SynE1-IL12 or MVA mock vector. Mice were injected three consecutive times with MVA-SynE1-IL12 or MVA-mock at a dose of 10^7ifu on days 0, 2, and 4, tumors were harvested, and the levels of M1 and M2 macrophages were evaluated by flow cytometry assay, demonstrating that MVA-mock can reduce M2 immunosuppressive cells but cannot reduce M1 proinflammatory cells. The simultaneous occurrence of M2 reduction and M1 increase was only achieved by treatment with MVA-SynE1-IL12 (Figure 8).

[0141] Example 9: Intratumoral (IT) treatment with MVA-SynE1-IL12 alone and in combination with anti-PD1 therapy is highly effective against checkpoint inhibitor (CPI)-resistant B16F10 tumors.

[0142] In this example, we investigated the therapeutic effect of MVA-SynE1-IL12 in the B16F10 model, a mouse tumor model known to be resistant to the activity of CPIs. Tumor-bearing mice (n=9 per group) were administered MVA encoding IL12 under the control of the SynE1 synthetic early promoter (MVA-SynE1-IL12) at a dose of 6x10e5ifu intratumorally (it) in the presence of anti-PD1 antibodies injected intraperitoneally (ip). Treatment began on day 0 for both groups of mice, randomly divided according to tumor volume. Injections of MVA were repeated every 4 days for a total of 8 times, and anti-PD1 treatment was performed twice a week until day 17. The results show that tumor growth is effectively controlled by treatment with MVA-SynE1-IL12 alone or in combination with MVA-SynE1-IL12 and anti-PD-1, compared to the control group that received anti-PD-1 monotherapy (Figure 10).

[0143] Example 10: MVA-SynE1-IL12 IT immunotherapy controls tumor growth of both injected and non-injected distant tumors.

[0144] To test the abscopal antitumor response induced by MVA-SynE1-IL12, a bilateral MC38 tumor implant model was used to evaluate whether MVA-SynE1-IL12 has antitumor activity against uninjected distant tumors. Tumor-bearing mice were injected with 6x10 IL-12 in the presence of anti-PD1 administered intraperitoneally (i.p.) into one of the two masses. 5 ifu or 10 7 Intratumoral injection of MVA-SynE1-IL12 was performed at a dose of 100x10 ... 5 ifu and 10 7 At the ifu dose, treatment resulted in eradication in 50% and 60% of animals, emphasizing that MVA-SynE1-IL12 can control cancer growth even at distant sites (Fig. 11d–f).

[0145] Example 11: Strong early in vitro activity of SynE promoters containing consensus sequence SEQ ID NO:1 or SEQ ID NO:3

[0146] In this example, the early activity of the P7.5 promoter is compared to SynE promoters containing different early elements including consensus SEQ ID NO:3 or the more generalized consensus SEQ ID NO:1: early elements Early-A (SEQ ID NO:43), Early-B (SEQ ID NO:44), Early-C (SEQ ID NO:45), or Early-D (SEQ ID NO:46), either combined or not with different late elements (Figures 12a and b).

[0147] For this purpose, HeLa cells were infected with MVA to provide all the viral functions necessary for poxvirus promoter expression and transfected with a plasmid encoding mIL12 under the control of various promoters. As a reference, a plasmid encoding mIL12 under the control of the P7.5 promoter was used. The DNA replication inhibitor cytosine β-D-arabinofuranoside (AraC) was added to the cells to inhibit MVA gene expression in the middle and late phases. This allows to analyze only the activity of the early promoter, even in the absence of viral functions supporting late expression (Chakrabarti S., Sisler JR, Moss B. Compact, synthetic, vacciniavirus early / late promoter for protein expression. Biotechniques.1997;23:1094-1097). All tested SynE promoters promote higher levels (more than 2-fold) of early expression of mIL12 compared to P7.5 (Figure 12c).

