Improved vaccinia virus vectors

Abstract

Disclosed herein are polynucleotides encoding fusion proteins, the fusion protein comprising a vaccinia virus envelope protein, such as A13 or a portion thereof, and at least one complement control protein, or a functional fragment thereof, such as CD35, CD55, CD59, CD46, CR1, Factor H, VCP, MOPICE, SPICE, and CCPH. Modified vaccinia virus vectors and vaccinia virus virions are also disclosed, as are therapeutic uses and methods for the treatment of cancer and / or proliferative diseases or disorders.

Description

[Technical field]

[0001] The present invention relates to the field of viral immunotherapy, for example, for the treatment of cancer and / or cell proliferative diseases or disorders using viruses. In particular, the present invention relates to engineered nucleic acid and genetically modified vaccinia viruses and their therapeutic uses and methods of using them to treat cancer and / or proliferative diseases or disorders. [Background technology]

[0002] Despite advances in new therapeutic approaches, survival of patients with many cancer types, particularly solid tumors, remains one of the greatest challenges in medicine today.

[0003] Oncolytic viruses are an attractive therapeutic approach for the treatment of cancer, especially those that may be resistant to current conventional therapies (Wong et al., (2010), Viruses 2, 78-106). Oncolytic viruses selectively infect and replicate in tumor cells, causing cell death and stimulating the immune system through both innate and adaptive immune responses. This is known as viral immunotherapy and is a rapidly growing area of ​​cancer research. However, despite extensive efforts to improve the selective activity of viruses against different tumor types, viral immunotherapy is limited because viruses are rapidly recognized and eliminated in vivo by the immune system before they can reach and infect target cells. To expand the use of viral immunotherapy beyond tumors where a viral bolus can be directly injected to more visceral or disseminated tumors, new approaches are needed to effectively address this issue.

[0004] Vaccinia virus has been effectively used to immunize against and ultimately eradicate smallpox. Additionally, modified forms have shown promise in human clinical trials for treating cancer or delivering antigens. The success of vaccinia vaccines in eradicating smallpox from the human population has eliminated the need for immunization against the disease, and therefore many people now lack immunity and antibodies to vaccinia virus. This makes vaccinia an interesting option for immunotherapy, but despite the lack of neutralizing antibodies at the first dose, vaccinia is rapidly inactivated when administered intravenously, mainly due to the action of components of the innate immune system against the viral envelope.

[0005] It is therefore desirable to obtain improved systems and viruses for therapeutic treatments, such as cancer therapy, for example by providing engineered vaccinia viruses capable of long-term exposure of the virus that is viable within the host organism. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention is directed to overcoming or at least alleviating one or more of the problems found in the prior art. [Means for solving the problem]

[0007] In general, the present invention provides novel engineered vaccinia viruses and nucleic acid molecules encoding same, compositions, and related uses and methods that can be used to treat diseases or conditions in vitro and / or in vivo. In particular, the present invention provides engineered viruses that can evade the immune response of an animal subject, for example when administered systemically in vivo, by engineering a complement control protein as a fusion with an exposed viral envelope protein. Thus, the present invention provides vaccinia viruses with extended viability in vivo, such as in the bloodstream of a subject or patient. In this way, the engineered vaccinia viruses of the present invention can be used to treat diseases or conditions in vitro and / or in vivo. The virus can survive in vivo for a sufficient period of time to maintain infection and spread to and within tumor cells distal to the target.

[0008] The viruses, compositions and methods of the present invention may be suitable for the treatment of any disease that may be treatable by providing a target cell with an active agent, in particular a therapeutic vaccinia virus according to the present invention.

[0009] The compositions and methods of the invention may be particularly useful in the treatment of cancer and / or proliferative diseases or disorders.

[0010] In one aspect, an isolated nucleic acid is provided that encodes a fusion polypeptide, the fusion polypeptide comprising a vaccinia virus envelope protein or a portion thereof and at least one complement control protein or a functional fragment thereof. Preferably, the functional fragment of the complement control protein is a fragment of the full-length complement control protein sufficient to directly or indirectly cause the destruction of a complement protein in a physiological system. In particular, the vaccinia virus envelope protein may be selected from A13 and A27 or a portion thereof. Most preferably, the vaccinia virus envelope protein is A13.

[0011] In various embodiments, the at least one complement control protein is selected from one or more of the group consisting of: (i) CD35, CD55, CD59, CD46, CR1, Factor H, VCP, MOPICE, SPICE, CCPH, C4 binding protein, CD35, Kaposi's sarcoma-associated herpesvirus Kaposica / KCP, Herpesvirus Saimiri (HVS) and HVS-CD59, Rhesus monkey rhadinovirus RCP-H and RCP-1, Murine gamma herpesvirus 68 (γHV-68) RCA, Influenza virus M1, EMICE and IMP and modified sequences thereof or functional fragments thereof; or (ii) CD35, CD55, VCP, mutated VCP, SPICE, CCPH and ORF4 or functional fragments thereof.

[0012] In an embodiment, at least one complement control protein or functional fragment thereof is fused to the transmembrane domain of a vaccinia virus MV envelope protein.

[0013] In an embodiment of this or any other aspect, the transmembrane region of the vaccinia virus envelope protein is not H3 or D8.

[0014] The vaccinia virus envelope protein A13 may be selected from the A13 protein sequences from Vaccinia Copenhagen virus, Camelpox virus, Smallpox virus, Cowpox virus, Taterapox virus, Monkeypox virus Zaire-96-I-16, Volepox virus, Akhmeta virus, Ectromelia virus, Orthopoxvirus Abatino virus, Skunkpox virus, Raccoonpox virus, Yokapox virus, Murmansk pox virus, NY_014 pox virus and Yabasa tumor virus or portions thereof. Vaccinia virus envelope protein A13 may comprise at least amino acids 2 to 21 of a sequence selected from one of SEQ ID NOs: 1 to 16, or a sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto.

[0015] Various embodiments of the present disclosure include A13 fused to VCP, A13 fused to two VCP proteins arranged in tandem, A13 fused to four VCP proteins arranged consecutively, A13 fused to a mutant VCP, A13 fused to CD55, A13 fused to CD3 and optionally, the mutated VCP comprises SEQ ID NO: 31; the CD55 comprises amino acids 35-284 of CD55 (e.g., SEQ ID NO: 71), the CD35 comprises amino acids 42-1584 of CD35 (e.g., SEQ ID NO: 72), the CCPH comprises amino acids 21-266 of CCPH (e.g., SEQ ID NO: 73), or the ORF4 comprises amino acids 22-268 of ORF4 (e.g., SEQ ID NO: 74). In an embodiment, the VCP protein is a poxvirus complement control protein or a modified poxvirus complement control protein selected from SPICE (SEQ ID NO: 32), MOPICE (SEQ ID NO: 33), EMICE (SEQ ID NOs: 75, 76, particularly SEQ ID NO: 76) or IMP (SEQ ID NOs: 77, 78, particularly SEQ ID NO: 78).

[0016] In embodiments, one or more complement control proteins or functional fragments thereof are fused to the C-terminus of the A13 protein, while in other embodiments, one or more complement control proteins or functional fragments thereof are fused to the N-terminus of the A27 protein.

[0017] The present disclosure encompasses nucleic acid molecules that encode polypeptides similar (but not identical) in function and / or sequence to those disclosed herein, and thus encompasses polynucleotides having the sequence of any one of SEQ ID NOs: 37-46 or 57-62, or polynucleotide sequences having at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to any of the specific sequences disclosed herein.

[0018] In aspects and embodiments of the present disclosure, there are provided engineered vaccinia virus vectors comprising one or more nucleic acids according to the present disclosure; in particular the isolated nucleic acids disclosed herein.

[0019] In embodiments, a nucleic acid encoding a fusion polypeptide comprising a vaccinia virus envelope protein or a portion thereof and at least one complement control protein or a functional fragment thereof is inserted into a vaccinia virus vector at a locus outside of the corresponding wild-type vaccinia virus envelope protein locus. For example, the fusion construct can be inserted into the thymidine kinase (TK) locus. In various embodiments, as a result of the insertion, the TK gene is deleted or inactivated.

[0020] In some embodiments, the nucleic acid encoding the fusion polypeptide is operably linked to the native promoter of the corresponding vaccinia virus envelope protein.

[0021] Suitably, according to embodiments, the engineered vaccinia virus genome is selected from one of the Copenhagen, Western Reserve, Wyeth, Lister or modified vaccinia Ankara strains. In some embodiments, the vaccinia virus strain is the Copenhagen or Western Reserve strain; in particular the Copenhagen strain.

[0022] In aspects and embodiments, there is provided a modified vaccinia virus virion comprising a nucleic acid according to the present disclosure.

[0023] In aspects and embodiments, there is provided a fusion polypeptide encoded by a nucleic acid according to the present disclosure. The polypeptide according to the present disclosure may have an amino acid sequence selected from any one of SEQ ID NOs: 47-56 or 63-68, or a sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto.

[0024] In aspects and embodiments, the nucleic acid, engineered vaccinia virus vector, Pharmaceutical compositions are provided that include the modified vaccinia virus virion or polypeptide and a pharma- ceutically acceptable carrier.

[0025] In various embodiments, the pharmaceutical compositions of the present disclosure may be formulated for systemic or local or topical administration.

[0026] Pharmaceutical compositions according to aspects and embodiments of the present disclosure that include modified vaccinia virus virions may include (i) about 1×10 3 ~Approx. 1×10 15 pfu; (ii) approximately 1 × 10 per ml 4 ~Approx. 1×10 14 pfu; or (iii) about 1 × 10 per ml 6 ~Approx. 1×10 12 The antibody may be formulated to provide a unit dose of pfu.

[0027] The present disclosure provides therapeutic compositions and methods for treating one or more diseases. In aspects and embodiments, an engineered vaccinia virus vector, an isolated nucleic acid, a modified vaccinia virus virion, or a pharmaceutical composition according to the present disclosure is provided for use in a method of treating cancer and / or a proliferative disease or disorder in a subject. Similarly, in aspects and embodiments, a method of treating cancer and / or a proliferative disease or disorder in a mammalian subject is provided, comprising administering to the subject a therapeutically effective amount of an engineered vaccinia virus vector, an isolated nucleic acid, a modified vaccinia virus virion, or a pharmaceutical composition according to the present disclosure.

[0028] In the therapeutic applications and methods of treatment according to the present disclosure, the cancer and / or proliferative disease or disorder is selected from lung cancer (e.g., lung adenocarcinoma), cervical cancer, breast cancer, heart cancer, colon cancer, prostate cancer, brain glioblastoma, pancreatic cancer, leukemia (e.g., acute monocytic leukemia), lymphoma, kidney cancer, colorectal cancer, bladder cancer, testicular cancer, gastrointestinal cancer, liver cancer (e.g., hepatocellular carcinoma) and / or glioblastoma. The present invention may also be useful in treating one or more of skin cancer (e.g., melanoma), head and / or neck cancer, gallbladder cancer, uterine cancer, stomach cancer, thyroid cancer, laryngeal cancer, lip and / or oral cavity cancer, pharyngeal cancer, eye cancer and bone cancer. In embodiments, the cancer may be a primary cancer, a secondary cancer or a metastasis. In particularly advantageous embodiments, the cancer is a metastasis.

[0029] The compositions according to the present disclosure may be administered systemically or locally to a subject. A suitable route of administration may be selected from intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, local (e.g., near a tumor, particularly the vasculature of the tumor or adjacent vasculature), percutaneous, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, or oral. In embodiments, the therapeutic composition may be administered in combination with one or more additional therapeutic agents or therapies. When used, the additional therapeutic agents or therapies may be administered simultaneously, separately, or sequentially with administration of the therapeutic or medical composition of the present disclosure.

[0030] It will be understood that any feature of one aspect or embodiment of the invention may be combined with any combination of features of any other aspect or embodiment of the invention, unless otherwise stated, and such combinations are within the scope of the invention as claimed. Thus, within the scope of this disclosure, it is expressly intended that the various aspects, embodiments, examples and alternatives described in the preceding paragraphs, clauses and claims and / or the following description and drawings, in particular their individual features, may be employed independently or in any combination. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination, so long as such features are not incompatible. More specifically, any embodiment of any aspect may form an embodiment of any other aspect, and all such combinations are specifically intended to be encompassed within the scope of the invention. The applicant reserves the right to modify the claims originally filed or to file new claims accordingly. This includes amending the claims originally filed to rely on and / or incorporate features of other claims, even if they were not originally claimed as such. Includes the right to rectification.

[0031] The invention is further illustrated by the accompanying drawings. [Brief description of the drawings]

[0032] [Figure 1] Schematic diagram of the complement activation pathway and its regulation by an engineered envelope protein composed of a complement regulatory protein domain (ENV-CRP). [Diagram 2] Schematic diagram of the vaccinia virus envelope showing the distribution and complexes of the envelope proteins. [Diagram 3] Sequence alignment of A13L proteins from various vaccinia virus species. Key: ref|YP_233014.1| (Vaccinia virus; SEQ ID NO: 1); ref|NP_570520.1| CMLV130 (Camelpox virus; SEQ ID NO: 2); ref|NP_042161.1| VARVgp117 (Smallpox virus; SEQ ID NO: 3); ref|NP_619928.1| CPXV145 protein (Cowpox virus; SEQ ID NO: 4); ref|YP_717441.1| Taterapox virus (SEQ ID NO: 5); ref|NP_536551.1| A14L (Monkeypox virus-Zaire-96-I-16; SEQ ID NO: 6); ref|YP_009281879.1| (Volepox virus; SEQ ID NO: 7); ref|YP_010085590.1| (Acumetavivirus; SEQ ID NO: 8); ref|NP_67163 4.1|EVM116 (Ectromeliavirus; SEQ ID NO: 9); ref|YP_010085800.1|Predicted A13L protein (Orthopoxvirus Abatinovirus; SEQ ID NO: 10); ref|YP_009282825.1|(Skunkpoxvirus; SEQ ID NO: 11); ref|YP_009143440.1|(Raccoonpoxvirus; SEQ ID NO: 12); ref|YP_004821469.1|(Yokapoxvirus; SEQ ID NO: 13); ref|YP_009408310.1|(Murmansk poxvirus; SEQ ID NO: 14); ref|YP_009408512.1|NY_014 poxvirus (SEQ ID NO: 15); and ref|NP_938359.1|(Yaba monkey tumor virus; SEQ ID NO: 16). [Figure 4]Sequence alignment of A27L proteins from various vaccinia virus species. Key: P20535.1 Vaccinia Copenhagen virus A27 (SEQ ID NO: 17); YP_233032.1 Vaccinia virus A27 (SEQ ID NO: 18); NP_619946.1 CPXV162 Cowpox virus A27 (SEQ ID NO: 19); NP_570536.1 CMLV146 Camelpox virus A27 (SEQ ID NO: 20); NP_042178.1 VARVgp134 Smallpox virus (SEQ ID NO: 21); YP_010085815.1 Orthopox Abatino virus (SEQ ID NO: 22); YP_ 010085606.1 Acmetavirus (sequence number 23); NP_671648.1 EVM129 Ectromeliavirus (sequence number 24); YP_717458.1 Taterapoxvirus (sequence number 25); NP_536566.1 A29L Monkeypoxvirus (sequence number 26); YP_009281894.1 Volepoxvirus (sequence number 27); YP_009143455.1 Raccoonpoxvirus (sequence number 28); YP_009282840.1 Skunkpoxvirus (sequence number 29). [Diagram 5] FIG. 1 is a schematic diagram showing the preparation of various vaccinia virus expression cassettes for expressing the fusion proteins of the present disclosure from the A13L locus of vaccinia virus. [Figure 6] FIG. 1 is a schematic diagram showing the preparation of various vaccinia virus expression cassettes for expressing the fusion proteins of the present disclosure from the J2R(TK) locus of vaccinia virus. [Figure 7]Viral infectivity assay after human serum incubation. (A) Parental control virus (WT; left column, blue) or a virus encoding an A13-CD55-V5 fusion protein according to an embodiment of the invention (vA13-CD55; right column, red) are pre-incubated with human serum for 30 or 60 minutes. The data show that in contrast to WT, the engineered virus of the invention (vA13-CD55) shows significantly increased resistance to serum-mediated neutralization and much higher infectivity after incubation with human serum. (B) Whole cell lysates of cells infected with WT or vA13-CD55 virus were probed with an antibody against the V5 affinity tag to reveal expression of the fusion protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs (e.g., cell culture, molecular genetics, nucleic acid chemistry, and biochemistry).

