Fusion proteins comprising surfactant protein-d and TNFSF members

Abstract

The present invention is in the field of immunology and oncology, in particular for the treatment, prevention or inhibition of proliferative diseases, in particular cancer, infectious diseases and conditions associated with TNF cytokine dysfunction. The present invention relates to a novel SPD-TNFSF fusion protein comprising a TNF-superfamily (TNFSF) ligand, or a receptor binding domain thereof, fused to a coiled-coil domain of a surface active protein-D (SPD). A trimeric or multimeric fusion protein comprising a plurality of the SPD-TNFSF fusion proteins is also provided. The invention also provides an expression vector (such as mRNA, plasmid or virus) comprising the 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 relates generally 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 conditions associated with TNF cytokine dysfunction. Embodiments include SPD-TNFSF fusion proteins containing a TNF-superfamily (TNFSF) ligand or its receptor binding domain, fused to the coiled-coil domain of surfactant protein-D (SPD), trimeric or multimeric fusion proteins containing multiple SPD-TNFSF fusion proteins. Embodiments also include expression vectors, such as mRNA, plasmids and viruses, comprising nucleotide sequences 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 are suitable for use in the treatment of cancer and infectious diseases, and more generally for the treatment of proliferative diseases and conditions associated with TNF cytokine dysfunction. Background Art

[0002] Cancer is caused by external factors (such as tobacco, infectious organisms, dietary habits, chemicals and radiation) and internal factors (such as genetic mutations, hormones, immune conditions and mutations caused by metabolism). More than 12 million individuals are diagnosed with cancer every year in the world. In industrialized countries, about one in every five people dies of cancer. Although there are a large number of chemotherapeutic drugs, they are usually ineffective, and there is still an unmet medical need for effective and less toxic therapies, especially for patients resistant to existing treatments.

[0003] Effective T cell responses require functional and optimal innate and adaptive immunity, which depends in particular on multiple specific interactions between different cells through soluble stimuli and surface adaptor / receptor binding. In this context, CD8+ cytotoxic T lymphocyte (CTL) responses play a key role in antitumor immunity (Ara et al., 2018, ImmunoTargets and therapy, 7, 55–61). Three signals are mentioned as key parameters. The first signal involves the binding of antigen-specific T cell receptors (TCR) to peptide-loaded major histocompatibility complexes (MHC) on antigen presenting cells (APCs). The second signal is generated by the engagement of costimulatory molecules, namely B7-1 (CD80) / B7-2 (CD86) and CD28 (e.g., T cell CD28 / APC CD80), followed by Ag-specific T cell–APC interactions. The third signal induced by cytokine secretion enhances and modifies responding effector CTLs.

[0004] The tumor necrosis factor (TNF) superfamily plays a key role as a co-stimulatory molecule in immunomodulation and anti-tumor immunity (Vinay et al., 2009, Cell Biol Int.; 33 (4): 453-465). It is well known that TNFSF members mediate immune responses through the shaping properties of T cells. Previous studies have allowed 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] Effective antigen recognition by antigen-specific T cells depends critically on the presence and function of specialized antigen presenting cells (APCs) such as B cells and dendritic cells (DCs). For example, although CD40 is expressed on many cell types (including macrophages, B cells, and DCs), its ligand CD40L is primarily expressed on activated CD4+T cells. CD40L is expressed on the surface of activated B, T, and NK cells, but is also expressed on 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 “permits” DCs to trigger CD8 T cell responses by upregulating co-stimulatory molecules. This CD40L-CD40 interaction leads to the activation of CD40-bearing cells, which then express adhesion (ICAM), co-stimulation (CD80 / CD86), and present MHC I and II molecules in addition to cytokines / chemokines (TNFα, IL6…). In the tumor microenvironment, adhesion molecules and cytokines / chemokines work together to induce the infiltration and activation of immune cells, which ultimately destroy tumor cells and transform the tumor from an immunosuppressive microenvironment to an immunoactive 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 co-stimulation through ligands expressed on APCs, lymphocytes, and innate immune cells. These different studies have highlighted the interest in developing TNFSF ligands to treat proliferative diseases such as cancer and conditions associated with TNF cytokine dysfunction such as infectious diseases.

[0006] In recent years, TNF superfamily ligands have become attractive candidates for the development of vaccines and immunotherapies, more specifically as alternative approaches to designing new molecular adjuvants (Gupta et al., 2013, Immunol Res., 57(1-3): 303-10). Adjuvants can alter the intensity, quality and function of innate and adaptive immune responses, focusing on the rapid induction of large numbers of CD8 T cells capable of protecting against specific diseases (Lauterbach et al., 2013, Front Immunol., 27; 4: 251). Recombinant MVA-CD40L has demonstrated the potential of CD40L adjuvanted viruses to induce rapid and strong antigen-specific CD8 T cell responses for the development of preventive and therapeutic vaccines against cancer and infectious diseases such as HIV / AIDS, Ebola and Marburg hemorrhagic fever, malaria and hepatitis C (Lauterbach et al., 2013, Front Immunol. 2013 Aug 27; 4: 251). A method of arming adenovirus with CD40L has also been developed to stimulate a beneficial immune response to treat tumors (Pesonen et al., 2012, Cancer Res., 72 (7)). In the same way, by providing a strong T-cell response, recombinant 4-1-BBL adenovirus is a promising adjuvant for human memory CD8 T cells and suggests that it is beneficial in antiviral treatment strategies (Bukczynski et al., 2004, Proc Natl Acad Sci USA; 101 (5): 1291-1296).

[0007] Those skilled in the art also recognize that TNFSF ligands generally require homopolymerization to fully activate response cells. TNFSF signal transduction, which is also well defined in structure, requires appropriate receptor clustering and at least trimerization. Although TNFSF ligands exist as trimer units by self-assembly, receptors are generally separated on the cell surface (Richards et al. 2020, Hum Vaccin Immunother., 16 (2): 377-387). The interaction between polymer TNFSF ligands and their corresponding receptors leads to accurate receptor clustering and is a prerequisite for generating signals to be transmitted to cells (Kucka et al., 2020, Front Cell Dev Biol., 8: 615141). As shown above, high-order oligomeric TNFSF ligands, particularly dodecamer TNFSF ligands, are more effective than single trimer TNFSF ligands in signal transduction (Haswell et al. 2001, Eur J Immunol., 31 (10): 3094-100).

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

[0009] Patent application WO01 / 49866 discloses the first recombinant fusion protein comprising a TNF cytokine and a dimerization or multimerization domain to increase the affinity of the ligand. However, due to the molecular weight of the multimerization domain, the multimerization of the TNF protein is quite inefficient. WO01 / 42298 discloses a method for constructing a stable biologically active fusion protein to express TNFSF (more particularly CD40L and RANKLE / TRANCE members) with a collectin (more particularly an SPD comprising a signal sequence, a collagen domain and a coiled coil neck domain). The large size of the resulting fusion protein makes it less likely to diffuse into the tumor, thereby limiting its potential activity. Although this property may be particularly suitable for preventing them from being cleared, these fusion proteins have limited CD40 agonist activity.

[0010] WO2009 / 007120 also discloses fusion proteins comprising TNFSF cytokines fused to the collectin trimerization domain. Although these fusion proteins can form multimeric proteins, they are limited to forming trimers due to the inability to self-assemble more than three monomer units, thereby reducing their potential agonist activity.

[0011] TNFSF receptors are ubiquitously expressed on normal cells, so TNFSF ligands have no or only limited binding selectivity and considerable toxicity. Although the TNFSF ligand axis remains of great interest in cancer immunotherapy, systemic TNFSF therapy is associated with severe dose-limiting toxicity and produces minimal clinical activity (Bremer et al., 2013, ISRN Oncol; 2013: 371854). In the first clinical studies of CD40 agonists, most patients presented with a cytokine release syndrome that produced fever, chills, and shivering (Vonderheide et al., 2007, J Clin Oncol., 25(7): 876–883). Evidence of hepatotoxicity is also commonly observed with CD40 agonists (Beatty et al., 2017, Expert Rev Anticancer Ther.; 17(2): 175-186). Therefore, selective enhancement of tumor-restricted activation and TNFSF-mediated T cell stimulation in the tumor environment continues.

[0012] Technical issues

[0013] One can expect that cancer will continue to be a serious global health threat for many years to come due to the large number of causal factors that can act together or separately to initiate or promote cancer development. In addition, malignant tumors, especially metastatic tumors, are often resistant to conventional therapies, explaining the high incidence of certain cancers.

[0014] Therefore, it is highly desirable to develop more effective methods to improve the prevention and treatment of cancer and more generally proliferative diseases and disorders associated with TNF cytokine dysfunction. Although it is known that multimeric complexes of TNFSF cytokines are difficult to prepare from recombinant monomeric forms, the present inventors have produced SPD-TNFSF fusion proteins that provide effective multimerization properties and enhance the agonistic properties of the ligand through optimal clustering of its receptors, and are independent of the cellular environment. The optimal form has a relatively small molecular size, allowing them to diffuse into tumors and be effectively eliminated by renal clearance. This relatively small size is also an advantage when inserted into vectors with limited capacity (such as some viruses). In fact, it is well known that tissue distribution or cancer penetration of smaller therapeutic agents is superior to larger therapeutic agents (Li et al. MAbs. 2016; 8 (1): 113-9). It is well known to those skilled in the art that the biodistribution of such molecules is thus enhanced.

[0015] Due to limited binding selectivity and deleterious off-target toxicities from systemic TNFSF receptor activation, there is a need to develop a novel targeted SPD-TNFSF fusion protein that improves T cell responses through possible synergistic effects of viral replication and TNFSF stimulation on tumor cells and immune cells.

[0016] Other and further aspects, features and advantages of the present invention will become apparent from the following description of presently preferred embodiments of the invention.These examples are given for the purpose of disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A and 1B show the expression levels of SPD-TNFSF constructs (pTG19965, pTG19966, pTG19967, pTG19968, pTG19969) by infected / transfected HeLa cells. Clarified supernatants containing different SPD-TNFSF molecules were loaded on SDS-PAGE under reducing and non-reducing conditions. Proteins were transferred to PDVF membranes and hybridized with HRP-conjugated anti-FLAG tags for immunodetection. Blot A: Different structures of SPD-CD40L. Blot B: pTG19967 constructs were loaded at different volumes. Arrows indicate oligomers larger than trimers.

[0018] Figure 2CD40L agonist activity of culture medium of HeLa cells infected with vaccinia virus and then transfected with different plasmids carrying cassettes encoding SPD-TNFSF constructs (pTG19965, pTG19966, pTG19967, pTG19968, pTG19969) and under the control of pH5.R promoter (i.e. poxvirus promoter) is shown. HEK cells modified to express the reporter enzyme SEAP under the control of CD40 inducible promoter were incubated with different dilutions of clarified supernatant containing SPD-TNFSF construct. After 20-24 hours of incubation, SEAP enzyme activity was measured in the culture medium. Negative controls were uninfected cells (medium) or cells infected and transfected with a plasmid without transgene (pTG19333) or with a plasmid encoding an unrelated FLAG-tagged protein (pTG19274). Results are reported as SEAP activity versus medium 1 / dilution and the mean and standard deviation of two measurements.

[0019] Figure 3 The CD40 binding activity of HeLa cell cultures infected with vaccinia virus and then transfected with different plasmids carrying a cassette encoding the SPD-TNFSF construct (pTG19325, pTG19344, pTG19965, pTG19966, pTG19967, pTG19968, pTG19969) and under the control of the pH5.R promoter (i.e., poxvirus promoter) is shown. CD40-Fc was coated on 96-well plates and different dilutions of the clarified supernatant containing the TNFSF construct were applied. The bound specific protein was detected with a non-blocking anti-CD40L monoclonal antibody followed by HRP-conjugated anti-IgG. The negative control was cells infected and transfected with a plasmid encoding an irrelevant FLAG-tagged protein (pTG19274). The results are reported as OD450nm versus medium 1 / dilution and the mean and standard deviation of duplicate measurements or a single measurement.

[0020] Figure 4The CD40 binding activity of HeLa cell culture media infected with vaccinia virus and then transfected with different plasmids (pTG19965, pTG19966, pTG19968, pTG20038, pTG20042) carrying a cassette encoding the SPD-TNFSF construct and under the control of the pH5.R promoter (i.e., a poxvirus promoter) is shown. CD40-Fc was coated on a 96-well plate and different dilutions of the clarified supernatant containing the TNFSF construct were applied. The bound specific protein was detected with a non-blocking anti-CD40L monoclonal antibody followed by HRP-conjugated anti-IgG. The negative control was untransfected infected cells (VVTG18058). The results are reported as OD450nm versus medium 1 / dilution.

[0021] Figure 5 The CD40L agonist activity of HeLa cell culture media infected with vaccinia virus and then transfected with different plasmids carrying cassettes encoding SPD-TNFSF constructs (pTG19965, pTG19966, pTG19968, pTG20038 to pTG20042) and under the control of the pH5.R promoter (i.e., poxvirus promoter) is shown. HEK cells modified to express the reporter enzyme SEAP under the control of the CD40 inducible promoter were incubated with different dilutions of the clarified supernatant containing the SPD-TNFSF construct. After 20-24 hours of incubation, the SEAP enzyme activity in the culture media was measured. Negative controls were untransfected infected cells (VVTG18058) or non-infected cells (insert culture media). Results are reported as SEAP activity versus culture media 1 / dilution.

[0022] Figure 6 Shows Figure 4 The CD40 binding activity of the culture medium of HeLa cells infected with COPTG19967, COPTG19968 and COPTG19969 was measured. The negative control was cells infected with VVTG18058. The results are reported as OD450nm versus culture medium 1 / dilution.

[0023] Figure 7 Shows Figure 3 The CD40 agonist activity of the culture medium of HeLa cells infected with COPTG19967, COPTG19968 and COPTG19969 was measured. Negative controls were culture medium of cells infected with VVTG18058 or non-infected cells (injection culture medium). Results are reported as SEAP activity versus culture medium 1 / dilution.

[0024] Figure 84-1BBL constructs (pTG20032 only encodes the 4-1BBL extracellular domain and pTG20033 encodes the SPD-4-1BBL fusion protein according to the present invention) are expressed by the expression level of HeLa cells infected / transfected. Under reducing and non-reducing conditions, the clarified supernatants containing the different constructs were loaded on SDS-PAGE. The proteins were transferred to PDVF membranes and hybridized with HRP-conjugated anti-FLAG tag monoclonal antibodies for immunodetection. The negative control was cells infected with a plasmid (pTG19274) encoding an unrelated FLAG-tagged protein.

[0025] Fig. 9 Shown by using Promega 4-1BB reporter cells, with 4-1BBL construct (pTG20032 encoding only 4-1BBL extracellular domain and encoding according to the invention SPD-4-1BBL fusion protein pTG20033) infection / transfection of HeLa cells of the culture medium 4-1BB agonist activity.Negative control is the cell infected with the plasmid (pTG19274) of the protein of irrelevant FLAG mark. Summary of the invention

[0026] The present invention relates to a SPD-TNFSF fusion protein, which comprises or consists 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.

[0027] In one embodiment, the SPD-TNFSF fusion protein comprises or consists of the following: 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, wherein the N-terminal domain and the coiled-coil neck domain of SPD are directly fused without amino acid residues in between. 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.

[0028] 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, wherein 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, wherein X is an amino acid and G is a glycine amino acid.

[0029] X can be the same or different in each repeat, for example but not limited to, a repeat can be GAA or a repeat can be GTA.

[0030] Alternatively or in combination, the SPD-TNFSF fusion protein further comprises a linker between the coiled-coil neck domain and the TNF-superfamily ligand or its receptor binding domain. The linker is a glycine / serine linker and has a length of 4-20 amino acids, preferably a length of 8-16 amino acids, and more preferably a length of 12 amino acids.

[0031] 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.

[0032] The N-terminal domain is at least 85%, preferably at least 90%, more preferably at least 95%, or 100% identical to the amino acid sequence in SEQ ID NO:1.

[0033] In one embodiment, the SPD-TNFSF fusion protein comprises or consists of a sequence selected from the group consisting of 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.

[0034] The present invention also relates to a trimer fusion protein comprising three SPD-TNFSF fusion proteins. The present invention also relates to a multimer fusion protein comprising a plurality of trimer fusion proteins and forming a hexamer, a dodecamer, an octadecamer or a higher order oligomer, preferably a hexamer, more preferably a dodecamer.

[0035] 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 the group 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, or SEQ ID NO: 21.

