A fusion protein comprising surfactant protein-D and a member of TNFSF

Abstract

The present invention is in the fields of immunology and oncology, and in particular is for treating, preventing, or inhibiting proliferative diseases, particularly cancer, infectious diseases, and disorders associated with dysfunction of TNF cytokines. The present invention relates to a novel SPD-TNFSF fusion protein comprising a TNF-superfamily (TNFSF) ligand fused to the coiled-coil domain of surfactant protein-D (SPD), or its receptor-binding domain. Also provided is a trimeric or multimeric fusion protein comprising a plurality of SPD-TNFSF fusion proteins. The present invention also provides an mRNA, plasmid, or expression vector as a virus comprising an isolated nucleotide sequence encoding the SPD-TNFSF fusion protein, and a cell or pharmaceutical composition comprising the same.

Description

Technical Field

[0001] The present invention generally relates to the fields of immunology and oncology, and more specifically, to compositions and methods for treating, preventing, or inhibiting proliferative diseases, particularly cancer, infectious diseases, and disorders associated with TNF cytokine dysfunction. Embodiments include a TNF-superfamily (TNFSF) ligand fused to the coiled-coil domain of surfactant protein-D (SPD), or an SPD-TNFSF fusion protein comprising its receptor-binding domain, a trimeric or multimeric fusion protein comprising a plurality of SPD-TNFSF fusion proteins. Embodiments also include expression vectors such as mRNA, plasmids, and viruses comprising a nucleotide sequence encoding one or more SPD-TNFSF fusion proteins. SPD-TNFSF fusion proteins, isolated nucleotide sequences encoding SPD-TNFSF fusion proteins, mRNA, plasmids, and viruses are suitable for pharmaceutical compositions and their use for treating cancer and infectious diseases, more generally, proliferative diseases and disorders associated with TNF cytokine dysfunction.

Background Art

[0002] Cancer is caused by both external factors (e.g., tobacco, infectious organisms, eating habits, chemicals, and radiation) and internal factors (e.g., genetic mutations, hormones, immune status, and mutations arising from metabolism). Every year, more than 12 million people worldwide are diagnosed with cancer. In developed industrial countries, approximately one in five people die from cancer. Although there is a vast number of chemotherapeutic drugs, they are often ineffective, and there remains an unmet medical need for effective and less toxic therapies, particularly for patients who are resistant to existing treatments.

[0003] Effective T cell responses require functional and optimal innate and adaptive immunity, which particularly rely on multiple specific interactions between different cells via soluble stimuli and surface linker / receptor binding. In this context, CD8+ cytotoxic T lymphocyte (CTL) responses play an important role in anti-tumor immunity (Ara et al., 2018, ImmunoTargets and therapy, 7, 55-61). Three signals are mentioned as important parameters. The first signal relates to the binding of the antigen-specific T cell receptor (TCR) to the major histocompatibility complex (MHC) loaded with peptides on antigen-presenting cells (APCs). The second signal results from the involvement of co-stimulatory molecules, namely B7-1 (CD80) / B7-2 (CD86) and CD28 (e.g., T cell CD28 / APC CD80), as a result of Ag-specific T cell-APC interactions. The third signal, induced by cytokine secretion, enhances and modifies the responding effector CTLs.

[0004] The tumor necrosis factor (TNF) superfamily as co-stimulatory molecules plays an important role in immune regulation and anti-tumor immunity (Vinay et al., 2009, Cell Biol Int.; 33(4):453-465). TNFSF members are well known to mediate immune responses through T cell-forming properties. Previous investigations have enabled the identification of the following TNFSF members: CD40L, 4-1-BBL, OX40L, CD70, TNF, GITRL, LIGHT, FASL, TWEAK, APRIL, RANKL, TRAIL, CD30L, NGF, Baff, LTβ, LTα, LTαβ2, TL1A, TLA, EDA.

[0005] Efficient antigen recognition by antigen-specific T cells is critically dependent on the presence and functionality of specialized antigen-presenting cells (APCs) such as B cells and dendritic cells (DCs). For example, CD40 is expressed in many cell types including macrophages, B cells, and DCs, while its ligand CD40L is expressed mainly in activated CD4+ T cells. CD40L is expressed on the surface of activated B, T, and NK cells, and also in adipocytes and basophils (Richards et al. Hum Vaccin Immunother. 2020;16(2):377-387). The direct interaction between CD40L expressed on CD4+ T cells and CD40 expressed on DCs "licenses" the DCs to prime CD8 T cell responses by upregulation of costimulatory molecules. Such CD40L-CD40 interactions result in the activation of CD40-bearing cells, which then show presentation of MHC I and II molecules in addition to adhesion (ICAM), costimulation (CD80 / CD86), cytokines / chemokines (TNFα, IL6, etc.). In the tumor microenvironment, adhesion molecules and cytokines / chemokines work together to induce infiltration and activation of immune cells, eventually destroying tumor cells and shifting the tumor from an immunosuppressive to an immune-responsive microenvironment (Richards et al. Hum Vaccin Immunother. 2020;16(2):377-387). Similarly, TNFSF receptors 4-1-BB, OX40, GITR, and CD27 are expressed on T cells and respond to costimulation by ligands expressed on APCs, lymphocytes, and innate immune cells. Such diverse studies have increased interest in the development of TNFSF ligands for the treatment of disorders associated with dysfunction of TNF cytokines such as proliferative diseases like cancer and infectious diseases.

[0006] In recent years, TNF superfamily ligands have emerged as attractive candidates for the development of vaccines and immunotherapies, more specifically, alternative approaches to the design of novel molecular adjuvants (Gupta et al., 2013, Immunol Res., 57(1-3):303-10). Adjuvants can alter the strength, quality, and functionality of innate and adaptive immune responses, with a focus on rapidly inducing large numbers of CD8 T cells that can protect against specific diseases (Lauterbach et al., 2013, Front Immunol., 27;4:251). Recombinant MVA-CD40L has already demonstrated the potential of CD40L adjuvant viruses to rapidly induce strong antigen-specific CD8 T cell responses for the development of prophylactic and therapeutic vaccines against cancer and infectious diseases such as HIV / AIDS, Ebola hemorrhagic fever, Marburg hemorrhagic fever, malaria, and hepatitis C (Lauterbach et al., 2013, Front Immunol. 2013 Aug 27;4:251). Approaches to arm adenoviruses with CD40L have also been developed to stimulate beneficial immune responses for cancer treatment (Pesonen et al., 2012, Cancer Res., 72(7)). Similarly, recombinant 4-1-BBL adenovirus has been shown to be a promising adjuvant for human memory CD8 T cells by providing a strong T cell response, suggesting its benefit in antiviral treatment strategies (Bukczynski et al., 2004, Proc Natl Acad Sci U S A.;101(5):1291-1296).

[0007] Those skilled in the art also recognize that TNFSF ligands generally require homo-oligomerization to fully activate responsive cells. Although this is also well-defined structurally, TNFSF signaling also requires appropriate receptor clustering and at least trimerization. TNFSF ligands exist as trimeric units by self-assembly, while receptors are usually separated on the cell surface (Richards et al. 2020, Hum Vaccin Immunother., 16(2):377-387). The interaction between multimeric TNFSF ligands and their corresponding receptors results in precise receptor clustering and is an essential condition for generating intracellular signaling (Kucka et al., 2020, Front Cell Dev Biol., 8:615141). As previously shown, higher-order oligomeric TNFSF ligands, particularly dodecameric TNFSF ligands, have higher signaling effects than single trimeric TNFSF ligands (Haswell et al. 2001, Eur J Immunol., 31(10):3094-100).

[0008] Due to the important role of TNFSF ligands in immune responses, more specifically, tumor responses, various strategies such as fusion with surfactant protein D as a lectin family member have been explored to enhance their agonist properties.

[0009] A first recombinant fusion protein comprising a TNF cytokine and a dimerization or multimerization domain to enhance the avidity of the ligand is disclosed in WO 01 / 49866. However, the multimerization of the TNF protein is rather inefficient due to the molecular weight of the multimerization domain. WO 01 / 42298 discloses a method for constructing a stable bioactive fusion protein for expression with a collectin, more specifically, a SPD comprising a signal sequence, a collagen domain, and a coiled-coil neck domain, with TNFSF, more specifically, CD40L and RANKL / TRANCE members. Due to the large size of the resulting fusion protein, it is difficult to diffuse into tumors, thereby limiting its potential activity. This property can be particularly useful for preventing clearance, but the CD40 agonist activity of these fusion proteins is limited.

[0010] WO 2009 / 007120 also discloses fusion proteins comprising a TNFSF cytokine fused to a collectin trimerization domain. These fusion proteins can form multimeric proteins, but are limited in forming trimers because they cannot self-assemble more than three monomeric units, and their potential agonist activity is reduced.

[0011] TNFSF receptors are ubiquitously expressed on normal cells, and as a result, TNFSF ligands have little or no binding selectivity and are quite toxic. Although the TNFSF ligand axis still attracts great interest in cancer immunotherapy, systemic administration of TNFSF is associated with severe dose-limiting toxicity and only modest clinical activity (Bremer et al., 2013, ISRN Oncol.; 2013:371854). In the first clinical trials of CD40 agonists, the majority of patients presented with cytokine release syndrome, which causes fever, rigors, and chills (Vonderheide et al., 2007, J Clin Oncol., 25(7):876-883). Evidence of hepatotoxicity is also commonly observed with CD40 agonists (Vonderheide et al., 2007, J Clin Oncol., 25(7):876-883). Thus, tumor-limited activation and selective enhancement of T cell stimulation via TNFSF in the tumor microenvironment have continued. SUMMARY OF THE INVENTION

[0012] Technical problem Cancer continues to be a profound global health threat for many years due to numerous etiological factors that can act together or separately to initiate or promote its development. Additionally, malignant tumors, particularly metastatic tumors, often exhibit resistance to conventional therapies, which accounts for the significant morbidity of some cancers.

[0013] Therefore, there is a significant need to develop a more effective approach for preventing and treating cancer, and more generally, proliferative diseases and disorders associated with TNF cytokine dysfunction. Multimeric complexes of TNFSF cytokines are known to be difficult to prepare from recombinant monomeric forms, but the inventors have created an SPD-TNFSF fusion protein that provides efficient multimerization properties and enhances the agonist properties of the ligand by optimal clustering of its receptor, independent of the cellular environment. The optimal form is relatively small in molecular size, allowing for efficient diffusion into tumors and efficient clearance by the kidneys. This relatively small size is also advantageous when inserting into vectors with limited capacity (e.g., some viruses). Indeed, it is well known that tissue distribution, i.e., penetration into cancer, is better for smaller therapeutic agents than for larger ones (Li et al. MAbs. 2016;8(1):113-9). Thus, those skilled in the art will be well aware that the in vivo distribution of such molecules is facilitated.

[0014] There is also a need to develop novel targeted SPD-TNFSF fusion proteins that improve T cell responses, perhaps by the synergistic action of viral replication and TNFSF stimulation on tumor cells and immune cells, due to the limitation of binding selectivity and harmful off-target toxicity by activation of systemic TNFSF receptors.

[0015] Other further aspects, features, and advantages of the present invention will become apparent from the following description of presently preferred embodiments of the invention. These embodiments are shown for the purpose of disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0016]

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[0017] Summary of the invention The present invention relates to an SPD-TNFSF fusion protein comprising, or consisting of, an N-terminal domain, a coiled-coil neck domain of surfactant protein-D (SPD) between the N-terminal domain and the C-terminal position, and a TNF-superfamily (TNFSF) ligand, or its receptor-binding domain at the C-terminal position.

[0018] In one embodiment, the SPD-TNFSF fusion protein comprises, consists of, or consists essentially of an N-terminal domain, a coiled-coil neck domain of surfactant protein-D (SPD) between the N-terminal domain and the C-terminus, and a TNF-superfamily (TNFSF) ligand or its receptor-binding domain at the C-terminus, and the N-terminal domain and the coiled-coil neck domain of SPD are directly fused without intervening amino acid residues. In one embodiment, the N-terminal domain of the SPD-TNFSF fusion protein and the coiled-coil neck domain of SPD consist of the sequence of SEQ ID NO: 54.

[0019] In one embodiment, the SPD-TNFSF fusion protein further comprises a collagen domain between the N-terminal domain and the coiled-coil neck domain of SPD. The collagen domain comprises, consists of, or consists essentially of 1 to 40 (GXX) repeats, preferably 3 to 30 (GXX) repeats, preferably 6 to 20 (GXX) repeats, more preferably 12 (GXX) repeats, where X is an amino acid and G is a glycine amino acid.

[0020] In each repeat, X may be the same or different, for example, but not limited to, the repeat may be GAA or the repeat may be GTA.

[0021] Alternatively, or in combination therewith, the SPD-TNFSF fusion protein further comprises a linker between the coiled-coil neck domain and the TNF-superfamily ligand or its binding domain. The linker is a glycine / serine linker and has a length of 4 to 20 amino acids, preferably 8 to 16, more preferably 12 amino acids.

[0022] The TNFSF ligand is preferably selected from CD40L, 4-1-BBL, OX40L, CD70, TNF, GITRL, LIGHT, FASL, TWEAK, APRIL, RANKL, TRAIL, CD30L, NGF, Baff, LTβ, LTα, LTαβ2, TL1A, TLA, EDA, more preferably CD40L or 4-1-BBL.

[0023] The N-terminal domain has at least 85%, preferably at least 90%, more preferably at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0024] In one embodiment, the SPD-TNFSF fusion protein comprises or consists of a sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.

[0025] The present invention further relates to a trimeric fusion protein comprising three SPD-TNFSF fusion proteins. The present invention also relates to a multimeric fusion protein comprising a plurality of trimeric fusion proteins and forming a hexamer, dodecamer, octamer or higher-order oligomer, preferably a hexamer, more preferably a dodecamer.

[0026] The present invention also relates to an isolated nucleotide sequence encoding an SPD-TNFSF fusion protein. In one embodiment, the isolated nucleotide sequence comprises or consists of a sequence selected from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

[0027] The present invention further relates to an expression vector such as mRNA, plasmid or virus comprising an isolated nucleotide sequence encoding an SPD-TNFSF fusion protein. In one embodiment, the virus is a oncolytic virus selected from the group consisting of poxvirus, herpesvirus, reovirus, Seneca Valley virus (SVV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), mobirvirus, retrovirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), measles virus, foamy virus, alphavirus, lentivirus, influenza virus, sindbis virus, rhabdovirus, picornavirus, coxsackievirus, parvovirus or chimeras thereof, and in particular, poxvirus is preferred. In a further embodiment, the poxvirus preferably belongs to the genus Orthopoxvirus selected from the group consisting of vaccinia virus, cowpox virus, canarypox virus and ectromelia virus or chimeras thereof, and in particular, vaccinia virus is preferred, and in particular, vaccinia virus selected from the group consisting of the Elstree strain, the Wyeth strain, the Copenhagen strain, the Lister strain, the Tian Tian strain and the Western Reserve strain is preferred. In another embodiment, the poxvirus belongs to the genus Leporipoxvirus selected from the group consisting of myxoma virus, rabbit fibroma virus and squirrel fibroma virus, and in particular, myxoma virus is preferred. In a preferred embodiment, the oncolytic poxvirus is a poxvirus deficient in modified thymidine kinase (TK) activity due to an inactivating mutation in the J2R viral gene. Alternatively, or in combination therewith, the oncolytic poxvirus is deficient in ribonucleotide reductase (RR) activity due to an inactivating mutation in the viral I4L and / or F4L gene.

[0028] Alternatively, or in combination therewith, the modified poxvirus may further have the M2L locus modified (preferably a modification that results in suppression of the expression of the viral m2 protein), resulting in a poxvirus lacking the m2 function (m2-deficient poxvirus).

[0029] In another embodiment, the virus is a non-oncolytic virus, preferably a poxvirus selected from the group consisting of vaccinia virus (PCPV), modified vaccinia virus Ankara (MVA), highly attenuated vaccinia virus strain (NYVAC), swinepox virus (SWPV), fowlpox virus (FPV) or chimeras thereof.

[0030] The present invention further relates to a method for producing the above virus, comprising: a) preparing production cells; b) transfecting or infecting the prepared production cells with the virus; c) culturing the transfected or infected production cells under appropriate conditions to allow virus production; d) recovering the produced virus from the culture of the production cells; and optionally e) purifying the recovered virus.

[0031] The present invention further relates to a cell comprising the nucleotide sequence, mRNA, plasmid or virus of the present invention.

[0032] The present invention also relates to SPD-TNFSF fusion proteins, trimeric fusion proteins, multimeric fusion proteins, nucleotide sequences, mRNAs, plasmids, viruses or cells for use in the treatment of proliferative diseases such as cancer as well as infectious diseases, inflammatory diseases, metabolic diseases, autoimmune diseases, degenerative diseases, apoptosis-related diseases and disorders associated with TNF cytokine dysfunction such as transplant rejection, more preferably in the medicine of proliferative diseases, preferably in treatment, and even more preferably in the treatment of cancer. The present invention further relates to SPD-TNFSF fusion proteins, trimeric fusion proteins, multimeric fusion proteins, nucleotide sequences, mRNAs, plasmids, viruses or cells in combination with one or more chemotherapeutic agents or immunotherapeutic agents effective for use in the treatment of cancer.

[0033] The present invention further provides a pharmaceutical composition comprising, or consisting of, the SPD-TNFSF fusion protein, nucleotide sequence, mRNA, plasmid, virus or cell of the present invention, optionally together with a pharmaceutically acceptable diluent, carrier, vehicle and / or excipient. In one embodiment, the pharmaceutical composition further comprises one or more effective chemotherapeutic agents or immunotherapeutic agents. In one embodiment, the pharmaceutical composition is used for the treatment of cancer. In one embodiment, the pharmaceutical composition for use is administered via a parenteral route, more preferably via an intravenous, subcutaneous or intramuscular route, and even more preferably via an intravenous route.

[0034] The present invention also relates to a method for treating cancer, comprising administering to a subject a therapeutically effective amount of an SPD-TNFSF fusion protein, nucleotide, mRNA, plasmid, virus, cell or pharmaceutical composition of the present invention.

[0035] Detailed description of the invention The present invention relates to a virus into which an SPD-TNFSF fusion protein and one or more molecules encoding one or more SPD-TNFSF fusion proteins have been inserted into the genome.

[0036] Definition As used throughout this specification, the terms "a" and "an" are used in the sense of meaning "at least one," "at least a first," "one or more," or "a plurality" of reference components or steps, unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0037] The term "one or more" refers to a number that is one or greater (e.g., 2, 3, 4, 5, etc.).