[0034] Unless otherwise indicated, the practice of the present invention employs conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA technology, chemical methods, pharmaceutical formulation, and animal delivery and treatment, which are within the capabilities of those of ordinary skill in the art. Such techniques are described in the literature, e.g., J. Sambrook, E. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology,ch.9,13,and 16,John Wiley&Sons,New York,NY);B.Roe,J.Crabtree,and A.Kahn,1996,DNA Isolation and Sequencing:Essential Techniques,John Wiley&Sons;JMPolak and James O'D.McGee,1990,In Situ Hybridization:Principles and Practice,Oxford University Press;MJGait(Editor),1984,Oligonucleotide Synthesis:A Practical Approach,IRL Press;and DMJLilley and JEDahlberg,1992,Methods of and Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press, each of which is incorporated herein by reference.

[0035] To facilitate the understanding of the present invention, certain terms are defined herein.

[0036] The terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably and refer to deoxyribonucleotide (DNA) or ribonucleotide (RNA) polymers in linear or circular configuration, in single-stranded or double-stranded form. For the purposes of the present invention, such DNA or RNA polymers may contain natural nucleotides, non-natural or synthetic nucleotides and mixtures thereof. Non-natural nucleotides may include analogs of natural nucleotides and nucleotides modified in the base, sugar and / or phosphate moieties (e.g., phosphorothioate backbones). Examples of modified nucleic acids are PNA and morpholino nucleic acids. In general, analogs of a particular nucleotide have the same base-pairing specificity, i.e., an analog of G will base-pair with C. For the purposes of the present invention, these terms should not be considered limiting with respect to the length of the polymer.

[0037] As used herein, a "gene" is a segment of nucleic acid (typically DNA) involved in the production of a polypeptide or ribonucleic acid gene product. It includes regions preceding and following the coding region (leader and trailer) and intervening sequences (introns) between individual coding segments (exons). Conveniently, the term refers to the regulatory sequences necessary for gene expression. Also included are regulatory sequences (e.g., enhancers, silencers, promoters, terminators, etc.) which may be adjacent to or distant from the associated coding sequence and the coding and / or transcribed regions which encode a gene product.

[0038] As used herein, the term "vector" is used to refer to a nucleic acid vector, e.g., a DNA vector such as a plasmid, an RNA vector, a virus or other suitable replicon (e.g., a viral vector). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO 1994 / 11026. The expression vectors of the invention may contain one or more additional sequence elements used for the expression of proteins and / or the integration of these polynucleotide sequences into the genome of a host cell, such as a mammalian cell (e.g., a human cell). Exemplary vectors that can be used for the expression of the antibodies and antibody fragments described herein include plasmids that contain control sequences such as promoter and enhancer regions that direct gene transcription. The vector may contain a nucleic acid that modulates the translation rate of a target gene or improves the stability or nuclear export of the mRNA resulting from gene transcription. These sequence elements may include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), ribosome skipping sequences such as picornavirus 2A sequences (T2A, F2A, E2A), and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector. The vectors described herein may also contain a polynucleotide encoding a marker for the selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as neomycin, geneticin, ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0039] As used herein, "A13L" refers to a vaccinia virus gene that encodes a protein of approximately 70 amino acids that is one of the major components of the vaccinia virus membrane (see FIG. 2, and e.g., Unger & Traktman (2004), J Virol., 78(16):8885-8901). It is essential for virion morphogenesis and is believed to play a key role in the transition from immature virions (IV) to intracellular mature virions (IMV). Examples of A13L in various species include YP_233014.1 vaccinia virus; NP_570520.1 CMLV130 camelpox virus; NP_042161.1 VARVgp117 smallpox virus; NP_619928.1 CPXV145 protein cowpox virus; YP_717441.1 taterapox virus; NP_536551.1 A14L monkeypox virus-Zaire-96-I-16; YP_009281879.1 volepox virus; YP_010085590.1 acmeta virus; NP_671634.1 YP_009408512.1 NY_014 poxvirus; and NP_938359.1 Yabasa tumor virus.

[0040] As used herein, "A27L" refers to a vaccinia virus gene that encodes a protein of approximately 110 amino acids that is one of the major components of the vaccinia virus membrane (Figure 2; see also, e.g., Vazquez et al., (1998), J Virol., 72(12):10126-10137). It is located on the surface of the intracellular mature virus (IMV) form and is thought to be essential for both release of the extracellular enveloped virus (EEV) from the cell and for virus spread. Examples of A27L in various species include P20535.1A27_VACCC RecName: Total length = 14 kDa fusion protein, ...fusion protein;YP_233032.1 IMV surface protein Vaccinia virus;NP_619946.1 CPXV162 protein Cowpox virus;NP_570536.1 CMLV146 Camelpox virus;NP_042178.1 Hypothetical protein VARVgp134 Smallpox virus;YP_010085815.1 Putative A27L protein Orthopoxvirus Abatino;YP_010085606.1 IMV surface protein Aquametavirus;NP_671648.1 EVM129 Ectromelia virus;YP_717458.1 IMV surface protein Taterapoxvirus;NP_536566.1 A29L Monkeypox virus Zaire-96-I-16;YP_009281894.1 imv surface protein volepoxvirus;YP_009143455.1 IMV surface protein raccoonpoxvirus;YP_009282840.1 imv surface protein skunkpoxvirus;YP_009408527.1 IMV surface protein NY_014poxvirus;YP_009408325.1 IMV surface protein Murmanskpoxvirus;YP_004821484.1 IMV surface protein Yokapoxvirus;YP_009408075.1 IMV membrane protein, fusion Eptesipoxvirus;YP_008658541.1 IMV cell attachment, fusion and microtubule transport Squirrelpox virus; YP_009389390.1 Putative fusion protein-like protein Parapoxvirus of Seals; YP_009177162.1 Type A inclusion body-like / fusion protein Turkeypox virus; YP_009112843.1 Putative fusion protein Parapoxvirus red deer / HL953; YP_009268837.1 IMV surface protein, fusion protein Pteropox virus; NP_044084.1 MC133L Molluscum contagiosum virus subtype 1; YP_009480655.1 IMV surface protein Sea otter pox virus; NP_957881.1 ORF104 fusion protein Orf virus; YP_010085255.1 P4c precursor Western grey pox kangaroopox)virus;YP_010085419.1 P4c precursor Eastern grey kangaroopox virus; NP_955288.1 CNPV265 Type A inclusion body-like / fusion protein Canarypox virus; YP_009046422.1 Type A inclusion body protein Pigeonpox virus; YP_009046188.1. Type A inclusion body protein Penguinpox virus; YP_009448114.1 Type A inclusion body protein Flamingopox virus FGPVKD09; NP_039154.1 Type A inclusion body protein Fowlpox virus; NP_958013.1 ORF104 fusion protein Bovine papular stomatitis virus; YP_005296322.1 MV attachment protein Cotia virus SPAn232; YP_003457410.1 Viral fusion peptide Pseudocowpox virus; YP_009329750.1 Putative fusion protein BeAn 58058 virus;NP_570274.1 hypothetical protein SWPVgp114 swinepox virus;NP_150551.1 hypothetical protein LSDVgp117 varicella virus NI-2490;NP_073502.1 117L protein Yaba-like disease virus; YP_227501.1 fusion protein Deerpox virus W-848-83; NP_938372.1 hypothetical protein YMTVg117L Yaba monkey tumor virus; NP_052004.1 hypothetical protein SFV_s115L Rabbit fibroma virus; NP_659689.1 hypothetical protein SPPV_112 Sheeppox virus; YP_001293308.1 hypothetical protein GTPV_gp112 Goatpox virus Pellor; NP_051829.1 hypothetical protein MYXV_gp119 Myxoma virus.

[0041] As used herein, "VCP" refers to a vaccinia virus gene that encodes a protein of approximately 243 amino acids that is the major protein secreted and expressed from the surface of vaccinia virus-infected cells (Girgis et al., (2008), J Virol., 82(9):4205-4214). It is similar in sequence to regulators of complement activation and is encoded by the smallpox virus, the smallpox complement enzyme inhibitor (SPICE). and is homologous to monkeypox complement inhibitor (MoPICE) (Liszewski et al. al.,(2006),J Immunol.,176(6):3725-3734). Its role is to protect the virus from attack by the host complement system by inhibiting complement produced by both the classical and alternative pathways (Figure 2, see, for example, Sahu et al. (see, e.g., et al., (1998), J Immunol., 160(11):5596-5604). Examples of VCPs from various species include: YP_232907.1 secreted complement binding C3b / C4b C3L Vaccinia virus: NP_570413.1 secreted complement binding protein Camelpox virus: NP_042056.1 secreted complement binding protein D15L B19L SPICE Smallpox virus: NP_619823.1 CPXV034 Cowpox virus: NP_671535.1 secreted complement binding protein Ectromelia virus: YP_010085695.1 putative C3L protein Orthopoxvirus Abatino: YP_010085478.1 complement binding protein Acmetavirus: NP_536444.1 D14L MOPICE monkeypox virus Zaire-96-I-16: YP_009282718.1 complement fixing Skunkpox virus: YP_009281772.1 complement fixing Volepox virus: YP_009143334.1 secreted complement fixing proteins C3b / C4b Raccoonpox virus: YP_717333.1 secreted proteins Taterapoxvirus: YP_005296214.1 complement fixing proteins Chothiavirus SPAn232: YP_009408407.1 complement fixing NY_014 poxvirus: YP_009408205.1 complement fixing Murmansk poxvirus; NP_051858.1 m144R myxoma virus.

[0042] As used herein, "herpesvirus complement control protein" refers to any of the following proteins: NP_570746.1 complement fixing protein macaque gammaherpesvirus 5; NP_040205.1 complement control protein homolog squirrel monkey gammaherpesvirus 2; YP_010084365.1 ORF4 retroperitoneal fibromatosis-associated herpesvirus; NP_040206.1 complement control protein homolog squirrel monkey gammaherpesvirus 2; NP_047979.1 complement control protein homolog ccph spider monkey gammaherpesvirus 3; YP_010084544.1 ORF4 pigtailed macaque rhadinovirus 2; YP_010084543.1 ORF4A Pigtailed macaque rhadinovirus 2;YP_009551812.1 Hypothetical protein Horseshoe bat gammaherpesvirus 1;YP_238307.1 JM4 Japanese macaque rhadinovirus;YP_001129351.1 ORF4;KCP Human gammaherpesvirus 8;YP_009408125.1 Secreted complement binding protein C3b / C4b Eptesipox virus;YP_009229839.1 Complement regulatory protein-like protein Myotis myotis gammaherpesvirus 8;YP_009552470.1 Complement regulatory protein Large brown bat gammaherpesvirus;NP_044845.1 Complement regulatory protein Murine gammaherpesvirus 4;YP_010085882.1 Complement regulatory protein Apodemus herpesvirus;YP_004207839.1 Complement regulatory protein Cricetidae gammaherpesvirus 2;YP_004207845.1 Complement regulatory protein A complement regulatory protein (similar to VCP) expressed by herpesviruses such as Cricetidae gammaherpesvirus 2.

[0043] In the context of the present invention, the term "amino acid" is used in the broadest sense and is meant to include naturally occurring L α-amino acids or residues. In this specification, the commonly used one-letter and three-letter abbreviations of natural amino acids are used: A=Ala; C=Cys; D=Asp; E=Glu; F=Phe; G=Gly; H=His; I=Ile; K=Lys; L=Leu; M=Met; N=Asn; P=Pro; Q=Gln; R=Arg; S=Ser; T=Thr; V=Val; W=Trp; and Y=Tyr (Lehninger, AL, (1975) Biochemistry, 2d ed., pp. 71-92, Worth Publishers, New York). The general term "amino acid" includes D-amino acids, retro-inverso amino acids as well as chemically modified amino acids such as amino acid analogs. The definition of amino acids further includes chemically synthesized compounds having properties known in the art to be characteristic of amino acids, such as the amino acids mentioned above, natural amino acids not normally incorporated into proteins, such as norleucine, and β-amino acids. For example, analogs or mimetics of phenylalanine or proline that allow the same conformational restriction of peptide compounds as natural phenylalanine or proline are included in the definition of amino acids. Such analogs and mimetics are referred to herein as "functional equivalents" of the respective amino acids. Other examples of amino acids are listed in Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., NY 1983, which is incorporated herein by reference.

[0044] The term "peptide" as used herein refers to multiple amino acids linked together in a linear or cyclic chain (e.g., in connection with viral envelope proteins or fusion proteins of the present disclosure). The term oligopeptide is typically used to describe peptides having from 2 to about 50 or more amino acids. Peptides larger than about 50 amino acids are often referred to as polypeptides or proteins. However, for purposes of the present invention, the term "peptide" is not limited to a particular number of amino acids and is used interchangeably with the terms "polypeptide" and "protein."

[0045] As used herein, in the context of a polypeptide / protein, the term "chimeric" or "fusion" refers to a polypeptide sequence comprising at least a first and a second polypeptide sequence covalently linked to each other (e.g., C-terminus and N-terminus), where the first and second polypeptide sequences do not naturally occur within the same polypeptide / protein. The first and second polypeptide sequences may each be a single protein domain or may comprise multiple protein domains that combine to perform a function. Typically, such first and second polypeptide sequences are linked to each other by an amino acid / peptide linker that may be of any suitable length, but is typically between 5 and 50 amino acids. Beneficially, such peptide linkers are relatively inert, i.e., do not interfere with the function of the fusion protein or the function of the first and second polypeptide sequences, and may comprise predominantly Gly and / or Ser residues. Similarly, the term "chimeric" or "fusion" may also be used to refer to a nucleic acid / polynucleotide sequence encoding a chimeric or fusion polypeptide / protein.

[0046] "Complement regulator proteins" or "complement control proteins" are proteins that play a role in regulating the (innate) immune complement system in humans and animals from overactivating and harming self-tissues. There are several soluble regulatory proteins, such as C1 inhibitor, C4b binding protein, factors H, B, D and I. In addition, membrane-bound complement control proteins (mCRPs) provide another complement control mechanism, including CD35 (complement receptor 1, CR1), CD46 (membrane cofactor protein, MCP), CD55 (decay accelerating factor, DAF) and CD59 (protectin). Complement control proteins are expressed in all cells of the human body, but the expression of these mCRPs varies between different tissue types. It has been hypothesized that mCRP expression between tissue types may also vary because different tissues face different immune interactions in the body (Qin et al., (2001), Mamm. Genome, 12:582-589).

[0047] In the context of the present disclosure, the terms "individual", "subject" or "patient" are used interchangeably to refer to an animal that may be suffering from a medical (pathological) condition and may respond to the molecules, compositions, methods, uses, medical treatments or therapeutic treatment regimens of the present disclosure. The animal is preferably a mammal, such as a human, non-human primate, cow, sheep, pig, dog, cat, rabbit, bat, mouse or rat. In particular, the subject may be a human, rabbit or mouse, and is in particular a human.

[0048] The viruses, nucleic acids, compositions, or agents of the present disclosure may be used to treat one or more diseases, infections, or disorders. As used herein, the terms "treat," "treating," or "therapy" in reference to a disease state or condition refer to a reduction in the severity of the disease or condition or pathogenic symptoms such that the therapy is effective to cure, inhibit, alleviate, reduce, or prevent the adverse effects of the disease or disorder being treated, or is sufficient to achieve a physiologically or biochemically detectable effect. Thus, an ameliorative, inhibitory, or preventive effect may be achieved with respect to the disease or disorder. In various embodiments, such treatment may include oncolysis or death of tumor cells, inhibition of tumor cell growth or metastasis, reduction of tumor size, and / or otherwise reversal or reduction of the malignant phenotype of tumor cells. For example, in the treatment of cancer, tumor growth may be reduced by up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, or up to about 10%. In embodiments, tumor growth may be reduced by about 10% to about 90%, about 20% to about 80%, or about 30% to about 70%. Thus, in some embodiments, the virus, nucleic acid, peptide, or composition (i.e., a therapeutic agent according to the present disclosure) may be manufactured into a medicament or incorporated or formulated into a pharmaceutical composition.