[0036] The present invention also relates to an expression vector, such as mRNA, plasmid or virus, comprising an isolated nucleotide sequence encoding the SPD-TNFSF fusion protein. In one embodiment, the virus is an oncolytic virus selected from poxvirus, herpesvirus, reovirus, Seneca Valley virus (SVV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), measles virus, retrovirus, adenovirus, adenovirus-associated virus (AAV), herpes simplex virus (HSV), measles virus, foamy virus, alpha virus, slow virus, influenza virus, Sindbis virus, rhabdovirus, picornaviridae, coxsackie virus, parvovirus or chimera thereof, particularly preferably poxvirus. In another embodiment, the poxvirus belongs to the genus Orthopoxvirus, preferably selected from vaccinia virus, cowpox virus, canarypox virus and mousepox virus or their chimeras, particularly preferably vaccinia virus, in particular selected from Elstree, Wyeth, Copenhagen, Lister, Tian Tian and Western Reserve strains of vaccinia virus. In another embodiment, the poxvirus belongs to the genus Leporipoxvirus, selected from myxoma virus, rabbit fibroma virus and squirrel fibroma virus, particularly preferably myxoma virus. In a preferred embodiment, the oncolytic poxvirus is a modified poxvirus, which is defective in thymidine kinase (TK) activity due to an inactivating mutation in the J2R viral gene. Alternatively or in combination, the oncolytic poxvirus has a ribonucleotide reductase (RR) activity defect due to an inactivating mutation in the viral I4L and / or F4L gene.

[0037] Alternatively or in combination, the modified poxvirus can be further modified in the M2L locus (preferably a modification that results in suppressed expression of the viral m2 protein), thereby generating a modified poxvirus deficient in m2 function (m2-deficient poxvirus).

[0038] In another embodiment, the virus is a non-oncolytic virus, preferably a poxvirus, selected from pseudocowpox virus (PCPV), modified vaccinia virus Ankara (MVA), highly attenuated vaccinia virus strain (NYVAC), Swinepox virus (SWPV), Fowlpox virus (FPV) or a chimera thereof.

[0039] The present invention also relates to a method for producing the virus, comprising the steps of a) preparing producer cells, b) transfecting or infecting the prepared producer cells with the virus, c) culturing the transfected or infected producer cells under suitable conditions to allow production of the virus, d) recovering the produced virus from the culture of the producer cells, and optionally e) purifying the recovered virus.

[0040] The present invention also relates to a cell comprising the nucleotide sequence, mRNA, plasmid or virus of the present invention.

[0041] The present invention also relates to the use of SPD-TNFSF fusion protein, trimer fusion protein, multimer fusion protein, nucleotide sequence, mRNA, plasmid, virus or cell for medicine, preferably for the treatment of proliferative diseases, such as cancer and disorders associated with dysfunction of TNF cytokines, such as infectious diseases, inflammatory diseases, metabolic diseases, autoimmune diseases, degenerative diseases, apoptosis-related diseases and transplant rejection, more preferably proliferative diseases, and even more preferably for the treatment of cancer. The present invention also relates to SPD-TNFSF fusion protein, trimer fusion protein, multimer fusion protein, nucleotide sequence, mRNA, plasmid, virus or cell in combination with one or more chemotherapeutic drugs or immunotherapy products for effective treatment of cancer.

[0042] 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, and optionally a pharmaceutically acceptable diluent, carrier, solvent and / or excipient. In one embodiment, the pharmaceutical composition further comprises one or more effective chemotherapeutic drugs or immunotherapy products. In one embodiment, the pharmaceutical composition is used to treat cancer. In one embodiment, the pharmaceutical composition used is administered by a parenteral route, more preferably by an intravenous, subcutaneous or intramuscular route, and even more preferably by an intravenous route.

[0043] The present invention also relates to a method of treating cancer in an individual, comprising administering a therapeutically effective amount of the SPD-TNFSF fusion protein, nucleotide, mRNA, plasmid, virus, cell or pharmaceutical composition of the present invention. DETAILED DESCRIPTION

[0044] The present invention relates to SPD-TNFSF fusion proteins and viruses comprising one or more molecules encoding one or more SPD-TNFSF fusion proteins inserted into their genome.

[0045] definition

[0046] As used throughout the application, the terms "a" and "an" are used in their sense to refer to "at least one", "at least a first", "one or more" or "a plurality" of the referenced component or step, unless the context clearly dictates otherwise. For example, the term "cell" includes a plurality of cells, including mixtures thereof.

[0047] The term "one or more" refers to a number that is one or more than one (eg, 2, 3, 4, 5, etc.).

[0048] The term "and / or" used herein includes the meanings of "and", "or" and "all or any other combinations of elements connected by this term".

[0049] As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0050] As used herein, the terms "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain"), when used to define products, compositions, and methods, are open-ended and do not exclude additional, unrecited elements or method steps. Thus, a polypeptide "comprises" an amino acid sequence when the amino acid sequence may be part of the final amino acid sequence of the polypeptide. Such a polypeptide may have up to several hundred additional amino acid residues. "Substantially consisting of" means excluding other components or steps of any substantial significance. Thus, a composition consisting essentially of said components does not exclude trace contaminants and pharmaceutically acceptable carriers. When such an amino acid sequence is ultimately present only with a few additional amino acid residues, the polypeptide "substantially consists of" said amino acid sequence. "Consisting of" means excluding other components or steps more than trace elements. For example, when a polypeptide does not contain any amino acids but comprises said amino acid sequence, the polypeptide "consists of" an amino acid sequence.

[0051] The terms "polypeptide", "peptide" and "protein" refer to a polymer of amino acid residues comprising at least nine or more amino acids bound by peptide bonds. The polymer may be linear, branched or cyclic and may contain naturally occurring and / or amino acid analogs, and it may be interrupted by non-amino acids. As a general indication, if an amino acid polymer exceeds 50 amino acid residues, it is preferably referred to as a "polypeptide" or "protein", while if it is 50 amino acids or less in length, it is referred to as a "peptide".

[0052] In the context of the present invention, the terms "nucleic acid", "nucleic acid molecule", "polynucleotide" and "nucleotide sequence" are used interchangeably to define any length of polydeoxyribonucleotides (DNA) (e.g., cDNA, genomic DNA, plasmids, vectors, viral genomes, isolated DNA, probes, primers and any mixture thereof) or polyribonucleotides (RNA) (e.g., mRNA, antisense RNA, siRNA) or mixed polyribo-polydeoxyribonucleotide polymers. They include single-stranded or double-stranded, linear or circular, natural or synthetic, modified or unmodified polynucleotides. In addition, polynucleotides may contain non-naturally occurring nucleotides and may be interrupted by non-nucleotide components.

[0053] The term "analog" or "variant" as used herein refers to a molecule (polypeptide or nucleic acid) that exhibits one or more modifications relative to a natural counterpart. Any modification is contemplated, including substitution, insertion and / or deletion of one or more nucleotide / amino acid residues. Preferred are analogs that retain a degree of 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% identity to the sequence of the natural counterpart.

[0054] In a general manner, the term "identity" refers to the amino acid to amino acid or nucleotide to nucleotide correspondence between two polypeptide or nucleic acid sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of spaces and the length of each space that need to be introduced for optimal alignment. There are a variety of computer programs and mathematical algorithms in the art that can be used to determine the percent identity between amino acid sequences, such as the Blast program provided by NCBI or ALIGN in the Protein Sequence and Structure Atlas (Dayhoffed, 1981, Suppl., 3:482-9). Programs for determining identity between nucleotide sequences can also be obtained in specialized databases (e.g., Genbank, Wisconsin Sequence Analysis Package, BESTFIT, FASTA, and GAP programs). For purposes of this description, "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%.

[0055] As used herein, the term "isolated" refers to a protein, polypeptide, peptide, polynucleotide, vector, etc. that is removed from its natural environment (i.e., separated from at least one other component with which it is naturally associated or found in nature). For example, a nucleotide sequence is isolated when it is separated from the sequences with which it is normally associated in nature (e.g., separated from a genome), but it may be associated with heterologous sequences.

[0056] The terms "obtained from", "originating from" or "originating from" are used to identify the original source of a component (eg, polypeptide, nucleic acid molecule), but are not meant to limit the method of making the component, which may be made, for example, by chemical synthesis or recombinant means.

[0057] As used herein, the term "host cell" should be broadly understood without any limitation on a specific tissue in a tissue, organ or separated cell. These cells can be a unique type of cell or a group of different types of cells, such as cultured cell lines, primary cells and dividing cells. 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, plants and mammals (eg, humans or non-human) cells and cells capable of producing viruses (oncolytic or non-oncolytic viruses) and / or fusion proteins for the present invention. The term also includes cells that may be or have been recipients of the vectors described herein and the offspring of such cells.

[0058] The terms "virus", "viral particle", "viral vector" and "virion" are used interchangeably and should be widely understood to mean a solvent comprising at least one element of a wild-type viral genome, and can be packaged into or packaged into a viral particle. Although the viral particle may contain or not contain nucleic acid (i.e., a viral genome), it is preferred that the virus comprises a DNA or RNA viral genome packaged into a viral particle (or a viral particle), and is infectious (i.e., able to infect and enter a host cell or individual). Ideally, 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 may be within endogenous viral genes (e.g., coding and / or regulatory sequences) and / or within intergenic regions. In addition, modifications may be silent or not (e.g., resulting in modified viral gene products). Modifications may be performed in a variety of ways known to those skilled in the art using conventional molecular biology techniques. Desirably, the modifications encompassed by the present invention affect, for example, the virulence, toxicity, pathogenicity or replication of the virus, compared to viruses without such modifications, but do not completely impair the infection and production of at least permitted cells.

[0059] The term "oncolytic virus" includes any naturally occurring, engineered or otherwise modified virus. As used herein, the term "oncolytic virus" refers to a virus that can selectively replicate in dividing cells (e.g., proliferating cells, such as cancer cells), with the purpose of slowing down the growth of the dividing cells and / or dissolving the dividing cells (in vitro or in vivo), while not showing or showing minimal replication in non-dividing cells (e.g., primary cells). Typically, an oncolytic virus contains a viral genome packaged into viral particles (or virions) and is infectious (i.e., capable of infecting and entering a host cell or individual). As used herein, the term encompasses DNA or RNA vectors (depending on the virus in question) and viral particles produced therefrom.

[0060] As used herein, the term "chimera", "virus chimera" or "virus chimera" refers to a virus obtained from homologous recombination between several different strains of a virus. Several chimeras obtained by mixing genomes from different poxviruses have been described and are available to those skilled in the art (e.g., CF189 chimeras obtained from ORF and pseudovaccinia virus (Choi et al., novel chimeric parapoxvirus CF189 as oncolytic immunotherapy for triple-negative breast cancer. Surgery Vol. 163, No. 2, February 2018, pp. 336-342); CF33 chimeras obtained from multiple strains of VV, vaccinia and rabbit pox (Chaurasiya, S. et al., 2020, Cancer Gene Ther 27, 125–135).

[0061] The term "non-oncolytic virus" encompasses any virus that is not defined as an oncolytic virus.

[0062] As used herein, the term "treatment" (and any form of therapy, such as "treating" or "treat") includes prevention (e.g., preventive measures in individuals at risk of having a pathological condition to be treated) and / or treatment (e.g., in individuals diagnosed with a pathological condition) ultimately associated with conventional treatment modalities. The result of treatment is slowing, curing, ameliorating, or controlling the progression of the target pathological condition. For example, if, following administration of a fusion protein as described herein, a virus as described herein, or a combination thereof as described herein, the individual shows an observable improvement in their clinical state, the subject is successfully treated for cancer.

[0063] As used herein, the term "administering" (or any form of administration, such as "administered") refers to the delivery of a therapeutic agent, such as a virus described herein, to a subject.

[0064] As used herein, the term "proliferative disease" includes any disease or condition caused by uncontrolled cell growth and spread, including cancer and some cardiovascular diseases (restenosis caused by proliferation of smooth muscle cells of the blood vessel wall, etc.). The term "cancer" can be used interchangeably with any of the terms "tumor", "malignant tumor", "neoplasm", etc. The term is intended to include any type of tissue, organ or cell, any stage of malignancy (e.g., from prelesion to stage IV).

[0065] As used herein, the term "disorder associated with TNF cytokine dysfunction" encompasses any disease, disorder or condition that results in TNF signaling dysfunction.

[0066] The term "individual" generally refers to an organism to which any product and method of the present invention is needed or to which any product and method of the present invention is beneficial. Typically, the organism is a mammal, particularly a mammal selected from livestock, farm animals, sports animals and primates. Preferably, the subject is a person who has been diagnosed with a proliferative disease or is at risk of a proliferative disease, such as cancer or a condition associated with TNF cytokine dysfunction. When it comes to human organisms, the terms "individual" and "patient" can be used interchangeably, and encompass males and females. The individual to be treated can be a newborn, infant, young adult, adult or elderly.

[0067] As used herein, the term "combination" or "association" refers to any possible arrangement of the various components (e.g., fusion protein or virus and one or more substances effective in anti-cancer therapy). This arrangement includes mixtures of the components as well as separate combinations for simultaneous or sequential administration. The present invention encompasses combinations containing equimolar concentrations of each component as well as combinations with very different concentrations. It should be understood that the optimal concentration of each component in the combination can be determined by a person skilled in the art.

[0068] As used herein, the term "chemotherapeutic agent" or "immunotherapeutic product" refers to a product comprising one or more antigens which, when properly delivered to an individual, are intended to induce or activate an immune response, whether specific or nonspecific, humoral or cellular.

[0069] SPD-TNFSF fusion protein

[0070] The term "fusion protein" as used herein refers to the covalent linkage of two or more polypeptides in a single protein chain, which is carried out genetically, i.e., by in-frame fusion of nucleic acid molecules encoding each of the polypeptides or fragments thereof. "In-frame fusion" means that the expression of the fused coding sequences produces a single polypeptide having functional properties derived from each of the original polypeptides. The fusion can be direct (i.e., without additional amino acid residues between them) or indirect (e.g., through a linker between the fusion polypeptides).

[0071] The term "SPD" or "surfactant protein D" as used herein refers to a functional fragment of natural surfactant protein D or its derivatives. SPD is preferably derived from a mammal, such as a human, mouse, rabbit, non-human primate or pig, preferably from a human or non-human primate, more preferably from a human.

[0072] In the context of the present invention, the SPD-TNFSF fusion protein comprises or consists of: a fusion of surfactant protein D (SPD) and a TNFSF ligand or its receptor binding domain. 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 amino acid residues in between. 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.

[0073] The N-terminal domain of the present invention comprises an amino acid sequence that is at least 85%, preferably at least 90%, more preferably at least 95%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 1, in particular amino acids 21-45 of native human SPD (SEQ ID NO: 22).

[0074] The coiled-coil neck domain of the present invention comprises an amino acid sequence that is at least 85%, preferably at least 90%, and more preferably at least 95% identical to the amino acid sequence shown in SEQ ID NO: 23, particularly amino acids 223-252 of native human SPD (SEQ ID NO: 22).

[0075] TNFSF ligands can be selected from TNF superfamily members, 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 its receptor binding. TNFSF ligands are preferably derived from mammals, such as humans, mice, rabbits, non-human primates or pigs, preferably derived from humans or non-human primates, more preferably derived from humans.

[0076] In a preferred embodiment, the member of TNFSF or its receptor binding domain is selected from:

[0077] CD40L, 4-1BBL, Baff, APRIL, EDA, GITRL, OX40L, CD70, TL1A, LIGHT, LTαβ2, RANKL, TWEAK, FASL, TRAIL, TNF, and LTα or their receptor binding domains;

[0078] Class II TNFSF members, preferably from CD40L, 4-1BBL, Baff, APRIL, EDA, OX40L, CD70, TWEAK, FASL, TRAIL and TNF or their receptor binding domains;

[0079] ● A TNFSF member involved in immune cell activation, preferably from CD40L, 4-1BBL, GITRL, OX40L, CD70, TL1A or its receptor binding domain;

[0080] ● a class II TNFSF member involved in immune cell activation, preferably from CD40L, 4-1BBL, OX40L, CD70 or a receptor binding domain thereof; or

[0081] CD40L, 4-1BBL or their receptor binding domains.

[0082] Most preferably, the member of TNFSF or its receptor binding domain is selected from CD40L or its receptor binding domain.

[0083] TNFSF Members

[0084] TNFSF comprises a number of structurally related members (also called ligands) that organize lymphoid tissue development, costimulate lymphocyte activation, and can increase lymphocyte survival and function or induce cell death, all by binding to their cognate receptors, forming the tumor necrosis factor receptor superfamily (TNFRSF).