[0038] The term "and / or" as used herein always includes the meanings of "and," "or," and "any and all or any other combination of the elements connected by said terms."

[0039] The term "about" or "approximately" as used herein means within 20%, preferably within 10%, more preferably within 5% of a given value or range.

[0040] As used herein, when used to define products, compositions, and methods, the term "comprising" (and any form of comprising such as "comprise" and "comprises"), "having" (and any form of having such as "have" and "has"), "including" (any form of including such as "includes" and "include"), or "containing" (any form of containing such as "contains" and "contain") is open-ended and does not exclude additional elements or method steps not recited. Thus, if an amino acid sequence can be part of the final amino acid sequence of a polypeptide, the polypeptide "comprises" that amino acid sequence. Such a polypeptide can have up to several hundred additional amino acid residues. "Consisting essentially of" means excluding other components or steps that are essentially important. Thus, a composition consisting essentially of the recited components excludes trace contaminants and pharmaceutically acceptable carriers. A polypeptide "consisting essentially of" a certain amino acid sequence means that the certain amino acid sequence is present with only a few additional amino acid residues ultimately added. "Consisting of" means excluding other components or steps that are not trace amounts. For example, if a polypeptide contains no amino acids other than the recited protein, the polypeptide "consists of" that amino acid sequence.

[0041] The terms "polypeptide", "peptide" and "protein" refer to polymers of amino acid residues comprising at least 9 or more amino acids linked via peptide bonds. The polymer can be linear, branched or cyclic, may comprise natural amino acids and / or amino acid analogs, and may have non-amino acids inserted. As a general indication, it is preferred that amino acid polymers of more than 50 amino acid residues be called "polypeptides" or "proteins", while those of 50 amino acids in length or less are called "peptides".

[0042] In the context of the present invention, the terms "nucleic acid", "nucleic acid molecule", "polynucleotide" and "nucleotide sequence" are used interchangeably and define polymers of any length of polydeoxyribonucleotides (DNA) (e.g., cDNA, genomic DNA, plasmid, vector, viral genome, isolated DNA, probe, primer and any mixture thereof) or polynucleotides (RNA) (e.g., mRNA, antisense RNA, siRNA) or hybrid polyribo-polydeoxyribonucleotides. They include single-stranded or double-stranded, linear or cyclic, natural or synthetic, modified or unmodified polynucleotides. Furthermore, the polynucleotide may comprise non-natural nucleotides and may have non-nucleotide components inserted.

[0043] The term "analog" or "variant", as used herein, refers to a molecule (polypeptide or nucleic acid) that exhibits one or more modifications relative to the natural counterpart. Any modification including substitution, insertion and / or deletion of one or more nucleotide / amino acid residues can be envisaged. Analogs that retain a sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, even more preferably at least 98% with the sequence of the natural counterpart are preferred.

[0044] Generally, the term "identity" refers to the amino acid-to-amino acid or nucleotide-to-nucleotide identity between two polypeptides or nucleic acid sequences. The percentage of identity between two sequences is a function of the number of identical positions the sequences share, taking into account the number of gaps that need to be introduced for optimal alignment and the length of each gap. To determine the percentage of identity between amino acid sequences, various computer programs and mathematical algorithms are available in the art, such as the Blast programs available at NCBI or ALIGN of Atlas of Protein Sequence and Structure (Dayhoffed, 1981, Suppl., 3: 482-9). Programs for determining identity between nucleotide sequences are also available in specialized databases (e.g., Genbank, Wisconsin Sequence Analysis Package, BESTFIT, FASTA and GAP programs). By way of illustration, "at least 80% identity" means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0045] As used herein, the term "isolated" refers to a protein, polypeptide, peptide, polynucleotide, vector, etc. that has been removed from its natural environment (i.e., separated from at least one other component with which it is naturally associated or found together in nature). For example, a nucleotide sequence is said to be isolated if it has been separated from the sequences with which it is normally associated in nature (e.g., separated from the genome), and heterologous sequences may be associated with it.

[0046] As used herein, the terms "obtained from," "originating in," or "originate in" are used to identify the original source of a component (e.g., a polypeptide, a nucleic acid molecule), but do not limit the method of making the component, and can be made, for example, by chemical synthesis or recombinant means.

[0047] As used herein, the term "host cell" should be understood broadly without limitation to a particular structure in a tissue, an organ, or an isolated cell. Such cells can be of a particular type of cell, such as a cultured cell line, a primary cell, and a dividing cell, or a different type of cell population. In the context of the present invention, the term "host cell" includes prokaryotic cells, lower eukaryotic cells such as yeast, and other eukaryotic cells such as insect cells, plant, and mammalian (e.g., human or non-human) cells, as well as cells capable of producing a virus (lytic virus or non-lytic virus) and / or a fusion protein for use in the present invention. The term also includes cells that can be or have been recipients of the vectors described herein, and progeny of such cells.

[0048] The terms "virus", "viral particle", "viral vector" and virion are used interchangeably and are widely understood to mean a vehicle comprising at least one element of a wild-type viral genome and can be packaged into or as a viral particle. A viral particle may or may not contain nucleic acid (i.e., a viral genome), but a virus comprises a DNA or RNA viral genome packaged in a viral particle (or virion) and is preferably infectious (i.e., capable of infecting and entering a host cell or subject). Desirably, the virus of the present invention comprises a DNA genome, most preferably a double-stranded DNA genome. In the context of the present disclosure, "virus" includes wild-type and engineered (modified) viruses. Modifications can be within endogenous viral genes (e.g., coding and / or regulatory sequences) and / or within intergenic regions. Further, the modifications may or may not be silent (e.g., resulting in a modified viral gene product). Modifications can be made in several ways known to those skilled in the art using conventional molecular biology techniques. Desirably, the modifications encompassed by the present invention affect, for example, pathogenicity, toxicity, virulence, or viral replication compared to a virus without such modifications, but do not completely impair infection and production even in cells with minimal tolerance.

[0049] The term "oncolytic virus" encompasses any naturally occurring virus, engineered or otherwise modified virus. As used herein, the term "oncolytic virus" does not replicate at all or replicates minimally in non-dividing cells (e.g., primary cells), but selectively replicates in dividing cells (e.g., proliferating cells such as cancer cells) in vitro or in vivo for the purpose of slowing down and / or lysing the growth of said dividing cells. Generally, an oncolytic virus contains a viral genome packaged in a virus particle (or virion) and is infectious (i.e., capable of infecting and entering a host cell or subject). As used herein, this term encompasses DNA or RNA vectors (depending on the virus of interest), as well as virus particles generated therefrom.

[0050] As used herein, the terms "chimeric", "viral chimera" or "chimera of viruses" refer to a virus obtained by homologous recombination between multiple different viral strains. Several chimeras obtained by mixing genomes from different poxviruses have been described and are available to those skilled in the art (e.g., the CF189 chimera obtained from ORF and vaccinia virus (Choi et al, Novel chimeric parapoxvirus CF189 as an oncolytic immunotherapy in triple-negative breast cancer. Surgery Volume 163, Issue 2, February 2018, Pages 336-342); the CF33 chimera obtained from multiple strains of VV, vaccinia, and rabbitpox (Chaurasiya, S. et al., 2020, Cancer Gene Ther 27, 125-135)).

[0051] The term "non-oncolytic virus" encompasses any virus not defined as an oncolytic virus.

[0052] The term "treatment" (and any form of treatment such as "treating" and "treat") as used herein encompasses prevention (e.g., preventive measures in a subject at risk of having a pathological condition to be treated) and / or therapy (e.g., in a subject diagnosed with a pathological condition), and ultimately relates to conventional treatment modalities. The result of treatment is to slow, cure, improve, or suppress the progression of the targeted pathological condition. For example, if a subject shows an observable improvement in their clinical condition after administration of a fusion protein described herein, a virus described herein, or a combination thereof described herein, it can be said that the subject has been successfully treated for cancer.

[0053] The term "administering" (or any form of "administering" such as "administered") as used herein refers to the delivery of a therapeutic agent, such as a virus described herein, to a subject.

[0054] The term "proliferative disorder" as used herein encompasses any disease or condition resulting from uncontrolled cell growth and spread, including cancer and certain cardiovascular diseases (such as restenosis caused by the proliferation of smooth muscle cells in the blood vessel wall). The term "cancer" can be used interchangeably with any of the terms such as "tumor", "malignant tumor", "neoplasm", etc. These terms are meant to include all types of tissues, organs, or cells, and all stages of malignant tumors (e.g., pre - lesion to stage IV).

[0055] The term "disorder associated with TNF cytokine dysfunction" as used herein encompasses any disease, disorder, or condition that results in TNF signaling dysfunction.

[0056] The term "subject" generally refers to an organism for which any of the products and methods of the present invention are required or may be beneficial. Generally, this organism is a mammal, particularly a mammal selected from the group consisting of domestic animals, agricultural animals, sport animals, and primates. Preferably, the subject is a human diagnosed with or at risk of a proliferative disease such as cancer or a disorder associated with TNF cytokine dysfunction. The terms "subject" and "patient" may be used interchangeably when referring to a human organism and include both males and females. The subject to be treated can be a neonate, infant, young adult, adult or elderly person.

[0057] The term "combination" or "associated", as used herein, refers to any possible arrangement of various components (e.g., a fusion protein or virus and one or more substances effective in anti-cancer therapy). Such arrangements include mixtures of the above components as well as separate combinations for co-administration or sequential administration. The present invention encompasses combinations comprising equimolar concentrations of each component as well as combinations of very different concentrations. It is understood that the optimal concentration of each component of the combination can be determined by those skilled in the art.

[0058] The term "chemotherapeutic agent" or "immunotherapeutic agent", as used herein, refers to a product comprising one or more antigens expected to induce or activate an immune response, whether specific or non-specific, humoral or cellular, when appropriately delivered to a subject.

[0059] SPD-TNFSF fusion protein As used herein, the term "fusion protein" means a covalent bond in a single protein chain of two or more polypeptides, which is effected by genetic means, i.e., by fusing in-frame nucleic acid molecules encoding each of said polypeptides or fragments thereof. "Fused in-frame" means that the expression of the fused coding sequences results in a single polypeptide having the functional properties derived from each of the original polypeptides. The fusion may be direct (i.e., without intervening additional amino acid residues) or indirect (e.g., via a linker between the polypeptides to be fused).

[0060] The term "SPD" or "surfactant protein D" as used herein refers to a functional fragment of native surfactant protein D or a derivative thereof. SPD is preferably of mammalian origin, such as of human, mouse, rabbit, non-human primate or porcine origin, preferably of human or non-human primate origin, more preferably of human origin.

[0061] In the context of the present invention, an SPD-TNFSF fusion protein comprises or consists of a fusion of surfactant protein D (SPD) and a TNFSF ligand, or a receptor-binding domain thereof. In the present invention, SPD comprises or consists of an N-terminal domain and a coiled-coil neck domain. In one embodiment, the N-terminal domain and the coiled-coil neck domain are directly fused without intervening amino acid residues. In one embodiment, the N-terminal domain and the coiled-coil neck domain of SPD in the SPD-TNFSF fusion protein consist of the sequence of SEQ ID NO: 54.

[0062] The N-terminal domain of the present invention comprises or consists of an amino acid sequence shown in SEQ ID NO: 1, in particular an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95% and most preferably 100% identity to amino acids 21 - 45 of native human SPD (SEQ ID NO: 22).

[0063] The coiled-coil neck domain of the present invention comprises an amino acid sequence shown in SEQ ID NO: 23, in particular, an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95% identity with amino acids 223 to 252 of native human SPD (SEQ ID NO: 22).

[0064] The TNFSF ligand can be selected from members of the TNF superfamily including, but not limited to, CD40L, 4-1-BBL, OX40L, CD70, TNF, GITRL, LIGHT, FASL, TWEAK, APRIL, RANKL, TRAIL, CD30L, NGF, Baff, LTβ, LTα, LTαβ2, TL1A, TLA, EDA, or their receptor-binding domains. The TNFSF ligand is preferably of mammalian origin such as human, mouse, rabbit, non-human primate or porcine origin, preferably of human or non-human primate origin, more preferably of human origin.

[0065] In a preferred embodiment, the member of TNFSF or its receptor-binding domain is · CD40L, 4-1BBL, Baff, APRIL, EDA, GITRL, OX40L, CD70, TL1A, LIGHT, LTαβ2, RANKL, TWEAK, FASL, TRAIL, TNF and LTα or their receptor-binding domains; · Preferably, a Category II TNFSF member from CD40L, 4-1BBL, Baff, APRIL, EDA, OX40L, CD70, TWEAK, FASL, TRAIL, and TNF or its receptor-binding domain; · Preferably, a TNFSF member involved in activation of immune cells from CD40L, 4-1BBL, GITRL, OX40L, CD70, TL1A or its receptor-binding domain; · Preferably, a Category II TNFSF member involved in activation of immune cells from CD40L, 4-1BBL, OX40L, CD70 or its receptor-binding domain; or ·CD40L, 4-1BBL or their receptor binding domains selected from

[0066] Most preferably, the member of TNFSF or its receptor binding domain is selected from CD40L or its receptor binding domain.

[0067] TNFSF member TNFSF is composed of many structurally related members (also called ligands), which organize the development of lymphoid tissues, co-stimulate lymphocyte activation, increase lymphocyte survival and function, or induce cell death by binding to cognate receptors that form the tumor necrosis factor receptor superfamily (TNFRSF).

[0068] TNFRSF can be divided into two categories by its ability to be activated by soluble ligand trimers of TNFSF (Kucka K, 2021, Front Cell Dev Biol. 11;8:615141).

[0069] Category I receptors of TNFRSF are strongly activated by soluble ligand trimers and include BaffR (ligand = Baff), DR3 (ligand = TL1A), GITR (ligand = GITRL), LTβR (ligand = LTαβ2 or LIGHT), TNFR1 (ligand = TNF or LTα).

[0070] Category II receptors of TNFRSF bind to soluble ligand trimers with high affinity but cannot be properly activated by them, and include CD40L, 4-1BBL, Baff, APRIL, EDA, OX40L, CD70, TWEAK, FASL, TRAIL, and TNF.

[0071] The limited reactivity of Category II TNFRs to soluble TNFL can be overcome by physically binding two or more soluble ligand trimers or immobilizing the soluble ligand molecules on the cell surface or extracellular matrix. In the context of the present invention, members of TNFSF that bind to Category II TNFRs are preferred because the ability of the fusion proteins according to the present invention is truly conditional and the activation of their cognate TNFRs depends on the presence in the tumor microenvironment of PD-L1-positive tumor cells.

[0072] Thus, in a preferred embodiment, the member of TNFSF contained in the fusion protein according to the present invention is preferably a Category II TNFSF member selected from CD40L, 4-1BBL, Baff, APRIL, EDA, OX40L, CD70, TWEAK, FASL, TRAIL, and TNF, or a receptor-binding domain thereof.

[0073] TNFSF members can also be classified by their known functions. In the context of the present invention, members of TNFSF involved in the activation of immune cells may preferably be included in the fusion proteins according to the present invention. Members of TNFSF involved in the activation of immune cells include CD40L, 4-1BBL, GITRL, OX40L, CD70, TL1A or their receptor-binding domains (Croft M. et al., 2017, Nat Rev Rheumatol.;13(4):217-233).

[0074] "CD40L", "CD40 Ligand", "CD40LG", "Tumor Necrosis Factor Superfamily Member 5", "TNFSF5", and "CD154" are used interchangeably herein and collectively refer to members of TNFSF that form a homotrimer and play a central role in initiating the adaptive immune response when interacting in trans with its receptor CD40. The interaction between CD40L and CD40 results in the activation of CD40-bearing cells, which then express MHC I and II molecules in addition to adhesion (ICAM), costimulation (CD80 / CD86), and cytokines / chemokines (TNFα, IL6, etc.). In the tumor microenvironment, adhesion molecules and cytokines / chemokines work together to induce infiltration and activation of immune cells, ultimately destroying tumor cells and eventually shifting the tumor from an immunosuppressive to an immune-responsive microenvironment (Richards et al., 2020, Hum Vaccin Immunother. 16(2):377-387). Thus, CD40L is a member of TNFSF involved in the activation of immune cells.

[0075] TNF and CD40L form a homotrimer and interact in trans with its receptor CD40 via its extracellular portion. CD40 is a category II receptor of TNFRSF.

[0076] Since CD40L is a member of TNFSF involved in the activation of immune cells and binds to a category II receptor of TNFRSF, it is a particularly preferred TNFSF member for the fusion protein according to the present invention.

[0077] The specific CD40L protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human CD40L is preferably used. Human CD40 corresponds to Entrez Gene ID 959, and the complete amino acid sequence of human CD40L can be found as GenBank accession number NP_000065.1 (version of January 17, 2022).

[0078] "4-1BBL", "4-1BB ligand", "CD137L", "tumor necrosis factor superfamily member 9", and "TNFSF9" are used interchangeably in the present invention and refer to a transmembrane cytokine that functions as a ligand for TNFRSF9 / 4-1BB, a costimulatory receptor molecule in T lymphocytes. This cytokine and its receptor are involved in the antigen presentation process and the generation of cytotoxic T cells. Thus, 4-1BBL is a TNFSF member involved in the activation of immune cells.

[0079] Its receptor 4-1BB is a category II receptor of TNFRSF.

[0080] Since 4-1BBL is a member of TNFSF that is involved in the activation of immune cells and binds to a category II receptor of TNFRSF, it is a particularly preferred TNFSF member for the fusion protein according to the present invention.

[0081] The specific 4-1BBL protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human 4-1BBL is preferably used. Human 4-1BBL corresponds to Entrez Gene ID 8744, and the complete amino acid sequence of human 4-1BBL can be found as GenBank accession number NP_003802.1 (version of February 27, 2022).

[0082] "OX40L", "OX40 ligand", "CD252", "CD134L", "tumor necrosis factor superfamily member 4", and "TNFSF4" are used interchangeably in the present invention and refer to an inducible molecule that is expressed in several cell types but is most importantly expressed on antigen-presenting cells (APCs). OX40L can cause signal transduction via its receptor OX40, resulting in various activities including the proliferation and accumulation of effector T cells and cytokine production. Thus, OX40L is a TNFSF member involved in the activation of immune cells.

[0083] The receptor OX40 is a category II receptor of TNFRSF.