[0049] Vaccinia virus Vaccinia virus belongs to the family Orthopoxvirus or Poxviridae, subfamily Chordopoxvirinae, and genus Orthopoxvirus, which is more homogeneous than other members of the subfamily Chordopoxvirinae and contains 11 distinct but closely related species, including vaccinia virus, variola virus (the causative agent of smallpox), cowpox virus, buffalopox virus, monkeypox virus, mousepox virus, and horsepox virus species, as well as others (see Moss, 1996). As described herein, certain embodiments of the invention may be extended to other members of the Orthopoxvirus genus as well as the genera Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Molluscipoxvirus and Yatapoxvirus. Genera in the Chordopoxvirinae subfamily are generally defined by serological means, including neutralization and cross-reactivity in laboratory animals.

[0050] Vaccinia virus is a large enveloped virus with a linear double-stranded DNA genome of about 190 kbp encoding approximately 250 genes. Unusually for a DNA virus, vaccinia is a non-integrating vector since it replicates only in the cytoplasm of the host cell. The vaccinia genome encodes the enzymes and proteins required for viral DNA replication. During replication, vaccinia produces several infectious forms that differ in the composition of the outer membrane: intracellular mature virions (IMV, also known as mature virions, MV), intracellular enveloped virions (IEV), cell-associated enveloped virions (CEV) and extracellular enveloped virions (EEV, also known as extracellular virions, EV). IMV is the most infectious form and is thought to be responsible for spread between hosts. Meanwhile, CEV is thought to play a role in cell-to-cell spread, and EEV is thought to be important for long-distance propagation within the host organism.

[0051] Vaccinia virus immunotherapy The advantage of using vaccinia virus for treatment is that the vaccinia vector is virus-depleted. These include the ability to carry up to 25 kb of foreign DNA without requiring deletion, a broad host range allowing infection of primary cultures and many different cell lines, cytoplasmic replication, and the viral genome does not splice the primary transcript. Viral replication within the host cell leads to cell lysis upon viral release.

[0052] The use of vaccinia virus as a clinical viral immunotherapy vector has shown promise for cancer treatment, but only limited clinical success has been achieved so far due to the lack of efficient systemic delivery and viral spread. Typically, viral immunotherapy vectors can be administered (a) systemically via intravenous injection or (b) locally by inoculation near the tumor ("locoregional" delivery) or by direct injection into the tumor ("intratumoral delivery"). Systemic administration of the virus would be beneficial to simultaneously treat both the primary tumor and disseminated, potentially undiagnosed metastatic lesions. However, viral immunotherapy is limited in efficacy to tumors that can be reached by local injection due to its low activity after systemic delivery. This rapid inactivation is primarily a result of the innate immune response of non-immune, non-antigen-primed individuals. The complement system is a major coordinator of the innate immune response and is a key defense mechanism against pathogens, recognizing viral membranes and opsonizing them, leading to recognition by immune cells or viral lysis. Enveloped RNA and DNA viruses are particularly susceptible to the action of the complement cascade.

[0053] Since metastasis is the main cause of death in cancer patients, a better solution is needed to avoid inactivation in bloodstream so that the virus can reach deep into the blood vessels of primary tumors or reach disseminated metastasis. Therefore, a method that can fully control the inactivation of vaccinia virus can improve the delivery of active therapeutic virus to target cells in tumors, which will be of great benefit to the treatment of cancer. Furthermore, delivery into tumors is complicated because it requires accurate imaging and localization of diseased tissue before injection, and because high interstitial pressure in tumors prevents local delivery through needles and may leak at the injection point. Therefore, improved vaccinia therapy can be achieved by modified vaccinia virus that can evade the host's immune system, along with the convenience of systemic delivery and improved activity of therapy.

[0054] Therefore, there is an urgent need for engineered vaccinia viruses that are more robust against complement attack and therefore can be delivered systemically and spread more easily from the point of infection in tumors to distant metastases.The present invention addresses this need and provides a solution to the systemic delivery of vaccinia viruses by modifying viral proteins, particularly viral envelope proteins, to promote tumor-targeted systemic delivery of the virus, intratumoral and intertumoral viral spread, and enhanced tumor-specific viral replication.Advantageously, the modified and engineered viruses of the present disclosure can be utilized as platform vectors for systemic delivery for viral immunotherapy.

[0055] Many of the genomes of poxviruses, including those of different strains of vaccinia virus, have been sequenced. The genome of the Western Reserve (WR) strain of vaccinia virus contains 218 potential open reading frames. Protein analysis of the intracellular mature virion (IMV) has shown it to contain at least 81 viral proteins, including structural proteins, enzymes, and transcription factors, including A2.5L, A3L, A4L, A5R, A6L, A7L, A9L, A10L, A12L, A13L, A14L, A14.5L, A15L, A16L, A17L, A18R, A21L, A22R, A23L, A24L, A25R, A26L, A27L, A28R, A29L, A30L, A31L, A32L, A33L, A34L, A35L, A36L, A37L, A38R, A39L, A40L, A41L, A42R, A43L, A44L, A45R, A46L, A47L, A48R, A49L, A50L, A51L, A52L, A53L, A54L, A55R, A56L, A57L, A58R, A60L, A61L, A62L, A63L, A64L, A65R, A66L, A67L, A68R, A69L, A70L, A71L, A72R, A73L, A74L, A75R, A76L, A77L, A78R, A79L, A80L, A81L, A82R, A83L, A84L, A85R, A86L, A87L, A88R, A89L, , A24R, A25L, A26L, A27L, A28L, A29L, A30L, A31R, A32L, A42R, A45R, A46R, B1R, C6L, D1R, D2R, D6R, D7 R, D8L, D11L, D12L, D13L, E1L, E4L, E6R, E8R, ElOR, E11L, F8L, F9L, F10L, F17R, G1L, G3L, G4L, G5R, G5. These include 5R, G7L, G9R, H1L, H2R, H3L, H4L, H5R, H6R, I1L, I2L, I3L, I5L, I6L, I7L, I8R, J1R, J3R, J4R, K4L, L1R, L3L, L4R, L5R, 02L. Among these are key proteins involved in binding, virion fusion and structural integrity. Their known functions include four attachment proteins A27, H3, D8, A26; eleven components of the entry fusion complex (EFC) A16, A21, A28, G3, G9, H2, J5, L5, O3, L1 and F9, as well as structural proteins such as A13. Proteins encoded by A27L, H3L, L1R and D8L have been identified as major immunogenic proteins, and intensive efforts have been made to engineer these proteins to avoid neutralization by the adaptive immune system (see, for example, WO 2020 / 086423A1). Proteins A27, H3 and D8 are adhesion molecules that bind to host glycosaminoglycans (GAGs), heparan sulfate (HS) and chondroitin sulfate (CS) and mediate endocytosis of the virus into the host cell. L1 protein is involved in virus maturation.

[0056] Various members of the Orthopoxvirus genus and other members of the Chordovirinae subfamily show conservation in the envelope proteins, as shown herein (see Figures 3 and 4, which show sequence alignments of A13 and A27, respectively).

[0057] The EEV form of the virus is released early in infection and has an additional membrane that protects the virus from complement by sequestering other proteins, such as the host complement proteins CD46, CD55, CD59 (Vanderplasschen et al. (1998) PNAS) and MHCI, in addition to those of the mature virion. A56 on the EEV envelope can also disulfide bond with the secreted vaccinia complement control protein VCP to further reduce complement-mediated destruction of the virus. This is thought to provide the virus with the ability to spread more widely and infect new tissues. In contrast, IMV has no natural or acquired ability to evade complement, and modifications of the viral envelope or proteins associated with the envelope can affect packaging and reduce the infectivity of the virus to target cells. As a result, many attempts to modify these proteins have failed to generate viable viruses (Paul et al., (2007), Viral Immunol.).

[0058] The present disclosure provides engineered vaccinia viruses that have an improved ability to evade an immune system, such as the innate immune system of a host animal subject. The present disclosure encompasses any suitably engineered oncolytic vaccinia virus strain. Potential vaccinia virus genomes that may be used in accordance with the present disclosure include Abatino macacapox virus, Aquametavirus, Camelpox virus 903, Camelpox virus CMG, Camelpox virus CMS, Camelpox virus CP1, Camelpox virus CP5, Camelpox virus M-96, Cowpox virus (Brighton Red), Cowpox virus (GRI-90 strain), Cowpox virus (Hamburg-1985 strain), Cowpox virus (Turkmenia-1974 strain), Elephantpox virus, Belo Horizonte virus, Ectromeliavirus ERPV, Ectromeliavirus Moscow, Ectromeliavirus Naval, Ectromeliavirus WH, Common marmoset (Callithrix jacchus orthopoxvirus, Monkeypox virus (Sierra Leone strain 70-0266), Monkeypox virus (Zaire strain 77-0666), Monkeypox virus-Zaire-96-I-16, Raccoonpox virus, Skunkpox virus, Taterapox virus, Aracatuba virus, Buffalopox virus, Cantagalo virus, Guarani P1 virus, Guarani P2 virus, Horsepox virus, Modified vaccinia-Ankara virus, Rabbitpox virus, Rabbitpox Virus Utrecht, SPAN 232 virus, Vaccinia virus Acambis 3000 MVA, Vaccinia virus Ankara, Vaccinia virus Copenhagen, Vaccinia virus Dalian I, Vaccinia virus GLV-1h68, Vaccinia virus IHD-J, Vaccinia virus L-IPV, Vaccinia virus LC16M8, Vaccinia virus LC16MO, Vaccinia virus Lister, Vaccinia virus LIVP, Vaccinia virus Mariana, Vaccinia virus Tashkent, Vaccinia virus Tian Tan, Vaccinia virus WAU86 / 88-1, Vaccinia virus Western Reserve, Vaccinia virus WR, Vaccinia virus WR 65-16, Vaccinia virus Wyeth, Variola major virus, Variola minor) virus, Variola virus-Human / India / Ind3 / 1967, Volepox virus, Alaskapox virus, Whalepox virus 1, Dolphinpox virus 1, Whalepox virus 2, Cowpox vaccinia virus, Catpox virus ITA1_PG, Catpox virus ITA2_BC, Orthopoxvirus GCP2010, Orthopoxvirus GCP2013, Orthopoxvirus NY99014 / 1999, Orthopoxvirus OH08 / 2008, Orthopoxvirus Tena Dona, Orthopoxvirus VPXV_CA85, Orthopoxvirus WA01960 / 2001, Todopox virus, Orthopoxvirus species (as listed in the NCBI taxonomy database).

[0059] In some embodiments, the vaccinia virus for use according to the present disclosure is based on the Copenhagen, Western Reserve, Wyeth, Lister or modified Vaccinia Ankara strain. Advantageously, the genetically modified virus is based on the Copenhagen or Western Reserve virus strain, in particular the Copenhagen-derived vaccinia virus. Such engineered viruses may be suitable for use and methods for the treatment of various cancers. The vaccinia viruses described herein may be administered to a patient, such as a mammalian patient (e.g., a human patient), to treat a variety of cell proliferation disorders, including a wide range of cancers.

[0060] Modified vaccinia virus Studies of vaccinia virus and modifications of the vaccinia virus genome have identified a number of engineered / modified viruses that have demonstrated potential in cancer therapy.

[0061] Wild-type vaccinia virus has no tumor selectivity, but viral strains have been engineered for selectivity against cancer cells by inactivating mutations or deletions of genes such as the viral thymidine kinase (gene J2R) or ribonucleotide reductase (F4L).

[0062] For example, engineered vaccinia viruses can be engineered to express the vaccinia virus genes: C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, J2R, F1L, F2L, F3L, B14R, B15R, B16R, B17L, B18R, B19R, B20R, K ORF A, K ORF B, B ORF E, B ORF F, B ORF G, B ORF H, B ORF I ... It has been identified that when engineered to contain deletions in one or more or all of G, B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R, and B29R, they exhibit one or more of significantly improved oncolytic activity, intratumoral replication, infectivity, immune evasion, tumor persistence, ability to integrate foreign DNA sequences, and suitability for large-scale manufacturing (see, for example, WO 2019 / 134049). Thus, aspects and embodiments of the present disclosure relate to engineered vaccinia viruses or viral nucleic acid genomes that contain deletions of one or more of the above genes, i.e., various wild-type vaccinia virus genes are deleted to enhance the oncolytic activity of the vaccinia virus. In various embodiments, at least one of the above genes is deleted from the recombinant vaccinia virus genome. In various embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the above genes are deleted from the recombinant vaccinia genome.

[0063] In various embodiments, the modified vaccinia virus contains at least a deletion of the B8R gene. The vaccinia virus B8R gene encodes a secreted protein with homology to gamma interferon receptor (IFN-g). The B8R protein binds to and neutralizes the antiviral activity of several species of gamma interferon, including human and rat gamma interferon, in vitro. The deletion of the B8R gene prevents the impairment of IFN gamma function in humans, while it is inactive in mice. Thus, the deletion of the B8R gene improves safety without reducing immunogenicity.

[0064] Where an engineered virus or viral genome of the present disclosure comprises a deletion of one or more endogenous genes, it will be understood that one or more heterologous genes, expression constructs or polynucleotide sequences may be inserted into the genome, for example by homologous recombination or other suitable mechanisms, as described elsewhere herein.

[0065] Various beneficial modifications to vaccinia virus for use in oncolytic therapy are known to those of skill in the art, some of which are described in this disclosure, but any other modifications that may be useful and obvious to those of skill in the art are also contemplated herein.

[0066] Some current clinical studies testing vaccinia virus as an oncolytic virus have a deletion in the viral thymidine kinase (TK) gene. This deletion makes the virus attenuated and dependent on the activity of cellular thymidine kinase for DNA replication and viral growth. Cellular thymidine kinase is expressed at low levels in most normal tissues, but at high levels in many cancer cells. This metabolic targeting allows TK viruses to grow efficiently in cells with high metabolic rates (e.g., healthy cells or tumor cells) and grow poorly in cells with low levels of thymidine kinase. However, some tumor cells are in a quiescent state (e.g., cancer stem cells), so some TK viruses may not be able to kill all cancer cell populations. In fact, many chemotherapy drugs are also largely ineffective against such quiescent cells.

[0067] Thus, in some embodiments, the engineered vaccinia virus vectors and viruses of the present disclosure may lack the TK gene or have the TK gene disabled or inactivated to promote activity (e.g., preferential replication) against rapidly dividing cells, particularly cancer cells, while in some alternative embodiments, the engineered viral vectors and viruses of the present disclosure may include TK to enable growth / replication in quiescent cancer cells.

[0068] In further embodiments, the oncolytic vaccinia virus may also (or alternatively in some embodiments) be engineered to lack vaccinia viral growth factor (VGF). VGF is a secreted protein produced early in the infection process, which acts as a mitogen to prime surrounding cells for infection. Thus, in some embodiments, the oncolytic vaccinia virus may be engineered to lack both VGF and TK activity.

[0069] Deletions have also been made in several other vaccinia virus genes, such as ribonucleotide reductase (F4L or RR). Indeed, it has been shown that double (or triple) deletions of viral genes such as RR and / or VGF together with TK confer strict tumor specificity to vaccinia virus.

[0070] A variety of different modified vaccinia virus strains have been developed, which may provide the vaccinia virus genome on which the present invention is based. Examples of suitable clinical and preclinical vaccinia viruses include, for example, the TK-deleted Wyeth strain Pexa-Vec (JX-5 94); vvDD, a Western Reserve strain lacking TK VGF; LV-1 h68, a Lister strain lacking TK, F14.5L, and A65R; and Tiantan, a Lister strain lacking TK. VG9-GMCSF, a Guang9 strain; ΔF4LΔJ2R, a Western Reserve strain deleted for F4L and TK; CVV, a Wyeth-derived strain isolated by recurrent selection deleted for TK; and deVV5, a chimeric virus of Wyeth, modified Ankara, Western Reserve, and Copenhagen strains deleted for TK. Other suitable candidate vaccinia viruses include, for example, those disclosed in US Patent Publication No. 20190218522A, such as the CF33 virus.