[0148] Drawing Terminology Tumor volume Days Percent Survival Untreated Secreted IL12 Intratumoral IL12 levels proteins hrs time tumor vs muscle Macrophages % M2 gated on F480+ % M2 in gated F480+ % M1 gated on F480+ % M1 in gated F480+ Right tumor Left tumor Early promoter activity FOC compared to P7.5

Claims

1. a) a first promoter comprising or consisting of: (i) the presence or absence of at least one viral early promoter element and at least one viral late promoter element, wherein the viral early promoter element is 1 N 2 AN 3 TGAAN 4 N 5 N 6 N 7 N 8 A (SEQ ID NO: 1), 1 , N 2 , N 4 , N 5 and N 6 are each independently selected from A or T, and N 3 is selected from C, G, or T, and N 7 is selected from C and A, and N 8 is selected from A, C and T, or (ii) at least one viral late promoter element and at least three viral early promoter elements; and b) a first nucleic acid sequence encoding at least one immunostimulatory protein; A recombinant orthopoxvirus vector comprising, in operable linkage,

2. 2. The recombinant orthopoxvirus vector of claim 1, wherein the orthopoxvirus is a species selected from the group consisting of Orthopoxvirus variola, Orthopoxvirus vaccinia, Orthopoxvirus simiae, Orthopoxvirus bovis, Orthopoxvirus muris, Orthopoxvirus cameli, Raccoonpox virus, or Taterapox virus, or the orthopoxvirus vaccinia is a subspecies modified vaccinia Ankara virus (MVA).

3. 3. The recombinant orthopoxvirus vector of claim 1 or 2, wherein the immunostimulatory protein is a pro-inflammatory protein, or a cytokine, or an interleukin of the interleukin-12 family, or interleukin-12 (IL-12).

4. The recombinant orthopoxvirus vector of claim 3, wherein the IL-12 is human single-chain IL-12 (sc-hIL-12), or has the amino acids set forth in SEQ ID NOs: 38 to 40, or has the amino acid sequence set forth in SEQ ID NO:

38.

5. 3. The recombinant orthopoxvirus vector of claim 1 or 2, further comprising a second nucleic acid sequence encoding one or more tumor antigens or antigenic fragments thereof.

6. The recombinant orthopoxvirus vector of claim 5 , wherein the second nucleic acid sequence is operably linked to the first promoter or the second promoter.