[0084] Since OX40L is a member of TNFSF that is involved in the activation of immune cells and binds to a category II receptor of TNFRSF, OX40L is a particularly preferred TNFSF member of the fusion protein according to the present invention.

[0085] The specific OX40L protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human OX40L is preferably used. Human OX40L corresponds to Entrez Gene ID 7292, and the complete amino acid sequence of the longest isoform of human OX40 can be found as GenBank accession number NP_003317.1 (version of March 16, 2022).

[0086] "CD70", "CD27L", "CD27LG", "tumor necrosis factor superfamily member 7", and "TNFSF7" are used interchangeably in the present invention and refer to molecules that can transmit signals to T cells through their interaction with the receptor CD27 to control their accumulation and reactivity, similar to those found in OX40, GITR, and DR3. Thus, CD70 is a member of TNFSF that is involved in the activation of immune cells.

[0087] The receptor CD27 is a category II receptor of TNFRSF.

[0088] Since CD70 is a member of TNFSF that is involved in the activation of immune cells and binds to a category II receptor of TNFRSF, CD70 is a particularly preferred TNFSF member of the fusion protein according to the present invention.

[0089] The specific CD70 protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human CD70 is preferably used. Human CD70 corresponds to Entrez Gene ID 970, and the complete amino acid sequence of the longest isoform of human CD70 can be found as GenBank accession number NP_001317261.1 (version of January 9, 2022).

[0090] "Baff", "B cell activating factor", "CD257", "tumor necrosis factor superfamily member 13b", "TNFSF13B", "tumor necrosis factor superfamily member 20", and "TNFSF20" are used interchangeably in the present invention and, although not exclusive, mainly refer to molecules that control the activity of B cells.

[0091] Baff binds to two receptors of TNFRSF: BaffR and TACI. BaffR is a category I receptor of TNFRSF, while TACI is a category II receptor of TNFRSF.

[0092] Since Baff is a member of TNFSF that binds to the category II receptor of TNFRSF (TACI), Baff is a particularly preferred TNFSF member of the fusion protein according to the present invention.

[0093] The specific Baff protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human Baff is preferably used. Human Baff corresponds to Entrez Gene ID 10673, and the complete amino acid sequence of the longest isoform of human Baff can be found as GenBank accession number NP_006564.1 (version of February 20, 2022).

[0094] "APRIL", "CD256", "tumor necrosis factor superfamily member 13", and "TNFSF13" are used interchangeably in the present invention and refer to ligands that have been found to be important for B cell development.

[0095] APRIL binds to two TNFRSF receptors: BCMA and TACI, both of which are TNFRSF category II receptors.

[0096] Since APRIL is a member of TNFSF that binds to TNFRSF category II receptors, APRIL is a preferred TNFSF member of the fusion proteins according to the present invention.

[0097] The specific APRIL protein that may be included in the fusion proteins according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human APRIL is preferably used. Human APRIL corresponds to Entrez Gene ID 8741, and the complete amino acid sequence of the longest isoform of human APRIL can be found as GenBank accession number NP_003799.1 (version of January 23, 2022).

[0098] "EDA-A1", "EDA-A2", "EDA", "ectodysplasin A", and "tumor necrosis factor ligand 7C" are used interchangeably in the present invention and refer to proteins involved in intercellular signaling during the development of ectodermal organs.

[0099] Its receptor EDAR is a TNFRSF category II receptor.

[0100] Since EDA is a member of TNFSF that binds to TNFRSF category II receptors, EDA is a preferred TNFSF member of the fusion proteins according to the present invention.

[0101] The specific EDA protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species in which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human EDA is preferably used. Human EDA corresponds to Entrez Gene ID 1896, and the complete amino acid sequence of the longest isoform of human EDA can be found as GenBank accession number NP_001390.1 (version of February 13, 2022).

[0102] "GITRL", "glucocorticoid-induced TNF receptor-related ligand", "tumor necrosis factor superfamily member 18", and "TNFSF18" are used interchangeably in the present invention and refer to an inducer molecule expressed in professional APCs and other cell types such as endothelial cells. Its receptor, glucocorticoid-induced TNF receptor-related protein (also known as GITR, TNFRSF18), can stimulate the activation of T cells, dendritic cells, and B cells. Thus, GITRL is a TNFSF member involved in the activation of immune cells.

[0103] Its receptor GITR is a category I receptor of TNFRSF.

[0104] Since it is a TNFSF member involved in the activation of immune cells, GITRL is a preferred TNFSF member of the fusion protein according to the present invention.

[0105] The specific GITRL protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species in which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human GITRL is preferably used. Human GITRL corresponds to Entrez Gene ID 8995, and the complete amino acid sequence of the longest isoform of human GITRL can be found as GenBank accession number NP_005083.3 (version of February 20, 2022).

[0106] "TL1A", "VEGI", "tumor necrosis factor superfamily member 15", and "TNFSF15" are used interchangeably in the present invention and refer to proteins that can be induced in antigen-presenting cells (APCs) such as dendritic cells and macrophages, as well as endothelial cells. Binding to its receptor, "delta receptor 3" (also known as "DR3", "TNFRSF25"), a stimulatory receptor expressed by T cells, can regulate the accumulation and / or responsiveness of effector T cells. Thus, TL1A is a TNFSF member involved in the activation of immune cells. This cytokine has also been found to inhibit the proliferation of endothelial cells and thus may function as an angiogenesis inhibitor.

[0107] Its receptor DR3 is a category I receptor of TNFRSF.

[0108] Since TL1A is a TNFSF member involved in the activation of immune cells, TL1A is a preferred TNFSF member of the fusion proteins according to the present invention.

[0109] The specific TL1A protein that may be included in the fusion proteins according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human TL1A is preferably used. Human TL1A corresponds to Entrez Gene ID 9966, and the complete amino acid sequence of the longest isoform of human TL1A can be found as GenBank accession number NP_005109.2 (version of February 27, 2022).

[0110] "LIGHT", "CD258", "tumor necrosis factor superfamily member 14", and "TNFSF14" are used interchangeably in the present invention and refer to proteins that bind to two receptors of TNFRSF: herpesvirus entry mediator (HVEM, also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14)) and lymphotoxin-β receptor (LTβR). The LIGHT-HVEM interaction is responsible for most of the immunostimulatory properties of LIGHT. When expressed on lymphocytes, NK cells, smooth muscle, and epithelium, HVEM acts as an important T cell costimulatory factor leading to activation, proliferation, and survival. With regard to antitumor immunosuppression, LIGHT-LTβR signaling has a broad role in influencing the sensitivity of cancer cells to the immune response, the repair function of chaotic tumor vasculature, and the suppression of cell trafficking and infiltration of effector cells into tumors (Skeate Joseph G. et al. TNFSF14: LIGHTing the Way for Effective Cancer Immunotherapy. TNFSF14: LIGHTing the Way for Effective Cancer Immunotherapy. May 2020. Vol. 11. Article 922).

[0111] LTβR is a category I receptor of TNFRSF. HVEM is a category I receptor of TNFRSF.

[0112] The specific LIGHT protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human LIGHT is preferably used. Human LIGHT corresponds to Entrez Gene ID 8740, and the complete amino acid sequence of the longest isoform of human LIGHT can be found as GenBank accession number NP_003798.2 (version of February 20, 2022).

[0113] "RANKL", "RANK ligand", "receptor activator of nuclear factor-kappa B ligand", "CD254", "tumor necrosis factor superfamily member 11", and "TNFSF11" are used interchangeably in the present invention and refer to the ligand of osteoprotegerin that functions as an important factor in osteoclast differentiation and activation. This protein has also been shown to be a survival factor for dendritic cells and to be involved in the regulation of T cell-dependent immune responses.

[0114] Its receptor RANK (or "receptor activator of nuclear factor-kappa B") is a category I receptor of TNFRSF.

[0115] The specific RANKL protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human RANKL is preferably used. Human RANKL corresponds to Entrez Gene ID 8600, and the complete amino acid sequence of the longest isoform of human RANKL can be found as GenBank accession number NP_003692.1 (version of February 27, 2022).

[0116] "TWEAK", "tumor necrosis factor superfamily member 12", and "TNFSF12" are used interchangeably in the present invention and refer to a cytokine that has a signal transduction function overlapping with TNF but shows a much broader tissue distribution. It exists in both membrane-bound and secreted forms and can induce apoptosis via multiple cell death pathways in a cell type-specific manner. It has also been found to act as a regulator of angiogenesis by promoting the proliferation and migration of endothelial cells.

[0117] Its receptor FN14 (also called TWEAKR) is a category II receptor of TNFRSF.

[0118] Since TWEAK is a member of TNFSF that binds to the category II receptor of TNFRSF, TWEAK is a preferred TNFSF member of the fusion protein according to the present invention.

[0119] The specific TWEAK protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human TWEAK is preferably used. Human TWEAK corresponds to Entrez Gene ID 8742, and the complete amino acid sequence of the longest isoform of human TWEAK can be found as GenBank accession number NP_003800.1 (version of February 20, 2022).

[0120] "FASL", "FAS ligand", "FASLG", "CD178", "CD95 ligand", "CD95L", "tumor necrosis factor superfamily member 6", and "TNFSF6" are used interchangeably in the present invention and refer to a transmembrane protein whose main function is to induce apoptosis induced by binding to its receptor FAS. The FAS / FASLG signaling pathway is essential for the regulation of the immune system, including activation-induced cell death (AICD) of T cells and cytotoxic T lymphocyte-induced cell death.

[0121] FAS is a category II receptor of TNFRSF.

[0122] Since FASL is a member of TNFSF that binds to the category II receptor of TNFRSF, FASL is a preferred TNFSF member of the fusion protein according to the present invention.

[0123] The specific FASL protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human TWEAK is preferably used. Human FASL corresponds to Entrez Gene ID 356, and the complete amino acid sequence of the longest isoform of human FASL can be found as GenBank accession number NP_000630.1 (version of February 27, 2022).

[0124] "TRAIL", "CD253", "tumor necrosis factor superfamily member 10", and "TNFSF10" are used interchangeably in the present invention and refer to proteins that preferentially induce apoptosis in malignantly transformed cells and tumor cells, but are expressed at significant levels in most normal tissues and do not show killing of normal cells. TRAIL binds to several members of the TNF receptor superfamily, including TNFRSF10A / TRAILR1, TNFRSF10B / TRAILR2, TNFRSF10C / TRAILR3, TNFRSF10D / TRAILR4, and possibly also TNFRSF11B / OPG. The activity of TRAIL can be regulated by binding to decoy receptors TNFRSF10C / TRAILR3, TNFRSF10D / TRAILR4, and TNFRSF11B / OPG that cannot induce apoptosis. The binding of TRAIL to its receptors has been shown to induce the activation of MAPK8 / JNK, caspase 8, and caspase 3.

[0125] Both TRAILR1 and TRAILR2 are category II receptors of TNFRSF.

[0126] Since TRAIL is a member of TNFSF that binds to category II receptors of TNFRSF, TRAIL is a preferred TNFSF member of the fusion protein according to the present invention.

[0127] The specific TRAIL protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human TRAIL is preferably used. Human TRAIL corresponds to Entrez Gene ID 8743, and the complete amino acid sequence of the longest isoform of human TRAIL can be found as GenBank accession number NP_003801.1 (version of March 17, 2022).

[0128] "TNF", "tumor necrosis factor", "TNFA", "TNFα", "tumor necrosis factor superfamily member 2", and "TNFSF2" are used interchangeably in the present invention and refer to multifunctional inflammatory cytokines involved in the regulation of a wide range of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation.

[0129] TNF binds to two receptors of TNFRSF: TNFR1 (also known as "TNFRSF1A") and TNFR2 (also known as "TNFRSF1B" or "TNFBR"). TNFR1 is a category I receptor of TNFRSF, and TNFR2 is a category II receptor of TNFRSF.

[0130] Since TNF is a member of TNFSF that binds to the category II receptor of TNFRSF, TNF is a preferred TNFSF member of the fusion protein according to the present invention.

[0131] The specific TNF protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human TNF is preferably used. Human TRAIL corresponds to Entrez Gene ID 8743, and the complete amino acid sequence of human TNF can be found as GenBank accession number NP_000585.2 (version of March 17, 2022).

[0132] "CD30L", "CD153", "TNFSF8", and "tumor necrosis factor superfamily member 8" are used interchangeably in the present invention and refer to proteins that exhibit limited expression in subpopulations of T and B cells activated in non-diseased states and regulate proliferation / apoptosis and antibody responses.

[0133] Its receptor TNFRSF8 (also called tumor necrosis factor receptor superfamily member 8 or CD30) is a category I receptor of TNFRSF.

[0134] The specific CD30L protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human CD30L is preferably used. Human CD30L corresponds to Entrez Gene ID 943, and the complete amino acid sequence of human CD30L can be found as GenBank accession number AH005843.2 (version of June 10, 2016).

[0135] "LTα", "lymphotoxin α", "TNFB", "TNFβ", "tumor necrosis factor superfamily member 1", and "TNFSF1" are used interchangeably in the present invention and refer to cytokines that are produced by lymphocytes, are highly inducible, secreted, and form a heterotrimer with lymphotoxin-β that anchors lymphotoxin α to the cell surface. This protein also mediates diverse inflammatory, immunostimulatory, and antiviral responses, is involved in the formation of secondary lymphoid organs during development, and plays a role in apoptosis.

[0136] LTα binds to TNFR1 (also known as "TNFRSF1A"), a category I receptor of TNFRSF.

[0137] The specific LTα protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human LTα is preferably used. Human LTα corresponds to Entrez Gene ID 4049, and the complete amino acid sequence of human LTα can be found as GenBank accession number NP_001153212.1 (version of March 10, 2022).

[0138] "LTβ", "lymphotoxin β", "TNFC", "tumor necrosis factor superfamily member 3", and "TNFSF3" are used interchangeably in the present invention and refer to a protein that immobilizes lymphotoxin-α on the cell surface via hetero-trimer formation. The predominant form on the lymphocyte surface is the lymphotoxin-α1 / β2 complex (also called "LTαβ2", for example, 1 molecule of α / 2 molecules of β), and this complex is the major ligand of the lymphotoxin-β receptor (also called "LTβR"), which is a category I receptor of TNFRSF. A minor complex is lymphotoxin-α2 / β1. LTB is an inducer of the inflammatory response system and is involved in the normal development of lymphoid tissues.

[0139] The specific LTβ protein that can be included in the fusion protein according to the present invention is preferably selected from the same species as the species for which the fusion protein is intended for therapeutic use. In particular, for use intended in humans, human LTβ is preferably used. Human LTβ corresponds to Entrez Gene ID 4050, and the complete amino acid sequence of human LTβ can be found as GenBank accession number NP_002332.1 (version of February 20, 2021).

[0140] Functional fragment or variant of TNFSF member The SPD-TNFSF fusion protein according to the present invention may comprise all wild-type members of TNFSF, or alternatively may comprise a functional fragment or a functional derivative thereof.

[0141] The "functional fragment" of a member of TNFSF refers to a fragment (i.e., a part of the amino acid sequence of all members of TNFSF) that has one or more insertions, deletions, terminal truncations, and / or substitutions and retains the function of all members of TNFSF, i.e., its ability to trimerize and bind to and activate its TNFRSF receptor.

[0142] In particular, TNFSF members exist as transmembrane proteins, but their transmembrane regions and intracellular regions are not required for trimerization and binding to and activation of their TNFRSF receptors.

[0143] Thus, in a preferred embodiment, the fragment of the TNFSF member contained in the SPD-TNFSF fusion protein according to the present invention is an extracellular fragment of the TNFSF member, i.e., a fragment lacking the transmembrane and intracellular portions of the TNFSF member. As long as the fragment retains its ability to trimerize, bind to its TNFRSF receptor, and activate, all or part of the extracellular domain may be included in the extracellular fragment.

[0144] In a preferred embodiment, the TNFSF ligand or its receptor-binding domain of the fusion protein is selected from human CD40L (SEQ ID NO: 24), particularly amino acids 119-261 of human CD40L (SEQ ID NO: 25).

[0145] In a more preferred embodiment, the TNFSF ligand or its receptor-binding domain of the fusion protein is selected from mouse CD40L (SEQ ID NO: 26), particularly amino acids 115-260 of mouse CD40L (SEQ ID NO: 27).

[0146] In another embodiment, the TNFSF ligand or its receptor-binding domain of the fusion protein is selected from human 4-1-BBL (SEQ ID NO: 28), particularly amino acids 80-254 of human 4-1-BBL (SEQ ID NO: 29).

[0147] In yet another embodiment, the TNFSF ligand or its receptor binding domain of the fusion protein is selected from mouse 4-1-BBL (SEQ ID NO: 30), particularly amino acids 139 to 309 of mouse 4-1-BBL (SEQ ID NO: 31).

[0148] In one embodiment, the SPD-TNFSF fusion protein comprises a collagen domain. Generally, the collagen domain comprises, or consists of, 1 to 40 (GXX) repeats, preferably 3 to 30 (GXX) repeats, preferably 6 to 20 (GXX) repeats, more preferably 12 (GXX) repeats, where X is an amino acid and G is a glycine amino acid, and in each repeat, X may be the same or different. The collagen domain may be located between the N-terminal domain and the coiled-coil neck domain of SPD. Examples of suitable collagen domains are shown in Table 1 below.

[0149]

Table 1

[0150] In one embodiment, an SPD-TNFSF fusion protein as described herein may further comprise a linker located between the coiled-coil neck domain and the TNFSF ligand or its binding domain. Generally, the linker is composed of a short stretch of amino acid residues such as glycine (Gly or G), serine (Ser or S), threonine (Thr or T), asparagine (Asn or N), alanine (Ala or A) and / or proline (Pro or P). The linker is preferably a glycine / serine linker, i.e., consisting essentially of the amino acids glycine and serine. The linker preferably has a length of 4 to 20 amino acids, particularly 4, 8, 12, 16 or 20 amino acids (e.g., 1, 2, 3 or 4 repeats of GGGS, GSGSG, or SGSGS, or 1 or 2 repeats of GSGSGSGSGS). More preferably, the length of the linker is 8 to 16 amino acids, even more preferably 12 amino acids. As a guide, a linker suitable for use in the context of the present invention comprises the amino acid sequence shown in SEQ ID NO: 38 (GGGSGGGSGGGS). Optimizing the size and sequence of the peptide linker between the two fusion partners is within the scope of those skilled in the art.