[0071] In various embodiments, oncolytic vaccinia viruses can be engineered to lack one or more genes involved in evading the host interferon (IFN) response, such as E3L, K3L, B18R, or B8R. B18R is known to neutralize secreted type I IFN. Other modifications that alter anti-apoptotic viral genes are deletions of SPI-1 and / or SPI-2. Attenuation of viruses for use as cancer-selective oncolytic agents includes deletion of A56R (hemagglutinin) in the background of Lister strain F14.5L and J2R. Strategies that promote mRNA decay by removing decapping enzymes D9 and D10 have also been used to limit activation of host defenses. Western Reserve Virus Delta 4 (A48R, B18R, C11R, and J2R), a compound deletion that acts in tandem on metabolic, growth, and signaling pathways, has also been generated (see, e.g., Guo et al., 2003). (see, al. (2019), J. Immunotherapy Cancer, 7, 6).

[0072] Modification of vaccinia virus to overcome complement-mediated neutralization Complement is a key component of the innate immune system, targeting viruses for neutralization and clearance from the circulation (Figure 1). Complement enhances the potency of antibodies, as demonstrated by the reduced neutralizing effect against smallpox in the absence of complement. Thus, complement is crucial in the innate immune response against vaccinia virus. Complement component C1 recognizes the Fc region of antibodies bound to viral epitopes and activates an enzymatic cascade of proteins that results in the formation of the C3 convertase C4b2b, which cleaves C3 and deposits the opsonic C3b fragment on the surface. Alternatively, C3 can be activated naturally by hydrolysis of an internal thioester bond and reaction with hydroxyl or amino groups on the surface of pathogens. Bound C3b functions as an opsonin for phagocytes and as a component of the C3 convertase C3bBb (the alternative pathway of complement activation). Further cleavage and binding of C3b leads to the formation of C5 convertase, cleavage of C5, and assembly of the membrane attack complex (C5b, 6, 7, 8, 9), which disrupts lipid bilayers such as the vaccinia envelope.

[0073] C3b can bind to normal cell membranes and unchecked activation can lead to inappropriate inflammation. Thus, mechanisms exist in animals to regulate complement activation. In humans, complement can be negatively regulated by several membrane regulators of complement activation (RCA). RCA downregulate complement activation in different ways (by inhibiting the formation of C3 convertase and promoting its decay by C and 35 (complement receptor 1) and CD55 (decay accelerating factor); catabolizing C3b and C4b through the action of CD35 and CD46 (membrane cofactor proteins) that serve as cofactors for the regulatory proteins H and I factors to inhibit the formation of C3 convertases C4b2a and C3bBb; and preventing the formation of the membrane attack complex by CD59 activity). Extracellular enveloped vaccinia virus (EEV) is resistant to complement by sequestering these proteins within its viral envelope, and it has been reported that incorporation of CD55 into A27 of IMV provides some complement protection.

[0074] The 14 kDa A27 protein mediates host cell proliferation via its N-terminal domain (residues 21–30). It binds to HS receptors on the vaccinia virus surface and interacts with the envelope protein A17 via its C-terminal domain to attach to the vaccinia virus envelope (see Figure 2). However, A27 has multiple roles in the viral lifestyle, in particular, A27 is involved in viral binding, fusion, and intracellular trafficking and wrapping of IMV. A27L deletion mutants are not defective in IMV production, but their EFCs lose polarity. This may underlie why such deletion variants are eight times less capable of mediating cell-cell fusion than wild-type (WT) virions (Gray et al., 2019).

[0075] Because of the multiple roles that A27 plays in the vaccinia virus life cycle, any alteration to the encoded protein may be deleterious to one or more vaccinia virus functions.

[0076] However, it has been surprisingly found, as disclosed herein, that certain mutations in A27 can provide the beneficial effect of complement evasion while maintaining suitable levels of viral growth and / or infectivity. Thus, the present disclosure advantageously provides fusion / chimeric proteins (and nucleic acids encoding same) comprising complement inhibitors together with A27 that are not detrimental to A27 function and thus do not result in loss of viral infectivity or undesired aggregation. Various embodiments of the present disclosure provide modified native vaccinia virus complement control protein VCP fused to the N-terminus of A27 and encoding nucleic acid molecules, particularly nucleic acid molecules encoding nucleic acids or genes that are operably linked to or under the control of the native A27L promoter. Furthermore, the present invention provides engineered viral genomes and engineered viral particles that include such fusion / chimeric A27L genes. Such engineered vaccinia viruses may exhibit greater inhibition of complement and / or improved infectivity compared to engineered vaccinia viruses expressing A27-CD55 fusion viral proteins.

[0077] Aspects and embodiments of the present disclosure also relate to fusion / chimeric proteins (and encoding nucleic acids) comprising complement inhibitors with the A13L gene. Thus, native or modified vaccinia virus complement control protein VCP (Liszewski et al., (2009), J. Immunol., 183:3150-3159) is provided that is fused with A13 protein to generate chimeric proteins. A13 is a structural protein encoded by the A13L late gene that is an integral component of the mature virion membrane. In light of the widespread / ubiquitous location of A13 protein on the surface of vaccinia virus (Figure 2), the inventors believe that its location within the virion envelope is not clearly polar, thus providing an opportunity to broadly decorate the IMV envelope with complement control proteins while leaving unaffected the proteins involved in membrane fusion and binding. To optimize the physiological effect of such chimeric proteins in combination with the activity of mature virions expressing such fusion proteins, VCP can be conveniently fused towards or at the N-terminus or towards or at the C-terminus of A13, depending on the desired effect. In this regard, the inventors have surprisingly discovered that A13 can be adapted for fusion with VCP, as described herein, and that both active components of the fusion can perform their intended natural functions. In particular, the fused VCP can bind to C3 and inhibit the activity of C3 convertase. Thus, the present disclosure also encompasses nucleic acid molecules encoding A13-VCP (or VCP-A13) fusion proteins, such as those described herein, in particular nucleic acids or genes operably linked to or under the control of the native A13L promoter. Furthermore, the present disclosure provides engineered viral genomes and engineered viral particles that contain the corresponding fusion / chimeric A13L genes.

[0078] In some preferred embodiments, the open reading frame of the complement control protein is fused towards the 3' end of the A13L gene (corresponding to the C-terminus of the expressed protein), The resulting fusion construct is flanked on both sides by native viral genomic sequences, including the A14L gene and the A13L promoter sequence at its 5' end and the A12L gene at its 3' end.

[0079] The invention further provides recombinant vaccinia virus nucleic acids, genomes and virions or modified sequences thereof, comprising heterologous nucleic acids encoding a complement activation modulator such as compstatin, CD55, CD59, CD46, CD35, Factor H, C4 binding protein, CD35, VCP, Kaposi's sarcoma-associated herpesvirus Kaposica / KCP, Herpesvirus saimiri (HVS) CCPH and HVS-CD59, Rhesus monkey rhadinovirus RCP-H and RCP-1, Murine gamma herpesvirus 68 (γHV-68) RCA, Influenza virus M1, SPICE, MOPICE, EMICE or IMP. For example, expression of a complement activation modulator from the viral genetic material within a host cell may provide the recombinant vaccinia virus with the ability to modulate complement activation in the host / subject, resulting in reduced complement-mediated virus neutralization compared to wild-type virus. In some embodiments, the heterologous nucleic acid carried by an engineered / recombinant vaccinia virus according to the present disclosure encodes a domain of human CD55, CD59, CD46, CD35, Factor H, C4 binding protein, CD35 or VCP or an activity modified sequence thereof or other identified modulators of complement activation. In some embodiments, the heterologous nucleic acid encodes a VCP or modified VCP protein (SEQ ID NO:30 and SEQ ID NO:31) or SPICE (SEQ ID NO:32, smallpox virus VCP encoded by the D15L gene), MOPICE (SEQ ID NO:33, monkeypox virus Zaire-96-I-16 VCP encoded by the D14L gene), EMICE (SEQ ID NO:75, 76; Ectromelia virus (ECTV) Moscow strain ECTV-MOS between nucleotides 27,564 and 26,776; Chen et al. al. (2003) Virology, 317:165-186) or another poxvirus complement control protein, such as IMP (SEQ ID NOs: 77, 78; CPV-IMP protein from cowpox described in Miller et al. (1997) Virology, 229:126-133).

[0080] Genetic modification of vaccinia virus Methods for inserting or deleting nucleic acid from a target genome include those described herein and those known in the art.

[0081] According to the present disclosure, an endogenous target viral gene can be mutated by recombination using a shuttle vector that encodes a modified protein and approximately 250-300 bp of homologous sequence flanking either side of the modification leading to homologous recombination.

[0082] Alternative methods for inserting or deleting nucleic acids into a targeted genome include CRISPR, zinc finger mediated gene editing, TALENS, and the like.

[0083] In other embodiments, mutations can be generated and selected by the process of natural selection under selective pressure to promote and identify desired phenotypes.

[0084] Any suitable method known to one of skill in the art may be used for nucleic acid delivery to achieve expression of the nucleic acid and / or viral genome in accordance with the present disclosure. For example, any suitable method capable of introducing a nucleic acid (e.g., DNA or RNA, including viral and non-viral vectors) into an organelle, cell, tissue or organism may be used. Such methods include injection (U.S. Pat. Nos. 5,994,624 and 5,981,274), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215); electroporation (U.S. Pat. No. 5,384,253), calcium phosphate precipitation (Graham and Van Der E. b, 1973; Rippe et al., 1990), using DEAE dextran followed by polyethylene glycol (Gopal, 1985), direct ultrasonic loading (Fechheimer et al., 1987); liposome-mediated transfection (Nicolau and Sene, 1982; Kato et al., 1991); particle bombardment (WO 94 / 09699; U.S. Pat. No. 5,610,042); agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. No. 5,302,523); Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055); PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. No. 4,684,611); or desiccation / inhibition-mediated DNA uptake (Potrykus et al., 1996; U.S. Pat. No. 5,684,611). These techniques include, but are not limited to, direct delivery of nucleic acids by nucleic acid synthesis via endothelial cell wall synthesis (E. et al., 1985). By applying such techniques, organelles, cells, tissues or organisms can be transformed stably or transiently as needed.

[0085] Transgene insertion In various embodiments, additional transgenes may be inserted into the nucleic acid or viral vector. For example, in embodiments, one, two or three transgenes may be inserted into the former locus of the deleted wild-type viral gene. Suitable candidate genes for deletion / replacement may include B8R or especially TK (also known as J2R). In some strains, in addition to the transgene present at the TK (and / or B8R) deletion site, the engineered viral genome may also have at least one transgene inserted into an additional locus on the vaccinia virus genome that is not the locus of the deleted TK (or other) gene. Preferably, the replacement / insertion is located at the locus of a gene that is not essential for vaccinia virus infection and replication. Some of such transgenes may beneficially encode cytokines such as GM-CSF; prodrug converting enzymes such as cytosine deaminase (CD); theranostic payloads such as sodium iodide symporter (NIS); affinity reagents such as bispecific antibodies; and imaging / detection agents such as luciferase, Renilla luciferase-GFP fusion protein, β-galactosidase, β-glucuronidase or green fluorescent protein.

[0086] In various embodiments of the engineered vaccinia viruses / viral genomes of the present disclosure, at least one transgene can be inserted toward the 5' and / or 3' border of a deleted viral locus or the 5' and / or 3' end of a truncated endogenous viral gene.

[0087] In various embodiments, at least three, four, five or more transgenes are inserted into the modified vaccinia virus genome.

[0088] In a particularly preferred embodiment, the oncolytic vaccinia virus into which the fusion / chimeric genes described herein are introduced is a Copenhagen strain oncolytic vaccinia virus that lacks a functional TK gene and has a transgene expressing human GM-CSF (e.g., a current clinical example is known as "Pexa-vec"). Thus, the present disclosure provides engineered vaccinia viruses, particularly engineered Copenhagen strain vaccinia viruses, that may have one or more beneficial effects / demonstrable improvements over prior art clinical examples, such as improved ability to evade the host immune system and thus improved infectivity and / or oncolytic activity.

[0089] Virus proliferation The present invention relates to vaccinia viruses, including those constructed with one or more gene deletions compared to the corresponding wild-type genome, such that the viruses have specific therapeutic potential, particularly against cancer cells. They exhibit desirable properties for use (ie, so-called "oncolytic viruses") and are low or non-toxic to non-cancerous cells.

[0090] By way of example, numerous different protocols are known to those of skill in the art that can be used to produce the recombinant vaccinia viruses described herein, and all such methods are contemplated for use in connection with the present disclosure.

[0091] First, any suitable molecular biology method can be employed to generate mutated / engineered viruses using recombinant DNA techniques, for example, vaccinia virus can be propagated using the methods described by Earl and Moss in Ausubel et al., 1994 or the methods described in WO 2013 / 022764.

[0092] For example, to generate mutations in the vaccinia virus genome, the nucleic acid molecules contained in the vector can code for native and modified polypeptides. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, plant viruses) and artificial chromosomes (e.g., YACs). A person skilled in the art should be fully competent to construct vectors by standard recombinant techniques, such as those described in Sambrook et al., (1989) and Ausubel et al., 1994, both of which are incorporated herein by reference. In addition to coding for modified polypeptides, the vector can also code for unmodified polypeptide sequences, such as tags or targeting molecules. To propagate the vector in a host cell, it preferably contains one or more origins of replication sites (often referred to as "ori"), which are specific nucleic acid sequences from which replication of polynucleotides is initiated. Alternatively, when the host cell is yeast, an autonomously replicating sequence (ARS) can be used.

[0093] In the context of expression of heterologous nucleic acid sequences, the term "host cell" refers to a prokaryotic or eukaryotic cell, including any transformable organism capable of replicating a vector and / or expressing a heterologous gene encoded by the vector. A host cell can be used as a recipient for a vector or a virus.

[0094] A host cell may be "transfected" or "transformed," which refers to the process by which exogenous nucleic acid, such as an engineered gene, vector, or viral genome, is transferred or introduced into the host cell. Transformed cells include the primary subject cell and its progeny. Host cells can be derived from prokaryotic or eukaryotic organisms, including yeast cells, insect cells, and mammalian cells, depending on whether the desired result is replication of the vector or expression of some or all of the nucleic acid sequence encoded by the vector. There are numerous cell lines and cultures that can be used as host cells, and they can be obtained, for example, through the American Type Culture Collection (ATCC; www.atcc.org). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. For example, a plasmid or cosmid can be introduced into a prokaryotic host cell for replication of many vectors.

[0095] Many host cells from a variety of cell types and organisms are available and will be known to those of skill in the art. Similarly, viral vectors can be used in combination with eukaryotic or prokaryotic host cells, particularly those cells that are permissive for replication or expression of the vector. Some vectors may use control sequences that allow them to replicate and / or express in both prokaryotic and eukaryotic cells. Those of skill in the art will further appreciate the conditions for culturing suitable host cells to maintain them and allow replication of the vector. Large scale production of vectors and expression of nucleic acids and their cognate polypeptides, proteins or peptides encoded by the vectors can be performed using a variety of methods. The techniques and conditions that may enable production are also understood and known.

[0096] The engineered vaccinia virus of the present disclosure can be produced by methods known to those skilled in the art. In certain embodiments, the modified oncolytic virus can be propagated in suitable host cells, for example, selected from HeLa cells, 293 cells, or Vero cells. Once the engineered viral virions are expressed and released from the host cells, they can be isolated from the host cells and stored under conditions that promote viral stability and integrity, minimizing loss of infectivity over time. In certain exemplary methods, the modified oncolytic virus is propagated in host cells using cell stacks, roller bottles, or perfusion bioreactors. In some examples, downstream methods for purification of the modified oncolytic virus can include filtration (e.g., depth filtration, tangential flow filtration, or a combination thereof), ultracentrifugation, or chromatographic capture.

[0097] The engineered vaccinia virus thus obtained can be stored, for example, by freezing or drying (such as lyophilization). In certain embodiments, prior to administration to a subject or to in vitro cells or cell culture medium, the stored engineered vaccinia virus can be reconstituted (if dried for storage) and diluted in a pharma- ceutically acceptable carrier for administration.

[0098] Therapeutic Peptides and Nucleic Acids An "active agent" or therapeutic molecule may include any suitable engineered vaccinia virus particle described herein or any polypeptide moiety expressed by an engineered viral genome according to the present disclosure. Such polypeptides (moieties) may include polypeptides that form structural components of an engineered vaccinia virus particle or polypeptides expressed in an engineered vaccinia virus genome, e.g., as free polypeptides released from a viral particle or a host cell. Particularly useful polypeptides include those that induce an immune response, such as an antigen or a cytokine.