[0085] TNFRSF can be divided into two different classes, depending on their ability to be activated by the soluble ligand trimer of TNFSF (Kucka K, 2021, Front Cell Dev Biol. 11; 8:615141).

[0086] The class I receptors of TNFRSF are strongly activated by soluble ligand trimers, including BaffR (ligand = Baff), DR3 (ligand = TL1A), GITR (ligand = GITRL), LTβR (ligand = LTαβ2 or LIGHT), and TNFR1 (ligand = TNF or LTα).

[0087] The class II receptors for TNFRSF, despite high affinity binding, cannot be properly activated by soluble ligand trimers including CD40L, 4-1BBL, Baff, APRIL, EDA, OX40L, CD70, TWEAK, FASL, TRAIL, and TNF.

[0088] The limited responsiveness of class II TNFRs to soluble TNFLs can be overcome by physical linkage of two or more soluble ligand trimers or, alternatively, by anchoring the soluble ligand molecules to the cell surface or extracellular matrix. In the context of the present invention, the binding of members of the TNFSF to class II TNFRs is preferred, since the capacity of the fusion proteins according to the invention is truly conditional, since the activation of their cognate TNFRs is dependent on the presence of PD-L1-positive tumor cells in the tumor microenvironment.

[0089] Therefore, in a preferred embodiment, the TNFSF member comprised in the fusion protein according to the invention is selected from the class II TNFSF members, preferably selected from CD40L, 4-1BBL, Baff, APRIL, EDA, OX40L, CD70, TWEAK, FASL, TRAIL and TNF or its receptor binding domain.

[0090] TNFSF members can also be classified according to their known functions. In the context of the present invention, TNFSF members involved in immune cell activation can preferably be included in the fusion protein according to the present invention. TNFSF members involved in immune cell activation include CD40L, 4-1BBL, GITRL, OX40L, CD70, TL1A or its receptor binding domain (Croft M. et al., 2017, Nat Rev Rheumatol.; 13 (4): 217-233).

[0091] "CD40L", "CD40 ligand", "CD40LG", "tumor necrosis factor superfamily member 5", "TNFSF5" and "CD154" are used interchangeably herein and refer to members of the TNFSF, which, when assembled into homotrimers and trans-interacting with its receptor CD40, play a key role in the initiation of adaptive immune responses. The CD40L-CD40 interaction leads to the activation of cells carrying CD40, which then express adhesion (ICAM), co-stimulatory (CD80 / CD86) and MHC I and II molecules as well as cytokines / chemokines (TNFα, IL6...). In the tumor microenvironment, adhesion molecules and cytokines / chemokines act together to induce infiltration and activation of immune cells, which ultimately destroy tumor cells and transform the tumor from an immunosuppressive microenvironment to an immunoactive microenvironment (Richards et al., 2020, Hum Vaccin Immunother. 16(2):377-387). Therefore, CD40L is a member of the TNFSF involved in the activation of immune cells.

[0092] As TNF, CD40L assembles into a homotrimer and interacts in trans with its receptor CD40 through its extracellular portion. CD40 is the class II receptor for TNFRSF.

[0093] CD40L is a member of TNFSF, which is involved in immune cell activation and binds to the class II receptor of TNFRSF. Therefore, it is a particularly preferred TNFSF member for the fusion protein of the present invention.

[0094] The specific CD40L protein that can be included in the fusion protein of 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 intended use in humans, human CD40L is preferably used. Human CD40L corresponds to Entrez gene ID 959, and the complete amino acid sequence of human CD40L can be found under GenBank accession number NP_000065.1 (version January 17, 2022).

[0095] "4-1BBL", "4-1BB ligand", "CD137L", "tumor necrosis factor superfamily member 9" and "TNFSF9" are used interchangeably herein and refer to transmembrane cytokines that act as ligands for TNFRSF9 / 4-1BB, which are co-stimulatory receptor molecules in T lymphocytes. This cytokine and its receptor are involved in the antigen presentation process and the production of cytotoxic T cells. Therefore, 4-1BBL is a TNFSF member involved in immune cell activation.

[0096] Its receptor 4-1BB is a class II receptor for TNFRSF.

[0097] 4-1BBL is a member of TNFSF, which is involved in immune cell activation and binds to the class II receptor of TNFRSF. Therefore, it is a particularly preferred TNFSF member for the fusion protein of the present invention.

[0098] The specific 4-1BBL protein that may be included in the fusion protein of 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 the intended use for 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 under GenBank accession number NP_003802.1 (version February 27, 2022).

[0099] "OX40L", "OX40 ligand", "CD252", "CD134L", "tumor necrosis factor superfamily member 4", and "TNFSF4" are used interchangeably herein to refer to an inducible molecule expressed on several cell types, although arguably the most important on antigen presenting cells (APCs). OX40L can trigger signaling through its receptor OX40, leading to a range of activities including the expansion and accumulation of effector T cells and their cytokine production. Thus, OX40L is a TNFSF member involved in immune cell activation.

[0100] Its receptor OX40 is a class II receptor for TNFRSF.

[0101] OX40L is a member of TNFSF, which is involved in immune cell activation and binds to the class II receptor of TNFRSF, and is therefore a particularly preferred TNFSF member for the fusion protein of the present invention.

[0102] The specific OX40L protein that can be included in the fusion protein of 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 intended use in humans, human OX40L is preferably used. Human OX40L corresponds to Entrez gene ID 7292, and the complete amino acid sequence of human OX40L can be found under GenBank accession number NP_003317.1 (version March 16, 2022).

[0103] "CD70", "CD27L", "CD27LG", "tumor necrosis factor superfamily member 7", and "TNFSF7" are used interchangeably herein and refer to a molecule that can provide signals to T cells to control their accumulation and responsiveness by interacting with its receptor CD27, similar to what is seen with OX40, GITR, and DR3. Thus, CD70 is a TNFSF member involved in the activation of immune cells.

[0104] Its receptor CD27 is a class II receptor for TNFRSF.

[0105] CD70 is a member of TNFSF, which is involved in immune cell activation and binds to the class II receptor of TNFRSF. Therefore, it is a particularly preferred TNFSF member for the fusion protein of the present invention.

[0106] The specific CD70 protein that can be included in the fusion protein of 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 intended use in humans, human CD70 is preferably used. Human CD70 corresponds to Entrez gene ID 970, and the complete amino acid sequence of human CD70 can be found under GenBank accession number NP_001317261.1 (version January 9, 2022).

[0107] "Baff", "B cell activating factor", "CD257", "tumor necrosis factor superfamily member 13b", "TNFSF13B", "tumor necrosis factor superfamily member 20" and "TNFSF20" are used interchangeably herein and refer to molecules that primarily, but not exclusively, control B cell activity.

[0108] It binds to two receptors for TNFRSF: BaffR and TACI. BaffR is a class I receptor for TNFRSF, while TACI is a class II receptor for TNFRSF.

[0109] As a TNFSF member that binds to the class II receptor of TNFRSF (TACI), BAFF is a preferred TNFSF member for the fusion protein of the present invention.

[0110] The specific Baff protein that can be included in the fusion protein of 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 intended use in humans, human Baff is preferably used. Human Baff corresponds to Entrez gene ID 10673, and the complete amino acid sequence of human Baff can be found under GenBank accession number NP_006564.1 (version February 20, 2022).

[0111] "APRIL," "CD256," "tumor necrosis factor superfamily member 13," and "TNFSF13" are used interchangeably herein and refer to a ligand that is important for B cell development.

[0112] It binds to two receptors for TNFRSF: BCMA and TACI, both of which are class II receptors for TNFRSF.

[0113] As a TNFSF member that binds to the class II receptor of TNFRSF, APRIL is a preferred TNFSF member for the fusion protein of the present invention.

[0114] The specific APRIL protein that can be included in the fusion protein of 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 intended use in humans, human APRIL is preferably used. Human APRIL corresponds to Entrez gene ID 8741, and the complete amino acid sequence of human APRIL can be found under GenBank accession number NP_003799.1 (version January 23, 2022).

[0115] "EDA-A1," "EDA-A2," "EDA," "ectodysplasin A," and "tumor necrosis factor ligand 7C" are used interchangeably herein and refer to proteins involved in cell-cell signaling during ectodermal organ development.

[0116] Its receptor EDAR is a class II receptor for TNFRSF.

[0117] As a TNFSF member that binds to the class II receptor of TNFRSF, EDA is a preferred TNFSF member for the fusion protein of the present invention.

[0118] The specific EDA protein that can be included in the fusion protein of 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 intended use in humans, human EDA is preferably used. Human EDA corresponds to Entrez gene ID 1896, and the complete amino acid sequence of human EDA can be found under GenBank accession number NP_001390.1 (version February 13, 2022).

[0119] "GITRL", "glucocorticoid-induced TNF receptor-related ligand", "tumor necrosis factor superfamily member 18" and "TNFSF18" are used interchangeably herein and refer to an inducible molecule expressed in professional APCs and other cell types such as endothelial cells. Its receptor, glucocorticoid-induced TNF receptor-related protein (GITR, also known as TNFRSF18), can stimulate T cell, dendritic cell and B cell activation. GITRL is therefore a TNFSF member involved in immune cell activation.

[0120] Its receptor GITR is a class I receptor for TNFRSF.

[0121] As a member of TNFSF involved in immune cell activation, GITRL is a preferred TNFSF member of the fusion protein of the present invention.

[0122] The specific GITRL protein that can be included in the fusion protein of 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 intended use in humans, human GITRL is preferably used. Human GITRL corresponds to Entrez gene ID 8995, and the complete amino acid sequence of human GITRL can be found under GenBank accession number NP_005083.3 (version February 20, 2022).

[0123] "TL1A", "VEGI", "tumor necrosis factor superfamily member 15" and "TNFSF15" are used interchangeably herein to refer to a protein that can be induced in APCs such as dendritic cells and macrophages, as well as in endothelial cells. Its binding to its receptor, the stimulatory receptor "death receptor 3" ("DR3", also known as "TNFRSF25") expressed on T cells, can modulate effector T cell accumulation and / or responsiveness. Thus, TL1A is a TNFSF member involved in the activation of immune cells. This cytokine has also been found to inhibit endothelial cell proliferation and may therefore function as an angiogenesis inhibitor.

[0124] Its receptor DR3 is a class I receptor for TNFRSF.

[0125] As a member of TNFSF involved in immune cell activation, TL1A is a preferred TNFSF member of the fusion protein of the present invention.

[0126] The specific TL1A protein that can be included in the fusion protein of 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 intended use in humans, human TL1A is preferably used. Human TL1A corresponds to Entrez gene ID 9966, and the complete amino acid sequence of human TL1A can be found under GenBank accession number NP_005109.2 (version February 27, 2022).

[0127] "LIGHT," "CD258," "tumor necrosis factor superfamily member 14," and "TNFSF14" are used interchangeably herein and refer to a protein that binds to two receptors for TNFRSF: the herpes virus entry mediator (HVEM, also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14)) and the lymphotoxin-β receptor (LTβR). The LIGHT-HVEM interaction is responsible for most of the immunostimulatory properties of LIGHT. HVEM is expressed on lymphocytes, NK cells, smooth muscle, and epithelial cells, and is an important T cell co-stimulator that leads to activation, proliferation, and survival. In the context of anti-tumor immune support, LIGHT-LTβR signaling has a wide range of effects, ranging from influencing the susceptibility of cancer cells to immune responses, acting to repair disrupted tumor vasculature, and to supporting the 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).

[0128] LTβR is a class I receptor for TNFRSF. HVEM is a class I receptor for TNFRSF.

[0129] The specific LIGHT protein that can be included in the fusion protein of 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 intended use in humans, human LIGHT is preferably used. Human LIGHT corresponds to Entrez gene ID 8740, and the complete amino acid sequence of human LIGHT can be found under GenBank accession number NP_003798.2 (version February 20, 2022).

[0130] "RANKL", "RANK ligand", "receptor activator of nuclear factor-κB ligand", "CD254", "tumor necrosis factor superfamily member 11" and "TNFSF11" are used interchangeably herein and refer to the ligand of osteoprotegerin, which functions as a key factor in osteoclast differentiation and activation. The protein has also been shown to be a dendritic cell survival factor and is involved in the regulation of T cell-dependent immune responses.

[0131] Its receptor RANK (or "receptor activator of nuclear factor-κB") is a class I receptor for TNFRSF.

[0132] The specific RANKL protein that may be included in the fusion protein of 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 intended use in humans, human RANKL is preferably used. Human RANKL corresponds to Entrez gene ID 8600, and the complete amino acid sequence of human RANKL can be found under GenBank accession number NP_003692.1 (version February 27, 2022).

[0133] "TWEAK", "tumor necrosis factor superfamily member 12", and "TNFSF12" are used interchangeably herein and refer to a cytokine that has overlapping signaling functions with TNF, but displays a broader tissue distribution. It exists in both membrane-bound and secreted forms and can induce apoptosis through multiple pathways of cell death in a cell type-specific manner. It has also been found to promote proliferation and migration of endothelial cells and thus acts as a regulator of angiogenesis.

[0134] Its receptor FN14 (also known as TWEAKR) is a class II receptor for TNFRSF.

[0135] As a TNFSF member that binds to the class II receptor of TNFRSF, TWEAK is a preferred TNFSF member for the fusion protein of the present invention.

[0136] The specific TWEAK protein that can be included in the fusion protein of 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 intended use in humans, human TWEAK is preferably used. Human TWEAK corresponds to Entrez gene ID 8742, and the complete amino acid sequence of human TWEAK can be found under GenBank accession number NP_003800.1 (version February 20, 2022).

[0137] "FASL", "FAS ligand", "FASLG", "CD178", "CD95 ligand", "CD95L", "tumor necrosis factor superfamily member 6", and "TNFSF6" are used interchangeably herein and refer to a transmembrane protein whose primary function is to induce apoptosis 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 cell death induced by cytotoxic T lymphocytes.

[0138] FAS is the class II receptor for TNFRSF.

[0139] As a TNFSF member that binds to the class II receptor of TNFRSF, FASL is a preferred TNFSF member for the fusion protein of the present invention.

[0140] The specific FASL protein that can be included in the fusion protein of 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 intended use in humans, human FASL is preferably used. Human FASL corresponds to Entrez gene ID 356, and the complete amino acid sequence of human FASL can be found under GenBank accession number NP_000630.1 (version February 27, 2022).

[0141] "TRAIL", "CD253", "tumor necrosis factor superfamily member 10", and "TNFSF10" are used interchangeably herein to refer to a protein that preferentially induces apoptosis in transformed and tumor cells, but does not appear to kill normal cells, despite being expressed at significant levels in most normal tissues. TRAIL binds to several members of the TNF receptor superfamily, including TNFRSF10A / TRAILR1, TNFRSF10B / TRAILR2, TNFRSF10C / TRAILR3, TNFRSF10D / TRAILR4, and possibly TNFRSF11B / OPG. The activity of TRAIL can be regulated by binding to the decoy receptors TNFRSF10C / TRAILR3, TNFRSF10D / TRAILR4, and TNFRSF11B / OPG, which are unable to induce apoptosis. Binding of TRAIL to its receptors has been shown to trigger activation of MAPK8 / JNK, caspase 8, and caspase 3.

[0142] Both TRAILR1 and TRAILR2 are class II receptors for TNFRSF.

[0143] As a TNFSF member that binds to the class II receptor of TNFRSF, TRAIL is a preferred TNFSF member for the fusion protein of the present invention.

[0144] The specific TRAIL protein that may be included in the fusion protein of 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 intended use in humans, human TRAIL is preferably used. Human TRAIL corresponds to Entrez gene ID 8743, and the complete amino acid sequence of human TRAIL can be found under GenBank accession number NP_003801.1 (version March 17, 2022).

[0145] "TNF," "tumor necrosis factor," "TNFA," "TNFα," "tumor necrosis factor superfamily member 2," and "TNFSF2" are used interchangeably herein and refer to a multifunctional proinflammatory cytokine that is involved in regulating a broad spectrum of biological processes, including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation.

[0146] TNF binds to two receptors for TNFRSF: TNFR1 (also known as "TNFRSF1A") and TNFR2 (also known as "TNFRSF1B" or "TNFBR"). TNFR1 is a class I receptor for TNFRSF, while TNFR2 is a class II receptor for TNFRSF.

[0147] As a TNFSF member that binds to the class II receptor of TNFRSF, TNF is a preferred TNFSF member for the fusion protein of the present invention.

[0148] The specific TNF protein that can be included in the fusion protein of 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 intended use in humans, human TNF is preferably used. Human TNF corresponds to Entrez gene ID 7124, and the complete amino acid sequence of human TNF can be found under GenBank accession number NP_000585.2 (version March 17, 2022).