[0151] In one embodiment, an SPD-TNFSF fusion protein as described herein comprises a collagen domain and a linker located at the N-terminus and C-terminus of the coiled-coil neck domain. A suitable SPD-TNFSF fusion protein comprises the amino acid sequence shown in SEQ ID NO: 11.

[0152] In the context of the present invention, it may be advantageous to include additional regulatory elements to promote the expression, transport and biological activity of the SPD-TNFSF fusion protein.

[0153] In the context of the present invention, the SPD-TNFSF fusion protein may further comprise a signal peptide at the N-terminus of the SPD-TNFSF fusion protein in order to affect the expression level, secretion, solubility, or other properties of the protein. Suitable signal peptides are known in the art. They may be derived from cellular polypeptides or viral polypeptides, such as those of immunoglobulins, tissue plasminogen activator, insulin, rabies glycoprotein, HIV viral envelope glycoprotein or measles virus F protein, or may be synthesized (see, for example, WO 2008 / 138649). By way of guidance, a signal peptide suitable for use in the context of the present invention comprises the amino acid sequence shown in SEQ ID NO: 39 (MLLFLLSALVLLTQPLGYLE), SEQ ID NO: 40 (MGLGLQWVFFVALLKGVHC) or SEQ ID NO: 41 (MGWSCIILFLVATATGVHS). Additionally, additional transmembrane domains can be envisaged to facilitate the immobilization of the TNFSF fusion protein in a suitable membrane of the cell (e.g., the plasma membrane). Transmembrane domains are generally inserted at the N-terminus or C-terminus of the protein. A vast variety of transmembrane domains are known in the art (see, for example, WO 99 / 03885).

[0154] In the context of the present invention, the SPD-TNFSF fusion protein may also further comprise a tag peptide (generally, a short peptide sequence recognizable by an available antiserum or compound) for subsequent expression, transport, or purification of the SPD-TNFSF protein or an infected host cell expressing such a fusion protein. The tag peptide can be detected by an immunodetection assay using an anti-tag antibody. A vast variety of tag peptides are available for use in the context of the present invention, including, but not limited to, a PK tag, a FLAG (DYKDDDK, SEQ ID NO: 42, GDYKDDDK, SEQ ID NO: 43, GSDYKDDDDK, SEQ ID NO: 44 or HHHHHHDYKDDDDKLVPRGS, SEQ ID NO: 45), a MYC tag (QKLISEEDL, SEQ ID NO: 46), a HIS tag (usually, a stretch of 4 to 10 histidine residues), an HA tag (YPYDVPDYA; SEQ ID NO: 47), an HSV tag (QPELAPEDPED; SEQ ID NO: 48), a VSV tag (YTDIEMNRLGK; SEQ ID NO: 49) and an e tag (U.S. Patent No. 6,686,152). By way of guidance, a tag peptide suitable for use in the context of the present invention comprises an amino acid sequence shown in SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO: 45. When multiple tags are used, the tag peptide may be located independently at the N-terminus or also at the C-terminus of the protein or also between or at either of these positions. If a signal peptide is already present at the N-terminus of the fusion protein, the tag peptide can preferably be inserted at the C-terminus of the fusion protein according to the present invention.

[0155] As another example, glycosylation can be modified to enhance the biological activity of the SPD-TNFSF fusion protein. Such modification can be achieved, for example, by mutating one or more residues within a glycosylation site.

[0156] The terms "oligomeric" or "multimeric", as used herein, refer to the ability to form a complex of at least two monomeric units. Association can be by specific interactions (requiring structural complementarity between the amino acid residues of the two partners at the binding site and one or more of electrostatic forces, hydrogen bonds, hydrophobic forces, and / or van der Waals forces to maintain the binding) or non-specific interactions (through one or more of the forces listed above but lacking structural complementarity). The oligomers of the SPD-TNFSF fusion protein are preferably formed by one or more intermolecular disulfide bonds involving one or more cysteine (Cys) residues on each polypeptide forming the oligomer, and thus disulfide bonds (i.e., intermolecular disulfide bonds) can be formed between oligomerized proteins. Preferably, the oligomer is formed between at least two SPD-TNFSF fusion proteins (dimer), three SPD-TNFSF fusion proteins (trimer), six SPD-TNFSF fusion proteins, or two trimers of the SPD-TNFSF fusion protein (hexamer), twelve SPD-TNFSF fusion proteins (dodecamer), eighteen SPD-TNFSF fusion proteins (octadecamer), or more than exactly eighteen SPD-TNFSF fusion proteins (higher-order oligomer).

[0157] The fusion protein can be a monomeric protein or a multimeric protein. Preferably, the fusion protein exists as a multimeric form consisting of three SPD-TNFSF fusion proteins self-assembled into trimeric SPD-TNFSF fusion proteins, which may be the same or different. Preferably, the multimeric form is a dodecameric form consisting of a collection of four identical trimeric forms.

[0158] Oncolytic virus The oncolytic virus of the present invention can be obtained from any member of the viruses currently identified as long as it is oncolytic by virtue of its property of selectively replicating and killing dividing cells as compared to non-dividing cells. It may be a naturally oncolytic virus, or may be engineered by modifying one or more viral genes so as to increase tumor selectivity and / or preferential replication in dividing cells, such as those involved in DNA replication, nucleic acid metabolism, host tropism, surface attachment, pathogenicity, lysis and spread (see, for example, Kirn et al., 2001, Nat. Med. 7: 781; Wong et al., 2010, Viruses 2: 78-106). It is also contemplated that one or more viral genes can be placed under the control of an event- or tissue-specific regulatory element (such as a promoter).

[0159] Exemplary oncolytic viruses include, but are not limited to, reovirus, Seneca Valley virus (SVV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), herpes simplex virus (HSV), mobirvirus, retrovirus, influenza virus, Sindbis virus, poxvirus, adenovirus, adeno-associated virus (AAV), measles virus, foamy virus, alphavirus, lentivirus, rhabdovirus, picornavirus, coxsackievirus, parvovirus, or chimeras thereof.

[0160] In one embodiment, the oncolytic virus of the present invention is obtained from reovirus. Representative examples include Reolysin (under development by Oncolytics Biotech; NCT01166542).

[0161] In one embodiment, the oncolytic virus of the present invention is obtained from Seneca Valley virus. Representative examples include NTX-010 (Rudin et al., 2011, Clin. Cancer. Res. 17(4): 888-95).

[0162] In one embodiment, the oncolytic virus of the present invention is obtained from vesicular stomatitis virus (VSV). Representative examples are described in the literature (e.g., Stojdl et al., 2000, Nat. Med. 6(7): 821-5; Stojdl et al., 2003, Cancer Cell 4(4): 263-75).

[0163] In one embodiment, the oncolytic virus of the present invention is obtained from Newcastle disease virus. Representative examples include, but are not limited to, 73-T PV701 and HDV-HUJ strains and those described in the literature (e.g., Phuangsab et al., 2001, Cancer Lett. 172(1): 27-36; Lorence et al., 2007, Curr. Cancer Drug Targets 7(2): 157-67; Freeman et al., 2006, Mol. Ther. 13(1): 221-8).

[0164] In one embodiment, the oncolytic virus of the present invention is obtained from a herpes virus. The Herpesviridae is a large family of DNA viruses composed of relatively large double-stranded linear DNA genomes encoding 100-200 genes encapsulated within an icosahedral capsid surrounded by a lipid bilayer, all having a common structure. Oncolytic herpes viruses can be derived from various types of HSV, with HSV1 and HSV2 being particularly preferred. The herpes virus may be genetically modified to restrict viral replication in tumors or to reduce cytotoxicity in non-dividing cells. For example, any viral gene involved in nucleic acid metabolism, such as the gene encoding thymidine kinase (Martuza et al., 1991, Science 252: 854-6), ribonucleotide reductase (RR) (Boviatsis et al., Gene Ther. 1: 323-31; Mineta et al., 1994, Cancer Res. 54: 3363-66), or uracil-N-glycosylase (Pyles et al., 1994, J. Virol. 68: 4963-72), can be inactivated. In another aspect, virus mutants lacking the function of genes encoding pathogenic factors such as the ICP34.5 gene are included (Chambers et al., 1995, Proc. Natl. Acad. Sci. USA 92: 1411-5). Representative examples of oncolytic herpes viruses include NV1020 (e.g., Geevarghese et al., 2010, Hum. Gene Ther. 21(9): 1119-28) and T-VEC (Andtbacka et al., 2013, J. Clin. Oncol. 31, abstract number LBA9008).

[0165] In one embodiment, the oncolytic virus of the present invention is obtained from a mobirvirus that can be obtained from the Paramyxoviridae family, and the measles virus is particularly preferred. Representative examples of oncolytic measles viruses include, but are not limited to, MV-Edm (McDonald et al., 2006; Breast Cancer Treat. 99(2): 177-84) and HMWMAA (Kaufmann et al., 2013, J. Invest. Dermatol. 133(4): 1034-42).

[0166] In one embodiment, the oncolytic virus of the present invention is obtained from an adenovirus. Methods for generating tumor-regressing adenoviruses are available in the art. In one embodiment, the cancer cell-lysing virus of the present invention is obtained from an adenovirus. Methods for designing cancer cell-lytic adenoviruses are available in the art. Advantageous strategies include replacement of the viral promoter with a tumor-selective promoter, or modification of the E1 adenovirus gene product to inactivate its binding function to p53 or retinoblastoma (Rb) proteins that are altered in tumor cells. In nature, the adenovirus E1B55 kDa gene cooperates with another adenovirus product to inactivate p53 (which is often dysregulated in cancer cells) and prevent apoptosis. Representative examples of oncolytic adenoviruses include ONYX-015 (e.g., Khuri et al., 2000, Nat. Med 6(8): 879-85), H101, also called Oncorine (Xia et al., 2004, Ai Zheng 23(12): 1666-70).

[0167] In one embodiment, the oncolytic virus of the present invention is obtained from an adeno-associated virus serotype including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, etc. or any other virus or serotype whose capsid protein sequence is substantially homologous to the AAV-2 capsid protein sequence.

[0168] In one embodiment, the oncolytic virus of the present invention is a poxvirus. As used herein, the term "poxvirus" refers to a virus belonging to the family Poxviridae, and poxviruses belonging to the Chordopoxvirus subfamily, more preferably the Orthopoxvirus genus, or chimeras thereof are particularly preferred. Genomic sequences of various poxviruses, such as vaccinia virus, cowpox virus, canarypox virus, and ectromelia virus genomes, are available in the art, and there are specialized databases such as Genbank (accession numbers NC_006998, NC_003663, NC_005309, NC_004105, respectively).

[0169] Advantageously, the oncolytic poxvirus is cowpox virus and can be derived from any cowpox strain such as CPXV_GER1980_EP4 (Genbank HQ420895), CPXV_GER2002_MKY (Genbank HQ420898), CPXV_GER1991_3 (Genbank DQ 437593), CPXV_FRA2001_A CY (Genbank HQ420894), CPXV_GR1990_2 (Genbank HQ420896), CPXV_UK2000_K2984 (Genbank HQ420900), CPXV_BR (Genbank AF482758.2 or NC 003663), and CPXV_NOR1994-MAN (Genbank HQ420899), CPXV_GER1998_2 (Genbank HQ420897), CPXV_GRI (Genbank X94355), CPXV_FIN2000_MAN (Genbank HQ420893), and CPXV_AUS1999_867 (Genbank HQ407377).

[0170] Desirably, the oncolytic poxvirus is an oncolytic vaccinia virus. Vaccinia virus is a member of the Poxviridae family, characterized by a 200 kb double-stranded DNA genome encoding a number of viral enzymes and factors enabling viral replication independently of host cell machinery. The majority of vaccinia virus particles are intracellular vaccinia virus particles with a single lipid envelope, and most are intracellular forms (IMV is intracellular mature virion) with a single lipid envelope, remaining in the cytoplasm of infected cells until lysis. As another infectious form, there are double-envelope particles (EEV: extracellular enveloped virion) that bud out from infected cells without lysing the infected cells.

[0171] It may be derived from any vaccinia virus strain, but the Elstree strain, Wyeth strain, Copenhagen strain, Lister strain, Tian Tian strain and Western Reserve strain are particularly preferred. The gene nomenclature used herein is that of the Copenhagen vaccinia strain. This nomenclature is used for homologous genes of other Poxviridae families as well, unless otherwise specified. However, the gene nomenclature may vary depending on the pox strain, but the correspondence between the Copenhagen strain and other vaccinia strains is generally available in the literature.

[0172] In one embodiment, the poxvirus belongs to the Chordopoxvirus subfamily, more preferably the Leporipoxvirus genus, preferably myxoma virus, rabbit fibroma virus or squirrel fibroma virus, more preferably myxoma virus (the genomic sequence is disclosed in Genbank under accession number NP_051868.1).

[0173] Preferably, the oncolytic virus of the present invention is modified by altering one or more viral genes. The modification preferably results in the synthesis (or lack of synthesis) of a defective protein that cannot ensure the activity of the protein produced by the unmodified gene under the conditions. The modification includes deletions, mutations and / or substitutions of one or more nucleotides (continuous or discontinuous) within the viral gene or its regulatory elements. The modification can be carried out in many ways known to those skilled in the art using conventional recombinant techniques. Exemplary modifications are disclosed in the literature, but those that modify viral genes involved in DNA metabolism, host pathogenicity, the IFN pathway (see, for example, Guse et al., 2011, Expert Opinion Biol. Ther. 11(5): 595-608) are particularly preferred.

[0174] More preferably, the oncolytic poxvirus of the present invention is modified by altering the thymidine kinase coding gene (locus J2R). The TK enzyme is involved in the synthesis of deoxyribonucleotides. Since nucleotide concentrations are generally low in normal cells, TK is required for viral replication in these cells, but is unnecessary in dividing cells containing high concentrations of nucleotides.

[0175] Alternatively, or in combination therewith, the oncolytic poxvirus of the invention is modified by altering at least one or both genes encoding ribonucleotide reductase (RR). In nature, this enzyme catalyzes the reduction of ribonucleotides to deoxyribonucleotides, which is an important step in DNA biosynthesis. The viral enzyme is similar to the mammalian enzyme, the subunit structure of which consists of two heterosubunits called R1 and R2 encoded by the I4L and F4L loci, respectively. The sequences of the I4L and F4L genes and their positions within the genomes of various poxviruses are available in public databases such as accession numbers DQ437594, DQ437593, DQ377804, AH015635, AY313847, AY313848, NC_003391, NC_003389, NC_003310, M-35027, AY243312, DQ011157, DQ011156, DQ011155, DQ011154, DQ011153, Y16780, X71982, AF438165, U60315, AF410153, AF380138, U86916, L22579, NC_006998, DQ121394 and NC_008291. In the context of the present invention, either or both of the I4L gene (encoding the R1 large subunit) or the F4L gene (encoding the R2 small subunit) can be inactivated.

[0176] Alternatively, or in combination therewith, the oncolytic poxvirus may be further modified at the M2L locus (preferably a modification that results in suppression of the expression of the viral m2 protein), thereby resulting in a modified poxvirus lacking the m2 function (m2-deficient poxvirus).

[0177] In one embodiment, the oncolytic poxvirus is further modified at the M2L locus and the J2R locus (modifications that preferably result in suppression of the expression of the viral tk protein), thereby resulting in an oncolytic poxvirus lacking both m2 and tk functions (m2-tk-poxvirus). In the context of the present invention, partial or complete deletion of the M2L locus and / or the J2R locus as well as insertion of foreign nucleic acids at the M2L locus and / or the J2R locus are contemplated for inactivating the m2 and tk functions.

[0178] Alternatively, or in combination therewith, the oncolytic poxvirus may be further modified at the M2L locus and the I4L and / or F4L loci (modifications that preferably result in suppression of the expression of the viral ribonucleotide reductase (rr) protein), thereby resulting in an oncolytic poxvirus lacking both m2 and rr functions (m2- and rr-deficient poxvirus). In the context of the present invention, the poxvirus can be modified, for example, by partial or complete deletion of the I4L gene (encoding the large subunit 1) or the F4L gene (encoding the small subunit r2) or both at the I4L locus and / or the F4L locus to obtain an rr-deficient poxvirus.

[0179] Also provided is an oncolytic poxvirus modified at the M2L locus, the J2R locus, and the I4L and / or F4L loci (M2L, J2R, and I4L loci; M2L, J2R, and F4L loci or M2L, J2R, I4L, and F4L loci), a modified triple-deficient virus), thereby resulting in an oncolytic poxvirus lacking m2, tk, and rr activities (m2-, tk-, rr-poxvirus).

[0180] Alternatively, or in combination therewith, other strategies can be pursued to further enhance the tumor specificity of the virus. Representative examples of suitable modifications include disrupting the VGF-encoding gene from the viral genome. VGF (representing VV growth factor) is a secreted protein that is expressed early after cell infection and whose function appears to be important for the spread of the virus in normal cells. Another example is disruption of the A56R gene encoding hemagglutinin, which is ultimately combined with tk deletion (Zhang et al., 2007, Cancer Res. 67: 10038-46). Disruption of interferon regulatory genes (e.g., the B8R or B18R genes) or the caspase-1 inhibitor B13R gene is also advantageous. Another suitable modification involves disruption of the F2L gene encoding the viral dUTPase, which is involved in both maintaining the fidelity of DNA replication and providing precursors for the production of TMP by thymidylate synthase (Broyles et al., 1993, Virol. 195: 863-5). The sequence of the vaccinia virus F2L gene is available in GenBank under accession number M25392.

[0181] In a preferred embodiment, the oncolytic virus of the present invention is a vaccinia virus lacking TK activity due to an inactivating mutation in the J2R gene. In another preferred embodiment, the oncolytic virus of the present invention is a vaccinia virus lacking both TK and RR activities due to inactivating mutations in both the J2R gene retained in the viral genome and the I4L and / or F4L genes (for example, as described in International Publication No. WO 2009 / 065546 and Foloppe et al., 2008, Gene Ther., 15: 1361-71). In another preferred embodiment, the oncolytic virus of the present invention is a vaccinia virus lacking TK, RR, and m2 activities, which results in inactivating mutations in both the J2R gene, the I4L and / or F4L genes, and the M2L gene. In another preferred embodiment, the oncolytic virus of the present invention is a dUTPase-deficient vaccinia virus due to an inactivating mutation in the F2L gene (for example, as described in International Publication No. WO 2009 / 065547), and finally, it is combined with the disruption of at least one or both of the TK and RR activities (resulting in viruses having inactivating mutations in F2L; F2L and the J2R gene; F2L and I4L; or F2L, J2R, and I4L).