[0099] The present invention also encompasses nucleic acid molecules that code for the peptide sequences of the present invention. It will be understood that, taking into account codon redundancy, many slightly different nucleic acid sequences can accurately code for each of the fusion proteins of the present invention, and each of these variants is encompassed within the scope of the present invention. Those skilled in the art can easily determine suitable nucleic acid sequences to code for each of the fusion proteins of the present invention, and can select suitable codon codes depending on the system in which the fusion protein is expressed (e.g., mouse, rabbit, or human). Any nucleic acid sequence that codes for the peptides of the present invention is encompassed by the present invention.

[0100] Considering that minor modifications to the primary sequence of the peptides / proteins of the invention may be made without substantially altering the scope of the claimed invention, the invention should be considered to further encompass any polypeptide sequence that is substantially identical to a specific amino acid sequence disclosed herein. For example, the claimed invention encompasses polypeptide sequences having at least 80% identity with the polypeptide sequence SEQ ID NOs disclosed herein; at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, or approximately 100% identity with the polypeptide sequence SEQ ID NOs explicitly disclosed herein.

[0101] Similarly, the claimed invention relates to polynucleotides having at least 70% identity to the polynucleotide SEQ ID NOs disclosed herein; at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, or approximately 100% identity to polynucleotide sequences encoding SEQ ID NOs expressly disclosed herein, particularly taking into account the effects of codon redundancy. It encompasses nucleotide sequences.

[0102] Advantageously, the engineered vaccinia virus genome according to the invention comprises at least one modified gene encoding a viral coat protein. In particular, the modified viral gene may be a fusion or chimeric gene comprising a polynucleotide sequence encoding a viral envelope protein and a heterologous polynucleotide sequence encoding a complement control protein. The heterologous polynucleotide is thus linked to the viral coat protein by a polynucleotide sequence encoding a covalent polypeptide linker sequence. Any suitable polypeptide linker sequence may be used, for example a suitable linker sequence may have from about 4 to about 50 amino acids, such as from about 5 to about 40 amino acids or from about 8 to about 30 amino acids, from about 10 to about 25 amino acids or from about 12 to about 20 amino acids.

[0103] Non-limiting examples of polypeptide linker sequences for joining the respective portions / halves of the fusion proteins of the present disclosure include linkers based primarily on Gly and Ser residues, such as those based on repetitive Gly3Ser or Gly4Ser sequences. Particularly suitable linker sequences include (Gly4Ser), (Gly4Ser)2 and (Gly4Ser)3 (SEQ ID NOs: 34, 35 and 36, respectively).

[0104] Expression of Nucleic Acids and Peptides The nucleic acid molecules according to the invention and, where appropriate, the engineered viruses of the invention can be produced by recombinant DNA technology and standard virus expression and purification procedures. Thus, the invention further provides nucleic acid constructs, such as expression vectors, comprising the nucleic acid molecules encoding the engineered viral coat proteins and / or heterologous (non-viral) proteins and their derivatives of the invention; and the nucleic acids encoding the peptides and derivatives of the invention. Typically, according to the present disclosure, the nucleic acid constructs of the present disclosure are incorporated into a suitable vaccinia virus vector for expressing the engineered polypeptides in concert with the production of engineered vaccinia virus particles. However, in some cases, it may be desirable for the expression of the engineered polypeptides of the present disclosure to be from a non-viral expression source.

[0105] For example, DNA encoding the relevant polypeptide can be inserted into a suitable expression vector (e.g., pGEM®, Promega Corp., USA), operably linked to an appropriate expression sequence, and transformed into a suitable host cell for protein expression according to conventional techniques (Sambrook J. et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY). Suitable host cells are cells that can be grown in culture and transformed with foreign DNA, including bacteria, fungal cells, and cells of higher eukaryotic origin, preferably mammalian cells (especially murine or human, etc.).

[0106] The term "operably linked," as applied to nucleic acid sequences of the present disclosure, such as expression vectors or constructs, indicates that the sequences are positioned so that they function in concert to achieve their intended purpose, i.e., a promoter sequence is capable of initiating transcription through the linked coding sequence to the termination sequence.

[0107] It will be appreciated that, depending on the application, the fusion proteins or other heterologous proteins of the invention may contain additional peptide sequences at the N-terminus and / or C-terminus to facilitate protein expression, cloning and / or peptide or RNA stability without altering the sequence of the final polypeptide sequence, such as the contemplated viral coat fusion peptide of the invention. Where deemed appropriate, N-terminal leader peptide sequences suitable for incorporation into the peptides of the invention are known to the skilled artisan from the available literature.

[0108] In some applications, it may be desirable to control the expression of the fusion polypeptide of the present invention or other heterologous polypeptides by using an inducible promoter or by using a modified wild-type / native viral promoter sequence. In this way, it may be possible to desirably change, for example, the expression pattern of the heterologous / fusion protein to occur, for example, at different stages of viral morphogenesis. In some embodiments, the native promoter sequence operably linked to one or more of the nucleic acid sequences encoding the heterologous or fusion protein is modified to improve or reduce expression, in particular to express the gene product earlier or later in viral morphogenesis than the wild-type promoter sequence on which it is based.

[0109] It is particularly desirable to express the chimeric or fusion peptides of the invention, such as engineered viral coat proteins, from vectors, especially viral vectors, suitable for in vivo or ex vivo use, e.g., for therapeutic applications (gene therapy). When the invention concerns a therapeutic method involving the use of engineered nucleic acid constructs for expression of proteins in vivo, the expression system selected must be capable of expressing the protein in the appropriate tissues / cells where the therapeutic effect will be exerted. Desirably, an expression system for use according to the invention is also capable of targeting the nucleic acid construct or peptide of the invention to the appropriate area, tissue or cell of the body where the virus assembly and / or the ultimate therapeutic treatment is intended.

[0110] Suitable medical applications and therapeutic methods may include the combined use of two or more different engineered viral vectors according to the present disclosure, either separately, sequentially or simultaneously, hi other embodiments, the present disclosure encompasses the use of one or more engineered viral vectors described herein in combination with one or more additional therapeutic agents.

[0111] As will be appreciated by those skilled in the art, strict adherence to the sequences provided is not necessary for the function of any promoter, as long as the functional elements (e.g. enhancers) and their spatial relationships are essentially maintained. In particular, the promoter sequences provided herein may contain flanking restriction sites for cloning into a vector. Where appropriate, the skilled artisan will know how to adapt these restriction sites to the particular cloning system used, as well as to make point mutations that may be necessary in the promoter sequence, for example to remove cryptic restriction sites.

[0112] Suitable induction systems may use small molecule induction such as the tetracycline control system (tet-on and tet-off), the radiation-inducible early growth response gene 1 (EGR1) promoter and other suitable induction systems known in the art.

[0113] Diseases and Disorders The agents, compositions, methods and uses of the invention may be particularly suitable for the treatment of a wide range of diseases and disorders, including, for example, any disease or disorder that would benefit from a targeted reduction in cell numbers associated with a disease phenotype or disease progression. In particular, diseases and disorders that may be treated according to the invention include cancers and / or proliferative or neoplastic diseases, such as cancers of hematopoietic origin or solid tumors.

[0114] Those skilled in the art will appreciate that proliferative disorders may be associated with: (1) pathological proliferation of normally quiescent or normally proliferating cells; (2) pathological migration of cells from their normal location (e.g., metastasis of tumor cells); (3) pathological expression of proteolytic enzymes, such as matrix metalloproteinases (e.g., collagenase, gelatinase, and elastase), which may lead to unwanted turnover of cell matrix; and / or (4) pathological angiogenesis, such as occurs in proliferative retinopathies and tumor metastasis. Exemplary proliferative disorders include: These include enhanced angiogenesis associated with cancer, benign neoplasms and disease states (defined above as pathological angiogenesis).

[0115] The compositions, medicaments, methods and uses of the invention may have beneficial effects in treating and / or reducing the symptoms of a wide range of proliferative diseases and disorders, for example by preventing cell proliferation and promoting cell death, particularly of pathogenic cells.

[0116] The present invention may have utility in multiple cancer types and / or have beneficial effects on tumor progression in vivo and / or in vitro (e.g., reversing tumor progression). In particular, the present invention may be useful in treating lung cancer (especially lung adenocarcinoma), cervical cancer, breast cancer, heart cancer, colon cancer, prostate cancer, brain glioblastoma, pancreatic cancer, leukemia (e.g., acute monocytic leukemia), lymphoma, kidney cancer, colorectal cancer, bladder cancer, testicular cancer, gastrointestinal cancer, liver cancer (e.g., hepatocellular carcinoma) and / or glioblastoma. The present invention may also be useful in treating one or more of skin cancer (e.g., melanoma), head and / or neck cancer, gallbladder cancer, uterine cancer, stomach cancer, thyroid cancer, laryngeal cancer, lip and / or oral cavity cancer, pharyngeal cancer, eye cancer, and bone cancer.

[0117] In some particular embodiments, the cancer is selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendiceal cancer, astrocytoma, atypical terato / rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, extrahepatic carcinoma, Ewing's sarcoma family, osteosarcoma and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumors, barretinoblastoma, bronchial tumors, and sarcoma. Kitt's lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasm, colon cancer, extrahepatic cholangiocarcinoma, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube cancer, osteofibrous histiocytoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brainstem glioma, hairy cell leukemia, hepatocellular carcinoma, Langerhans cell histiocytosis, Hodgkin's lymphoma, hypopharyngeal Head cancer, pancreatic islet cell tumors, pancreatic neuroendocrine tumors, Wilms tumor and other childhood kidney tumors, Langerhans cell histiocytosis, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer, midline carcinoma, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, myelodysplastic syndrome, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low-grade ovarian cancer, pancreatic neuroendocrine tumors, The cancer may be selected from any one or more of the group consisting of endocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi's sarcoma, rhabdomyosarcoma, Sezary syndrome, small intestine cancer, soft tissue sarcoma, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenstrom's macroglobulinemia.

[0118] Furthermore, the present disclosure also encompasses the therapeutic use of the therapeutic agents and compositions of the present invention and methods for inhibiting or preventing the local invasiveness or metastasis or both of any kind of primary cancer.For example, the primary cancer can be melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, liver cancer, retinoblastoma, astrocytoma, glioblastoma, gum cancer, tongue cancer, leukemia, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, brain cancer, colon cancer or bladder cancer.In certain embodiments, the primary cancer can be liver cancer.For example, the liver cancer can be hepatocellular carcinoma (HCC) and / or metastasis to the liver.

[0119] Additionally, the present disclosure may be used to prevent cancer or treat pre-cancerous or pre-malignant cells, including metaplasia, dysplasia, and hyperplasia. It may also be used to inhibit unwanted, but benign, cells, such as squamous cell metaplasia, dysplasia, benign prostatic hyperplasia, and hyperplastic lesions. In some embodiments, the disclosed uses and methods, including the therapeutic agents disclosed herein, can halt, interrupt or slow the progression of cancer or to more severe forms of cancer.

[0120] Thus, the present invention provides agents and compositions for use in medicine, in particular for use in the treatment of cancers selected from lung cancer (particularly lung adenocarcinoma or lung squamous cell carcinoma), bladder cancer, cervical cancer, breast cancer, colon cancer, brain glioblastoma, pancreatic cancer, acute monocytic leukemia, kidney cancer, colorectal cancer, skin cancer (e.g. melanoma), gastric cancer, thyroid cancer, bone cancer and liver cancer. Methods of treating such diseases are also provided. The uses and methods may comprise administering the agents according to the present invention to a patient in need thereof.

[0121] The compositions, medicaments, methods and uses of the present invention may provide benefits in the treatment of any or all of such diseases and disorders.

[0122] therapeutic composition The fusion peptides, nucleic acids or engineered virus particles (e.g., "therapeutic agents") of the invention may be incorporated into pharmaceutical compositions for use in the treatment of animals; preferably humans. The therapeutic peptides, nucleic acids or engineered viruses (or derivatives thereof) of the invention may be used to treat one or more diseases or infectious diseases, depending on the activity / properties of the engineered nucleic acid construct, fusion peptide or virus. In various embodiments, nucleic acids encoding therapeutic peptides may be inserted into expression constructs / vectors, in particular vaccinia virus vectors, and incorporated into pharmaceutical formulations / medicines for the same purpose.

[0123] As will be appreciated by those skilled in the art, potential therapeutics such as those according to the present disclosure may be tested in animal models such as rabbits or mice before they can be approved for use in human subjects. Thus, the fusion proteins or viruses of the present invention may be expressed in vivo in rabbits or mice, or ex vivo in rabbit or mouse cells and human / human cells. In accordance with the present invention, appropriate expression cassettes and expression constructs / vectors specific to each animal system may be designed. Thus, the engineered fusion proteins of the present disclosure may be modified according to their known or identified ability to inhibit the complement pathway in the selected animal host.

[0124] The therapeutic peptides and nucleic acids of the invention are particularly suitable for the treatment of diseases, conditions and / or infections that can be targeted (and treated) intracellularly, for example, by targeting vaccinia virus to animal cells, and may also be suitable for in vitro and ex vivo applications. As used herein, the terms "therapeutic agent" and "active agent" include peptides and nucleic acids encoding the peptides of the invention as well as viral particles comprising the peptides and / or nucleic acids described herein. Therapeutic nucleic acids of the invention include modified / engineered vaccinia virus genomes that include a nucleic acid sequence encoding a viral coat fusion protein according to the present disclosure and, optionally, one or more additional heterologous genes and / or proteins.

[0125] Therapeutic uses and applications of the therapeutic agents of the present invention include any disease, disorder or other condition that can be treated by expressing an engineered vaccinia virus in the subject being treated.

[0126] According to aspects and embodiments of the present invention, particularly preferred diseases include cancer and other proliferative diseases or disorders, as disclosed elsewhere herein.

[0127] One or more additional pharma- ceutically acceptable "carriers" (such as diluents, adjuvants, excipients, or vehicles) may be combined with the therapeutic agents of the invention in a pharmaceutical composition. The pharmaceutical preparations and compositions of the present invention are formulated to comply with regulatory standards and can be administered orally, intravenously, topically or by other standard routes. As used herein, the term "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the composition. The phrases "pharmacologically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce allergic or similar untoward reactions when administered to humans.

[0128] According to the present invention, the therapeutic agent may be manufactured as a medicament or formulated into a pharmaceutical composition. When administered to a subject, the therapeutic agent is suitably administered as a component of a composition that includes a pharma-ceutically acceptable vehicle. The molecules, compounds and compositions of the present invention may be administered by any convenient route, for example, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intravaginal, transdermal, rectal, inhalation or topical administration to the skin. Administration may be systemic or local. Known delivery systems also include, for example, encapsulation in microgels, liposomes, microparticles, microcapsules, capsules, etc., any of which may be used in some embodiments to administer the agents of the present invention. Any other suitable delivery system known in the art is also contemplated for use in the present invention.

[0129] Acceptable pharmaceutical vehicles can be liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin (peanut oil, soybean oil, mineral oil, sesame oil, etc.). Pharmaceutical vehicles can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, auxiliary substances, stabilizers, thickening agents, lubricants and coloring agents can also be used. When administered to a subject, the pharma- ceutically acceptable vehicle is preferably sterile. Water is a suitable vehicle, especially when the compound of the present invention is administered intravenously. Saline and aqueous dextrose and glycerol solutions can also be used as liquid vehicles, especially for injectable solutions. Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, etc. The present compositions, if desired, can also contain minor amounts of wetting agents, emulsifying agents, or buffering agents.

[0130] The medicaments and pharmaceutical compositions of the present invention can take the form of a liquid, solution, suspension, lotion, gel, tablet, pill, pellet, powder, modified release formulation (e.g., slow or sustained release), suppository, emulsion, aerosol, spray, capsule (e.g., capsule containing a liquid or powder), liposome, microparticle, or any other suitable formulation known in the art. Other examples of suitable pharmaceutical vehicles can be found in Remington's Pharmaceutical Sciences, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, see e.g., pages 1447-1676.