[0149] "CD30L," "CD153," "TNFSF8," and "tumor necrosis factor superfamily member 8" are used interchangeably herein and refer to a protein that exhibits restricted expression in activated T- and B-cell subsets and regulates proliferation / apoptosis and antibody responses under non-pathological conditions.

[0150] Its receptor TNFRSF8 (also known as tumor necrosis factor receptor superfamily member 8 or CD30) is a class I receptor for TNFRSF.

[0151] The specific CD30L protein that can be included in the fusion protein of 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 intended use 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 under GenBank accession number AH005843.2 (version June 10, 2016).

[0152] "LTα", "lymphotoxin alpha", "TNFB", "TNFβ", "tumor necrosis factor superfamily member 1", and "TNFSF1" are used interchangeably herein to refer to a cytokine produced by lymphocytes that is highly inducible, secreted, and forms a heterotrimer with lymphotoxin-β, thereby anchoring lymphotoxin-α to the cell surface. The protein also mediates a variety of inflammatory, immunostimulatory, and antiviral responses, participates in the formation of secondary lymphoid organs during development, and plays a role in apoptosis.

[0153] LTα binds to TNFR1 (also known as "TNFRSF1A"), the class I receptor for TNFRSF.

[0154] The specific LTα protein that can be included in the fusion protein of 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 intended use 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 under GenBank accession number NP_001153212.1 (version March 10, 2022).

[0155] "LTβ", "lymphotoxin beta", "TNFC", "tumor necrosis factor superfamily member 3" and "TNFSF3" are used interchangeably herein to refer to a protein that anchors lymphotoxin-α to the cell surface by forming a heterotrimer. The major form on the surface of lymphocytes is the lymphotoxin-α1 / β2 complex (also called "LTαβ2", e.g., 1 molecule of α / 2 molecules of β), which is the major ligand for the lymphotoxin-β receptor (also called LTβR), a class I receptor for TNFRSF. The minor complex is lymphotoxin-α2 / β1. LTB is an inducer of the inflammatory response system and is involved in the normal development of lymphoid tissue.

[0156] The specific LTβ protein that can be included in the fusion protein of 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 intended use 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 under GenBank accession number NP_002332.1 (version February 20, 2021).

[0157] Functional fragments or variants of TNFSF members

[0158] Although the SPD-TNFSF fusion protein according to the present invention may include the entire wild-type member of TNFSF, it may alternatively include a functional fragment or a functional derivative thereof.

[0159] A "functional fragment" of a TNFSF member refers to a fragment (i.e., a partial amino acid sequence of a TNFSF integral member) having one or more insertions, deletions, truncations and / or substitutions, and which retains the function of the TNFSF integral member, i.e., its ability to trimerize and bind to and activate its TNFRSF receptor.

[0160] In particular, TNFSF members exist as transmembrane proteins, but their transmembrane and intracellular regions are not required for trimerization and binding and activation of their TNFRSF receptors.

[0161] Therefore, in a preferred embodiment, the TNFSF member fragment contained in the SPD-TNFSF fusion protein of the present invention is an extracellular fragment of the TNFSF member, i.e., a fragment without the transmembrane and intracellular parts of the TNFSF member. All or part of the extracellular domain may be contained in the extracellular fragment, provided that the fragment retains its ability to trimerize and bind to and activate its TNFRSF receptor.

[0162] In a preferred embodiment, the TNFSF ligand or its receptor of the fusion protein binds to amino acids 119-261 selected from human CD40L (SEQ ID NO: 24), in particular human CD40L (SEQ ID NO: 25).

[0163] In a preferred embodiment, the TNFSF ligand or its receptor binding domain of the fusion protein is selected from murine CD40L (SEQ ID NO: 26), in particular amino acids 115-260 of murine CD40L (SEQ ID NO: 27).

[0164] In another embodiment, the TNFSF ligand or receptor binding domain thereof of the fusion protein is selected from amino acids 80-254 of human 4-1-BBL (SEQ ID NO: 28), in particular human 4-1-BBL (SEQ ID NO: 29).

[0165] In yet another embodiment, the TNFSF ligand or receptor binding domain thereof of the fusion protein is selected from the group consisting of murine 4-1-BBL (SEQ ID NO: 30), in particular amino acids 139-309 of murine 4-1-BBL (SEQ ID NO: 31).

[0166] In one embodiment, the SPD-TNFSF fusion protein includes 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, wherein X is an amino acid, G is a glycine amino acid, and wherein each X in each repeat 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:

[0167]

[0168]

[0169] Table 1. Collagen domains and corresponding (GXX) repeat numbers. It should be understood that "6-N-Gly" means that the collagen domain under consideration contains 6 (GXX) repeats and no N-glycosylation sites, while "6+N-Gly" means that the collagen domain under consideration contains 6 (GXX) repeats, including N-glycosylation sites. It should be understood that "12" or "12bis" means that the collagen domain under consideration contains 12 (GXX) repeats, however "12" and "12bis" present different sequences.

[0170] In one embodiment, the SPD-TNFSF fusion protein described herein may further comprise a linker located between the coiled-coil neck domain and the TNFSF ligand or its receptor binding domain. Typically, the linker consists 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., essentially composed of glycine and serine amino acids. The linker is preferably 4-20 amino acids in length, in particular 4, 8, 12, 16 or 20 amino acids (e.g., 1, 2, 3 or 4 repeats of GGGS, GGSSG or SGSGS, or 1 or 2 repeats of GSGSGSGSGS). More preferably, the linker is 8 to 16 amino acids in length, even more preferably 12 amino acids. For guidance, a suitable linker for use in 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 two fusion partners is within the capabilities of one skilled in the art.

[0171] In one embodiment, the SPD-TNFSF fusion protein described herein comprises a collagen domain and a linker, wherein the collagen domain and the linker are located at the N-terminus and C-terminus of the coiled-coil neck domain, respectively. A suitable SPD-TNFSF fusion protein comprises the amino acid sequence shown in SEQ ID NO:11.

[0172] In the context of the present invention, it may be advantageous to include additional regulatory elements to facilitate expression, trafficking and biological activity of the SPD-TNFSF fusion protein.

[0173] In the context of the present invention, the SPD-TNFSF fusion protein may additionally comprise a signal peptide at the N-terminus of the SPD-TNFSF fusion protein to influence the expression level, secretion, solubility or other properties of the protein. Suitable signal peptides are known in the art. They may be obtained from cellular polypeptides or viral polypeptides, such as polypeptides of immunoglobulins, tissue plasminogen activator, insulin, rabies glycoprotein, HIV viral envelope glycoprotein or measles virus F protein, or may be synthetic (see, for example, WO2008 / 138649). For guidance, signal peptides suitable for use in the present invention include the amino acid sequences shown in SEQ ID NO: 39 (MLLFLLSALVLLTQPLGYLE), SEQ ID NO: 40 (MGLGLQWVFFVALLKGVHC) or SEQ ID NO: 41 (MGWSCIILFLVATATGVHS). It is also conceivable to add a transmembrane domain to facilitate anchoring of the TNFSF fusion protein in the appropriate membrane of the cell (e.g., the plasma membrane). The transmembrane domain is usually inserted at the N-terminus or C-terminus of the protein. Various transmembrane domains are known in the art (see, for example, WO99 / 03885).

[0174] In the context of the present invention, the SPD-TNFSF fusion protein may further comprise a tag peptide (generally, a short peptide sequence that can be recognized by available antisera or compounds) for subsequent expression, transport or purification of the SPD-TNFSF protein or infected host cells expressing such a fusion protein. The tag peptide can be detected by immunodetection analysis using anti-tag antibodies. A variety of tag peptides can be used in the context of the present invention, including but not limited to PK tags, FLAG (DYKDDDK, SEQ ID NO: 42, GDYKDDDK, SEQ ID NO: 43, GSDYKDDDDK, SEQ ID NO: 44 or HHHHHHDYKDDDDKLVPRGS, SEQ ID NO: 45), MYC tags (QKLISEEDL, SEQ ID NO: 46), HIS tags (usually a fragment of 4 to 10 histidine residues), HA tags (YPYDVPDYA; SEQ ID NO: 47), HSV tags (QPELAPEDPED; SEQ ID NO: 48), VSV tags (YTDIEMNRLGK; SEQ ID NO: 49) and e tags (US 6,686,152). For guidance, suitable tag peptides suitable for use in the present invention include the amino acid sequences shown in SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO: 45. The tag peptide can be independently positioned at the N-terminus of the protein, or at its C-terminus, or inside the protein, or when multiple tags are used, it can be positioned at any of these positions. When a signal peptide already exists at the N-terminus of the fusion protein, it is preferred to insert a tag peptide at the C-terminus of the fusion protein of the present invention.

[0175] As another example, glycosylation can be altered to increase the biological activity of the SPD-TNFSF fusion protein. For example, such modification can be achieved by mutating one or more residues within the glycosylation site.

[0176] As used herein, the term "oligomerization" or "polymerization" refers to the ability of at least two monomer units to form a complex. The binding may be specific (requiring structural complementarity between the amino acid residues of the two partners at the binding site, and requiring one or more types of electrostatic forces, hydrogen bonds, hydrophobic forces and / or van der Waals forces to maintain binding) or non-specific (interacting through one or more types of the above forces, 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, so that disulfide bonds can be formed between oligomeric proteins (i.e., intermolecular disulfide bonds). 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 a trimer of two SPD-TNFSF fusion proteins (hexamer), twelve SPD-TNFSF fusion proteins (dodecamer), eighteen SPD-TNFSF fusion proteins (octadecamer) or strictly greater than eighteen SPD-TNFSF fusion proteins (highly ordered oligomer).

[0177] The fusion protein may be a monomeric protein or a multimeric protein. Preferably, the fusion protein is present in a multimeric form, which is composed of three SPD-TNFSF fusion proteins self-assembled in a trimer SPD-TNFSF fusion protein, and the trimer SPD-TNFSF fusion proteins may be identical or different. Preferably, the multimeric form is a dodecamer form composed of an assembly of four identical trimer forms.

[0178] Oncolytic viruses

[0179] The oncolytic virus of the present invention can be obtained from any virus member currently identified, provided that the virus has oncolytic effect, i.e., it tends to selectively replicate and kill dividing cells compared with non-dividing cells. It can be a natural virus of natural oncolysis, or it can be engineered by modifying one or more viral genes to increase tumor selectivity and / or preferential replication in dividing cells, such as genes involved in DNA replication, nucleic acid metabolism, host tropism, surface attachment, toxicity, cracking and diffusion (see, e.g., Kirn et al., 2001, Nat. Med. 7: 781; Wong et al., 2010, Viruses 2: 78-106). It is also conceivable that one or more viral genes are placed under the control of an event or tissue-specific regulatory element (e.g., promoter).

[0180] Exemplary oncolytic viruses include, but are not limited to, reovirus, Senecavirus (SVV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), herpes simplex virus (HSV), measles virus, retrovirus, influenza virus, Sindbis virus, poxvirus, adenovirus, adenovirus-associated virus (AAV), morbillivirus, foamy virus, alphavirus, lentivirus, rhabdovirus, picornaviridae, coxsackievirus, parvovirus, or chimeras thereof.

[0181] In one embodiment, the oncolytic virus of the present invention is obtained from a reovirus. Representative examples include Reolysin (developed by Oncolytics Biotech; NCT01166542).

[0182] In one embodiment, the oncolytic virus of the present invention is from Seneca virus. Representative examples include NTX-010 (Rudin et al., 2011, Clin. Cancer. Res. 17(4):888-95).

[0183] 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).

[0184] 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 strains 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).

[0185] In one embodiment, the oncolytic virus of the present invention is obtained from herpes virus. Herpesviridae is a large family of DNA viruses, all of which have a common structure and consist of a relatively large double-stranded linear DNA genome encoding 100-200 genes, which are encapsulated in an icosahedral capsid, and the capsid is wrapped by a lipid bilayer membrane. Although the oncolytic herpes virus can be derived from different types of HSV, HSV1 and HSV2 are particularly preferred. Herpes virus can be genetically modified to limit viral replication in tumors or reduce its cytotoxicity in non-dividing cells. For example, any viral gene involved in nucleic acid metabolism can be inactivated, such as genes 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). Another aspect relates to viral mutants that are defective in the function of genes encoding virulence factors, such as the ICP34.5 gene (Chambers et al., 1995, Proc. Natl. Acad. Sci. USA 92:1411-5). Representative examples of oncolytic herpes viruses include NV1020 (eg, Geevarghese et al., 2010, Hum. Gene Ther. 21(9): 1119-28) and T-VEC (Andtbacka et al., 2013, J. Clin. Oncol. 31, Abstract No. LBA9008).

[0186] In one embodiment, the oncolytic virus of the present invention is obtained from the genus Morbillivirus, which can be obtained from the family Paramyxoviridae, and 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).

[0187] In one embodiment, the oncolytic virus of the present invention is obtained from adenovirus. The method of engineering oncolytic adenovirus is available in this area. Advantageous strategies include replacing viral promoters with tumor selective promoters or modifying E1 adenovirus gene products to inactivate the binding function of p53 or retinoblastoma (Rb) protein changed in tumor cells. In the natural environment, adenovirus E1B55kDa gene synergizes with another adenovirus product to inactivate p53 (p53 is often dysregulated in cancer cells), thereby preventing apoptosis. Representative examples of oncolytic adenoviruses include ONYX-015 (e.g., Khuri et al., 2000, Nat.Med 6 (8): 879-85) and H101, also known as Oncorine (Xia et al., 2004, Ai Zheng 23 (12): 1666-70).

[0188] 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, and its capsid protein sequence is substantially homologous to AAV-2 or the capsid protein sequence.

[0189] In one embodiment, the oncolytic virus of the present invention is a poxvirus. The term "poxvirus" as used herein refers to a virus of the Poxviridae family, particularly preferably a poxvirus of the subfamily of the Chordopoxviridae subfamily, more preferably a virus of the genus Orthopoxvirus or its chimera. Various poxviruses, such as vaccinia virus, cowpox virus, canarypox virus (Canarypox virus) and mousepox virus genome sequences can be obtained in the art and specialized databases such as Genbank (accession numbers are NC_006998, NC_003663, NC_005309, NC_004105, respectively).

[0190] Advantageously, the oncolytic poxvirus is a vaccinia virus and can be from any vaccinia strain, for example CPXV_GER1980_EP4 (Genbank HQ420895), CPXV_GER2002_MKY (Genbank HQ420898), CPXV_GER1991_3 (Genbank DQ 437593), CPXV_FRA2001_ACY (Genbank HQ420894), CPXV_GR1990_2 (Genbank HQ420896), CPXV_UK2000_K2984 (Genbank HQ420900), CPXV_BR (Genbank AF482758.2 or NC003663) 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).

[0191] Ideally, the oncolytic poxvirus is an oncolytic vaccinia virus. Vaccinia virus is a member of the poxvirus family, characterized by a 200kb double-stranded DNA genome that encodes many viral enzymes and factors, allowing the virus to replicate independently of the host cell machinery. Most vaccinia virus particles are intracellular (IMV stands for intracellular mature virions), have a single lipid envelope, and remain in the cytosol of infected cells until cell lysis. Another form of infection is a double envelope particle (EEV stands for extracellular enveloped virion), which buds out of infected cells without lysing it.

[0192] Although it can be derived from any vaccinia virus strain, Elstree, Wyeth, Copenhagen, Lister, Tian Tian and Western Reserve strains are particularly preferred. The gene nomenclature used herein is that of the Copenhagen vaccinia strain. Unless otherwise indicated, it is also used herein for homologous genes of other Poxviridae. However, the gene nomenclature varies according to the vaccinia strain, but the correspondence between Copenhagen and other vaccinia strains is generally available in the literature.

[0193] In one embodiment, the poxvirus belongs to the subfamily Chordatapoxvirinae, more preferably to the genus Leporopoxvirus, preferably myxoma virus, rabbit fibroma virus or squirrel fibroma virus, more preferably myxoma virus (whose genome sequence is published in Genbank under accession number NP_051868.1).

[0194] Preferably, the oncolytic virus of the present invention is modified by changing one or more viral genes. The modification preferably results in the synthesis of defective proteins, which cannot ensure the activity (or lack of synthesis) of proteins produced by unmodified genes under normal conditions. Modification covers the deletion, mutation and / or substitution of one or more nucleotides (continuous or non-continuous) in viral genes or their regulatory elements. Modification can be carried out in a variety of ways known to those skilled in the art using conventional recombinant techniques. Some exemplary modifications are disclosed in the literature, particularly preferably those changes involving DNA metabolism, host virulence, IFN pathways, etc. The modification of viral genes (see, for example, Guse et al., 2011, Expert Opinion Biol. Ther. 11 (5): 595-608).