[0182] Non-oncolytic virus In one embodiment, the non-oncolytic virus of the present invention is a poxvirus. Exemplary non-oncolytic poxvirus includes, but is not limited to, pseudocowpox virus (PCPV), modified vaccinia virus Ankara strain (MVA), highly attenuated vaccinia virus strain (NYVAC), swinepox virus (SWPV), fowlpox virus (FPV), or chimeras thereof.

[0183] In one embodiment, the non-oncolytic poxvirus of the invention is obtained from vaccinia virus Ankara (PCPV). Representative examples of PCPV strains suitable for use herein include, but are not limited to, YG2828 (GenBank accession number LC230119), F07.801 R (GenBank accession number JF773693), F10.3081 C (GenBank accession number JF773695), F07.798R (GenBank accession number JF773692), F99.177C (GenBank accession number AY453678), IT1303 / 05 (GenBank accession number JF800906), F00.120R (GenBank accession number GQ329669; Tikkanen et al., 2004, J. Gen. Virol. 85: 1413-8) and TJS (also called VR634; GenBank accession number GQ329670; Friedman-Kien et al., 1963, Science 140: 1335-6; available from the ATCC under accession number VR634). Such strains may have morphological, structural and / or genetic differences from each other, e.g., differences in ITR length, number of putative genes and / or GC-rich content (see, e.g., Hautaniemi et al., 2010, J. Gen. Virol. 91: 1560-76). In a preferred embodiment, the PCPV virus of the invention is obtained from the wild-type TJS identified by the ATCC reference number ATCC VR-634 (trademark) or a virus strain of the same or similar name, as well as functional fragments and variants thereof.

[0184] In one embodiment, the non-oncolytic poxvirus of the invention is obtained from the highly attenuated vaccinia virus strain NYVAC (Tartaglia et al., 1992, Virol. 188(l):217-32, US5,494,807). By way of example, NYVAC is a strain of highly attenuated vaccinia virus derived from a plaque-cloned isolate of the Copenhagen vaccine strain by precisely deleting 18 open reading frames (ORFs) from the viral genome.

[0185] In one embodiment, the non-oncolytic poxvirus of the invention is obtained from modified vaccinia virus Ankara by an attenuated phenotype (Mayr et al., 1975, Infection 3: 6-14; Sutter and Moss, 1992, Proc. Natl. Acad. Sci. USA 89: 10847-51). The nucleotide sequence of the MVA genome and the amino acid sequences of the encoded viral proteins are available in the art, for example, from Antoine et al. (1998, Virol, 244 : 365-96) and GenBank (accession number U94848).

[0186] In one embodiment, the non-oncolytic poxvirus of the invention is obtained from swinepox virus (SWPV). The nucleotide sequence and amino acid sequences of the SWPV genome are available in the art, for example, from Alfonso et al. (2002, Virol, 76(2):783-90) and GenBank accession numbers (NC_003389.1 and MW036632).

[0187] In one embodiment, the non-oncolytic poxvirus of the invention is obtained from fowlpox virus (FPV). The nucleotide sequence of the fowlpox virus genome is available in the art, for example, from Alfonso et al. (2000, Virol., 74(8): 3815-383) and GenBank accession number (AF198100).

[0188] Expression of one or more nucleic acid molecules encoding an SPD-TNFSF fusion protein inserted into the viral genome The nucleic acid molecule encoding SPD-TNFSF can be readily obtained by standard molecular biology techniques (e.g., PCR amplification, cDNA cloning, chemical synthesis) using sequence data available in the art and the information provided herein. Analogs and fragments can be made using standard techniques of molecular biology.

[0189] In one embodiment, the nucleic acid molecule encoding SPD-TNFSF may be independently inserted at any position of the viral genome, but non-essential loci are particularly preferred. Insertion into the virus can be carried out by conventional molecular biology, for example, as described in Sambrook et al. (2001, Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory). Insertion into an adenovirus vector or a poxvirus vector can be carried out by homologous recombination as described in Chartier et al. (1996, J. Virol. 70: 4805-10) and Paul et al. (2002, Cancer gene Ther. 9: 470-7), respectively. For example, the TK, RR and F2L genes and the intergenic regions are particularly suitable for insertion into oncolytic vaccinia virus, and the E3 and E4 regions are suitable for insertion into tumor regression adenovirus.

[0190] Furthermore, the coding nucleotide sequence can be optimized to provide high-level expression in a particular host cell or subject. It has actually been observed that the codon usage patterns of organisms are highly non-random and can vary significantly between different hosts. For example, therapeutic genes are of bacterial or lower eukaryotic origin (e.g., suicide genes) and may have codon usage patterns that are inappropriate for efficient expression in higher eukaryotic cells (e.g., human). In general, codon optimization is performed by replacing one or more "natural" (e.g., bacterial or yeast) codons corresponding to codons with low usage frequency in the host organism of interest with one or more codons encoding the same amino acid with high usage frequency. It is not necessary to replace all natural codons corresponding to codons with low usage frequency.

[0191] In addition to codon usage optimization, expression in a host cell or subject can be further improved by additional modification of the nucleotide sequence. For example, various modifications can be envisioned to prevent the presence of regions where clusters of rare non-optimal codons are concentrated and / or to suppress or modify "negative" sequence elements that are expected to have an adverse effect on expression levels. Such negative sequence elements include, but are not limited to, regions with a very high GC content (>80%) or a very low GC content (<30%); AT-rich or GC-rich sequence stretches; unstable direct or inverted repeats; RNA secondary structures; and / or internal potential regulatory elements such as internal TATA boxes, chi sites, ribosome entry sites, and / or splicing donor / acceptor sites.

[0192] In the present invention, each of the one or more nucleic acid molecules encoding the above-described SPD-TNFSF fusion protein inserted into the genome of the virus of the present invention is operably linked to regulatory elements appropriate for its expression in a host cell or subject. As used herein, the term "regulatory element" or "regulatory sequence" refers to any element that enables, contributes to, or regulates the expression of a coding nucleic acid molecule in a given host cell or subject, including replication, amplification, transcription, splicing, translation, stability, and / or transport of the nucleic acid or its derivative (i.e., mRNA). As used herein, "operably linked" means that the linked elements are arranged so as to function in concert for the intended purpose. For example, a promoter is operably linked to a nucleic acid molecule if it affects transcription of the nucleic acid molecule from initiation to termination in a permissive host cell.

[0193] Those skilled in the art will understand that the selection of regulatory sequences can depend on factors such as the nucleic acid molecule itself, the virus into which it is inserted, the host cell or subject, and the desired expression level. Promoters are particularly important. In the context of the present invention, it can be constitutive, which directs the expression of a nucleic acid molecule in many types of host cells, or specific to a particular host cell (e.g., liver-specific regulatory sequences), or regulated in response to a particular event or exogenous factor (e.g., temperature, nutrient additives, hormones, etc.), or dependent on the phase of the viral cycle (e.g., late or early). Also, in order to optimize virus production and avoid potential toxicity of the expressed polypeptide, a promoter that is suppressed during the production step in response to a particular event or exogenous factor can also be used.

[0194] Suitable promoters for constitutive expression in mammalian cells include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter (U.S. Patent No. 5,168,062), the RSV promoter, the adenovirus major late promoter, the phosphoglycerate kinase (PGK) promoter (Adra et al., 1987, Gene 60: 65-74), the thymidine kinase (TK) promoter of herpes simplex virus (HSV)-1, and the T7 polymerase promoter (International Publication No. 98 / 10088). The vaccinia virus promoter is particularly adapted for expression in poxviruses. Representative examples include, but are not limited to, vaccinia p7.5K, pH5R, p11K7.5 (Erbs et al., 2008, Cancer Gene Ther. 15(1): 18-28), pSE, pTK, p28, p11, pB2R, pF17R, pA14L, pSE / L, pA35R, and pK1L promoters, as well as synthetic promoters such as those described by Chakrabarti et al. (1997, Biotechniques 23: 1094-7; Hammond et al, 1997, J. Virol Methods 66: 135-8; and Kumar and Boyle, 1990, Virology 179: 151-8), and early / late chimeric promoters. Suitable promoters for oncolytic measles virus include, but are not limited to, any promoter that directs the expression of the measles transcription unit (Brandler and Tangy, 2008, CIMID 31: 271). Suitable promoters for expression can be tested in vitro (e.g., in a suitable cultured cell line) or in vivo (e.g., in a suitable animal model or subject).

[0195] Those skilled in the art will understand that the regulatory elements that control the expression of the nucleic acid molecule inserted into the viral genome may further comprise additional elements for proper initiation, regulation and / or termination of transcription (e.g., polyA transcription termination sequences), additional elements for mRNA transport (e.g., nuclear localization signal sequences), additional elements for processing (e.g., splicing signals), as well as additional elements for stability (e.g., introns and non-coding 5' and 3' sequences), and additional elements for translation (e.g., initiator Met, tricistronic leader sequences, IRE ribosome binding sites, signal peptides, etc.).

[0196] Where applicable, it may be advantageous to include additional regulatory elements to promote the expression, transport and biological activity of at least one of the genes (i.e., SPD-TNFSF fusion protein) inserted into the viral genome of the virus of the present invention.

[0197] An approach that can be carried out in the context of the present invention is to bind the gene product encoded by the virus of the present invention to an exogenous agent such as a cytotoxic agent and / or a labeling agent. As used herein, the term "cytotoxic agent" refers to a compound that is directly toxic to cells, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof), and that inhibits its reproduction or growth. As used herein, the term "labeling agent" refers to a detectable compound. The labeling agent may be detectable by itself (e.g., a radioisotope label or a fluorescent label), or in the case of an enzyme label, it may catalyze the chemical modification of a detectable substrate compound. The binding can be effected by a gene fusion between the gene product (SPD-TNFSF) and the exogenous agent.

[0198] In a preferred embodiment, the oncolytic virus of the present invention is a vaccinia virus (preferably derived from the Copenhagen strain) lacking both TK and RR activities (e.g., due to inactivating mutations in both the J2R and I4L genes of the virus) into which a nucleic acid molecule encoding an SPD-TNFSF fusion protein has been inserted into the genome. Desirably, these elements are each placed under the transcriptional control of the pH5R promoter. Preferably, the nucleic acid molecule encoding the SPD-TNFSF fusion protein is inserted into the J2R (TK) locus of the viral genome.

[0199] In another preferred embodiment, the oncolytic virus of the present invention is a vaccinia virus (preferably derived from the Copenhagen strain) lacking TK, RR, and M2L activities (e.g., due to inactivating mutations in the J2R, I4L, and M2L genes of the virus) into which a nucleic acid molecule encoding an SPD-TNFSF fusion protein has been inserted into the genome. Desirably, these elements are each placed under the transcriptional control of the pH5R promoter. Preferably, the nucleic acid molecule encoding the SPD-TNFSF fusion protein is inserted into the J2R (TK) locus of the viral genome.

[0200] Alternatively, the oncolytic virus of the present invention is a vaccinia virus (preferably derived from the Wyeth strain) lacking TK activity (due to an inactivating mutation in the J2R gene of the virus) into which a nucleic acid molecule encoding an SPD-TNFSF fusion protein has been inserted into the genome.

[0201] Additional therapeutic polypeptide / gene The virus according to the present invention may encode only the SPD-TNFSF fusion protein of the present invention (as defined above), or may further encode another nucleic acid molecule encoding a polypeptide of interest.

[0202] The another nucleic acid molecule encoding the polypeptide of interest as described above is preferably a foreign nucleic acid (also referred to as a recombinant gene, transgene, or nucleic acid).

[0203] In the context of the present invention, the "foreign nucleic acid" inserted into the viral genome is not found in the naturally occurring viral genome or is not expressed thereby. However, the foreign nucleic acid may be homologous or heterologous to the subject into which the recombinant virus is introduced. More specifically, it may or may not be of human origin (e.g., bacteria, yeast or virus origin excluding poxviruses). Advantageously, the recombinant nucleic acid is a nucleic acid sequence that can at least partially (by hybridization) bind to complementary cellular nucleic acids (e.g., DNA, RNA, miRNA) present in diseased cells for the purpose of encoding a polypeptide or inhibiting a gene involved in the disease. Such a recombinant nucleic acid may be a natural gene or a part thereof (e.g., cDNA), or any variant thereof obtained by mutation, deletion, substitution and / or addition of one or more nucleotides.

[0204] In an advantageous embodiment, the polypeptide of interest is a therapeutic polypeptide. Thus, in this advantageous embodiment, the virus according to the invention (as defined above) further comprises another nucleic acid molecule inserted into its genome that encodes a therapeutic polypeptide.

[0205] "Therapeutic polypeptide" means a polypeptide for therapeutic or prophylactic purposes that, when appropriately administered to a subject, has a beneficial effect on the course or symptoms of the condition to be treated or prevented.

[0206] The therapeutic polypeptide is preferably selected from the group consisting of immunomodulatory polypeptides (preferably immunostimulatory polypeptides), antigenic polypeptides, suicide gene products, antibodies, functional derivatives of antibodies, functional fragments of antibodies, and any combination thereof.

[0207] In a preferred embodiment, the therapeutic polypeptide is an immunostimulatory polypeptide, preferably a cytokine such as interleukin, chemokine, interferon, tumor necrosis factor, colony stimulating factor; an APC-exposing protein; an agonist of a stimulatory immune checkpoint; an antagonist of an inhibitory stimulatory checkpoint; and any combination thereof.

[0208] Immunomodulatory polypeptide The term "immunomodulatory polypeptide" refers to a polypeptide that targets components of signaling pathways that can be directly or indirectly involved in the regulation of the immune response. "Regulation" of the immune response means any alteration in the activity of cells of the immune system or such cells (e.g., T cells). Such regulation can include stimulation or inhibition of the immune system manifested by increases or decreases in the number of various cell types, increases or decreases in the activity of these cells, or other changes that can occur within the immune system. Preferably, such a polypeptide can at least partially downregulate an inhibitory pathway (antagonist), and / or at least partially upregulate a stimulatory pathway (agonist); such pathways are, in particular, immune pathways that exist between antigen-presenting cells (APCs) or cancer cells and effector T cells.

[0209] The immunomodulatory polypeptides that can be expressed by the vectors according to the invention can act at every step of T cell-mediated immunity, including clonal selection of antigen-specific cells, activation of T cells, proliferation, transport to antigen and inflammation sites, execution of direct effector functions, and signaling via cytokines and membrane ligands. Each of these steps is regulated by the interplay of stimulatory and inhibitory signals that finely tune the response.

[0210] Suitable immunomodulatory polypeptides and methods of using them are described in the literature. Exemplary immunomodulatory polypeptides include, but are not limited to, the following: · Cytokines such as interleukin, chemokine, interferon, tumor necrosis factor, colony stimulating factor; · APC-exposed protein; · Agonist of stimulatory immune checkpoint; · Antagonist of inhibitory immune checkpoint different from PD-L1; and · Any combination thereof.

[0211] In one embodiment, the immunomodulatory polypeptide expressed by the vector according to the present invention is a cytokine, preferably selected from the group consisting of: · Interleukin (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-36), IFNα, IFNγ and granulocyte macrophage colony stimulating factor (GM-CSF); · Chemokine (e.g., ΜIPIα, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19 and CCL21), · Interferon (e.g., IFNα, IFNγ), · Tumor necrosis factor (e.g., TNFα), and · Colony stimulating factor (e.g., granulocyte macrophage colony stimulating factor (GM-CSF)).

[0212] When the immunostimulatory polypeptide is a cytokine, it is preferably an interleukin or a colony stimulating factor, and GM-CSF is particularly preferred.

[0213] In another embodiment, the immunomodulatory polypeptide expressed by the vector according to the present invention is an agonist of a stimulatory immune checkpoint or an antagonist of an inhibitory immune checkpoint.

[0214] "Immune checkpoint" refers to proteins that, under normal physiological conditions, prevent uncontrolled immune responses and thus are directly or indirectly involved in immune pathways important for maintaining self-tolerance and / or tissue protection. Immune checkpoints can be classified into two categories: stimulatory immune checkpoints and inhibitory immune checkpoints respectively. "Stimulatory immune checkpoint" refers to an immune checkpoint involved in upregulation of the immune response, and "inhibitory immune checkpoint" refers to an immune checkpoint involved in downregulation of the immune response.

[0215] Stimulatory immune checkpoints include CD28, ICOS, CD137 (4-1BB), OX40, CD70, CD40, and GITR. Agonists of stimulatory immune checkpoints are preferably selected from agonist antibodies against human ICOS ligand (ICOSL), 4-1BB ligand (4-1BBL), OX40 ligand (OX40L), CD70, CD40 ligand (CD40L), GITR ligand (GITRL), and human ICOS (for example, International Publication No. WO2018 / 187613), CD137 (4-1BB) (for example, International Publication No. WO2005 / 035584), OX40 (for example, US Patent No. 7,291,331 and International Publication No. WO03 / 106498), CD70 (for example, International Publication No. WO2012 / 004367), CD40 (for example, International Publication No. WO2017 / 184619), or GITR (for example, International Publication No. WO2017 / 068186). Some agonists of stimulatory immune checkpoints are TNFSF members. When an agonist of such a stimulatory immune checkpoint is further encoded by the vector according to the present invention, it is preferably different from the TNFSF member or its receptor-binding domain of the fusion protein according to the present invention.