[0131] In some embodiments, the therapeutic compositions or medicaments of the present invention are formulated according to conventional procedures as pharmaceutical compositions suitable for oral administration (more appropriately for humans). Compositions for oral delivery can be in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs. Thus, in various embodiments, the pharma-ceutically acceptable vehicle can be a capsule, tablet or pill.

[0132] Orally administered compositions may contain one or more agents, such as sweeteners, e.g., fructose, aspartame, or saccharin, flavorings, e.g., peppermint, wintergreen oil, or cherry, colorings, and preservatives, to provide a pharma- ceutically palatable preparation. When the composition is in tablet or pill form, it may be coated to delay disintegration and absorption in the digestive tract, allowing for sustained release of the active agent over an extended period of time. Selectively permeable membranes surrounding the osmotically active driving compound are also suitable for orally administered compositions. In these dosage forms, fluid from the environment surrounding the capsule is absorbed by the driving compound, which expands and forces the drug or drug composition through an opening. These dosage forms can provide an essentially zero order delivery profile, as opposed to the spiked profiles of immediate release formulations. Time-delay materials, such as glycerol monostearate or glycerol stearate, may also be used. Oral compositions may include standard vehicles, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Such vehicles are preferably of pharmaceutical grade. For oral formulations, the location of release is the stomach, the small intestine (duodenum, jejunum or ileum) or the large intestine. Those skilled in the art can prepare formulations that do not dissolve in the stomach but release the substance in the duodenum or elsewhere in the intestine. Suitably, the release avoids the deleterious effects of the stomach environment by protecting the peptide (or derivative) or releasing the peptide (or derivative) beyond the stomach environment, such as into the intestine. To ensure full gastric resistance, a coating that is impermeable to at least pH 5.0 is essential. Examples of more common inactive ingredients used as enteric coatings include cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac, which may be used as blend films.

[0133] Surfactants may be added as wetting agents to facilitate dissolution of therapeutic agents into aqueous environments. Surfactants may include anionic surfactants such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate, sodium dioctyl sulfonate, etc. Cationic surfactants may be used, which may include benzalkonium chloride or benzethomium chloride. Potential nonionic surfactants that may be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. When used, these surfactants may be present alone or as a mixture of different ratios in the formulation of peptide or nucleic acid or derivative.

[0134] Typically, compositions for intravenous administration comprise sterile isotonic aqueous buffer. Optionally, the composition may also include a solubilizing agent.

[0135] Mixtures of the viral particles or nucleic acids described herein may be prepared in water suitably mixed with one or more excipients, carriers or diluents. Dispersions may also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the forms may be sterile and sufficiently fluid to be injectable with a suitable syringe. Suitably, the composition must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (such as, for example, glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Suitable fluidity may be achieved by, for example, the addition of a coating such as lecithin. This can be achieved by the use of a surfactant, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is desirable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0136] For example, for parenteral administration in an aqueous solution, the solution can be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those skilled in the art.

[0137] Another suitable route of administration of the therapeutic compositions of the present invention is by pulmonary or nasal delivery.

[0138] Additives such as the fatty acids, oleic acid, linoleic acid and linolenic acid may be included to enhance cellular uptake of the therapeutic agents of the invention.

[0139] The therapeutic agents of the present invention, in some embodiments, may be formulated into compositions for topical application to the skin of a subject.

[0140] In an embodiment of the present invention, a therapeutic composition may contain only one therapeutic agent of the present invention or may contain two or more, e.g., two complementary therapeutic agents of the present invention. For example, inhibition of different proteins involved in the complement pathway of a target animal may be achieved by multiple engineered proteins of the present disclosure. In particular, an engineered virus according to the present disclosure may express two chimeric / fusion proteins each comprising a heterologous protein or protein domain that targets a different stage or a different complement pathway, fused to an expressed vaccinia protein, such as two different viral envelope / structural proteins. When two (or more) therapeutic agents are contemplated, the different peptides or encoding nucleic acid constructs, such as viral vectors or viral particles, may be incorporated into the same pharmaceutical composition or may be manufactured separately. When two (or more) pharmaceutical compositions are manufactured for administration to the same individual, it will be understood that the compositions may be administered simultaneously, sequentially or separately, as indicated or required.

[0141] Methods for Delivery of Engineered Vaccinia Viruses Vaccinia virus infection immunization is typically very safe, resulting in mild or asymptomatic infection in healthy individuals. Complications and adverse effects are generally only seen in vulnerable individuals, such as immunocompromised individuals. As a vaccine, the virus is typically administered by multiple skin puncture techniques using a bifurcated needle. However, to efficiently deliver the virus to solid tumors, a different administration route is required.

[0142] Any suitable delivery method for administering a therapeutic agent, engineered vaccinia virus, or therapeutic composition of the invention may be used in accordance with the present disclosure. The preferred route of administration will be determined by a skilled physician and may depend on one or more factors.

[0143] The delivery of the therapeutic agent or composition of the present invention can be systemic or local. For example, the route of administration can be determined by the location and / or nature of the disease, e.g., cancer to be treated, and can include intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, local (e.g., near the tumor, particularly the vasculature of the tumor or adjacent vasculature), percutaneous, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, and oral administration, as described, for example, in U.S. Pat. No. 5,543,158, U.S. Pat. No. 5,641,515, and U.S. Pat. No. 5,399,363. Typically, the systemic route of administration is Convenient forms may include oral, parenteral, intranasal, sublingual, rectal, transdermal administration or any combination thereof.

[0144] In various embodiments, the therapeutic composition may be administered directly to the site of the tumor. In particularly beneficial embodiments, the therapeutic composition of the present disclosure may be delivered by injection into the vascular system of a subject.

[0145] In some embodiments, (continuous) administration / administration over a long period of time is preferred, which may be achieved by any suitable mechanism, such as, for example, implanting a catheter into the tumor or tumor vasculature. Advantageously, the dose of therapeutic composition by continuous perfusion may be equivalent to the dose administered by single or multiple injections, and is adjusted over the period of perfusion. Furthermore, it is contemplated that limb perfusion may be used to administer the therapeutic composition of the present invention, particularly in the treatment of melanoma and sarcoma.

[0146] Injection of the nucleic acid construct may be delivered by a syringe or other method used for injecting solutions, so long as the expression construct (e.g., a virus) can pass through a needle of the particular gauge required for injection. In some embodiments, needle-free injection systems, such as those described in U.S. Patent No. 5,846,233, may be used, as known to those of skill in the art.

[0147] Therapeutic Treatment Regimens and Dosages Treatment regimens may vary and often depend on the type and / or location of the tumor, the stage / progression of the disease, and / or the health and age of the patient. For example, some tumors may respond better to treatment with localized and / or highly concentrated doses of the therapeutic composition, while other diseases (and individuals) may benefit from more widespread and / or lower doses and / or longer administration of the therapeutic agent. A skilled clinician can determine the appropriate therapeutic treatment in each situation.

[0148] In various aspects and embodiments, the present disclosure provides therapeutic uses and methods for treating a subject by administration of one or more engineered vaccinia viruses as disclosed herein. By contacting a target cell or a first target cell population with the engineered vaccinia virus, the virus can infect the target cell / first target cell population and reproduce in those target cells, causing a toxic effect sufficient to kill the target cell. Thus, the present disclosure provides uses and methods for reducing the size and / or growth of a tumor by killing the target infected cell through the toxic effect caused by the engineered virus in the target cancer cell. The uses and methods include administering to an individual or a cancer cell or a first target cell population of an individual a therapeutically effective amount of the engineered vaccinia virus of the present disclosure or a pharmaceutical composition containing the engineered vaccinia virus of the present invention or other therapeutic agent as described above. According to the therapeutic method of the present invention, when a first target cell (or a first target cell population) is infected, the vaccinia virus can reproduce in the first target cell (population), and when new virus is released from the first target cell (or first target cell population), a second target cell or a second target cell population can be infected with the released virus. Thus, advantageously, the use of such engineered viruses does not require direct administration or infection of all target cells in a tumor or subject with an initial population of engineered vaccinia viruses. Rather, by allowing the engineered virus to maintain its ability to grow in the target cells while avoiding rapid destruction by the host's (individual's) immune system, it is possible to infect and destroy many more target cancer cells than would be possible with an initial single dose of therapeutic agent. Furthermore, by utilizing the natural ability of the vaccinia virus to invade and infect nearby or distant target cells, the use and method of the present invention may enable the destruction of cancer cells to which it is not easy or possible to directly administer a therapeutic agent.

[0149] Thus, the present disclosure provides a method for the treatment of cancer cells comprising administering an engineered vaccinia virus according to the present disclosure to a first cancer cell. Further provided is a method of treating a cancer or tumor or inhibiting the growth and / or proliferation of a second cancer cell comprising administering to the first cancer cell infected with the virus and subsequently infecting a second cancer cell with the virus. The second cancer cell may be separate from the first cancer cell; for example, the second cancer cell may be in a solid tumor separate from the solid tumor in which the first cancer cell is located; or the second cancer cell may be a metastasis of the first cancer cell.

[0150] An effective amount of an engineered vaccinia virus or pharmaceutical composition thereof of the present disclosure may include an amount sufficient to lead to oncolysis / killing of a target cancer cell and / or inhibition of the lysis or proliferation of a target cancer cell or inhibition or reduction in the growth or size of a target cancer cell or tumor. Reduction in the growth of a tumor or target cancer cell may be manifested, for example, as cell death or a slowing of the replication or growth rate of a tumor comprising the cell or an increased survival time of a subject containing the cancer cell.

[0151] Thus, in some embodiments, there is provided a therapeutic use and method of treating a subject having a cancer or tumor, comprising administering to the subject an effective amount of an engineered vaccinia virus of the present disclosure. The effective amount in such uses and methods may include an amount that reduces the growth rate or spread of the cancer or extends the survival time of the subject. In some embodiments, the tumor may be completely or substantially eradicated. The uses and methods of the present disclosure may comprise administering to a subject or individual an engineered vaccinia virus. Administration may be by any suitable means, such as injection or oral ingestion. Administration may be systemic or local, for example directly at the site of the tumor. In one embodiment, systemic delivery is preferred.

[0152] Therapeutic compositions and / or therapies may include various "unit doses," which may be defined as containing a predetermined amount of a therapeutic agent or composition. Identifying the appropriate amount to be administered and the specific route of administration and formulation is within the skill of one of ordinary skill in the clinical arts. The unit dose may be administered, for example, as a single injection or tablet, but may also be administered over an extended period of time, such as by continuous infusion over a period of time.

[0153] For embodiments involving engineered vaccinia viruses, the unit dose can be conveniently described in terms of plaque forming units (pfu) of the viral construct rather than weight or molar concentration. The unit dose can alternatively be expressed as virus particles, infectious virus (PFU) or tissue culture inhibitor dose 50% (TCID50). The unit dose can be 1×10 per mL, per kg, per dosage form (e.g., per tablet or per injection). 3 ~1×10 15 PFU range; e.g., about 1 x 10 4 , about 1×10 6 , about 1×10 8 , about 1×10 10 , about 1×10 12 Or about 1 x 10 14 Delivery of such dosages can be direct to the tumor or tumor site, or delivery can be systemic.

[0154] In certain embodiments, the modified oncolytic virus can be administered in one or more doses over a set period of time (e.g., 12 hours or 24 hours), which together constitute a "unit dose." In other embodiments, each individual administration can be considered a "unit dose."

[0155] Although an engineered vaccinia virus according to the present disclosure may survive in vivo longer than a comparable vaccinia virus not having the modifications described herein, the therapeutic compositions and / or medicaments of the present disclosure may be administered in multiple doses as deemed appropriate (e.g., 2, 3, 4, 5, 6 or more doses) within a suitable treatment period (e.g., over a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days, weeks, months or years as deemed suitable). The frequency of administration of the engineered vaccinia virus or pharmaceutical compositions described herein can be determined by a skilled physician, but may be, for example, once daily, twice daily, once weekly, once every two weeks, once monthly, once every two months, once every three months, once every six months, etc.

[0156] Furthermore, according to the therapeutic treatment regimen that may be devised, the unit dose may vary depending on the stage or type of treatment, and taking into account the size and / or age of the subject or the type of cancer or tumor that is intended to be treated. For example, in some embodiments, the first dose of the engineered vaccinia virus, therapeutic agent or pharmaceutical composition of the present disclosure administered to the subject may be less than or more than the second, third or subsequent doses that may be administered. In other embodiments, the dose during the first administration period may be less than or more than the dose administered during the second, third or subsequent administration period. The skilled practitioner can determine the duration of such administration periods, for example, about one day, about one week, about one month, or intermediate or longer periods.

[0157] In some examples, the subject being treated may be administered one or more additional therapeutic agents or may undergo one or more additional conventional therapies (such as radiation therapy or chemotherapy) or complementary therapies. For example, in some embodiments, the subject may be placed on a reduced carbohydrate diet, such as a ketogenic diet, prior to, concurrently with, and / or after administration of a therapeutic composition disclosed herein.

[0158] Combination therapy In various embodiments, the uses and methods of the present disclosure include administering a therapeutic agent of the present disclosure (e.g., an engineered vaccinia virus or engineered vaccinia virus vector disclosed herein) or a pharmaceutical composition containing a therapeutic agent of the present disclosure in combination with one or more additional therapies or therapeutic agents. As disclosed herein, the one or more additional therapies or therapeutic agents may be administered simultaneously, sequentially or separately (before or after) a therapeutic agent in accordance with the present disclosure.

[0159] The additional therapeutic agent may be an engineered vaccinia virus or an engineered vaccinia virus vector according to the present disclosure, such that one or two engineered vaccinia viruses and / or one or two viral vectors of the present invention are administered in combination. Other examples of additional therapies or therapeutic agents include, but are not limited to, chemotherapy, radiation therapy, oncolytic virotherapy with additional viruses, treatment with immunomodulatory proteins, anti-cancer drugs, or any combination thereof.

[0160] In certain embodiments, the additional therapeutic agent is an anti-cancer agent or cancer therapy. Anti-cancer agents may include, but are not limited to, chemotherapeutic agents, radiotherapy agents, cytokines, immune checkpoint inhibitors, anti-angiogenic agents, apoptosis inducers, anti-cancer antibodies, and / or anti-cyclin-dependent kinase agents. Cancer therapies may include chemotherapy, biological therapy, radiation therapy, immunotherapy, hormonal therapy, anti-vascular therapy, cryotherapy, toxin therapy, and / or surgery, or a combination thereof.

[0161] Combining the modified oncolytic vaccinia virus with chemotherapy advantageously achieves synergistic effects with the additional therapeutic agent, allowing the typical dose of the additional therapeutic agent alone to be reduced. Thus, the therapeutic uses and methods of the present disclosure may reduce the toxicity associated with chemotherapy without reducing the therapeutic benefits of the present invention.

[0162] In various embodiments, the treatment of cancer may involve surgery. Such surgery may include resection, in which all or a portion of the cancerous tissue is physically removed (e.g., by resection) and / or otherwise destroyed from the subject. Tumor resection refers to the physical removal of at least a portion of the tumor. Surgical treatment includes laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery) in addition to tumor resection. In some embodiments, it may be determined that the tumor (or a portion thereof) needs to be removed by resection or that the tumor is not suitable for resection. In such situations, embodiments of the present invention provide for treating a subject with a therapeutic agent or composition of the present disclosure to improve the outcome of the resection or to allow for the resection of the tumor or a portion thereof. Thus, the therapeutic procedures of the present disclosure may increase the resectability of the tumor, for example, by reducing the tumor margins or removing the involved portion. Additional treatments following resection may help eliminate microscopic residual disease at the tumor site.

[0163] The therapeutic agents of the present disclosure may be used in particular in combination with (i.e., before and / or after) surgical treatment of cancer, for example, to treat and / or destroy tumor margins surrounding the site of cancerous tissue. In embodiments, after removing part or all of the cancer cells, tissue, or tumor, one or more therapeutic agents of the present disclosure, for example, engineered vaccinia viruses, may be administered to the site of removal by perfusion, direct injection, or local application to the area, for example, the cell wall surrounding the cavity or space left by tumor removal. Depending on the treatment regimen, such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, 5, or 6 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

[0164] How to determine validity Aspects and embodiments of the present disclosure also encompass methods of determining the efficacy of a therapeutic agent (e.g., an engineered vaccinia virus) of the present disclosure. For example, the method may include determining the infectivity, antitumor activity, or amount of tumor-specific viral replication of an engineered vaccinia virus of the present disclosure. To this end, such engineered vaccinia viruses may further comprise a reporter gene construct capable of identifying the engineered virus of the present disclosure. Such reporter gene constructs may express reporter proteins such as, for example, luciferase, Renilla luciferase-GFP fusion protein, β-galactosidase, β-glucuronidase, or green fluorescent protein.