[0195] More preferably, the oncolytic poxvirus of the present invention is modified by changing the thymidine kinase encoding gene (locus J2R). The TK enzyme is involved in the synthesis of deoxyribonucleotides. TK is required for the virus to replicate in normal cells because these cells usually have low concentrations of nucleotides, while it is unnecessary in dividing cells containing high nucleotide concentrations.

[0196] Alternatively, or in combination, the oncolytic poxvirus of the present invention is modified by altering at least one gene or two genes encoding ribonucleotide reductase (RR). In nature, the enzyme catalyzes the reduction of ribonucleotides to deoxyribonucleotides, which represents a key step in DNA biosynthesis. The viral enzyme is similar to the mammalian enzyme in subunit structure and consists of two heterologous subunits, encoded by the I4L and F4L loci, referred to as R1 and R2, respectively. The sequences of the I4L and F4L genes and their locations in various poxvirus genomes are available in public databases, for example, by accession numbers DQ437594, DQ437593, DQ377804, AH015635, AY313847, AY313848, NC_003391, NC_003389, NC_003310, M-35027, AY24 3312, 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, the I4L gene (encoding the R1 large subunit) or the F4L gene (encoding the R2 small subunit) or both can be inactivated.

[0197] Alternatively or in combination, the oncolytic poxvirus can be further modified in the M2L locus (preferably a modification that results in suppressed expression of the viral m2 protein), thereby generating a modified poxvirus deficient in m2 function (m2-deficient poxvirus).

[0198] In one embodiment, the oncolytic poxvirus is further modified in the M2L locus and the J2R locus (preferably a modification that results in the inhibition of the expression of the viral tk protein), thereby generating an oncolytic poxvirus that is defective in 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 and insertion of foreign nucleic acids into the M2L locus and / or the J2R locus are contemplated to inactivate the m2 and tk functions.

[0199] Alternatively or in combination, the oncolytic poxvirus may be further modified in the M2L locus and the I4L and / or F4L locus (preferably a modification resulting in suppressed expression of the viral ribonucleotide reductase (rr) protein), thereby generating an oncolytic poxvirus defective in both m2 and rr functions (m2 and rr defective poxvirus). In the context of the present invention, the poxvirus may be modified in the I4L gene (encoding the r1 large subunit) or the F4L gene (encoding the r2 small subunit) or both to provide an rr-deficient poxvirus. For example, by partially or completely deleting the I4L and / or F4L loci.

[0200] Also provided are oncolytic poxviruses modified in the M2L locus, the J2R locus, and the I4L and / or F4L loci (triple defective viruses modified in the M2L, J2R, and I4L loci; the M2L, J2R, and F4L loci or the M2L, J2R, I4L, and F4L loci), thereby generating oncolytic poxviruses defective in m2, tk, and rr activity (m2-, tk-rr-poxviruses).

[0201] Alternatively, or in combination, other strategies may also be adopted to further increase viral tumor specificity. Representative examples of suitable modifications include destroying the VGF encoding gene of the viral genome. VGF (for VV growth factor) is a secretory protein that is expressed early after cell infection, and its function seems to be important for the spread of the virus in normal cells. Another example is to destroy the A56R gene encoding hemagglutinin, which is ultimately combined with tk deletion (Zhang et al., 2007, Cancer Res.67:10038-46). The destruction of interferon regulatory genes (such as B8R or B18R genes) or caspase-1 inhibitor B13R genes may also be advantageous. Another suitable modification includes destroying the F2L gene encoding viral dUTPase, which is involved in maintaining the fidelity of DNA replication and providing precursors to produce TMP by thymidylate synthase (Broyles et al., 1993, Virol.195:863-5). The sequence of the vaccinia virus F2L gene is available from GenBank under accession number M25392.

[0202] In a preferred embodiment, the oncolytic virus of the present invention is a vaccinia virus with a defective TK activity, which is produced by an inactivation mutation in the J2R gene. In another preferred embodiment, the oncolytic virus of the present invention is a vaccinia virus with defects in both TK and RR activity, which is produced by an inactivation mutation in the J2R gene and I4L and / or F4L gene carried by the viral genome (e.g., as described in WO2009 / 065546 and Foloppe et al., 2008, Gene Ther., 15: 1361-71). In a further preferred embodiment, the oncolytic virus of the present invention is a vaccinia virus with defects in TK, RR and m2 activity, resulting in inactivation mutations in J2R gene, I4L and / or F4L gene and M2L gene. In another preferred embodiment, the oncolytic virus of the present invention is a dUTPase-deficient vaccinia virus, which is produced by an inactivating mutation in the F2L gene (e.g., as described in WO2009 / 065547), ultimately combined with the disruption of at least one or both of the TK and RR activities (generating a virus having an inactivating mutation in F2L; F2L and J2R genes; F2L and I4L; or F2L, J2R and I4L).

[0203] non-oncolytic viruses

[0204] In one embodiment, the non-oncolytic virus of the present invention is a poxvirus. Exemplary non-oncolytic virus poxviruses include, but are not limited to, pseudovaccinia virus (PCPV), modified vaccinia virus Ankara (MVA), highly attenuated vaccinia virus strain (NYVAC), swine pox virus (SWPV), fowl pox virus (FPV), or chimeras thereof.

[0205] In one embodiment, the non-oncolytic poxvirus of the present invention is obtained from pseudovaccinia virus (PCPV). Representative examples of PCPV strains suitable for use herein include, but are not limited to, YG2828 (GenBank Accession No. LC2301 19), F07.801 R (GenBank Accession No. JF773693), F10.3081 C (GenBank Accession No. JF773695), F07.798R (GenBank Accession No. JF773692), F99.177C (GenBank Accession No. AY453678), IT1303 / 05 (GenBank Accession No. JF800906), F00.120R (GenBank Accession No. GQ329669; Tikkanen et al., 2004, J. Gen. Virol. 85:1413-8), and TJS (also known as VR634; GenBank Accession No. GQ329670; Friedman-Kien et al., 1963, Science 140:1335-6; available under ATCC Accession No. VR634). These strains may differ from each other morphologically, structurally and / or genetically, for example, in terms of ITR length, number of predicted 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 present invention is obtained from ATCC reference number ATCC VR-634. TM Identified wild-type TJS strain or virus strain with the same or similar name and functional fragments and variants thereof.

[0206] In one embodiment, the non-oncolytic poxvirus of the present invention is obtained from a highly attenuated vaccinia virus strain (NYVAC) (Tartaglia et al., 1992, Virol. 188 (1): 217-32, US5,494,807). For the purpose of illustration, NYVAC is a highly attenuated vaccinia virus strain obtained from a plaque clone isolate of the Copenhagen vaccine strain by precisely deleting 18 open reading frames (ORFs) from the viral genome.

[0207] In one embodiment, the non-oncolytic poxvirus of the present invention is obtained from a modified vaccinia virus Ankara (MVA) because it has a highly 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 sequence of the encoded viral protein are available in the art, for example from Antoine et al. (1998, Virol, 244: 365-96) and GenBank (Accession No. U94848).

[0208] In one embodiment, the non-oncolytic poxvirus of the present invention is obtained from swine poxvirus (SWPV). The nucleotide sequence and amino acid sequence of the SWPV genome are available in the art, such as Alfonso et al. (2002, Virol, 76 (2): 783-90) and GenBank accession numbers (NC_003389.1 and MW036632).

[0209] In one embodiment, the non-oncolytic poxvirus of the present invention is obtained from fowl pox virus (FPV). The nucleotide sequence of the fowl pox virus genome is available in the art, for example, Alfonso et al. (2000, Virol., 74 (8): 3815-383) and GenBank accession number (AF198100).

[0210] Expression of one or more nucleic acid molecules encoding the SPD-TNFSF fusion protein inserted into the viral genome.

[0211] Nucleic acid molecules encoding SPD-TNFSF can be readily obtained by standard molecular biology techniques (eg, PCR amplification, cDNA cloning, chemical synthesis) using sequence data available in the art and the information provided herein. Analogs and fragments can be generated using standard techniques of molecular biology.

[0212] In one embodiment, the nucleic acid molecule encoding SPD-TNFSF can be independently inserted into any position of the viral genome, and non-essential loci are particularly preferred. Insertion into the virus can be carried out by conventional molecular biological methods, such as described by Sambrook et al. (2001, Molecular Cloning Laboratory Manual, Cold Spring Harbor Laboratory). Insertion into an adenovirus vector or a poxvirus vector can be carried out by homologous recombination as described by Chartier et al. (1996, J. Virol. 70: 4805-10) and Paul et al. (2002, Cancer gene Ther. 9: 470-7), respectively. For example, TK, RR and F2L genes and intergenic regions are particularly suitable for insertion into oncolytic vaccinia viruses, and E3 and E4 regions are suitable for insertion into oncolytic adenoviruses.

[0213] In addition, the coding nucleotide sequence can be optimized to provide high-level expression in a specific host cell or individual. In fact, it has been observed that the codon usage pattern of an organism is highly non-random, and the use of codons may be significantly different between different hosts. For example, therapeutic genes can come from bacteria or lower eukaryotic sources (such as suicide genes), so there is an inappropriate codon usage pattern for effective expression in higher eukaryotic cells (such as people). Generally, codon optimization is carried out by replacing one or more "natural" (for example, bacteria or yeast) codons (corresponding to codons that are not commonly used in the target host organism) with one or more more commonly used codons encoding the same amino acid. It is not necessary to replace all natural codons corresponding to codons that are not frequently used, because even partial replacement can also achieve the increase of expression.

[0214] Further optimization of codon usage can further improve expression in host cells or individuals by additional modification of the nucleotide sequence. For example, various modifications can be envisioned to prevent rare non-optimal codons from gathering in concentrated areas and / or to suppress or modify "negative" sequence elements that are expected to negatively affect expression levels. Such negative sequence elements include, but are not limited to, regions with very high (>80%) or very low (<30%) GC content; AT-rich or GC-rich sequence segments; unstable direct or reverse repeats; RA secondary structures; and / or internal hidden regulatory elements, such as internal TATA boxes, chi sites, ribosome entry sites, and / or splice donor / acceptor sites.

[0215] According to the present invention, each of the one or more nucleic acid molecules encoding the SPD-TNFSF fusion protein inserted into the viral genome of the present invention is operably linked to a suitable regulatory element for its expression in a host cell or individual. As used herein, the term "regulatory element" or "regulatory sequence" refers to any element that allows, helps or regulates the expression of a coding nucleic acid molecule in a given host cell or individual, including replication, repetition, transcription, splicing, translation, stability and / or transport of a nucleic acid or its derivative (i.e., mRNA). As used herein, "operably linked" means that the connected elements are arranged so that they work synergistically to achieve their intended purpose. For example, if a promoter achieves transcription from the start of transcription to the terminator of a nucleic acid molecule in a permissive host cell, then the promoter is operably linked to the nucleic acid molecule.

[0216] Those skilled in the art will appreciate that the selection of regulatory sequences may depend on factors such as the nucleic acid molecule itself, the virus into which it is inserted, the host cell or individual, the desired expression level, and the like. Promoters are particularly important. In the context of the present invention, it may be constitutively directed to express nucleic acid molecules in many types of host cells, or specific to certain host cells (e.g., liver-specific regulatory sequences), or regulated in response to specific events or exogenous factors (e.g., by temperature, nutritional additives, hormones, etc.) or according to the stage of the viral cycle (e.g., late or early stages). In order to optimize viral production and avoid the potential toxicity of expressed polypeptides, promoters that are suppressed in response to specific events or exogenous factors in the production step may also be used.

[0217] Promoters suitable for constitutive expression in mammalian cells include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter (US 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 (WO 98 / 10088). The vaccinia virus promoter is particularly suitable for expression in poxviruses. Representative examples include, but are not limited to, vaccinia virus 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 in 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. Promoters suitable for oncolytic measles viruses 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 in an individual).

[0218] Those skilled in the art will appreciate that the regulatory elements controlling the expression of nucleic acid molecules inserted into the viral genome may further comprise additional elements for proper initiation, regulation and / or termination of transcription (e.g., polyA transcription termination sequence), mRNA transport (e.g., nuclear localization signal sequence), processing (e.g., splicing signals) and stability (e.g., introns and non-coding 5' and 3' sequences), translation (e.g., initiator Met, tripartite leader sequence, IRES ribosome binding site, signal peptide, etc.).

[0219] When appropriate, it may be advantageous to include additional regulatory elements to facilitate the expression, trafficking and biological activity of at least one gene inserted into the viral genome of the virus of the invention (ie, the SPD-TNFSF fusion protein).

[0220] The method that can be carried out in the context of the present invention is to couple the gene product encoded by the virus of the present invention with an external 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, preventing their reproduction or growth, such as a toxin (e.g., an enzymatically active toxin or fragment thereof of bacterial, fungal, plant or animal origin). As used herein, a "labeling agent" refers to a detectable compound. The labeling agent itself can be detectable (e.g., radioisotope labeling or fluorescent labeling), or in the case of enzyme labeling, can catalyze the chemical modification of a detectable substrate compound. Coupling can be performed by genetic fusion between the gene product (SPD-TNFSF) and the external agent.

[0221] In a preferred embodiment, the oncolytic virus of the present invention is a vaccinia virus (preferably from the Copenhagen strain) that is defective in both TK and RR activity (e.g., produced by inactivating mutations in the viral J2R and I4L genes), and a nucleic acid molecule encoding the SPD-TNFSF fusion protein is inserted into its genome. Ideally, the elements are placed under the transcriptional control of the pH5R promoter, respectively. Preferably, the nucleic acid molecule encoding the SPD-TNFSF fusion protein is inserted into the J2R (TK) locus of the viral genome.

[0222] In another preferred embodiment, the oncolytic virus of the present invention is a vaccinia virus (preferably from the Copenhagen strain) that is defective in TK, RR and M2L activity (e.g., produced by inactivating mutations in the viral J2R, I4L and M2L genes), and a nucleic acid molecule encoding the SPD-TNFSF fusion protein is inserted into its genome. Ideally, the elements are placed under the transcriptional control of the pH5R promoter, respectively. Preferably, the nucleic acid molecule encoding the SPD-TNFSF fusion protein is inserted into the J2R (TK) locus of the viral genome.

[0223] In one option, the oncolytic virus of the present invention is a vaccinia virus (preferably from the Wyeth strain) that is defective in TK activity (generated by an inactivating mutation in the viral J2R gene), into whose genome a nucleic acid molecule encoding the SPD-TNFSF fusion protein is inserted.

[0224] Additional therapeutic peptides / genes

[0225] Although the virus according to the invention may only encode the SPD-TNFSF fusion protein of the invention (as defined above), it may also encode another nucleic acid molecule encoding a polypeptide of interest.

[0226] The another nucleic acid molecule encoding the target polypeptide is preferably a foreign nucleic acid (also called a recombinant gene, a transgene or a nucleic acid).

[0227] In the context of the present invention, the "foreign nucleic acid" inserted into the viral genome is not found or expressed in the naturally occurring viral genome. However, the foreign nucleic acid can be homologous or heterologous to the individual into which the recombinant virus is introduced. More specifically, it can be of human origin or non-human origin (for example, of bacterial, yeast or viral origin other than poxvirus). Advantageously, the recombinant nucleic acid encodes a polypeptide or a nucleic acid sequence that is capable of at least partially (by hybridization) binding to a complementary cellular nucleic acid (for example, DNA, RNA, miRNA) present in a diseased cell to inhibit the gene involved in the disease. This recombinant nucleic acid can be a natural gene or a portion thereof (for example, cDNA), or any variant thereof obtained by mutation, deletion, substitution and / or addition of one or more nucleotides.

[0228] In an advantageous embodiment, the polypeptide of interest is a therapeutic polypeptide.Thus, in this advantageous embodiment, the virus of the invention (as defined above) further comprises another nucleic acid molecule encoding a therapeutic polypeptide inserted into its genome.

[0229] "Therapeutic polypeptide" refers to a polypeptide of therapeutic or prophylactic interest that, when properly administered to an individual, results in a beneficial effect on the course or symptoms of the pathological condition to be treated or prevented.

[0230] The therapeutic polypeptide is preferably selected from the group consisting of an immunomodulatory polypeptide (preferably an immunostimulatory polypeptide), an antigenic polypeptide, a suicide gene product, a functional derivative of an antibody, a functional fragment of an antibody, and any combination thereof.