[0216] Inhibitory immune checkpoints include PD-1, SIRPa, CD47, PD-L2, LAG3, Tim3, BTLA, and CTLA4. Antagonists of inhibitory immune checkpoints are preferably human ·PD-1 (e.g., those described in WO 2004 / 004771; WO 2004 / 056875; WO 2006 / 121168; WO 2008 / 156712; WO 2009 / 014708; WO 2009 / 114335; WO 2013 / 043569; and WO 2014 / 047350, particularly nivolumab, pembrolizumab, and semaprimab), ·SIRPa (e.g., WO 2019 / 023347), ·CD47 (e.g., WO 2020 / 019135), ·PD-L2 (e.g., WO 2019 / 158645), ·LAG3 (e.g., WO 2018 / 071500), ·Tim3 (e.g., WO 2020 / 093023), ·BTLA (e.g., WO 2010 / 106051), and ·CTLA4 (e.g., those described in, inter alia, U.S. Patent No. 8,491,895, WO 2000 / 037504, WO 2007 / 113648, WO 2012 / 122444, and WO 2016 / 196237, particularly ipilimumab sold by Bristol Myer Squibb under the trade name Yervoy® (see, e.g., U.S. Patent No. 6,984,720; U.S. Patent No. 8,017,114), MK-1308 (Merck), AGEN-1884 (Agenus Inc.; WO 2016 / 196237), and tremelimumab (AstraZeneca; see U.S. Patent No. 7,109,003 and U.S. Patent No. 8,143,379) and single-chain anti-CTLA4 antibodies (see, e.g., WO 97 / 20574 and WO 2007 / 123737) selected from antagonist antibodies of

[0217] Antigenic polypeptide The term "antigenicity" refers to the ability to induce or stimulate a measurable immune response in a subject into which the virus of the invention encoding the polypeptide being antigenic (as described herein) has been introduced. The immune response stimulated or induced against the antigenic polypeptide expressed by the virus can be humoral and / or cellular (e.g., production of antibodies, cytokines and / or chemokines involved in the activation of effector immune cells). The stimulated or induced immune response usually contributes to a protective effect in the administered subject. An enormous variety of direct or indirect biological assays are available in the art for evaluating the antigenicity of a polypeptide in vivo (in animal or human subjects) or in vitro (e.g., in biological samples). For example, the ability of a particular antigen to stimulate innate immunity can be examined by measuring NK / NKT cells (e.g., their representation and activation levels), as well as IFN-related cytokine and / or chemokine production cascades, activation of TLR (Toll-like receptor), and other markers of innate immunity (Scott-Algara et al., 2010 PLOS One 5(1), e8761; Zhou et al., 2006, Blood 107, 2461-2469; Chan, 2008, Eur. J. Immunol. 38, 2964-2968). The ability of a particular antigen to stimulate a cell-mediated immune response can be determined, as described herein, by, for example, conventional bioassays (e.g., ELISpot, multi-parameter flow cytometry, ICS (intracellular cytokine staining), multiplex technology or cytokine profile analysis using ELISA) for the quantification of cytokines produced by activated T cells, including those derived from CD4+ T cells and CD8+ T cells, determination of the proliferative capacity of T cells (e.g., T cell proliferation assay by [3H] thymidine incorporation assay), assay of the cytotoxic ability of antigen-specific T lymphocytes in sensitized subjects, or identification of lymphocyte subpopulations by flow cytometry, and by immunization of appropriate animal models.

[0218] The term "antigenic polypeptide" is intended to encompass not only natural antigens, but also fragments thereof (e.g., epitopes, immunogenic domains, etc.) and variants thereof, as long as such fragments or variants can be targets of an immune response. Preferred antigenic polypeptides for use herein are tumor-associated antigens. Selecting one or more antigenic polypeptides appropriate for the treatment of a particular condition is within the scope of those skilled in the art.

[0219] In one embodiment, the antigenic polypeptide encoded by the recombinant modified virus is a cancer antigen (also referred to as a tumor-associated antigen or TAA) that is associated with cancer and / or functions as a marker for cancer. Cancer antigens include, for example, those that are normally silent (i.e., not expressed) in healthy cells, those that are expressed at low levels or only at specific stages of differentiation, those that are transiently expressed such as embryonic and fetal antigens, and those that are due to mutations in cellular genes such as oncogenes (e.g., the activated ras oncogene), proto-oncogenes (e.g., the ErbB family), or proteins resulting from chromosomal translocations, and encompass various categories of polypeptides.

[0220] Many tumor-associated antigens are known in the art. Exemplary tumor antigens include, but are not limited to, colorectal-associated antigen (CRC), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), BAGE, GAGE or MAGE antigen families, p53, mucin antigens (e.g., MUC1), HER2 / neu, p21ras, hTERT, Hsp70, iNOS, tyrosine kinase, mesothelin, c-erbB-2, alpha-fetoprotein, AM-1, and any immunogenic epitopes or variants thereof.

[0221] Tumor-associated antigens may also include neoepitopes / antigens that arise during the carcinogenic process in cancer cells and contain one or more mutations in amino acid residues relative to the corresponding wild-type antigen. Generally, this antigen is found in cancer cells or tissues obtained from a patient, but not in samples of normal cells or tissues obtained from the patient or a healthy individual.

[0222] Tumor-associated antigens can also include antigens encoded by pathogenic organisms that can induce pathological conditions in a subject (particularly, a subject with chronic infection), such as RNA and DNA tumor viruses (e.g., human papillomavirus (HPV), hepatitis C virus (HCV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), etc.) and bacteria (e.g., Helicobacter pylori).

[0223] In another embodiment, the antigenic polypeptide encoded by the virus of the present invention is a vaccine antigen intended to provide therapeutic or prophylactic protection against infectious diseases when delivered to a human or animal subject. A number of vaccine antigens are known in the art. Exemplary vaccine antigens include, but are not limited to, cellular antigens, viral antigens, bacterial antigens, or parasitic antigens. An example of a cellular antigen is the mucin 1 (MUC1) glycoprotein. Examples of viral antigens include, for example, hepatitis A, B, C, D, and E viruses, immunodeficiency viruses (e.g., HIV), herpes viruses, cytomegalovirus, varicella-zoster virus, papillomavirus, Epstein-Barr virus, influenza virus, parainfluenza virus, coxsackievirus, picornavirus, rotavirus, respiratory syncytial virus, rhinovirus, rubella virus, papovavirus, mumps virus, measles virus, and antigens derived from rabies virus. Some non-limiting examples of HIV antigens include gp120, gp40, gp160, p24, gag, pol, env, vif, vpr, vpu, tat, rev, nef tat, nef. Some non-limiting examples of human herpes virus antigens include gH, gL, gM, gB, gC, gK, gE or gD, or immediate-early proteins such as ICP27, ICP47, ICP4, ICP36 derived from HSV1 or HSV2. Some non-limiting examples of cytomegalovirus antigens include gB. Some non-limiting examples of Epstein-Barr virus (EBV) include gp350. Some non-limiting examples of varicella-zoster virus antigens include gp1, 11, 111, and IE63. Some non-limiting examples of hepatitis C virus antigens include the env E1 or E2 protein, core protein, NS2, NS3, NS4a, NS4b, NS5a, NS5b, p7. Some non-limiting examples of human papillomavirus (HPV) antigens include L1, L2, E1, E2, E3, E4, E5, E6, E7.Antigens derived from other viral pathogens such as respiratory syncytial virus (e.g., F and G proteins), parainfluenza virus, measles virus, mumps virus, flavivirus (e.g., yellow fever virus, dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus) and influenza virus cells (e.g., HA, NP, NA, or M proteins) can also be used in the present invention. Examples of bacterial antigens include antigens derived from, for example, TB, leprosy, mycobacteria causing pneumonia, aerobic gram-negative bacilli, mycoplasma, staphylococcus, streptococcus, salmonella, chlamydia, neisseria, etc. Examples of parasitic antigenic polypeptides include antigens such as malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis, and filariasis.

[0224] Antibody and its antigen-binding fragment or derivative Any antibody or antigen-binding fragment or derivative thereof having therapeutic activity, particularly an antibody or antigen-binding fragment or derivative that affects the regulation of cell surface receptors, such as anti-HER2 antibodies (e.g., trastuzumab), anti-EGFR antibodies (e.g., cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab), anti-VEGF antibodies (e.g., bevacizumab and ranibizumab) or antigen-binding fragments or derivatives thereof, may be further encoded by the virus of the present invention.

[0225] In the context of the present invention, "antibody" ("Ab") is used in the broadest sense and is preferably as defined in the "General Definition" section above.

[0226] The antibody is preferably a monoclonal antibody, preferably a humanized or chimeric antibody.

[0227] Representative examples of antigen-binding fragments are known in the art and include Fab, Fab’, F(ab’)2, dAb, Fd, Fv, scFv, ds-scFv and diabody. A particularly useful antibody fragment is a single-chain antibody (scFv) comprising the two domains VL and VH of the Fv fragment, which is ultimately fused with a linker to form a single protein chain.

[0228] Production of SPD-TNFSF fusion protein In one embodiment, the virus can also be used in the context of the present invention to produce by recombinant means one or more SPD-TNFSF fusion proteins that it encodes. It can advantageously comprise one or more additional elements that allow for the maintenance, propagation or expression of a nucleic acid molecule encoding an SPD-TNFSF fusion protein in a host cell. Such additional elements can comprise a marker gene (e.g., by cell auxotrophic complementation or antibiotic resistance) to facilitate the identification and isolation of the production host cell. Suitable marker genes include, but are not limited to, dihydrofolate reductase (dhfr) that confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77: 3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt that confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072); neo that confers resistance to aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1); zeo that confers resistance to zeomycin, kana that confers resistance to kanamycin; hygro that confers resistance to hygromycin (Santerre et al., 1984, Gene 30: 147). Recombinant viruses lacking functional TK (e.g., due to the insertion of a nucleic acid molecule encoding an SPD-TNFSF protein into the J2R(TK) locus) can be selected in a medium containing bromodeoxyuridine (BrdU). In fact, TK-viruses are insensitive to the BrdU agent, while in TK+ viruses the agent inhibits DNA synthesis. It is also possible to rely on reporter luminescence or colorimetric systems based, for example, on GFP (green fluorescent protein), luciferase and β-galactosidase.

[0229] Methods for the recombinant production of SPD-TNFSF fusion proteins are conventional in the art. Generally, such methods comprise (a) introducing the virus described herein into a suitable production cell to create a transfected or infected production cell, (b) culturing the transfected or infected production cell in vitro under conditions suitable for its growth, (c) recovering one or more SPD-TNFSF fusion proteins from the cell culture, and (d) optionally, purifying the recovered SPD-TNFSF fusion protein. In the context of the present invention, the production cell is preferably a human or non-human eukaryotic cell. Preferred production cells include, but are not limited to, hamster cell lines such as BHK-21 (ATCC CCL-10), CV-1 (African green monkey kidney cell line), COS (e.g., COS-7) cells, Chinese hamster ovary (CHO) cells, mouse NIH / 3T3 cells, mouse NSO myeloma cells, human cell lines such as HeLa (ATCC-CRM-CCL-2™ or ATCC-CCL-2.2™), Vero cells, HEK293 cells (Graham et al., 1997, J. Gen. Virol. 36: 59-72), HER96 and PERC.6 cells (Fallaux et al., 1998, Human Gene Ther. 9: 1909-17), avian cells (e.g., chicken, duck cells described herein and in International Publication Nos. WO 2005 / 042728, WO 2006 / 108846, WO 2008 / 129058, WO 2010 / 130756, WO 2012 / 001075, etc.), and primary chicken embryo fibroblast (CEF) prepared from chicken embryos obtained from fertilized eggs.

[0230] The production cells can be cultured in ordinary fermentation bioreactors, flasks, and Petri plates. The culture can be carried out at a temperature, pH, and oxygen content suitable for the given host cells. Here, various methods known for producing proteins in eukaryotic cells will not be described in detail. The production of the SPD-TNFSF fusion protein can be secreted intracellularly or preferably extracellularly (e.g., in the culture medium) of the production cells.

[0231] Before use according to the present invention, the virus can be at least partially isolated. Various purification steps can be envisaged, including clarification, enzymatic treatment (e.g., benzonase, protease), chromatography, and filtration steps. Suitable methods are described in the art (e.g., WO 2007 / 147528; WO 2008 / 138533, WO 2009 / 100521, WO 2010 / 130753, WO 2013 / 022764).

[0232] Next, the SPD-TNFSF fusion protein can be purified by well-known purification methods. The conditions and techniques used to purify a particular protein depend on factors such as expression conditions, net charge, molecular weight, hydrophobicity, hydrophilicity, etc., and are apparent to those skilled in the art. Further, the level of purification varies depending on the intended use. If necessary, especially when the SPD-TNFSF fusion protein is not secreted extracellularly from the producing cells or is not fully secreted, it can be recovered by standard lysis procedures including freeze-thaw, sonication, mechanical disruption, use of lysing agents, etc. If secreted, it can be recovered directly from the culture medium. Various purification steps can be envisioned, including but not limited to clarification (e.g., ammonium sulfate precipitation, acid extraction), enzymatic treatment (e.g., benzonase, protease), chromatography (e.g., reverse phase, size exclusion, ion exchange, affinity, phosphocellulose, hydrophobic interaction or hydroxyapatite chromatography, etc.) and filtration steps. Suitable methods are described in the art (e.g., WO 2007 / 147528; WO 2008 / 138533, WO 2009 / 100521, WO 2010 / 130753, WO 2013 / 022764). Desirably, the SPD-TNFSF fusion protein recombinantly produced from the virus of the present invention is at least partially purified in the sense that it is substantially free of other cellular materials. Further, the SPD-TNFSF fusion protein can be formulated according to conditions conventionally used in the art (e.g., WO 2009 / 073569).

[0233] Therapeutic use The present invention also provides a composition comprising a therapeutically effective amount of the SPD-TNFSF fusion protein, the virus of the present invention, optionally together with a pharmaceutically acceptable vehicle. Such a composition can be administered one or more times, by the same or different routes.

[0234] "Therapeutically effective amount" corresponds to an amount of the SPD-TNFSF fusion protein or virus sufficient to bring about one or more beneficial results. Such a therapeutically effective amount can vary as a function of various parameters, in particular, the mode of administration; the disease state; the age and weight of the subject; the ability of the subject to respond to treatment; the type of concomitant treatment; the frequency of treatment; and / or the need for prophylaxis or treatment. In the case of prophylactic use, the SPD-TNFSF fusion protein, virus or composition of the invention is administered in a dosage sufficient to prevent and / or delay the onset and / or establishment and / or recurrence of a pathological condition (e.g., a proliferative disease such as cancer) in a subject at particular risk. In the case of "therapeutic" use, the SPD-TNFSF fusion protein, virus or composition of the invention is administered to a subject diagnosed with a pathological condition (e.g., a proliferative disease such as cancer or a disorder associated with TNF cytokine dysfunction) for the purpose of treating the disease, ultimately in combination with one or more conventional therapies. In particular, a therapeutically effective amount is one that results in an observable improvement in the clinical condition, e.g., stabilization of the disease state (i.e., does not worsen), delay or slowing of disease progression or severity, improvement or alleviation of the disease state, extension of survival, better response to standard treatment, improvement in QOL, reduction in mortality, reduction in the number of tumors, reduction in tumor size, reduction in the number or extent of metastases, extension of remission period, etc., compared to the baseline state or the state expected if the subject had not received treatment. A therapeutically effective amount may also be an amount necessary to elicit the expression of an effective non-specific (natural) and / or specific immune response such as a tumor immune response. In general, the expression of an immune response, particularly a T cell response, can be evaluated using in vitro, appropriate animal models, or biological samples taken from the subject. For example, tumor monitoring can be performed using techniques commonly used in the laboratory (e.g., flow cytometry, histology). Also, various available antibodies can be used to identify various immune cell populations involved in the anti-tumor response present in a treated subject, such as cytotoxic T cells, activated cytotoxic T cells, natural killer cells and activated natural killer cells.Improvement of the clinical condition can be readily evaluated by relevant clinical measurements routinely used by physicians or other skilled medical practitioners.

[0235] The term "pharmaceutically acceptable vehicle" is intended to include any carrier, solvent, diluent, excipient, adjuvant, dispersion medium, coating agent, antibacterial and antifungal agents, absorbent, etc. that are compatible with administration to mammalian, particularly human subjects.

[0236] The SPD-TNFSF fusion protein, virus or its composition can be placed in a solvent or diluent suitable for human or animal use. The solvent or diluent is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength. Representative examples include sterile water, physiological saline (e.g., sodium chloride), Ringer's solution, glucose, trehalose or sucrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions (see, for example, Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins, latest edition).

[0237] In one embodiment, the SPD-TNFSF fusion protein, virus or its composition is appropriately buffered for human use. Suitable buffers include, but are not limited to, phosphate buffers (e.g., PBS), bicarbonate buffers and / or Tris buffers that can maintain a physiological pH or a slightly basic pH (e.g., about pH 7 to about pH 9).

[0238] The SPD-TNFSF fusion protein, virus or its composition may also contain other pharmaceutically acceptable excipients to provide desirable pharmaceutical or pharmacodynamic properties, including, for example, osmotic pressure, viscosity, clarity, color, sterility, stability, dissolution rate of the formulation, modification or maintenance of release or absorption in human or animal subjects, facilitation of transport across the blood-brain barrier or penetration in specific organs.

[0239] In one embodiment, the viral composition may also include, at the time of administration, one or more adjuvants that can stimulate immunity (particularly, T cell-mediated immunity) via toll-like receptors (TLRs) such as TLR-7, TLR-8, and TLR-9, or promote infection of tumor cells. Non-limiting examples include alum, mineral oil emulsions such as Freund's complete and incomplete adjuvants (IFA), lipopolysaccharide or its derivatives (Ribi et al., 1986, Immunology and Immunopharmacology of Bacterial Endotoxins, Plenum Publ. Corp., NY, p407-419), saponins such as QS21 (Sumino et al., 1998, J.Virol. 72: 4931; International Publication No. 98 / 56415), imidazoquinoline compounds such as imiquimod (Suader, 2000, J. Am Acad Dermatol. 43:S6), S-27609 (Smorlesi, 2005, Gene Ther. 12: 1324), and related compounds as described in International Publication No. 2007 / 147529, cytosine phosphate guanosine oligodeoxynucleotides such as CpG (Chu et al., 1997, J. Exp. Med. 186: 1623; Tritel et al., 2003, J. Immunol. 171: 2358), and cationic peptides such as IC-31 (Kritsch et al., 2005, J. Chromatogr Anal. Technol. Biomed. Life Sci. 822: 263-70).

[0240] In one embodiment, the SPD-TNFSF fusion protein, virus or composition of the present invention can be formulated, in particular, for the purpose of improving stability under production conditions and long-term storage (i.e., at least 6 months, preferably at least 2 years) at freezing (e.g., -70 °C, -20 °C), refrigeration (e.g., 4 °C) or ambient temperature. In the art, various virus formulations are available in either frozen, liquid or lyophilized form (e.g., WO 98 / 02522, WO 01 / 66137, WO 03 / 053463, WO 2007 / 056847 and WO 2008 / 114021, WO 2016087457, etc.). Solid (e.g., dry powder or lyophilized) compositions can be obtained by processes including vacuum drying and lyophilization (see, e.g., WO 2014 / 053571). By way of example, buffer formulations containing NaCl and / or sugar are particularly suitable for the storage of viruses (e.g., S01 buffer: 342.3 g / L sucrose, 10 mM Tris, 1 mM MgCl2, 150 mM NaCl, 54 mg / L, Tween 80; ARME buffer: 20 mM Tris, 25 mM NaCl, 2.5% glycerol (w / v), pH 8.0; S520 buffer: 100 g / L sucrose, 30 mM Tris, pH 7.6; S08 buffer: 10 mM Tris, 50 mM NaCl, 50 g / L sucrose, 10 mM sodium glutamate, pH 8.0).