7. Each of the viral early promoter elements is independently selected from the group consisting of: (i) the vaccinia virus (VV) p7.5 early promoter element; (ii) Nucleic acid sequence AAN 1 N 2 AN 3 TGAAN 4 N 5 N 6 N 7 N 8 A viral early promoter element comprising or consisting of N 1 , N 2 , N 4 , N 5 and N 6 are each independently selected from A or T, and N 3 is selected from C, G, or T, and N 7 is selected from C and A, and N 8 is selected from A, C and T; Or, Nucleic acid sequence AAN 1 N 2 AN 3 TGAAN 4 N 5 N 6 N 7 N 8 AN 9 TCTAATTTATTGN 10 AN 11 A viral early promoter element comprising or consisting of NGG (SEQ ID NO: 2), 1 , N 2 , N 4 , N 5 and N 6 are each independently selected from A or T, and N 3 is selected from C, G, or T, and N 7 is selected from C and A, and N 8 is selected from A, C, and T, and N 9 is selected from G and T, and N 10 is selected from C and T, and N 11 is selected from A and C; Or, Nucleic acid sequence AAN 1 N 2 AN 3 TGAAN 4 N 5 N 6 N 7 N 8 A viral early promoter element comprising or consisting of AGTCTAATTTATTGCACGG (SEQ ID NO: 3), 1 , N 2 , N 4 , N 5 and N 6 are each independently selected from A or T, and N 3 is selected from C, G, or T, and N 7 is selected from C and A, and N 8 is selected from A, C and T; Or, The early promoter element has the nucleic acid sequence set forth in SEQ ID NOs: 4 to 11 and SEQ ID NOs: 43 to 46, or has the nucleic acid sequence set forth in any one of SEQ ID NOs: 4, 5, 43, 44, 45 and 46, or has the nucleic acid sequence set forth in SEQ ID NO: 4; and / or wherein each of the viral late promoter elements is independently selected from the group consisting of: (i) the vaccinia virus (VV) p7.5 late promoter element; (ii) Nucleic acid sequence TTTN 1 N 2 N 3 N 4 N 5 N 6 N 7 N 8 N 9 TTTTTN 10 N 11 N 12 N 13 N 14 N 15 N 16 A viral late promoter element comprising or consisting of ATAAATA (SEQ ID NO: 41), 1 From N 9 are each independently selected from A, C, G, T, or absent, and N 10 From N 16 each of is independently selected from A, C, G, T or absent; Or, Nucleic acid sequence TTTN 1 N 2 N 3 N 4 N 5 N 6 N 7 N 8 N 9 TTTTTN 10 N 11 N 12 N 13 N 14 N 15 N 16 A viral late promoter element comprising or consisting of ATAAATA (SEQ ID NO: 42), 1 is selected from C or T, and N 2 and N 9 are independently selected from A, G, or T; 3 , N 4 and N 8 are each independently selected from A or T, and N 5 is selected from G, T or absent, and N 6 is selected from A, T, or absent, and N 7 is selected from A, G, T or absent, and N 10 is selected from C, G, or T, and N 11 is selected from A, G, or T, and N 12 is selected from A or C, and N 13 is selected from T or absent, and N 14 is selected from G or absent, and N 15 is selected from A, C, or T, and N 16 is selected from A, C, or T; Or, The viral late promoter element has the nucleic acid sequence of any one of SEQ ID NOs: 12 to 16, or has the nucleic acid sequence of SEQ ID NO:

12. wherein each late promoter element and / or each early promoter element is linked by a nucleic acid linker (or links are not present) that is 5, 6, 7, or 8 nucleotides in length; Or, The first promoter comprises or consists of a nucleic acid sequence set forth in SEQ ID NOs: 18 to 30; The recombinant orthopoxvirus vector according to claim 1 or 2.

8. A cell comprising the recombinant orthopoxvirus vector described in claim 1.

9. a) a recombinant orthopoxvirus vector according to claim 1 or a cell according to claim 8, and b) a plurality of two or more orthopoxvirus vectors according to claim 1 encoding different therapeutic proteins; c) a pharmaceutically acceptable carrier, and d) a recombinant viral vector comprising a nucleic acid sequence encoding a checkpoint inhibitor, with or without a nucleic acid encoding a checkpoint inhibitor, or a checkpoint inhibitor; A composition comprising:

10. 10. The composition of claim 9, wherein the checkpoint inhibitor is selected from the group consisting of a CTLA-4 inhibitor, a PD-1 inhibitor, and a PD-L1 inhibitor, and preferably the PD-1 inhibitor is an anti-PD1 antibody selected from the group consisting of nivolumab, atezolizumab, pembrolizumab, cemiplimab, durvalumab, and avelumab.

11. A pharmaceutical comprising the recombinant orthopoxvirus vector described in claim 1.

12. A pharmaceutical for treating, ameliorating or preventing cancer, comprising the recombinant orthopoxvirus vector described in claim 1.

13. The pharmaceutical composition of claim 12, wherein the cancer is breast cancer, small intestine cancer, stomach cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, lung cancer, colon cancer, prostate cancer, B-cell lymphoma, Burkitt lymphoma, or Hodgkin lymphoma.

14. The method of claim 12, wherein the recombinant orthopoxvirus vector, cell, or composition is administered directly into the tumor tissue by direct injection or by catheter before or during surgery.

15. The pharmaceutical composition of claim 12, wherein the recombinant orthopoxvirus vector and the recombinant viral vector comprising a nucleic acid sequence encoding a checkpoint inhibitor, the nucleic acid encoding a checkpoint inhibitor, or the checkpoint inhibitor are administered simultaneously or sequentially.

Images