[0231] In a preferred embodiment, the therapeutic polypeptide is an immunostimulatory polypeptide, preferably selected from cytokines, such as interleukins, chemokines, interferons, tumor necrosis factors, colony stimulating factors; APC-exposing proteins; agonists of stimulatory immune checkpoints; antagonists of inhibitory immune checkpoints; and any combination thereof.

[0232] Immunomodulatory peptides

[0233] The term "immunomodulatory polypeptide" refers to a polypeptide that targets a component of a signaling pathway that may be directly or indirectly involved in regulating an immune response. "Modulating" an immune response refers to any change in the activity of an immune system cell or such a cell (e.g., a T cell). Such modulation includes stimulating or inhibiting the immune system, which may be demonstrated by increasing or decreasing the number of various cell types, increasing or decreasing the activity of these cells, or any other changes that may occur in the immune system. Preferably, such a polypeptide is capable of at least partially downregulating an inhibitory pathway (antagonist) and / or at least partially upregulating a stimulatory pathway (agonist); in particular, an immune pathway present between an antigen presenting cell (APC) or cancer cell and an effector T cell.

[0234] The immunomodulatory polypeptides that can be expressed by the vectors of the present invention can act at any step of T cell-mediated immunity, including clone selection of antigen-specific cells, T cell activation, proliferation, transport to antigens and inflammatory sites, execution of direct effector functions and signal transduction by cytokines and membrane ligands. Each of these steps is regulated by balancing stimulatory and inhibitory signals that finely regulate the response.

[0235] Suitable immunomodulatory polypeptides and methods of using them are described in the literature. Exemplary immunomodulatory polypeptides include, but are not limited to:

[0236] Cytokines, such as interleukins, chemokines, interferons, tumor necrosis factor, and colony-stimulating factor;

[0237] APC exposed proteins;

[0238] agonists that stimulate immune checkpoints;

[0239] Antagonists of inhibitory immune checkpoints other than PD-L1; and

[0240] and any combination thereof.

[0241] In one embodiment, the immunomodulatory polypeptide expressed by the vector of the present invention is a cytokine, preferably selected from;

[0242] interleukins (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), IFNa, IFNg, and granulocyte macrophage colony-stimulating factor (GM-CSF);

[0243] Chemokines (e.g., MIP1α, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL2, CCL19, and CCL21),

[0244] Interferons (e.g. IFNα, IFNγ),

[0245] Tumor necrosis factor (eg, TNFα), and

[0246] Colony stimulating factors (eg, granulocyte macrophage colony stimulating factor (GM-CSF)).

[0247] When the immunostimulatory polypeptide is a cytokine, it is preferably an interleukin or a colony stimulating factor, and GM-CSF is particularly preferred.

[0248] In another embodiment, the immunomodulatory polypeptide expressed by the vector of the invention is an agonist of a stimulatory immune checkpoint or an antagonist of an inhibitory immune checkpoint.

[0249] The term "immune checkpoint" refers to proteins that are directly or indirectly involved in immune pathways, which under normal physiological conditions are crucial for preventing uncontrolled immune responses and therefore for maintaining self-tolerance and / or tissue protection. Immune checkpoints can be classified into two different categories, namely: stimulatory and inhibitory immune checkpoints. "Stimulatory immune checkpoints" refer to immune checkpoints that are involved in the upregulation of immune responses, while "inhibitory immune checkpoints" are involved in the downregulation of immune responses.

[0250] Stimulatory immune checkpoints include CD28, ICOS, CD137 (4-1BB), OX40, CD70, CD40 and GITR, and the agonists of stimulatory immune checkpoints are preferably selected from human ICOSL, 4-1BBL, OX40L, CD70, CD40L, GITRL and agonist antibodies against human ICOS (e.g., WO2018 / 187613), CD137 (4-1BB) (e.g., WO2005 / 035584), OX40 (e.g., US 7,291,331 and WO03 / 106498), CD70 (e.g., WO2012 / 004367), CD40 (e.g., WO2017 / 184619) or GITR (e.g., WO2017 / 068186). Since some agonists of stimulatory immune checkpoints are TNFSF members, when such agonist of stimulatory immune checkpoints 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.

[0251] Inhibitory immune checkpoints include PD-1, SIRPa, CD47, PD-L2, LAG3, Tim3, BTLA and CTLA4, and the antagonists of the inhibitory immune checkpoints are preferably selected from human antagonist antibodies:

[0252] PD-1 (e.g. those described in WO2004 / 004771, WO2004 / 056875, WO2006 / 121168, WO2008 / 156712, WO2009 / 014708, WO2009 / 114335, WO2013 / 043569 and WO2014 / 047350, in particular nivolumab, pembrolizumab and cemiplimab),

[0253] SIRPa (e.g. WO2019 / 023347),

[0254] CD47 (e.g. WO2020 / 019135),

[0255] PD-L2 (e.g. WO2019 / 158645),

[0256] LAG3 (e.g. WO2018 / 071500),

[0257] Tim3, (e.g. WO2020 / 093023)

[0258] BTLA (e.g. WO2010 / 106051), and

[0259] CTLA4 (e.g., those described in US 8,491,895, WO2000 / 037504, WO2007 / 113648, WO2012 / 122444, and WO2016 / 196237, among others, and in particular by Bristol Myer Squibb as Marketed are ipilimumab (see, e.g., US 6,984,720; US 8,017,114), MK-1308 (Merck), AGEN-1884 (Agenus; WO2016 / 196237) and tremelimumab (AstraZeneca; US 7,109,003 and US 8,143,379) and single-chain anti-CTLA4 antibodies (see, e.g., WO97 / 20574 and WO2007 / 123737).

[0260] Antigenic Peptide

[0261] The term "antigenicity" refers to the ability to induce or stimulate a measurable immune response in an individual, wherein the virus of the present invention (as described herein) encoding a polypeptide with antigenicity has been introduced into the individual. The immune response stimulated or induced by the antigenic polypeptide expressed by the virus can be humoral and / or cellular (e.g., producing antibodies, cytokines and / or chemokines that participate in the activation of effector immune cells). The immune response stimulated or induced generally contributes to the protective effect in the individual administered. There are a variety of direct or indirect biological assays in the art that can be used to assess the antigenic properties of polypeptides in vivo (animal or human individuals) or in vitro (e.g., in biological samples). For example, the ability of a particular antigen to stimulate innate immunity can be measured, for example, by measuring NK / NKT cells (e.g., representation and activation levels), as well as IFN-related cytokine and / or chemokine production cascades, activation of TLRs (for Toll-like receptors), 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 by quantitative determination of cytokine production by activated T cells (including cytokines derived from CD4+ and CD8+ T cells) by conventional bioassays (e.g., by ELISpot, multiparameter flow cytometry, ICS (for intracellular cytokine staining), cytokine profiling using multiplex techniques or ELISA to characterize and / or quantify T cells), by determining the proliferative capacity of T cells (e.g., T cell proliferation assay by [3H] thymidine incorporation assay), by determining the cytotoxic capacity of antigen-specific T lymphocytes of sensitized individuals or by identifying lymphocyte subsets by flow cytometry, and by immunization of appropriate animal models, as described herein.

[0262] The term antigenic polypeptide is considered to encompass native antigens as well as fragments thereof (e.g., epitopes, immunogenic domains, etc.) and variants, provided that such fragments or variants are capable of being targets for an immune response. Preferred antigenic polypeptides for use herein are tumor-associated antigens. It is within the capabilities of those skilled in the art to select one or more antigenic polypeptides suitable for treating a particular pathological condition.

[0263] 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), which is associated with cancer and / or serves as a cancer marker. Cancer antigens encompass various types of polypeptides, such as those that are usually silent (i.e., not expressed) in healthy cells, those that are expressed only at low levels or at certain stages of differentiation, and those that are temporarily expressed, such as embryonic and fetal antigens, and those produced by cellular gene mutations, such as oncogenes (e.g., activated ras oncogenes), proto-oncogenes (e.g., ErbB family), or proteins produced by chromosomal translocations.

[0264] 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 family, p53, mucin antigens (e.g., MUC1), HER2 / neu, p21ras, hTERT, Hsp70, iNOS, tyrosine kinase, mesothelin, c-erbB-2, alpha-fetoprotein, AM-1, etc., and any immunogenic epitope or variant thereof.

[0265] Tumor-associated antigens may also encompass new epitopes / antigens that appear during the carcinogenesis process in cancer cells and contain one or more amino acid residue mutations relative to the corresponding wild-type antigen. Typically, they are found in cancer cells or tissues obtained from patients, but not in normal cells or tissue samples obtained from patients or healthy individuals.

[0266] Tumor-associated antigens may also encompass antigens encoded by pathogenic organisms that are capable of inducing malignant conditions in individuals, especially chronically infected individuals, 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).

[0267] In another embodiment, the antigenic polypeptide encoded by the virus of the present invention is a vaccine antigen, which, when delivered to a human or animal individual, is intended to provide therapeutic or preventive protection against infectious diseases. Many vaccine antigens are known in the art. Exemplary vaccine antigens include, but are not limited to, cellular antigens, viral, bacterial or parasitic antigens. Cellular antigens include mucin 1 (MUC1) glycoproteins. Viral antigens include, for example, antigens from: hepatitis A, B, C, D and E viruses, immunodeficiency viruses (e.g., HIV), herpes viruses, cytomegaloviruses, varicella zoster viruses, papilloma viruses, Epstein Barr viruses, influenza viruses, parainfluenza viruses, coxsackie viruses, picorna viruses, rotaviruses, respiratory syncytial viruses, rhinoviruses, rubella viruses, papoviruses, mumps viruses, measles viruses and rabies viruses. 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 protein, such as asICP27, ICP47, ICP4, ICP36 from HSV1 or HSV2. Some non-limiting examples of cytomegalovirus antigens include gB. Some non-limiting examples derived from EpsteinBarr 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 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. According to the present invention, antigens derived from other viral pathogens may also be used, 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). Bacterial antigens include, for example, antigens from mycobacteria that cause TB, leprosy, pneumococcus, aerobic gram-negative bacilli, mycoplasma, staphylococcus, streptococcus, salmonella, chlamydia, neisseria, etc. Parasite antigenic polypeptides include, for example, antigens from malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis and filariasis.

[0268] Antibodies and their antigen-binding fragments or derivatives

[0269] The virus of the present invention may further encode any antibody or antigen-binding fragment or derivative thereof having therapeutic activity, including anti-tumor antibodies or antigen-binding fragments or derivatives thereof, in particular antibodies or antigen-binding fragments or derivatives thereof that affect 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.

[0270] In the context of the present invention, "antibody" ("Ab") is used in the broadest sense and is preferably as defined above in the "General Definitions" section.

[0271] The antibody is preferably a monoclonal antibody, preferably humanized or chimeric.

[0272] 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), which comprises two domains of the Fv fragment, VL and VH, fused together to ultimately form a single protein chain with a linker.

[0273] Preparation of SPD-TNFSF fusion protein

[0274] In one embodiment, the virus can also be used in the context of the present invention to produce one or more SPD-TNFSF fusion proteins encoded by it by recombinant methods. It may advantageously contain one or more additional elements that enable the maintenance, propagation or expression of nucleic acid molecules encoding SPD-TNFSF fusion proteins in host cells. These additional elements include marker genes to facilitate the identification and isolation of production host cells (for example, by complementation of cell auxotrophy or by antibiotic resistance). Suitable marker genes include, but are not limited to, dihydrofolate reductase (dhfr), which 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, which confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); zeo, which confers resistance to zeomycin, kana, which confers resistance to kanamycin; and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Recombinant viruses lacking functional TK (e.g., produced by inserting a nucleic acid molecule encoding the SPD-TNFSF protein into the J2R (TK) locus) can be selected using medium containing bromodeoxyuridine (BrdU). In fact, TK- viruses are insensitive to the BrdU drug, while the drug interferes with DNA synthesis in TK+ viruses. It is also possible to rely on reporter gene luminescence or colorimetric systems, such as those based on GFP (green fluorescent protein), luciferase, and β-galactosidase.

[0275] Methods for recombinantly producing SPD-TNFSF fusion proteins are routine in the art. Generally, such methods include (a) introducing the viruses described herein into suitable production cells to produce transfected or infected production cells, (b) culturing the transfected or infected production cells in vitro under conditions suitable for their growth, (c) recovering one or more SPD-TNFSF fusion proteins from the cell culture, and (d) optionally, purifying the recovered SPD-TNFSF fusion proteins. In the context of the present invention, the production cells are preferably human or non-human eukaryotic cells. Preferred production cells include, but are not limited to, hamster cell lines, such as BHK-21 (ATCC CCL-10), CV-1 (African 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 TM or ATCC-CCL-2.2 TM ), 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 and duck cells described herein and in WO2005 / 042728, WO2006 / 108846, WO2008 / 129058, WO2010 / 130756, WO2012 / 001075, etc.), and primary chicken embryo fibroblasts (CEFs) prepared from chicken embryos obtained from fertilized eggs.

[0276] The production cells can be cultured in conventional fermentation bioreactors, flasks and petri dishes. The culture can be carried out at a temperature, pH and oxygen content suitable for a given host cell. No attempt is made here to describe in detail the various methods known for producing proteins in eukaryotic cells. The production of the SPD-TNFSF fusion protein can be intracellular or, preferably, secreted outside the production cell (e.g., in the culture medium).

[0277] Prior to use according to the invention, the virus may be at least partially isolated. Various purification steps may be envisioned, including clarification, enzyme treatment (e.g., benzonase, protease), chromatography and filtration steps. Suitable methods are described in the art (e.g., WO2007 / 147528; WO2008 / 138533, WO2009 / 100521, WO2010 / 130753, WO2013 / 022764).

[0278] The SPD-TNFSF fusion protein can then be purified by well-known purification methods. The conditions and techniques used to purify a particular protein will depend on factors such as expression conditions, net charge, molecular weight, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art. In addition, the level of purification will depend on the intended use. If desired, particularly when the SPD-TNFSF fusion protein is not secreted outside the production cell or is incompletely secreted, it can be recovered by standard lysis methods, including freeze-thaw, sonication, mechanical disruption, use of lysis agents, etc. If secreted, it can be recovered directly from the culture medium. Various purification steps are contemplated, including, but not limited to, clarification (e.g., ammonium sulfate precipitation, acid extraction), enzyme 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., WO2007 / 147528; WO2008 / 138533, WO2009 / 100521, WO2010 / 130753, WO2013 / 022764). Ideally, the SPD-TNFSF fusion protein recombinantly produced from the virus of the invention is at least partially purified, meaning that it is substantially free of other cellular material. In addition, the SPD-TNFSF fusion protein can be formulated according to conditions routinely used in the art (e.g., WO2009 / 073569).

[0279] Therapeutic Uses

[0280] The present invention also provides a composition comprising a therapeutically effective amount of the SPD-TNFSF fusion protein of the present invention, a virus, and optionally a pharmaceutically acceptable solvent. Such a composition can be administered once or multiple times, and by the same or different routes.

[0281] A "therapeutically effective amount" corresponds to an amount of SPD-TNFSF fusion protein or virus sufficient to produce one or more beneficial results. Such a therapeutically effective amount may vary as a function of a variety of parameters, in particular the mode of administration; the disease state; the age and weight of the individual; the ability of the individual to respond to treatment; the kind of concurrent treatment; the frequency of treatment; and / or the need for prevention or treatment. When it comes to preventive uses, the SPD-TNFSF fusion protein, virus or composition of the invention is administered in a dose sufficient to prevent or delay the onset and / or establishment and / or recurrence of a pathological condition (e.g., a proliferative disease such as cancer), especially in an at-risk individual. For "therapeutic" uses, the SPD-TNFSF fusion protein, virus or composition of the invention is administered to an individual diagnosed with a pathological condition (e.g., a proliferative disease such as cancer or a disorder associated with TNF cytokine dysfunction) with the goal of treating the disease, ultimately in combination with one or more conventional treatment modalities. In particular, a therapeutically effective amount can be the amount necessary to cause an observable improvement in the clinical state relative to the baseline state or relative to the expected state if not treated, such as stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression or severity, improvement or alleviation of the disease state, prolonged survival, better response to standard treatment, improvement in quality of life, reduced mortality, reduction in the number of tumors, reduction in tumor size, reduction in the number or range of metastases, increase in remission time, etc. A therapeutically effective amount can also be the amount necessary to cause the development of an effective nonspecific (innate) and / or specific immune response (e.g., tumor immune response). Typically, the development of an immune response, particularly a T cell response, can be assessed in vitro, in a suitable animal model, or using a biological sample collected from an individual. For example, techniques routinely used in laboratories (e.g., flow cytometry, histology) can be used for tumor monitoring. People can also use a variety of available antibodies to identify different immune cell populations that participate in anti-tumor responses present in the treated individual, such as cytotoxic T cells, activated cytotoxic T cells, natural killer cells, and activated natural killer cells. Improvement in clinical status can be readily assessed by any relevant clinical measure commonly used by a physician or other skilled healthcare provider.