[0165] The method may include (i) administering to the subject a therapeutically effective amount of a therapeutic agent (such as an engineered vaccinia virus or pharmaceutical composition according to the present disclosure, which virus, vector or nucleic acid can further express a reporter protein), alone or in combination with an additional therapeutic agent; (ii) collecting a first biological sample from the subject immediately after administration of the virus, vector or nucleic acid and determining the level of luciferase reporter in the first biological sample; (iii) collecting a second biological sample from the subject after administration in step (i) and determining / detecting the level of reporter in the second biological sample, where if the level of luciferase in step (iii) is higher than in step (ii), the therapeutic agent (e.g., engineered vaccinia virus) is determined to be effective (e.g., exhibits infectivity, anti-tumor activity, and / or exhibits tumor-specific viral replication). The second biological sample is collected a predetermined time after the first sample is collected, such as up to one day, up to one week, or up to one month after the first sample is collected. In embodiments, the second sample may be collected about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, about 24 hours, about 2 days, about 4 days, about 7 days, about 14 days, about 1 month, or about 2 months after administration in step (i).

[0166] In some embodiments, the method of determining the efficacy of a therapeutic agent (e.g., an engineered virus of the present disclosure) may further comprise detecting the level of one or more cytokines in steps (ii) and (iii). The cytokines may be selected from one or more of IL-2, IL-7, IL-8, IL-10, IFN-γ, GM-CSF, TF-a, IL-6, IL-4, IL-5, and IL-13.

[0167] Methods according to these aspects and embodiments may further include comparing the efficacy of a therapeutic agent (e.g., an engineered vaccinia virus) according to the present disclosure to a comparative therapeutic agent (e.g., a vaccinia virus) that includes a reporter construct and is identical to the engineered therapeutic agent (e.g., a vaccinia virus) of the present disclosure, except that it lacks the fusion protein and optional additional heterologous gene expression constructs described herein. Suitably, the comparative or control vaccinia virus The reporter construct of the virus expresses a reporter that is different from that of the engineered therapeutic agent (e.g., vaccinia virus) according to the present disclosure. Thus, in some embodiments, the increase in reporter gene expression between steps (ii) and (iii) is compared to the difference in reporter gene expression of a comparative or control therapeutic agent (e.g., vaccinia virus).

[0168] Thus, the method may further comprise: (iv) administering to the subject (which may be the same or different from the subject of step (i)) a therapeutically effective amount of a comparison or control therapeutic agent (e.g., a vaccinia virus) expressing a reporter construct; (v) obtaining a first biological sample from the subject immediately after administration of the therapeutic agent (e.g., a virus) of step (iv) and determining the level of the reporter in the first biological sample; and (vi) collecting a second biological sample from the subject after administration in step (iv) and determining / detecting the level of the reporter in the second biological sample, wherein steps (iv)-(vi) may be performed simultaneously with steps (i)-(iii).

[0169] Other exemplary techniques known to those skilled in the art for detecting and monitoring viral load following administration of the engineered therapeutics and vaccinia virus of the present disclosure may alternatively be used. An exemplary alternative technique is real-time quantitative PCR, where the step of determining viral load involves performing real-time quantitative PCR of the collected biological sample to detect the engineered virus, rather than determining the amount of a reporter construct.

[0170] kit In embodiments, the present disclosure provides a kit for administering an engineered vaccinia virus, an engineered vaccinia virus vector, or a nucleic acid as described herein. In certain embodiments, the kit of the present disclosure may include an engineered vaccinia virus, an engineered vaccinia virus vector, or a nucleic acid, or a (pharmaceutical) composition comprising such a therapeutic agent, as described above. In certain embodiments, the kit of the present disclosure may further include one or more components, such as instructions for use, devices, and additional reagents, as well as components, such as tubes, containers, and syringes, for carrying out the methods disclosed above. In various embodiments, the kit of the present disclosure may further include one or more active agents that may be administered in combination with the engineered virus (separately, sequentially, or simultaneously), such as at least one selected from the group consisting of anti-cancer agents, immunomodulatory agents, or any combination thereof.

[0171] In some embodiments, kits according to the present disclosure may include one or more containers housing an engineered virus, and / or an engineered viral vector, and / or a nucleic acid or protein according to the present invention, one or more additional active agents, and / or any of the reagents described herein.

[0172] In some embodiments, the kit may further comprise an apparatus or device for administering the therapeutic agent of the present disclosure (e.g., an engineered vaccinia virus) and / or any additional active agents to a subject. Suitable apparatus or devices may include one or more of a hypodermic needle, an intravenous needle, a catheter, a needle-free injection device, an inhaler, and / or a liquid dispenser.

[0173] The instructions for use of the kit may suitably include what is to be contained in the kit and how it should be properly used; for example, how the various components of the kit should be administered to an individual (including timing, concentrations and amounts), appropriate methods of administration and whether the individual needs to be monitored during use or treatment.

[0174] Virulence and immunogenicity of the host organism The toxicity and immunogenicity (immunotoxicity) of heterologous peptides expressed in a host organism can be improved by optimizing the primary peptide sequence to match that of the native host peptide. It has been proposed that this can be reduced.

[0175] Polypeptides comprising the fusion / chimeric proteins according to the invention may not be at most 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11% or 10% identical to endogenous / native peptide sequences found in the host organism; for example, the peptides of the invention may not be approximately 1%-25%, approximately 3%-20%, or approximately 5%-15% identical to endogenous peptide sequences of the host organism. A host organism may be considered to be the animal subject to which the virus or polypeptide is administered or in which the polypeptide is expressed.

[0176] Sequence identity can be assessed in any way known to those skilled in the art, such as using the algorithms described in Lipman & Pearson (1985), Science 227, pp 1435; or sequence alignment.

[0177] As used herein, "percent identity" means that the percentage of amino acid residues (or bases in the context of a nucleic acid sequence) is the same when the two sequences are compared when aligned. The amino acid sequences are not identical, but rather have amino acid substitutions, deletions or additions compared to the reference sequence. In the context of the present invention, since the subject protein is considered to be modular, i.e., to include several different domains or effector and auxiliary sequences (such as NLS sequences, peptide expression sequences and viral coat protein sequences), sequence identity can be conveniently assessed for each domain / module of the peptide separately, including the possibility that the polypeptide may be a fusion / chimeric protein, compared to any homologous endogenous or native peptide domain / module known in the host organism. This is considered to be an acceptable approach, since relatively short peptide fragments (epitopes) of any host-expressed peptide, when expressed in the host organism in vivo, may play a role in determining immunogenicity, such as through self / non-self peptide recognition. As an example, a 100 amino acid peptide sequence in which a first host protein domain is directly fused to a second host protein domain, and neither protein domain sequence is altered by mutation (but the fusion protein is not a native protein), may be considered to be 100% identical to the host peptide sequence. For purposes of this assessment, it does not matter whether such a separate fusion domain represents only a fragment of a larger native protein sequence expressed in the host, or whether the fusion itself is not naturally occurring. However, if one of the 100 amino acids is altered from the native sequence, the altered sequence is considered to be 99% identical to the host's native protein sequence.

[0178] Thus, the degree of sequence identity between a query sequence and a reference sequence may be determined by any means available to one skilled in the art, such as sequence alignment.

[0179] If a peptide sequence is designed / engineered to match (or best match) an endogenous host sequence, such as mouse or human, it may be considered "murine" or "humanized". In practice, "humanization" or e.g. "murine" is intended to reduce the possibility of foreign epitopes in the peptide sequence of the invention. Similarly, the peptide sequence may of course also be adapted to other animal expression systems, such as rabbit. Such deliberate sequence alterations are desirably performed within the constraint of achieving a fully active (or at least suitably viable) viral coat protein that can be used to assemble viral particles for displaying the fusion / chimeric protein on the surface of the vaccinia virus.

[0180] To improve sequence identity, the preferred "host-matched" fusion / chimeric protein sequence for use in humans may be replaced with a suitable mouse analogue protein for mouse studies. Similarly, to further improve host optimization, the relevant peptide sequences are preferably selected from human and mouse sequences for expression in human or mouse, respectively. . EXAMPLES

[0181] The invention is further illustrated by the following non-limiting examples.

[0182] Unless otherwise noted, commercially available reagents and standard techniques in molecular biology and biochemistry were used.

[0183] Materials and Equipment The following procedure used by the applicant is described in Sambrook, J. et al., 1989 (supra): analysis of restriction enzyme digestion products on agarose gels and preparation of phosphate buffered saline. General purpose reagents, oligonucleotides, chemicals and solvents were purchased from Merck Life Science Ltd (UK). Enzymes and polymerases were obtained from New England Biolabs (NEB Inc.; UK).

[0184] pUC57-Amp A27L fusion and pUC57-Amp A13L fusion (GENEWIZ) as described in Table 1. BSC-1 cells (ATCC, Catalog No. CCL-26), HeLa cells (ATCC, Catalog No. CCL-2). VACV-COP ΔJ2R(TK) vaccinia virus strain. Lipofectamine 2000 transfection reagent (ThermoFisher, Catalog No. 11668027). DMEM medium (Gibco™ 41965039), FBS (Gibco™ 10500064), DPBS (Gibco™ 20012027). G418 (Gibco™ 11811031), ultrasonic water bath, 6-well tissue culture plate (Corning™ 3516), 6 cm dish (ThermoScientific™ 150288), 15 ml polypropylene tube (Falcon™ 352196), disposable scraper, Dounce homogenizer, sterile 2 ml microcentrifuge tubes.

[0185] method Cell preparation and infection with wild-type vaccinia virus BSC-40 cells (2 x 10 5cells / well) (RRID:CVCL_3656), a continuous line of African green monkey kidney cells derived from BSC-1 cells (Hruby, et al., 1979), were seeded into wells of a 6-well tissue culture plate in complete DMEM medium and incubated (overnight at 37°C, 5% CO2) until 50-80% confluent. An aliquot of up to 100 μl of the parent virus (Copenhagen strain) was thawed and sonicated for 1 min in an ultrasonic water bath to disperse viral aggregates. Virus was diluted to 1.2 × 10 4 The medium was removed from the confluent cell monolayers and the cells were infected with 0.5 ml of diluted vaccinia virus (0.03 pfu / cell) and incubated at 37°C for 3 h. The infected monolayers were mixed every 15 min. After 3 h, the inoculum was washed once and then replaced with 2 ml of complete DMEM (DMEM, 10% FBS, 1% DMEM with non-essential amino acids, 1% sodium pyruvate, 1% Glutamax).

[0186] Transfection with gene-targeting plasmids For each well to be transfected, 250 μl of serum-free medium was added to two sterile tubes. 6 μl of Lipofectamine 2000 was added dropwise directly to the serum-free medium in tube A, mixed by inversion, and then incubated for 5 minutes. 3 μg of DNA was added to the second tube (tube B) containing 250 μl of serum-free medium, and tube B was placed in a 200-well plate. The contents of tube B were mixed with the contents of tube A by dropping it dropwise into tube A and then incubated at room temperature for 15 min. The entire volume of the Lipofectamine 2000 / DNA mixture was then dropped onto the infected cells in complete DMEM medium 3 h postinfection. The dishes were gently rocked to ensure even distribution. The transfection mixture was removed after at least 12 h of incubation and replaced with complete DMEM medium containing 2.8 mg / ml G418, then incubated at 37°C (5% CO2) for 24–48 h. After 48–72 h, the cells were detached from the wells using a cell scraper and transferred to a 2 ml screw-top sterile microcentrifuge tube. The cell suspension was then lysed by performing three freeze-thaw cycles: freezing at -80°C, thawing in a 37°C water bath, and vortexing. The cell lysate was stored at -80°C until needed.

[0187] Plaque purification After integration of the whole gene targeting plasmid into the vaccinia genome, the virus was purified by infecting BSC-40 cells seeded in 6-well dishes. The infected cell lysate was thawed and centrifuged to sediment cell debris. When BSC-40 cells were 50-80% confluent, wells were infected twice with 1 μl of the infection lysate and twice with its 10-fold dilution in serum-free medium. After 1 h, the inoculum was replaced with complete DMEM medium containing 2.8 mg / ml G418 and incubated at 37°C (5% CO2) for 24-48 h. After 48 h, cells were washed with PBS and then plaques were harvested using a pipette tip. Plaques were dissolved in 100 μl of 1 mM Tris pH 9 and frozen / thawed three times. Plaque picking was performed in triplicate.

[0188] Final viruses were recovered through three rounds of plaque purification as described above, but further purification was performed if clonal purity was not achieved. Target gene recombination in the candidate viruses was verified by PCR assay and sequencing.

[0189] Example 1 Introduction of modified mature virion (MV) genes A13L and A27L into the vaccinia virus genome by homologous recombination Complement regulatory proteins were engineered in fusion with vaccinia envelope proteins to inhibit complement-mediated lysis or opsonization of virions and uptake by macrophages. The candidate envelope proteins were A13 and A27. As discussed above, the A27 protein is localized within the vaccinia envelope as a complex with the A17 protein and has a polar distribution relative to the virus particle, whereas A13 has a non-polar distribution and is found throughout the envelope.

[0190] Each virion envelope gene was modified by introducing DNA encoding a fusion protein at either the amino or carboxy terminus of the A13 and A27 genes, depending on which end is exposed on the virion surface, and the resulting fusion protein genes were confirmed to be driven by the native A13L and A27L promoters, respectively. In some embodiments, a V5 tag was also fused to the protein and used to facilitate detection of the expressed product. In some embodiments, the fusion between the viral gene and the respective fusion partner gene was established by insertion of a Gly / Ser linker peptide. As described herein, various linkers based on Gly / Ser are envisaged, but it will be understood that any suitable peptide linker may be used instead, the main requirement being that the linker sequence allows for proper folding, assembly and presentation of each protein of the fusion pair on the surface of the virion, and that the linker does not interfere with the intended function of each protein domain. In some embodiments, the linker sequence is optional, such that the fusion protein / nucleic acid partners may be fused directly to each other without the use of an additional linker sequence.

[0191] In this embodiment, a (Gly4Ser) linker is used, i.e., a linker peptide based on four adjacent Gly residues followed by a Ser residue, and this pattern can be repeated one or more times as required. In these examples of the invention, a single Gly4Ser linker peptide was used to fuse the A13 protein sequence to the N-terminus of each transgene sequence, and a (Gly4Ser)3 linker was used to fuse the A27 protein sequence to the C-terminus of each transgene sequence, as described below.

[0192] In some exemplary embodiments, targeting constructs encoding viral genes A13L or A27L (with or without modifications, i.e., wild-type or mutant sequences of each protein) were linked to the intended fusion partner by appropriate linker sequences (if used), flanked by approximately 250 bps of homologous regions derived from the vaccinia genome, synthesized by Genewiz, and cloned into a plasmid (pUC57) that contains both positive mCherry-Neo selection cassettes. However, it will be understood that the exact length of the flanking sequences is somewhat arbitrary, and that, for example, homologous sequences of 200 bp or more may be used instead. The requirement is that the length of homology on both sides of the transgene construct is sufficient to direct homologous recombination with the target viral genome.

[0193] In some embodiments, the flanking sequences for homologous recombination with the appropriate vaccinia virus genome correspond to (i.e., are homologous to) the sequence regions flanking the natural locus of the respective A13L or A27L viral genes, as appropriate, for insertion of the engineered fusion construct in place of the corresponding wild-type gene (see, e.g., FIG. 5). In these examples, the resulting modified vaccinia viruses do not contain the wild-type A13L and / or A27L genes (due to the variant strains produced (see below)), and therefore the A13 and / or A27 proteins produced in the viral envelope are all engineered fusion proteins.