[0241] In certain embodiments, the viral compositions of the invention can be formulated to ensure proper distribution or sustained release in vivo. For example, they can be formulated in liposomes. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are described, for example, in J. R. Robinson's “Sustained and Controlled Release Drug Delivery Systems”, ed., Marcel Dekker, Inc., New York, 1978.

[0242] The appropriate dosage of the SPD-TNFSF fusion protein can be tailored as a function of various parameters and can be routinely determined by one of ordinary skill in the art in light of the relevant circumstances. The appropriate dosage of the SPD-TNFSF fusion protein can vary from 0.001 to about 100 mg / kg, depending on the route of administration of the protein and the quantification technique used. As a general guideline, the amount of SPD-TNFSF fusion protein present in a sample can be determined by conventional titer measurement techniques, such as UV absorbance or ELISA.

[0243] The appropriate dosage of the virus can be tailored as a function of various parameters and can be routinely determined by one of ordinary skill in the art in light of the relevant circumstances. The appropriate dosage of the virus can vary from about 10 4 to about 10 13 vp (viral particles), iu (infectious units) or pfu (plaque forming units). As a general guideline, a dosage of vaccinia virus of about 10 4 to about 10 13 pfu is suitable, preferably about 10 6 pfu to about 10 11 pfu, more preferably about 10 7 pfu to about 5×10 9 pfu, and particularly for human use about 10 8pfu~about 10 9 PFU is particularly preferred. The amount of virus present in the sample can be determined by conventional titer measurement techniques, such as counting the number of plaques after infection of permissive cells (e.g., BHK-21 or CEF) using permissive cells, immunostaining (e.g., using anti-viral antibodies; Caroll et al., 1997, Virology 238: 198-211), measurement of A260 absorbance (vp titer), or also quantitative immunofluorescence (iu titer).

[0244] Administration The SPD-TNFSF fusion protein, virus or composition of the present invention can be administered once (e.g., bolus injection) or multiple times. In the case of multiple administrations, they can be carried out by the same or different routes and at the same or different sites. Also, administration can be carried out in sequential administration cycles that are repeated after a drug-free period. The interval between each administration can be from several hours to 1 year (e.g., 24 hours, 48 hours, 72 hours, weekly, every two weeks, monthly or annually). The interval can also be irregular (e.g., according to the progression of the tumor). The dosage is variable for each administration within the above range.

[0245] In the context of the present invention, any of the conventional administration routes including parenteral, topical or mucosal routes are applicable. The parenteral route is intended for administration as injection or infusion. Common types of parenteral injection are intravenous (into a vein), intraarterial (into an artery), intradermal (into the dermis), subcutaneous (under the skin), intramuscular (into muscle), and intratumoral (into or near the tumor). Infusion is generally carried out by the intravenous route. Mucosal administration includes, but is not limited to, oral / gastrointestinal, intranasal, intratracheal, intralung, intravaginal or rectal routes. Topical administration can also be carried out using transdermal means (e.g., patches, etc.). Administration can be carried out using conventional syringes and needles (e.g., Quadrafuse needles), or any compound or device available in the art that can facilitate or improve the delivery of the active agent in the subject. Preferred administration routes for the virus include intravenous and intratumoral routes.

[0246] In the context of the present invention, the virus can be administered one or several times (e.g., 2, 3, 4, 5, 6, 7, or 8 times, etc.) at a dose within the range of 10 7 ~5×10 9 pfu. The time interval between each administration can vary from about 1 day to about 8 weeks, preferably from about 2 days to about 6 weeks, more preferably from about 3 days to about 4 weeks, and even more preferably from about 1 week to about 3 weeks (e.g., every 2 weeks, etc.). A preferred treatment scheme involves 2 to 5 (e.g., 3) intravenous or intratumoral administrations of oncolytic vaccinia virus at a dose of 10 8 or 10 9 pfu at intervals of about 1 or 2 weeks.

[0247] The present invention also relates to a method for treating proliferative diseases such as cancer, or infectious diseases, inflammatory diseases, metabolic diseases, autoimmune diseases, degenerative diseases, apoptosis-related diseases, and disorders associated with TNF cytokine dysfunction such as transplant rejection, comprising administering a virus as described herein to a subject in need thereof.

[0248] In one embodiment, the present invention also relates to a method for treating proliferative diseases such as cancer, comprising administering a virus as described herein to a subject in need thereof.

[0249] In one embodiment, the present invention also relates to a method for inhibiting tumor cell growth in vivo, comprising administering a virus as described herein to a subject in need thereof.

[0250] In one embodiment, the present invention also relates to a method for enhancing the immune response against tumor cells, comprising administering a virus as described herein to a subject in need thereof.

[0251] In one embodiment, the administration of the virus for use in the present invention induces, stimulates and / or redirects an immune response. In particular, the administration induces a defensive T cell or B cell response in the host being treated, for example, against the virus or, ultimately, against the product encoded by the SPD-TNFSF nucleic acid molecule inserted into the viral genome (if present). The defensive T cell response can be mediated via CD4+ or CD8+, or both CD4+ and CD8+ cells. The B cell response can be measured by ELISA, and the T cell response can be evaluated from any sample (e.g., blood, organs, tumors, etc.) taken from the immunized animal or subject by conventional ELISpot, ICS assays.

[0252] In one embodiment, the administration of the oncolytic virus is also capable of changing the tumor microenvironment for the purpose of enhancing the activity of effector cells, particularly effector T lymphocytes, within the tumor and / or promoting at least partial Treg depletion. Tumor infiltrating cells can be readily identified, for example, by conventional immunostaining assays.

[0253] A higher therapeutic effect can be demonstrated, with particular preference for a longer survival period, as described above in connection with the term "therapeutically effective amount".

[0254] Examples of disorders that can be treated using the SPD-TNFSF fusion protein, virus, composition, or method of the present invention include, but are not limited to, the following: - Proliferative diseases · Cancers, such as bone cancer, liver cancer, pancreatic cancer, gastric cancer, colon cancer, esophageal cancer, oropharyngeal cancer, lung cancer, head and neck cancer, skin cancer, melanoma, uterine cancer, endometrial cancer, cervical cancer, ovarian cancer, breast cancer, rectal cancer, cancer of the anal area, prostate cancer, lymphoma, endocrine cancer, thyroid cancer, soft tissue sarcoma, chronic or acute leukemia, bladder cancer, kidney cancer, central nervous system (CNS) neoplasms, glioma, glioblastoma, etc. Preferred cancers that can be treated with the SPD-TNFSF fusion protein or virus of the present invention generally include cancers responsive to immunotherapy. Non-limiting examples of preferred cancers for treatment include melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory adenocarcinoma of the prostate), breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer), and liver cancer (e.g., hepatocellular carcinoma). · Cardiovascular diseases such as restenosis - Disorders associated with TNF cytokine dysfunction: · Infectious diseases, such as HIV infection, especially chronic viral diseases, such as hepatitis A, B or C, herpes, tuberculosis, Epstein - Barr virus, cytomegalovirus, John Cunningham virus and human papillomavirus, yellow fever, dengue fever, flavivirus, influenza virus, hemorrhagic infectious diseases (Marburg or Ebola virus), and severe acute respiratory syndrome (SARS), bacterial infectious diseases, such as Legionnaires' disease (Legionella), sexually transmitted diseases (e.g., chlamydia or gonococcal infection), gastric ulcer (Helicobacter), cholera (Vibrio), diphtheria, Escherichia coli, staphylococcus, salmonella or streptococcus (tetanus) infections; infections caused by protozoal pathogens such as malaria, sleeping sickness, leishmaniasis; toxoplasmosis, i.e., infections caused by Plasmodium, Trypanosoma, Leishmania and Toxoplasma; or fungal infections caused by, for example, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis or Candida albicans; · Inflammatory diseases, such as celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease (COPD), irritable bowel disease, atherosclerosis, arthritis, ankylosing spondylitis, Crohn's disease, colitis, chronic active hepatitis, dermatitis and psoriasis; · Metabolic diseases, such as diabetes, cystinosis, dyslipidemia, hyperthyroidism, hypothyroidism, hyperlipidemia, hypolipidemia, galactosemia, obesity, Gaucher's disease and phenylketonuria; · Autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis and Sjögren's syndrome · Degenerative diseases, such as neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, macular degeneration, multiple sclerosis, muscular dystrophy, Niemann - Pick disease, neuronal ceroid lipofuscinosis, osteoporosis · Apoptosis-related diseases · Transplant rejection

[0255] The SPD-TNFSF fusion protein, virus, composition or method according to the present invention can be associated with one or more substances or therapies effective in anti-cancer therapy, and the present invention also relates to a method comprising the step of delivering an additional cancer therapy to a subject. In the context of the present invention, the additional cancer therapy comprises surgery, radiotherapy, chemotherapy, immunotherapy, hormone therapy, or a combination thereof. In a preferred embodiment, the method of the present invention comprises administration of one or more substances effective in anti-cancer therapy. Among the pharmaceutical substances effective in anti-cancer therapy that can be used in association with or in combination with the SPD-TNFSF fusion protein, virus, composition, or method according to the present invention, more specifically, the following can be mentioned: - Alkylating agents, such as mitomycin C, cyclophosphamide, busulfan, ifosfamide, isosfamide, melphalan, hexamethylmelamine, thiotepa, chlorambucil, or dacarbazine; - Antimetabolites, such as gemcitabine, capecitabine, 5-fluorouracil, cytarabine, 2-fluorodeoxycytidine, methotrexate, idatrexate, tomudex or trimethoprim; - Topoisomerase II inhibitors, such as doxorubicin, epirubicin, etoposide, teniposide or mitoxantrone; - Topoisomerase I inhibitors, such as irinotecan (CPT-11), 7-ethyl-10-hydroxy-camptothecin (SN-38) or topotecan; - Mitotic inhibitors, such as paclitaxel, docetaxel, vinblastine, vincristine or vinorelbine; - Platinum derivatives, such as cisplatin, oxaliplatin, spiroplatinum or carboplatinum; - Tyrosine kinase receptor inhibitors, such as sunitinib (Pfizer) and sorafenib (Bayer); - Anti-neoplastic antibodies, in particular antibodies that affect the regulation of cell surface receptors, such as trastuzumab, cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, bevacizumab and ranibizumab; - EGFR (epidermal growth factor receptor) inhibitors, such as gefitinib, erlotinib and lapatinib; and - Immunomodulatory agents, such as alpha, beta or gamma interferon, interleukin (in particular, IL-2, IL-6, IL-10 or IL-12) or tumor necrosis factor.

[0256] Also, the SPD-TNFSF fusion protein, expression vector, composition, or method according to the present invention can also be used in connection with radiotherapy.

[0257] The present invention also provides a kit comprising different containers (e.g., sterile glass or plastic vials) for each viral dose to be administered. Optionally, the kit can include an apparatus for administering the active agent. The kit can also include an accompanying document containing information regarding the composition or individual components and dosage forms in the kit.

[0258] Materials and methods One skilled in the art can recognize or confirm, without performing more than routine experimentation, numerous equivalents to the specific methods and reagents described herein, including alternatives, modifications, additions, deletions, alterations and substitutions. Such equivalents are considered to be within the scope of the present invention and are encompassed by the following claims.

[0259] Viruses and plasmids VVTG18058 (empty VACV, VACV control, or unarmed control VACV) is a vaccinia virus (Copenhagen strain) lacking the J2R and I4L genes. VVTG18058 was used as an unarmed control virus. VVTG18058 was produced on chicken embryo fibroblasts (CEF). Titration was performed by plaque assay on Vero cells.

[0260] Plasmid pTG19274 is a plasmid encoding an irrelevant FLAG-tagged molecule. This plasmid pTG1927 was used as a negative control.

[0261] Plasmid pTG19333 is a plasmid without a transgene. This plasmid pTG19333 was used as a negative control.

[0262] Plasmid pTG19325 is a plasmid encoding a fusion protein SPD (M1 - G257, P35247 numbering)-CD40L (from P29965 numbering H47~L261) construct (SEQ ID NO: 50).

[0263] Plasmid pTG19344 is a plasmid encoding a soluble CD40L (G116~L261) (SEQ ID NO: 51) consisting of a heterologous signal peptide followed by the extracellular domain of CD40L.

[0264] Plasmid pTG19965 is a plasmid encoding a heterologous signal peptide upstream of human SPD (A21~G257)-CD40L (N119~L261). This construct (SEQ ID NO: 52) contains a heterologous signal peptide, a short-chain form of CD40L, and a FLAG tag at the C-terminus.

[0265] Plasmid pTG20032 is a plasmid encoding a 4-1-BBL (D80~E254) extracellular domain (SEQ ID NO: 53) consisting of a heterologous signal peptide followed by the extracellular domain of 4-1-BBL.

[0266] Transfer plasmids carrying various SPD-TNFSF constructs according to the present invention are listed in Table 2 below.

[0267]

Table 2

[0268] COPTG19967 is a recombinant Copenhagen vaccinia virus double deletion (tk- and rr-) in which the expression sequence of pTG19967 is inserted into the J2R locus.

[0269] COPTG19968 is a recombinant Copenhagen vaccinia virus double deletion (tk- and rr-) in which the expression sequence of pTG19968 is inserted into the J2R locus.

[0270] COPTG19969 is a recombinant Copenhagen vaccinia virus double deletion (tk- and rr-) in which the expression sequence of pTG19969 is inserted into the J2R locus.

[0271] Infection / transfection experiments The method for expressing the recombinant protein is the infection / transfection method. Such a method consists of infecting cells, for example, HeLa cells with vaccinia virus (poxvirus), and transfecting the above cells with a plasmid encoding the gene of interest under the regulatory control of a poxvirus promoter. Such a method enables the expression of the gene of interest intracellularly. The expression product can be recovered in the supernatant for further analysis.

[0272] For the purpose of selecting the most effective SPD-TNFSF construct for vectorization with the VACV (vaccinia virus) genome, co-infection / transfection in HeLa cells was performed.

[0273] Briefly, 2 days before infection, cells were seeded in 6-well plates at 4E+05 cells / well / 3 mL complete medium (DMEM 4 Gibco ref.41966-029; glutamine 2 mM; gentamicin 40 μg / mL; 10% fetal bovine serum (FBS)).

[0274] Before infection, the culture medium was removed and replaced with 400 μL of a vaccinia virus preparation (VVTG18058) in PBS+(PBS + 1% cation) corresponding to an MOI of 1. After 30 minutes at room temperature (RT), the virus inoculum was removed and replaced with 1.2 mL of complete medium without FBS. The plates were incubated at 37 °C, 5% CO2 for 2 hours. Next, transfection was performed by adding 1 μg of plasmid DNA formulated with 4.5 μL of Lipofectamine 2000 (Invitrogen, 11668 - 027) to each well according to the supplier's protocol. pTG19274 (encoding an irrelevant FLAG-tagged molecule) was used as a negative control. Infection / transfection was performed in triplicate. The plates were incubated at 37 °C, 5% CO2 for 48 hours. Next, the culture supernatant was collected, centrifuged, and filtered through a 0.1 μm filter to remove all virus particles and cell debris. The clarified supernatant was stored at -80 °C until use.

[0275] Expression experiments using recombinant viruses Four days before infection, HeLa cells were seeded in 6-well plates at 1.5E+06 cells / well / 2 mL of complete medium (DMEM 4 Gibco ref.41966 - 029; glutamine 2 mM; gentamicin 40 μg / mL; 10% FBS). The cells were infected with one of the following viruses: COPTG19968, COPTG19967, COPTG19969, or VVTG18058 at an MOI of 0.1. After a 30-minute incubation, the culture medium was discarded and replaced with 2 mL of DMEM; glutamine 2 mM; gentamicin 40 μg / mL. The cells were incubated at 37 °C, 5% CO2 for 48 hours, after which the culture supernatant was collected and processed as described above.

[0276] Immunoblot of CD40L and 4-1BBL 25 μL of the sample (clarified supernatant) from infection / transfection was treated with Laemmli buffer (Biorad, 161-0747) with (reducing) or without (non-reducing) β-mercaptoethanol. In the case of reducing conditions, the sample was heated at 95 °C for 3 minutes. Next, the sample was loaded onto a polyacrylamide gel (TGX 4–15% Stain Free Biorad) and electrophoresed in Tris Glycin SDS buffer (Biorad 161-0772). Western blotting was performed using a Transblot Turbo System (Biorad) set on the Midi Program High Molecular weight. Next, using the Ibind Flex Western System (Invitrogen ref SLF2000), the blot was incubated with an anti-FLAG-HRP conjugated antibody (Sigma A8592) at 2 μg / mL. The positive control was the culture medium obtained from infection / transfection with an unrelated plasmid (pTG19274) encoding a FLAG-tagged recombinant protein. The blot was incubated with an HRP substrate (Amersham ECL Prime Western blotting detection) and luminescence was recorded using a Chemidoc device.

[0277] ELISA of CD40 CD40-Fc was coated onto a Medisorp (Nunc) 96-well ELISA plate at 0.5 μg / mL in 50 mM carbonate buffer pH 9.6. The clarified supernatant from the infection / transfection experiment was added to the first well of the ELISA plate after 10-fold dilution, and further serial 2-fold dilutions were performed directly on the plate with ELISA saturation buffer. Bound CD40L was detected by adding non-competitive anti-human CD40L (MCA1561 Biorad) diluted 1000-fold in saturation buffer. Next, anti-mouse immunoglobulin-HRP conjugated antibody (Dako P0447) diluted 2000-fold was added to each well. Finally, HRP substrate (3,3’,5,5’-tetramethylbenzidine) TMB was added to each well, and the absorbance at 450 nm was measured using a TECAN microplate reader. The optical density (OD) at 450 nm was plotted against 1 / culture supernatant dilution factor using GraphPad prism software.

[0278] CD40 agonist activity HEK-Blue CD40L cells (Invivogen: hkb-cd40) are recombinant cells transformed to express both human CD40 and a reporter enzyme (secreted embryonic alkaline phosphatase: SEAP) under the transcriptional control of the CD40-inducible promoter. When CD40 is activated, SEAP is produced and its enzyme activity is measured in the culture medium according to the supplier's recommendations. SEAP activity is proportional to CD40 agonist activity.