[0282] The term "pharmaceutically acceptable vehicle" is intended to include any and all carriers, solvents, diluents, excipients, adjuvants, dispersion media, coatings, antibacterial and antifungal agents, absorbents, and the like, compatible with administration in mammals, and particularly human subjects.

[0283] The SPD-TNFSF fusion protein, virus, or combination thereof can be placed in a solvent or diluent suitable for human or animal use. The solvent or diluent is preferably isotonic, hypotonic, or slightly 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 physiologically balanced saline solutions (e.g., see the latest edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins Publishers).

[0284] In one embodiment, the SPD-TNFSF fusion protein, virus, or composition thereof is appropriately buffered for human use. Suitable buffers include, but are not limited to, phosphate buffer (e.g., PBS), bicarbonate buffer, and / or Tris buffer capable of maintaining a physiological or slightly alkaline pH (e.g., from about pH 7 to about pH 9).

[0285] The SPD-TNFSF fusion protein, virus, or composition thereof may also contain other pharmaceutically acceptable excipients to provide desired pharmaceutical or pharmacodynamic properties, including, for example, molar osmotic pressure concentration, viscosity, clarity, color, sterility, stability, dissolution rate of the formulation, improving or sustaining release or absorption in human or animal subjects, facilitating transport across the blood barrier or penetration of specific organs.

[0286] In one embodiment, the viral composition may also include one or more adjuvants capable of stimulating immunity (especially T cell-mediated immunity) or promoting tumor cell infection after administration, for example, through Toll-like receptors (TLRs), such as TLR-7, TLR-8 and TLR-9, including but not limited to alum, mineral oil emulsions, such as Freund's complete and incomplete emulsions (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; WO98 / 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 described, for example, in WO 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).

[0287] In one embodiment, the SPD-TNFSF fusion protein, virus or composition of the present invention can be formulated to achieve the purpose of improving its stability, especially under conditions of manufacture and long-term storage (i.e., at least 6 months, preferably at least two years) at frozen (e.g., -70°C, -20°C), refrigerated (e.g., 4°C) or ambient temperature. In the art, various viral preparations can be obtained in frozen, liquid or lyophilized form (e.g., WO98 / 02522, WO01 / 66137, WO03 / 053463, WO2007 / 056847 and WO2008 / 114021, WO2016087457, etc.). Solid (e.g., dry powder or lyophilized) compositions can be obtained by methods including vacuum drying and freeze drying (see, e.g., WO2014 / 053571). For illustrative purposes, buffer formulations containing NaCl and / or sugars are particularly suitable for preserving 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).

[0288] In certain embodiments, the viral composition of the present invention can be formulated to ensure proper distribution or delayed release in vivo. For example, it can be formulated into 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 preparations have been described by JR Robinson in "Sustained and Controlled Release Drug Delivery Systems" edited by Marcel Dekker, Inc., for example, in 1978.

[0289] The appropriate dosage of the SPD-TNFSF fusion protein can be adjusted as a function of a variety of parameters and can be routinely determined by the practitioner according to the relevant circumstances. The appropriate dosage of the SPD-TNFSF fusion protein varies between 0.001 and about 100 mg / kg, depending on the route of administration of the protein and the quantitative technique used. As a general guide, the amount of SPD-TNFSF fusion protein present in a sample can be determined by conventional titration techniques, such as UV absorption or ELISA.

[0290] The appropriate dose of the virus can be adjusted as a function of various parameters and can be routinely determined by the practitioner according to the relevant circumstances. Depending on the virus used and the quantitative technique, the appropriate dose of the virus is about 104 to about 10 13 The dose of vaccinia virus varies between VP (virion), iu (infectious unit) or pfu (plaque forming unit). As a general guide, the dose of vaccinia virus ranges from about 10 4 To about 10 13 pfu is suitable, preferably from about 10 6 pfu to about 10 11 pfu, more preferably from about 10 7 pfu to about 5x10 9 A particularly preferred dose is about 10 8 pfu to about 10 9 pfu, especially for human use. The amount of virus present in a sample can be determined by conventional titration techniques, such as by counting the number of plaques after infection of permissive cells using permissive cells (e.g., BHK-21 or CEF), immunostaining (e.g., using antiviral antibodies; Carroll et al., 1997, Virology 238: 198-211), by measuring A260 absorbance (vp titer), or still by quantitative immunofluorescence (iu titer).

[0291] Application

[0292] The SPD-TNFSF fusion protein, virus or composition of the present invention can be administered in a single dose (e.g., bolus injection) or multiple doses. If multiple administrations are given, they can be given by the same or different routes and can be given at the same site or at an alternative site. It can also be given by a continuous administration cycle repeated after a rest period. The interval between each administration can be from a few hours to a year (e.g., 24 hours, 48 ​​hours, 72 hours, weekly, biweekly, monthly or annually). The interval can also be irregular (e.g., after tumor progression). The dose for each administration can vary within the above range.

[0293] Any conventional route of administration is applicable to the present invention, including parenteral, topical or mucosal routes. Parenteral routes are intended to be used as injection or infusion. Common parenteral injection types are intravenous (entering vein), intraarterial (entering artery), intradermal (entering dermis), subcutaneous (under the skin), intramuscular (entering muscle) and intratumoral (entering tumor or near it). Infusion is usually given by intravenous route. Mucosal administration includes but is not limited to oral / digestive tract, intranasal, intratracheal, intrapulmonary, intravaginal or intrarectal routes. Topical administration can also be carried out using transdermal means (such as patches, etc.). Administration can use conventional syringes and needles (such as Quadrafuse injection needles) or any compound or device that can promote or improve the delivery of active agents in individuals available in the art. Preferred routes of administration of viruses include intravenous and intratumoral routes.

[0294] In the context of the present invention, a virus may be 7 Up to 5x10 9 The dosage range of pfu can be administered once or multiple times (e.g., 2, 3, 4, 5, 6, 7 or 8 times, etc.). The time interval between each administration can vary from about 1 day to about 8 weeks, advantageously from about 2 days to about 6 weeks, preferably from about 3 days to about 4 weeks, and even more preferably from about 1 week to about 3 weeks (e.g., every two weeks). A preferred treatment regimen comprises intravenous or intratumoral administration of 10 8 or 10 9 pfu of oncolytic vaccinia virus 2 to 5 times (e.g., 3 times).

[0295] The present invention also relates to a method of treating a proliferative disease such as cancer or a condition associated with TNF cytokine dysfunction such as infectious diseases, inflammatory diseases, metabolic diseases, autoimmune diseases, degenerative diseases, apoptosis-related diseases and transplant rejection, which comprises administering a virus as described herein to an individual in need thereof.

[0296] In one embodiment, the invention also relates to a method of treating a proliferative disease, such as cancer, comprising administering a virus as described herein to an individual in need thereof.

[0297] In one embodiment, the invention also relates to a method of inhibiting tumor cell growth in vivo, the method comprising administering a virus as described herein to an individual in need thereof.

[0298] In one embodiment, the invention also relates to a method for enhancing an immune response to tumor cells, the method comprising administering a virus as described herein to an individual in need thereof.

[0299] In one embodiment, administration of the virus used in the present invention will trigger, stimulate and / or redirect an immune response. In particular, administration induces a protective T or B cell response in the treated host, for example against the virus or ultimately against the product encoded by the SPD-TNFSF nucleic acid molecule (if any) inserted into the viral genome. The protective T response can be CD4+ or CD8+ or CD4+ and CD8+ cell-mediated. B cell responses can be measured by ELISA, and T cell responses can be assessed by conventional ELISpot, ICS assays from any sample collected from the immunized animal or individual (e.g., blood, organs, tumors, etc.).

[0300] In one embodiment, the administration of oncolytic viruses also allows for changes in the tumor microenvironment with the goal of enhancing the activity of effector cells, particularly effector T lymphocytes, in the tumor and / or promoting the exhaustion of at least some Tregs. Tumor infiltrating cells can be easily identified, for example, by conventional immunostaining assays.

[0301] As mentioned above, in connection with the term "therapeutically effective amount", higher therapeutic efficacy may be demonstrated, with longer survival being particularly preferred.

[0302] Examples of conditions that can be treated using the SPD-TNFSF fusion proteins, viruses, compositions or methods of the invention include, but are not limited to:

[0303] √ Proliferative diseases

[0304] o Cancer, such as bone cancer, liver cancer, pancreatic cancer, stomach 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, anal region cancer, prostate cancer, lymphoma, endocrine system cancer, thyroid cancer, soft tissue sarcoma, chronic or acute leukemia, bladder cancer, kidney cancer, central nervous system (CNS) tumors, glioma, glioblastoma, etc. Preferred cancers that can be treated using the SPD-TNFSF fusion protein or virus of the present invention include cancers that are generally responsive to immunotherapy. Non-limiting examples of preferred cancers to be treated include melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate cancer), breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer), and liver cancer (e.g., hepatocellular carcinoma).

[0305] ○ Cardiovascular disease, such as restenosis

[0306] √ Conditions associated with TNF cytokine dysfunction:

[0307] o infectious diseases, such as HIV infection, in particular 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 virus or Ebola virus) and severe acute respiratory syndrome (SARS), bacterial infectious diseases, such as Legionnaires' disease (Legionella), sexually transmitted diseases (such as chlamydia or gonorrhea), gastric ulcers (Helicobacter), cholera (Vibrio), diphtheria, Escherichia coli, Staphylococcus, Salmonella or Streptococcus (tetanus) infection; infections with protozoan pathogens, such as malaria, sleeping sickness, leishmaniasis; toxoplasmosis, i.e. infection with Plasmodium, Trypanosoma, Leishmania and Toxoplasma; or fungal infections, such as caused by Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis or Candida albicans;

[0308] 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;

[0309] ○ Metabolic diseases, such as diabetes, cystinosis, dyslipidemia, hyperthyroidism, hypothyroidism, hyperlipidemia, hypolipidemia, galactosemia, obesity, Gaucher disease, and phenylketonuria;

[0310] ○Autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome

[0311] 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

[0312] ○Diseases related to cell apoptosis

[0313] ○Transplant rejection

[0314] The SPD-TNFSF fusion protein, virus, composition or method according to the invention can be associated with one or more substances or therapies effective in anti-cancer therapy, and the invention also relates to a method comprising the step of delivering an additional cancer therapy to an individual. In the context of the present invention, the additional cancer therapy comprises surgery, radiation, chemotherapy, immunotherapy, hormone therapy or a combination thereof. In a preferred embodiment, the method of the invention comprises the 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 conjunction or in combination with the SPD-TNFSF fusion protein, virus, composition or method of the invention, more particular mention can be made of:

[0315] √ Alkylating agents, such as mitomycin C, cyclophosphamide, busulfan, ifosfamide, ifosfamide, melphalan, hexamethylmelamine, thiotepa, chlorambucil, or dacarbazine;

[0316] √ Antimetabolites, such as gemcitabine, capecitabine, 5-fluorouracil, cytarabine, 2-fluorodeoxycytidine, methotrexate, idatrexate, tomudex, or trimetrexate;

[0317] Topoisomerase II inhibitors, such as doxorubicin, epirubicin, etoposide, teniposide, or mitoxantrone;

[0318] Topoisomerase I inhibitors, such as irinotecan (CPT-11), 7-ethyl-10-hydroxy-camptothecin (SN-38), or topotecan;

[0319] √ Antimitotic drugs, such as paclitaxel, docetaxel, vinblastine, vincristine, or vinorelbine;

[0320] √ Platinum derivatives, such as cisplatin, oxaliplatin, spiroplatin, or carboplatin;

[0321] √Tyrosine kinase receptor inhibitors, such as sunitinib (Pfizer) and sorafenib (Bayer);

[0322] √ Anti-tumor antibodies, especially those that affect the regulation of cell surface receptors, such as trastuzumab, cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, bevacizumab, and ranibizumab.

[0323] √EGFR (epidermal growth factor receptor) inhibitors, such as gefitinib, erlotinib, and lapatinib; and

[0324] √ Immunomodulators, such as interferon alpha, beta, or gamma, interleukins (especially IL-2, IL-6, IL-10, or IL-12), or tumor necrosis factor;

[0325] The SPD-TNFSF fusion protein, expression vector, composition, or method of the present invention can also be used in combination with radiation therapy.

[0326] The present invention also provides a kit comprising different containers (e.g., sterile glass or plastic bottles) for each viral dose to be administered. Optionally, the kit may include a device for administering the active agent. The kit may also include a package insert containing information about the composition or individual components and dosage forms in the kit.

[0327] Materials and methods

[0328] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific methods and reagents described herein, including substitutions, variations, additions, deletions, modifications, and substitutions. Such equivalents are considered to be within the scope of the invention and are covered by the appended claims.

[0329] Viruses and plasmids

[0330] 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.

[0331] Plasmid pTG19274 is a plasmid encoding an unrelated FLAG-tagged molecule. Plasmid pTG19274 was used as a negative control.

[0332] Plasmid pTG19333 is a plasmid without a transgene. Plasmid pTG19333 was used as a negative control.

[0333] Plasmid pTG19325 is a plasmid encoding the fusion protein SPD (M1-G257, P35247 numbering)-CD40L (H47 to L261 of P29965 numbering) construct (SEQ ID NO: 50).

[0334] Plasmid pTG19344 is a plasmid encoding soluble CD40L (G116 to L261) consisting of a heterologous signal peptide followed by the CD40L extracellular domain (SEQ ID NO: 51).

[0335] Plasmid pTG19965 is a plasmid encoding a heterologous signal peptide upstream of human SPD (A21 to G257)-CD40L (N119 to L261). This construct (SEQ ID NO: 52) contains a heterologous signal peptide, a shorter form of CD40L, and a C-terminal FLAG tag.

[0336] Plasmid pTG20032 is a plasmid encoding the 4-1-BBL (D80 to E254) extracellular domain, which consists of a heterologous signal peptide followed by the 4-1-BBL extracellular domain (SEQ ID NO: 53).

[0337] The transfer plasmids of the present invention carrying different SPD-TNFSF constructs are described in Table 2 below.

[0338]

[0339]

[0340] Table 2. Design of SPD-TNFSF constructs of the invention and corresponding plasmids. ([N-terminus] is defined by SEQ ID NO: 1, [coiled-coil neck domain] is defined by SEQ ID NO: 23, [CD40L] is defined by SEQ ID NO: 24, [4-1BBL] is defined by SEQ ID NO: 28, and the collagen domain is defined according to Table 1).

[0341] COPTG19967 is a double-deleted (tk- and rr-) recombinant Copenhagen vaccinia virus in which the expression sequence of pTG19967 has been inserted into its J2R locus.

[0342] COPTG19968 is a double-deleted (tk- and rr-) recombinant Copenhagen vaccinia virus in which the expression sequence of pTG19968 has been inserted into its J2R locus.

[0343] COPTG19969 is a double-deleted (tk- and rr-) recombinant Copenhagen vaccinia virus in which the expression sequence of pTG19969 has been inserted into its J2R locus.

[0344] Infection / transfection experiments

[0345] A method for expressing recombinant proteins is an infection / transfection method. This method involves infecting cells, such as HeLa cells, with vaccinia virus (poxvirus) and transfecting the cells with a plasmid encoding the gene of interest under the control of a poxvirus promoter. This method allows the encoded gene of interest to be expressed intracellularly. The expression product can be recovered in the supernatant for further analysis.

[0346] The purpose of co-infection / transfection in HeLa cells was to select the most efficient SPD-TNFSF construct to be vectorized in the VACV (vaccinia virus) genome.

[0347] Briefly, two days before infection, cells were seeded in 6-well plates at a density of 4E+05 cells / well / 3 mL complete medium (DMEM 4 Gibcoref. 41966-029; glutamine 2 mM; gentamicin 40 μg / mL; 10% fetal bovine serum (FBS)).

[0348] Prior to infection, the medium was removed and replaced with 400 μL of vaccinia virus preparation (VVTG18058) in PBS+ (PBS+1% cations), equivalent 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 for 2 hours at 37°C and 5% CO2. Transfection was then performed according to the supplier's protocol by adding 1 μg of plasmid DNA prepared with 4.5 μL of Lipofectamine 2000 (Invitrogen, 11668-027) to each well, and pTG19274 (encoding a molecule with an irrelevant FLAG tag) was used as a negative control. Infection / transfection was performed in triplicate. The plates were incubated for 48 hours at 37°C and 5% CO2. The culture supernatant was then collected, centrifuged and filtered on a 0.1 μm filter to remove all viral particles and cell debris. The clarified supernatant was stored at -80°C until use.