[0279] Measurements were performed according to the supplier's instructions. Briefly, 50,000 HEK-Blue CD40L cells in 90 μL were dispensed into a 96-well plate and incubated with 20 μL of serial dilutions of the clarified supernatant prepared by the above infection / transfection. After incubation at 37 °C, 5% CO2 for 24 hours, 40 μL of the culture medium was transferred together with 160 μL of SEAP substrate (Invivogen: hb-det2) and incubated at 37 °C for 3 hours. The absorbance at 620 nm was measured using a microplate reader, and the optical density was plotted against 1 / supernatant dilution factor using GraphPad prism software.

[0280] 4-1BB agonist activity The 4-1BB Bioassay Promega kit (JA2351) was used according to the supplier's instructions. Briefly, 25 μL of effector cells / well were mixed with 25 μL of medium. Next, serial dilutions of 25 μL of clarified supernatant were added to each well. The cells were then incubated at 37 °C and 5% CO2 for 6 hours. 75 μL / well of Reconstituted Bio-Glo (Promega, G7941) was added, and luminescence was recorded using a Berthold reader and MikroWin 2000 software. Using GraphPad prism software, luminescence was plotted against 1 / supernatant dilution rate, and four-parameter logistic curve analysis was performed.

[0281] Vectorization and virus production Recombinant viruses were generated using the same plasmids as those used in the above infection / transfection experiments. Briefly, chicken embryo fibroblasts (CEFs) were infected with a parental virus encoding GFP at the J2R (TK) locus and lacking the I4L (RR) gene. The infected cells were transfected with a transfer plasmid carrying an expression cassette flanked by recombinant harm (DNA sequences homologous to the upstream and downstream J2R (TK) loci). Recombinant viruses were selected by picking "white" (i.e., GFP-negative) lytic plaques under a binocular microscope. The expression cassette was confirmed by PCR amplification and subsequent DNA sequencing.

[0282] For in vivo experiments, recombinant virus was produced using CEF cultured on F500 (MOI 0.05, 72 hours). The cell suspension containing the virus was homogenized using a homogenizing mixer equipped with an in-line chamber. Next, large cell residues were removed by depth filtration using a filter with a pore size of 5 μm. Thereafter, the clarified virus suspension was concentrated and diafiltered with formulation buffer (50 g / L sucrose, 50 mM NaCl, 10 mM Tris, 10 mM sodium glutamate, pH 8) using tangential flow filtration and a hollow fiber microfiltration filter with a pore size of 0.2 μm. The purified virus was dispensed and stored at -80 °C until use.

Example

[0283] Examples / Results Expression of novel constructs HeLa cells were infected and transfected with transfer plasmids (pTG19965, pTG19966, pTG19967, pTG19968, pTG19969) carrying various SPD-TNFSF constructs as described above. Expression of the recombinant SPD-TNFSF protein in the culture supernatant was evaluated by immunoblotting using an anti-FLAG tag for detection. Figures 1A and 1B show that all SPD-TNFSF fusion proteins were expressed at the expected monomer size under reducing conditions and showed varying degrees of oligomerization under non-reducing conditions. Note that oligomers not locked by disulfide bonds migrated as monomers under denaturing conditions of electrophoresis.

[0284] Also, it is particularly advantageous that the fusion protein according to the present invention has a smaller molecular size compared to existing fusion proteins.

Table 3

[0285] Interestingly, the construct encoded by pTG19967 also showed distinct oligomerization under non-reducing conditions, with the trimer being the major band (∼75 kDa), although hexamers (∼150 kDa) and higher-order oligomers were also clearly visible.

[0286] It is also particularly interesting that the fusion protein can form multimers without being restricted to the trimeric form as compared to the prior art disclosures.

[0287] CD40 agonist activity of various SPD-TNFSF constructs The CD40 agonist activity of each construct was evaluated in HEK blue CD40L cells and compared to the proteins encoded by pTG19344 (soluble CD40L extracellular domain) and pTG19325. The construct encoded by pTG19965 was found to be slightly improved compared to the constructs encoded by pTG19325 and pTG19344, indicating that changes in the peptide signal and / or truncation of the CD40L domain affected the expression level and / or agonist activity of the molecule. This effect was highly amplified by the addition of a (GGGS)×3 linker between the coiled-coil neck domain and CD40L in the collagen domain complete deletion type (construct encoded by pTG19967) or without the addition (construct encoded by pTG19966). Miniaturization of the collagen domain from 59 (GXX) repeats of the SPD full length (i.e., the construct encoded by pTG19965) to 12 (GXX) repeats (i.e., the constructs encoded by pTG19968, pTG19999) showed the best CD40 agonist activity among all other constructs. This result indicates that there is an optimal size of the collagen domain to obtain the best CD40 agonist effect of the fusion. The collagen portions of the constructs encoded by pTG19968 and pTG19969 contain N-glycosylation sites, which may have some effect on the assembly and / or structure of the oligomers and thus the CD40 agonist activity of the recombinant molecules. The present results do not show a significant effect of N-glycosylation.

[0288] Binding to CD40 To examine the CD40-binding ability of all SPD-TNFSF constructs, an ELISA assay using immobilized recombinant human CD40 was set up. In this assay, a non-competitive anti-CD40L was used to detect the formation of CD40L / CD40 complexes on the ELISA plate. In this assay, the clarified supernatants generated from the constructs encoded by the infection / transfection of pTG19965 and pTG19325 had the same slight CD40-binding activity at a 1 / 10 dilution. Constructs with a modified SPD collagen domain showed a clear improvement in CD40 in the same ranking as observed in the CD40 agonist assay (i.e., the constructs encoded by pTG19968 > pTG19969 > pTG19967 > pTG19966). These results indicate that the superior CD40 agonist activity observed in the collagen constructs is due, at least in part, to better binding to CD40.

[0289] Effect of collagen domain According to the present invention, other numbers of (GXX) repeats before and after the 12 (GXX) repeats tested were evaluated. Thus, different lengths or properties of the collagen (GXX) repeats were tested by i) shortening the original 12 (GXX) repeats into two constructs each containing 6 (GXX) repeats (constructs encoded by pTG20038 with N-glycosylation sites and constructs not containing N-glycosylation sites (encoded by pTG20038)), ii) selecting another 12 (GXX) repeats lacking N-glycosylation sites at another location in the SPD collagen domain (construct encoded by pTG20041), and iii) increasing the number of (GXX) repeats to 19 (construct encoded by pTG20040) and 30 (construct encoded by pTG20042).

[0290] All of these constructs were expressed, by the above-described infection / transfection method, in parallel with the constructs encoded by pTG19968, pTG19966, and pTG19965 as references. The resulting culture media were tested for CD40 binding by ELISA (Figure 4) and CD40 agonist assay (Figure 5).

[0291] In both the CD40 binding by ELISA (Figure 4) and the CD40 agonist assay (Figure 5), these constructs were shown to be at best equivalent to the construct encoded by pTG19968. Regardless of the presence or absence of N-glycosylation sites, the two constructs with six (GXX) repeats had slightly lower biological activity than the construct encoded by pTG19968. The two constructs with twelve (GXX) repeats (different from the construct encoded by pTG19968), nineteen (GXX) repeats (different from the construct encoded by pTG19968), and nineteen (GXX) repeats were clearly less bioactive than the construct encoded by pTG19968. Finally, the construct with thirty (GXX) repeats had low biological activity of the construct and was equivalent in biological activity to the construct encoded by pTG19966.

[0292] Collectively, these results indicate that several alternative constructs (e.g., the constructs encoded by pTG19969, pTG19967, pTG20038, pTG20039) can be considered to be approximately equivalent, but that the construct encoded by pTG19968 is the best among those tested.

[0293] Vectorization of pTG19968, pTG19969 and pTG19967 The selected constructs (i.e., the constructs encoded by pTG19968, pTG19969, and pTG19967) were inserted into the J2R (TK) locus of the vaccinia virus genome by homologous recombination. HeLa cells were infected with the recombinant virus, and the culture medium obtained 48 hours after infection was tested for CD40 binding by ELISA (Figure 6) and CD40 agonist assay (Figure 7). The EC50 for CD40 binding increased approximately 10-fold (Figure 5, Figure 7) (i.e., binding was lower) for COPTG19968 and COPTG19969 compared to their infection / transfection counterparts.

[0294] Note that from the shape of the curves, it was not even possible to calculate the exact EC50 for COPTG19968 and COPTG19969. However, COPTG19967 showed much better CD40 agonist activity than COPTG19968 and COPTG19969. Among the SPD-TNFSF constructs vectorized with vaccinia virus, that encoded by COPTG19967 showed the best CD40 agonist activity.

[0295] 4-1-BBL expression and 4-1-BB agonist activity of SPD-TNFSF constructs The potential of the SPD-TNFSF fusion proteins was extended to another member of TNFSF, 4-1BBL. In the construct encoded by pTG19968 as the fusion showing the best agonist activity in this infection / transfection setting, the CD40L ectodomain was replaced with the 4-1BBL ectodomain to generate the construct encoded by pTG20033. Also, only the 4-1BBL ectodomain was cloned into the same backbone plasmid to generate construct pTG20032, which was further used as a reference. These two 4-1BBL molecules: the 4-1BBL ectodomain alone encoded by pTG20032, or the 4-1BBL ectodomain fused to the modified SPD with 12 (GXX) repeats (the construct encoded by pTG20033) were expressed at the same level by the infected / transfected cells (Figure 8). Furthermore, the pattern of oligomerization observed under non-reducing conditions was similar for both modified SPDs with the 12 (GXX) repeat construct (i.e., the CD40L and 4-1BBL fusion). The 4-1BB agonist activity of the modified SPD with the 12 (GXX) repeat-4-1BBL construct was clearly superior to that of 4-1BBL alone (Figure 9), although the increase in activity was smaller than that observed for the equivalent CD40L construct.

[0296] References

Table 4

Claims

1. - An N-terminal domain, - A coiled-coil neck domain of surfactant protein-D (SPD) between the N-terminal domain and the C-terminal position, and - A TNF-superfamily (TNFSF) ligand or its receptor-binding domain at the C-terminal position An SPD-TNFSF fusion protein comprising the above.

2. The SPD-TNFSF fusion protein according to claim 1, wherein the fusion protein further comprises a collagen domain between the N-terminal domain and the coiled-coil neck domain of SPD.

3. The SPD-TNFSF fusion protein according to claim 2, wherein the collagen domain comprises 1 to 40 repeats of (GXX), preferably 3 to 30 repeats of (GXX), more preferably 6 to 20 repeats of (GXX), and even more preferably 12 repeats of (GXX), where X is an amino acid and G is a glycine amino acid.

4. The SPD-TNFSF fusion protein according to any one of claims 1 to 3, wherein the fusion protein further comprises a linker between the coiled-coil neck domain and the TNF-superfamily ligand or its receptor-binding domain.

5. The SPD-TNFSF fusion protein according to claim 4, wherein the linker is a glycine / serine linker and has a length of 4 to 20 amino acids, preferably 8 to 16, and more preferably 12 amino acids.

6. The SPD-TNFSF fusion protein according to any one of claims 1 to 5, wherein the TNF-superfamily ligand is selected from CD40L, 4-1-BBL, CD70, OX40L, TNF, GITRL, LIGHT, FASL, TWEAK, APRIL, RANKL, TRAIL, CD30L, NGF, BAFF, LTβ, LTα, LTαβ2, TL1A, TLA, EDA, and more preferably, selected from CD40L or 4-1-BBL.

7. The SPD-TNFSF fusion protein according to any one of claims 1 to 6, wherein the N-terminal domain has at least 85%, preferably at least 90%, more preferably at least 95% identity with the amino acid sequence shown in SEQ ID NO:

1.

8. The SPD-TNF-SF fusion protein according to any one of claims 1 to 7, comprising an array selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or consisting of an array selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:

11.

9. A trimeric fusion protein comprising three fusion proteins according to any one of claims 1 to 8.

10. A multimeric fusion protein comprising a plurality of trimeric fusion proteins according to claim 9, forming a hexamer, dodecamer, octadecamer or higher-order oligomer, preferably a hexamer, more preferably a dodecamer.

11. An isolated nucleotide sequence encoding the fusion protein according to any one of claims 1 to 8.

12. The isolated nucleotide sequence according to claim 11, comprising SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:

21.

13. An mRNA comprising the nucleotide sequence according to claim 11.

14. A plasmid comprising the nucleotide sequence according to claim 11 or 12.

15. A virus comprising the nucleotide sequence according to claim 11 or 12.

16. The virus according to claim 15, wherein the virus is an oncolytic virus or a non-oncolytic virus.

17. The virus according to claim 16, wherein the oncolytic virus is selected from the group consisting of poxvirus, herpesvirus, reovirus, Seneca Valley virus (SVV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), mobiliovirus, retrovirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), measles virus, foamy virus, alphavirus, lentivirus, influenza virus, Sindbis virus, rhabdovirus, picornavirus, coxsackievirus, parvovirus, or chimeras thereof.

18. The virus according to claim 17, wherein the oncolytic virus is a poxvirus belonging to the genus Orthopoxvirus, and is preferably selected from the group consisting of vaccinia virus, cowpox virus, canarypox virus, and ectromelia virus.

19. The virus according to claim 18, wherein the oncolytic poxvirus is a vaccinia virus, particularly a vaccinia virus selected from the group consisting of the Elstree strain, the Wyeth strain, the Copenhagen strain, the Lister strain, the Tian Tian strain, and the Western Reserve strain.

20. The virus according to claim 17, wherein the oncolytic virus is a poxvirus belonging to the genus Leporipoxvirus, and is preferably selected from the group consisting of myxoma virus, rabbit fibroma virus, and Shope fibroma virus, and is preferably myxoma virus.

21. The virus according to any one of claims 18 to 20, wherein the oncolytic poxvirus is a thymidine kinase (TK) -deficient virus caused by an inactivating mutation in the J2R virus gene.

22. The virus according to claim 21, wherein the oncolytic poxvirus is a ribonucleotide reductase (RR) -deficient virus caused by an inactivating mutation in the viral I4L and / or F4L gene.

23. The virus according to claim 21 or 22, wherein the oncolytic poxvirus is an m2 -deficient virus caused by an inactivating mutation in the M2L virus gene.

24. The virus according to claim 16, wherein the non - oncolytic virus is a poxvirus.

25. The virus according to claim 24, wherein the poxvirus is selected from the group consisting of pseudocowpox virus (PCPV), modified vaccinia virus Ankara (MVA), highly attenuated vaccinia virus strain (NYVAC), swinepox virus (SWPV), fowlpox virus (FPV), or chimeras thereof.

26. a) a step of preparing production cells; b) a step of transfecting or infecting the prepared production cells with a virus; c) a step of culturing the transfected or infected production cells under appropriate conditions for virus production; d) a step of recovering the produced virus from the culture of the production cells; and optionally e) a step of purifying the recovered virus. A method for producing the virus according to any one of claims 15 to 25.

27. A cell comprising the nucleotide sequence according to claim 11 or 12, or the mRNA according to claim 13, or the plasmid according to claim 14, or the virus according to any one of claims 15 to 25.

28. The SPD-TNFSF fusion protein according to any one of claims 1 to 8, the trimeric fusion protein according to claim 9, the multimeric fusion protein according to claim 10, the nucleotide sequence according to claim 11 or 12, the mRNA according to claim 13, the plasmid according to claim 14, the virus according to any one of claims 15 to 25, or the cell according to claim 27, for use in medicine.

29. The SPD-TNFSF fusion protein according to any one of claims 1 to 8, the trimeric fusion protein according to claim 9, the multimeric fusion protein according to claim 10, the nucleotide sequence according to claim 11 or 12, the mRNA according to claim 13, the plasmid according to claim 14, the virus according to any one of claims 15 to 25, or the cell according to claim 27, for use in the treatment of proliferative diseases such as cancer and infectious diseases, inflammatory diseases, metabolic diseases, autoimmune diseases, degenerative diseases, apoptosis-related diseases, and disorders associated with TNF cytokine dysfunction such as transplant rejection.

30. The SPD-TNF-SF fusion protein according to any one of claims 1 to 8, the trimeric fusion protein according to claim 9, the multimeric fusion protein according to claim 10, the nucleotide sequence according to claim 11 or 12, the mRNA according to claim 13, the plasmid according to claim 14, the virus according to any one of claims 15 to 25, or the cell according to claim 27, for use in the treatment of cancer.

31. The SPD-TNF-SF fusion protein according to any one of claims 1 to 8, the trimeric fusion protein according to claim 9, the multimeric fusion protein according to claim 10, the nucleotide sequence according to claim 11 or 12, the mRNA according to claim 13, the plasmid according to claim 14, the virus according to any one of claims 15 to 25, or the cell according to claim 27, in combination with one or more chemotherapeutic agents or immunotherapeutic agents effective for use in the treatment of cancer.

32. The SPD-TNF-SF fusion protein according to any one of claims 1 to 8, the trimeric fusion protein according to claim 9, the multimeric fusion protein according to claim 10, the nucleotide sequence according to claim 11 or 12, the mRNA according to claim 13, the plasmid according to claim 14, the virus according to any one of claims 15 to 25, or the cell according to claim 27, comprising optionally a pharmaceutically acceptable diluent, carrier, vehicle and / or excipient, or a pharmaceutical composition consisting of the SPD-TNF-SF fusion protein according to any one of claims 1 to 8, the trimeric fusion protein according to claim 9, the multimeric fusion protein according to claim 10, the nucleotide sequence according to claim 11 or 12, the mRNA according to claim 13, the plasmid according to claim 14, the virus according to any one of claims 15 to 25, or the cell according to claim 27 and optionally a pharmaceutically acceptable diluent, carrier, vehicle and / or excipient.

33. The pharmaceutical composition according to claim 32, wherein the pharmaceutical composition further comprises one or more effective chemotherapeutic agents or immunotherapeutic agents.

34. The pharmaceutical composition according to claim 32 or 33, for use in the treatment of cancer.

35. The pharmaceutical composition according to any one of claims 32 to 34, wherein the pharmaceutical composition is administered via a parenteral route, more preferably via an intravenous, subcutaneous, or intramuscular route, and even more preferably via an intravenous route.

36. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the fusion protein according to any one of claims 1 to 8, the trimeric fusion protein according to claim 9, the multimeric fusion protein according to claim 10, the nucleotide sequence according to claim 11 or 12, the mRNA according to claim 13, the plasmid according to claim 14, the virus according to any one of claims 15 to 25, or the cell according to claim 27, or the pharmaceutical composition according to any one of claims 32 to 34.

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