[0349] Expression experiments using recombinant viruses

[0350] The day before infection, HeLa cells were seeded in 6-well plates at a density of 1.5E+06 cells / well / 2mL complete medium (DMEM 4Gibcoref.41966-029; glutamine 2mM; gentamicin 40μg / mL; 10% FBS). Cells were infected with one of the following viruses at an MOI of 0.1: COPTG19968, COPTG19967, COPTG19969, or VVTG18058. After 30 minutes of culture, the medium was discarded and replaced with 2mL DMEM; glutamine 2mM; gentamicin 40μg / mL. Cells were cultured at 37°C and 5% CO2 for 48 hours, and then the culture supernatant was recovered and processed as described above.

[0351] CD40L and 4-1BBL immunoblotting

[0352] 25 μL samples (clarified supernatant) of infection / transfection were treated with Laemmli buffer (Biorad, 161-0747) containing (reduced) or not containing (non-reduced) β-mercaptoethanol. The samples were heated at 95°C for 3 minutes under reducing conditions. The samples were then loaded onto polyacrylamide gels (TGX 4-15% Stain Free Biorad) and migrated in Tris GlycineSDS buffer (Biorad 161-0772). Western blotting was performed using the Transblot Turbo system (Biorad), which was set on the Midi Program High Molecular weight. The blot was then incubated with 2 μg / mL of anti-FLAG-HRP binding antibody (Sigma A8592) using the Ibind FlexWestern System (Invitrogen ref SLF2000). The positive control was the culture medium of infection / transfection, which contained an unrelated plasmid (pTG19274) encoding a recombinant protein with a FLAG tag. The blots were incubated with HRP substrate (Amersham ECL Prime Western Blot Detection) and luminescence was recorded by a Chemidoc instrument.

[0353] CD40 ELISA

[0354] CD40-Fc was coated on a Medisop (Nunc) 96-well ELISA plate at 0.5 μg / mL in 50 mM carbonate buffer (pH 9.6). The clarified supernatant of the infection / transfection experiment was diluted 10 times, added to the first well of the ELISA plate, and further diluted twice in ELISA saturation buffer directly on the plate. The bound CD40L was detected by adding a non-competitive anti-human CD40L (MCA1561 Biorad) diluted 1000 times in saturation buffer. Then an anti-mouse immunoglobulin-HRP conjugated antibody (Dako P0447) diluted 2000 times 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, and a graph of the culture supernatant at 450 nm optical density (OD) was plotted against 1 / dilution using GraphPadPrism software.

[0355] CD40 agonist activity

[0356] HEK-Blue CD40L cells (Invivogen: hkb-cd40) are recombinant cells transformed to express human CD40 and a reporter enzyme (secreted embryonic alkaline phosphatase: SEAP) under the transcriptional control of a CD40 inducible promoter. Upon CD40 activation, SEAP is produced and its enzymatic activity is measured in the culture medium as recommended by the supplier. SEAP activity is proportional to CD40 agonist activity.

[0357] The measurements were performed according to the instructions of the supplier. Briefly, 50,000 HEK-Blue CD40L cells in 90 μL were distributed in a 96-well plate and incubated with 20 μL of serially diluted clear supernatant produced by the above infection / transfection. After incubation for 24 hours at 37°C and 5% CO2, 40 μL of the culture medium was transferred 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 relationship between optical density and 1 / supernatant dilution was plotted using GraphPad Prism software.

[0358] 4-1BB agonist activity

[0359] 4-1BB Bioassay Promega kit (JA2351) was used according to the instructions of the supplier. In brief, 25 μL effector cells / well were mixed with 25 μL culture medium. Then 25 μL of serially diluted clarified supernatant was added to each well. The cells were then incubated at 37 ° C, 5% CO2 for 6 hours. 75 μL / well of reconstructed Bio-Glo (Promega, G7941) was added, and luminescence was recorded using Berthold reader and MikroWin 2000 software. GraphPad Prism software was used to draw a curve of luminescence to 1 / supernatant dilution, and a four-parameter logistic curve analysis was performed.

[0360] Vectorization and virus production

[0361] Recombinant viruses were generated using the same plasmid as used in the above infection / transfection experiments. In brief, chicken embryo fibroblasts (CEF) were infected with the parental virus encoding GFP at the J2R (TK) locus and the I4L (RR) gene was deleted. The infected cells were transfected with a transfer plasmid carrying an expression cassette flanked by recombinant harms (J2R (TK) locus upstream and downstream DNA sequence homologs). Recombinant viruses were selected by binocular selection of "white" (i.e., GFP negative) lysis plaques. The expression cassette was checked by PCR amplification and subsequent DNA sequencing.

[0362] For in vivo experiments, recombinant viruses are produced on CEF cultured in F500 (MOI 0.05, 72 hours). The cell suspension containing the virus is homogenized using a homogenizer equipped with an in-line chamber. Then a 5 micron pore size filter is used to remove large cell debris by deep filtration. Subsequently, the clarified virus suspension is concentrated and dialyzed with a formula buffer (sucrose 50g / L, NaCl 50mM, Tris 10mM, sodium glutamate 10mM, pH 8), using tangential flow filtration and 0.2 μm pore size hollow fiber microfiltration filter. The purified virus is divided and stored at -80 ° C until use.

[0363] Examples / Results

[0364] Expression of new constructs

[0365] HeLa cells were infected and transfected with transfer plasmids (pTG19965, pTG19966, pTG19967, pTG19968, pTG19969) carrying the above-mentioned different SPD-TNFSF constructs. The expression of recombinant SPD-TNFSF protein in the culture supernatant was detected by immunoblotting using anti-FLAG tag. Figure 1 A and 1B show that all SPD-TNFSF fusion proteins are expressed at the expected monomer size under reducing conditions and show varying degrees of oligomerization under non-reducing conditions. Note that under the denaturing conditions of electrophoresis, oligomers that are not locked by disulfide bonds migrate as monomers.

[0366] It is also particularly advantageous that the fusion proteins of the present invention have a smaller molecular size compared to existing fusion proteins.

[0367]

[0368] Table 3. Molecular sizes of SPD-TNFSF constructs of the invention.

[0369] Interestingly, the construct encoded by pTG19967 also showed clear oligomerization under non-reducing conditions, with the trimer being the major band (at -75 kDa), but hexamers (-150 kDa) and higher oligomers were also clearly visible.

[0370] It is also particularly interesting that, in contrast to prior art disclosures, the fusion protein can form multimers and is not limited to trimers.

[0371] CD40 agonist activity of different SPD-TNFSF constructs

[0372] The CD40 agonist activity of each construct was evaluated on HEK blue CD40L cells and compared to the proteins encoded by pTG19344 (soluble form of CD40L extracellular domain) and pTG19325. A slight improvement was observed for the construct encoded by pTG19965 relative to the constructs encoded by pTG19325 and pTG19344, suggesting that changes in the peptide signal and / or shortening of the CD40L domain have an impact on the expression level and / or agonist activity of the molecule. This benefit was highly amplified by the complete deletion of the collagen domain with the addition (pTG19967 encoded construct) or without the addition (pTG19966 encoded construct) of a (GGGS) x3 linker between the coiled-coil neck domain and CD40L. The collagen domain reduced from 59 (GXX) repeats of the full-length form of SPD (i.e., the construct encoded by pTG19965) to 12 (GXX) repeats (i.e., the construct encoded by pTG19968, pTG19969) demonstrated the best CD40 agonist activity compared to all other constructs. This result suggests that there is an optimal size of collagen domain to obtain the best CD40 agonist effect of the fusion protein. The collagen portion of the constructs encoded by pTG19968 and pTG19969 contains N-glycosylation sites, which may have some effect on oligomer assembly and / or structure and therefore on the CD40 agonist activity of the recombinant molecule. The results of the present invention do not have a significant effect of N-glycosylation.

[0373] Binding to CD40

[0374] An ELISA assay using immobilized recombinant human CD40 was established to investigate the CD40 binding capacity of all SPD-TNFSF constructs. In this assay, the formation of CD40L / CD40 complexes on ELISA plates was detected using non-competitive anti-CD40L. In this assay, the clarified supernatants generated from constructs encoded by infection / transfection with pTG19965 and pTG19325 had the same slight CD40 binding activity at a 1 / 10 dilution. Constructs with modified SPD collagen domains showed a clear improvement in CD40, ranking identical to that observed in the CD40 agonist assay (i.e., constructs encoded by pTG19968>pTG19969>pTG19967>pTG19966). These results suggest that the observed better CD40 agonist activity of the collagen constructs may be due, at least in part, to better binding to CD40.

[0375] The role of collagen domains

[0376] According to the present invention, other numbers of (GXX) repeats around the 12 (GXX) repeats tested have been evaluated. Therefore, different lengths or properties of collagen (GXX) repeats were tested by i) shortening the original 12 (GXX) repeats to two constructs each containing 6 (GXX) repeats (with (pTG20038 encoded construct) and without (pTG20038 encoded construct) N-glycosylation sites) ii) selecting another 12 (GXX) repeats lacking N-glycosylation at another position of the SPD collagen domain (pTG20041 encoded construct), and iii) increasing the number of (GXX) repeats to 19 (pTG20040 encoded construct) and 30 (pTG20042 encoded construct).

[0377] All these constructs have been expressed by the infection / transfection method described above and in parallel with the constructs encoded by pTG19968, pTG19966 and pTG19965 as reference. Figure 4 ) and CD40 agonist assay ( Figure 5 ) The obtained culture medium was tested for CD40 binding.

[0378] By ELISA ( Figure 4 ) and CD40 agonist assay ( Figure 5 ) demonstrated that the constructs were at most equivalent to the construct encoded by pTG19968. The two constructs with 6 (GXX) repeats, with or without N-glycosylation sites, had slightly less potent biological activities than the construct encoded by pTG19968. The biological activities of the two constructs with 12 (GXX) repeats (different from the construct encoded by pTG19968) and 19 (GXX) repeats were significantly less potent than the biological activity of the construct encoded by pTG19968. Finally, the biological activity of the construct with 30 (GXX) repeats was less potent and comparable to the biological activity of the construct encoded by pTG19966.

[0379] Together these results demonstrate that the construct encoded by pTG19968 is the best construct among those tested, although some alternative constructs (e.g., those encoded by pTG19969, pTG19967, pTG20038, pTG20039) can be considered nearly equivalent.

[0380] Vectorization of pTG19968, pTG19969 and pTG19967

[0381] 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. The recombinant viruses were used to infect HeLa cells and the expression of proteins was detected by ELISA ( Figure 6 ) and CD40 agonist assay ( Figure 7 ) were used to measure CD40 binding in culture medium obtained 48 hours after infection. The EC50 for CD40 binding of COPTG19968 and COPTG19969 was increased (i.e., the binding rate was reduced) by approximately ten-fold compared to the infected / transfected counterparts ( Figure 5 , Figure 7 ).

[0382] Note that the curve shape does not even allow the calculation of accurate EC50s for COPTG19968 and COPTG19969. However, COPTG19967 produced some CD40 agonist activity that was much better than the activity of COPTG19968 and COPTG19969. Among the SPD-TNFSF constructs vectored in vaccinia virus, the construct encoded by COPTG19967 produced the best CD40 agonist activity.

[0383] 4-1-BBL expression and 4-1-BB agonist activity of SPD-TNFSF constructs

[0384] The potential of SPD-TNFSF fusion protein extends to 4-1BBL as another member of TNFSF. In the construct encoded by pTG19968, the CD40L extracellular domain is replaced by one of 4-1BBL as a fusion protein with optimal agonist activity in an infection / transfection environment to produce a construct encoded by pTG20033. The 4-1BBL extracellular domain is also cloned separately into the same backbone plasmid to produce a construct pTG20032 further used as a reference. Two 4-1BBL molecules: 4-1BBL extracellular domains encoded by pTG20032 alone or fused with a modified SPD (construct encoded by pTG20033) with 12 (GXX) repeats are expressed at the same level by infected / transfected cells ( Figure 8 ). In addition, the oligomerization pattern observed under non-reducing conditions was similar to that of the modified SPD construct with 12 (GXX) repeats (i.e., CD40L and 4-1BBL fusion). The 4-1BB agonist activity of the modified SPD with 12 (GXX) repeats 4-1BBL construct was significantly better than that of 4-1BBL alone ( Fig. 9 ), although the increase in activity was less than that observed for the equivalent CD40L construct.

[0385] References

[0386] Adra et al.,1987,Gene 60:65-74

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Claims

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

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 (GXX) repeats, preferably 3 to 30 (GXX) repeats, preferably 6 to 20 (GXX) repeats, more preferably 12 (GXX) repeats, wherein 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 4-20 amino acids, preferably a length of 8-16 amino acids, more preferably a length of 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, more preferably 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%, and more preferably at least 95% identity to the amino acid sequence in SEQ ID NO:

1.

8. The SPD-TNFSF fusion protein according to any one of claims 1 to 7, comprising or consisting of a sequence selected from the group consisting of 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 claims 1 to 8.

10. A multimeric fusion protein comprising a plurality of trimeric fusion proteins according to claim 9, which form hexamers, dodecamers, octadecamers or higher-order oligomers, preferably hexamers, more preferably dodecamers.

11. An isolated nucleotide sequence encoding a fusion protein as defined in any one of claims 1 to 8.

12. An isolated nucleotide sequence according to claim 11, wherein the sequence comprises or consists 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 as defined in claim 11.

14. A plasmid comprising a nucleotide sequence as defined in claim 11 or 12.

15. A virus comprising the nucleotide sequence defined in 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 poxvirus, herpesvirus, reovirus, Senecavirus (SVV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), measles virus, retrovirus, adenovirus, adenovirus-associated virus (AAV), herpes simplex virus (HSV), measles virus, foamy virus, alpha virus, lentivirus, influenza virus, Sindbis virus, rhabdovirus, picornaviridae, coxsackievirus, parvovirus or a chimera thereof.

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

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

20. The virus of claim 17, wherein the oncolytic virus is a poxvirus and belongs to the genus Leporopoxvirus, preferably selected from the group consisting of myxoma virus, rabbit fibroma virus and squirrel fibroma virus, preferably myxoma virus.

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

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

23. The virus according to any one of claims 21 or 22, wherein the oncolytic poxvirus is an m2 function-deficient virus, the m2 function deficiency being caused by an inactivating mutation in the M2L viral 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), swine pox virus (SWPV), fowl pox virus (FPV) or chimeras thereof.

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

27. A cell comprising a nucleotide sequence as defined in claim 11 or 12, or an mRNA as defined in claim 13, or a plasmid as defined in claim 14, or a virus as defined in any one of claims 15 to 25.

28. The SPD-TNFSF fusion protein as defined in 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 as defined in 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 disorders associated with dysfunction of TNF cytokines, such as infectious diseases, inflammatory diseases, metabolic diseases, autoimmune diseases, degenerative diseases, apoptosis-related diseases and transplant rejection.

30. Use of a SPD-TNFSF fusion protein as defined in any one of claims 1 to 8, a trimeric fusion protein according to claim 9, a multimeric fusion protein according to claim 10, a nucleotide sequence according to claim 11 or 12, an mRNA according to claim 13, a plasmid according to claim 14, a virus according to any one of claims 15 to 25, or a cell according to claim 27 for the treatment of cancer.

31. Use of a SPD-TNFSF fusion protein as defined in any one of claims 1 to 8, a trimeric fusion protein according to claim 9, a multimeric fusion protein according to claim 10, a nucleotide sequence according to claim 11 or 12, an mRNA according to claim 13, a plasmid according to claim 14, a virus according to any one of claims 15 to 25, or a cell according to claim 27 in combination with one or more chemotherapeutic drugs or immunotherapeutic products for the effective treatment of cancer.

32. A pharmaceutical composition comprising or consisting of: The SPD-TNFSF fusion protein as defined in 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, solvent and / or excipient.

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

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

35. The pharmaceutical composition for use 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 an individual, comprising administering to the subject a therapeutically effective amount of a fusion protein as defined in any one of claims 1 to 8, a trimeric fusion protein according to claim 9, a multimeric fusion protein according to claim 10, a nucleotide sequence according to claim 11 or 12, an mRNA according to claim 13, a plasmid according to claim 14, a virus according to any one of claims 15 to 25, or a cell according to claim 27, or a pharmaceutical composition according to any one of claims 32 to 34.

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