[0194] In some applications, it may be desirable for the engineered vaccinia virus to maintain wild-type A13 and / or A27 expression levels; for example, improving viral assembly and / or modulating the viral effect on the complement cascade. Thus, in some alternative embodiments of the invention, the A13L or A27L fusion construct is recombined into the J2R / TK gene (Figure 6). In this way, (most of) the TK coding sequence is removed, which may be beneficial for reasons already discussed and known to those skilled in the art. However, in this case, at least some of the A13 or A27 proteins expressed by the virus are wild-type, since wild-type copies of the respective genes (A13L and / or A27) are still present. On the other hand, the presence of wild-type A13 and A27 genes provides a template for homologous recombination with the inserted A13L / A27L fusion construct, which is generally undesirable. Thus, plasmid constructs used to insert the A13L and A27L fusion sequences specifically into the TK locus may contain A13L and A27L nucleotide sequences that are codon-optimized for expression in human cells, thereby reducing the possibility of unintended recombination with the wild-type gene sequence.

[0195] BSC40 cells were transfected with the linearized targeting construct using Lipofectamine 2000 and subsequently infected (individually) with various vaccinia virus strains, including Copenhagen (COP), Western Reserve (WR) and Lister, to achieve recombination with the viral genome of each vaccinia strain. The resulting cell lysates were titrated on a BSC40 cell layer overlaid with 1.0% methylcellulose or bacto agar / DMEM containing G418. mCherry-positive clones were plaque-purified by three or four rounds of selection of fluorescent plaques with G418.

[0196] Correct insertion of the transgene gene fusion was confirmed by PCR and gene sequencing.

[0197] Constructs To prepare fusion constructs for homologous recombination, the following A13 gene fusions (Table 1) were synthesized by Genewiz and inserted into the pUC57-Amp plasmid.

[0198] [Table 1] TIFF2025503515000003.tif43152

[0199] Similarly, to prepare additional fusion constructs for homologous recombination, the following A27 gene fusions (Table 2) were synthesized by Genewiz and inserted into the pUC57-Amp plasmid.

[0200] [Table 2]

[0201] In this example, the A13 and A27 protein sequences from the Vaccinia Copenhagen strain were used. However, the A13L and A27L genes from any other desired Vaccinia strain may also be used. The child sequences can be used to engineer other vaccinia virus strains, especially since all gene sequences are orthologous and are expected to behave similarly. For demonstration, sequence alignments of A13 and A27 from different vaccinia strains are shown in Figures 3 and 4, respectively, showing how well the sequences of these genes are conserved throughout the viral genome. Furthermore, in some embodiments, the A13 and A27 protein sequences can be modified / mutated if it is desired to modify the natural behavior of the protein in some way or to achieve, for example, efficient or optimal fusion with a fusion partner.

[0202] Various other embodiments include fusion constructs that do not have a GGGGS linker between the respective domains, as well as fusion constructs that have flexible or structured linkers different from those described herein, provided that each member of the fusion pair is capable of performing its intended function.

[0203] To generate gene targeting constructs, each insert was excised and cloned into a suitable targeting vector, specifically a plasmid containing a positive mCherry-Neo selection cassette and appropriate homologous recombination sequences for recombination into the desired locus of vaccinia virus, such as pUC57.

[0204] Gene targeting by homologous recombination, followed by virus selection and purification, yielded the following 29 engineered vaccinia viruses in which Env protein 1 is A13 and Env protein 2 is A27 (Table 3). In each of the following embodiments, the A13L fusion construct was inserted into the corresponding A13L locus and the A27L fusion construct was inserted into the corresponding A27L locus to generate the engineered virus.

[0205] [Table 3] TIFF2025503515000006.tif43153

[0206] Example 2 Virus purification and quantification HeLa cells were grown in T175 flasks containing complete DMEM with 5% FBS. When 50–70% confluent, cells were washed once with PBS and infected with 5 ml of serum-free medium containing virus (MOI=0.02). After 1.5 h, the inoculum was removed and replaced with 20 ml of complete medium. At 48–72 h postinfection, cells were scraped from the medium and spun at 1,500 rpm for 5 min at 4°C. The cell pellet was washed twice with cold PBS and then resuspended in 10 ml of 10 mM Tris-HCl pH 9 and incubated on ice for 15 min. The suspension was lysed on ice using a Dounce homogenizer.

[0207] Cell nuclei were pelleted at 1,500 rpm for 5 min at 4° C. and the supernatant (referred to as post-enucleation supernatant, PNS) was loaded into ultracentrifuge tubes with an equal volume of 36% sucrose (w / v) in 10 mM Tris-HCl pH 9.

[0208] Virus was pelleted at 24,000 rpm for 90 min, the supernatant discarded, resuspended in 1 mM Tris-HCl pH 9, and aliquots frozen at -80°C. Aliquots were titrated on BSC40 cells. BSC40 cells were grown to confluence in 6-well plastic plates, after which aliquots of virus were diluted 10-fold in 1 ml of serum-free medium. The samples in the 6-well plastic plates were then washed with serum-free medium and the inoculum was added. After 1.5 h, the medium was removed, the wells were washed twice with PBS, and overlaid with a layer of 1% methylcellulose in DMEM containing 5% FBS. After 3 days, the overlay was aspirated and the wells were washed twice with PBS before fixing with 4% formalin. The wells were stained with 1% crystal violet in 20% ethanol for 30 min at room temperature, washed with water, and dried.

[0209] Plaques were counted and titers were calculated as plaque forming units per ml.

[0210] Example 3 Protein expression by Western blot Western blots were performed to determine the expression of full strength envelope protein-complement regulatory protein fusions.

[0211] Cells were washed once with ice-cold PBS and wells were lysed with 1x lithium dodecyl sulfate buffer supplemented with 0.5 M DTT and 1x protease / phosphatase inhibitor mix (Thermo Fisher Scientific). Cell lysates were sonicated for 1 min to reduce viscosity. Proteins were separated on 4-12% NuPAGE Bis-Tris gels (Thermo Fisher Scientific) and analyzed by blotting. The membrane was transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in Tris-buffered saline (TBS) for 1 hour, then incubated overnight at 4°C with anti-V5 rabbit antibody (Clone Poly29038 Biolegend) diluted in 5% non-fat milk in TBS containing 0.1% Tween 20 (TBST). The membrane was washed three times with TBS, then treated with goat anti-rabbit IgG H&L (HRP) (Abcam ab6721) diluted 1 / 3000 in TBST containing 5% non-fat milk for 1 hour at room temperature. The membrane was washed three times with TBS containing 0.1% Tween 20 (TBST) and twice with water.

[0212] Bound proteins were detected using Novex™ HRP chromogenic substrate (TMB) (Invitrogen™ WP20004).

[0213] Example 4 Viral growth analysis Plaque morphology In some cases, fusion of envelope proteins can alter viral replication, infectivity and spread, which can be evident, for example, from changes in plaque morphology and can be determined using the following methods.

[0214] Virus was titrated by serial dilution on BSC40 cells and overlaid with a 1% methylcellulose semisolid medium layer as described above. An appropriate dilution that yielded 20-50 plaques per well of a 6-well tissue culture plate was established and used to determine the average plaque size.

[0215] Representative images of plaques formed by each virus strain in BSC40 cells were visualized with crystal violet 4 days after infection. The diameters of at least 125 individual plaques from such low density titrations were determined approximately 48 hours post-infection (hpi) using NIH ImageJ software. Quantitative analysis of infectious virus (plaque forming units per ml) present in culture supernatants of the indicated virus strains at 24 hours post-infection is then determined using a standard plaque assay in BSC40 cells.

[0216] The results (not shown) show that none of the viruses containing the modified A13L gene exhibited a growth defect that reduced plaque size compared to the unmodified gene and the A13L-EGFP fusion virus.

[0217] Step growth analysis The replicative capacity of the candidate viruses was also determined by performing growth curves.

[0218] For single-step curves, HeLa cells were infected at a multiplicity of infection of 10 (MOI=10) in 6-well plates, whereas for multi-step curves, cells were infected at an MOI of 0.02. Cells from individual wells were harvested in 1 ml serum-free medium at 0, 2, 4, 6, 8, 12, 16 and 24 hours post-infection (single-step) or 0, 8, 12, 24, 48, 72 and 96 hours post-infection (multi-step). For multi-step growth, the released virus population was collected with an overlaying medium layer, after which the cells were harvested in 1 ml medium. The supernatants and scraped cells were each freeze-thawed three times separately and centrifuged at 13,000k for 30 min, and the resulting clarified supernatants were titrated in triplicate on BSC40 cells. The resulting infectivities were plotted (pfu / ml) and the data revealed that all A13 fusion protein-expressing viruses exhibited levels of intracellular growth and cell-to-cell spread comparable to the parental viruses (i.e., equivalent viruses lacking the relevant A13L fusion gene construct).

[0219] Example 5 In vitro complement inhibition assay Human serum assay The ability of protein fusion variants of vaccinia mature virions (MV) to evade serum complement inhibition was investigated in human serum.

[0220] Virus (2×10 4 ~2×10 7 pfu) were incubated for 60 min at 37°C in serum-free medium containing 50% pooled human complement serum (Merck S1764). Control serum samples were heat inactivated for 30 min at 56°C. To determine the level of infectivity, samples were serially diluted in 1 ml of serum-free DMEM and added to confluent BSC40 cells in 6-well plastic plates for 1 h. After this, the medium was removed, the wells were washed twice with PBS and overlaid with a layer of 1% methylcellulose in DMEM containing 5% FBS. After 2 days, the overlay was aspirated and the wells were washed twice with PBS before fixing with 4% formaldehyde (Merck). The wells were stained with 1% crystal violet in 15% ethanol for 30 min at room temperature, washed with water and dried.

[0221] The plaques were counted and the neutralization rate was calculated.

[0222] Pooled human serum inhibited the infectivity of wild-type Copenhagen virus and A13L-EGFP mutant virus strains by more than 80%, but less than 10% inhibition of viral infectivity was observed when viruses were incubated with heat-inactivated serum.

[0223] Corresponding experiments were performed with each of the following engineered viruses described herein expressing the following fusions: A13-VCP, A13-VCPmut, A13-CD55, A13-CD35, A13-compstatin, A13-CCPH, and A13-ORF4. Results (not shown) indicate that each of the test viruses expressing the complement inhibitory fusion proteins of the present disclosure can significantly retain infectivity in the presence of pooled human serum containing complement. Figure 7 is provided as an example and shows the results of incubating either vA13-CD55 / A13-CD55 or the corresponding wild type (WT) virus with human serum, demonstrating that the engineered viruses of the present disclosure exhibit a significant increase in resistance to serum-mediated neutralization. Additionally, improved retention of infectivity was observed for VCP-A27, SPICE-A27, CD55-A27, CD35-A27, compstatin-A27, CCPH-A27 and ORF4-A27 viruses compared to wild-type and reporter viruses, demonstrating that the engineered vaccinia viruses of the present disclosure have improved complement evasion in human serum. Furthermore, the results indicate that either the A13 or A27 envelope proteins are suitable candidates for fusion proteins designed to evade complement-induced inactivation.

[0224] Rabbit Complement Assay The ability of MV protein fusion variants to evade serum complement inhibition was also investigated in rabbit serum.

[0225] Virus (2×10 4 ~2×107 pfu) were incubated in 1 ml PBS containing 50% rabbit complement (Merck S7764) for 60 min at 37°C. Control serum samples were heat inactivated at 56°C for 30 min. To determine the level of infectivity, samples were serially diluted in 1 ml serum-free DMEM and added to confluent BSC40 cells in 6-well plastic plates for 1 h. After this, the medium was removed, the wells were washed twice with PBS and overlaid with a layer of 1% methylcellulose in DMEM containing 5% FBS. After 2 days, the overlay was aspirated and the wells were washed twice with PBS before fixing with 4% formaldehyde. The wells were incubated at room temperature with 1% crystal violet in 15% ethanol. The stain was left in the dark for 30 minutes, washed with water and dried.

[0226] The plaques were counted and the neutralization rate was calculated.

[0227] Similar to the above assay using human serum, rabbit serum inhibited the infectivity of wild-type, A27-GFP and A13-GFP viruses by more than 80%, whereas incubation of the viruses with heat-inactivated serum resulted in only less than 10% inhibition. The results show that the tested mutant viruses (A13-VCP, A13-VCPmut, A13-CD55, A13-CD35, A13-compstatin, A13-CCPH and A13-ORF4) were all able to significantly retain infectivity in the presence of complement-containing rabbit serum. This effect was also observed with VCP-A27, SPICE-A27, CD55-A27 and CD35-A27, compstatin-A27, CCPH-A27 and ORF4-A27 viruses, again demonstrating that the engineered vaccinia viruses of the present disclosure exhibit improved complement evasion in rabbit serum.

[0228] antibody neutralization The ability of MV fusion mutants to evade antibody neutralization was investigated.

[0229] BSC40 cells were seeded in 6-well plates and infected when they reached confluence. 4pfu) were diluted in 1 ml serum-free medium in the presence or absence of 50% rabbit complement serum and incubated for 1 h at 37°C with 40 μg of the following antibodies: 9503-2057 (BioRad), PA1-7258 (Invitrogen), both anti-vaccinia; and control rabbit IgG antibody ab37415 (Abcam). Serial dilutions were prepared in 1 ml serum-free medium and added to wells containing BSC40. After 2 h, the inoculum was removed, the wells were washed twice with PBS, and overlaid with a layer of 1% methylcellulose in complete DMEM containing 5% FBS. After 2 days, the overlay was aspirated and the wells were washed twice with PBS before fixing with 4% formaldehyde. The wells were stained with 1% crystal violet in 15% ethanol for 30 min at room temperature, washed with water, and dried.

[0230] The plaques were counted and the neutralization rate was calculated.

[0231] The results show that the fusion construct reduces antibody-mediated neutralization in the presence of complement.

[0232] Many modifications to the above examples can be made without departing from the scope of the invention as defined in the appended claims.

[0233] Observations and Conclusions Vaccinia virus fusion proteins / chimeric genes are designed to be expressed intravirally and localized within the viral envelope. The fusion proteins contain one portion (or domain) that is a native (or engineered) viral protein sequence and one portion (or domain) that is a heterologous gene.

[0234] As disclosed herein, the viral gene portion of the fusion / chimeric gene may encode vaccinia virus proteins A13 or A27, which are expressed and found in different distribution patterns in the viral envelope: A13 is widely distributed around the viral envelope, while A27 shows a more biased distribution.

[0235] In these examples, the heterologous gene is a complement regulator (or complement control) protein, such as VCP, SPICE, etc., which are known to inhibit complement activity, as described elsewhere herein. Therefore, the A13 and / or A27 fusion proteins may be It was hypothesized that engineered vaccinia virus particles might be able to avoid complement-mediated inactivation, provided that such a fusion protein was suitably expressed and displayed on the viral envelope such that the VCP (or a functionally equivalent domain) of the vaccinia virus could interact with complement.

[0236] Fusion / chimeric nucleic acid construct sequences encoding fusion proteins containing the relevant vaccinia envelope proteins, A13 and / or A27, with one or various complement inhibitory peptide domains have been produced and used to express engineered vaccinia virus particles. Studies of the resulting engineered viruses have demonstrated that nucleic acids encoding complement inhibitory fusion proteins can be inserted into various vaccinia virus vectors and the encoded fusion proteins are expressed and displayed on the envelope of the engineered vaccinia virus.

[0237] As will be appreciated by those skilled in the art, appropriate design of the fusion / chimeric nucleic acid sequence allows the insertion of a heterologous chimeric gene construct into any desired location within a viral vector. In these studies, the chimeric gene construct was inserted into the thymidine kinase (TK) locus (J2R) while generating a vaccinia virus TK deletion mutant. The vaccinia virus TK deletion mutant must rely on the host cell's TK activity for replication, which is significantly more prevalent in proliferating cells compared to quiescent cells, and may therefore exhibit beneficial behavior for disease therapy, particularly with respect to the treatment of proliferative disorders (such as cancer). By inserting the chimeric gene of the present disclosure into the TK locus, the viral vector retains the wild-type A13L and / or A27L genes and associated expression elements (unless further modified), such that the engineered virus in these aspects and embodiments may express a mixture of wild-type envelope proteins (i.e., A13 or A27) and the corresponding heterologous fusion proteins comprising the A13 or A27 protein sequences fused to a complement control protein sequence. In this way, the beneficial properties of wild-type virus replication, assembly and infectivity can, in some embodiments, be better maintained than in a comparable engineered virus in which all of the expressed A13 or A27 protein sequences are fused to a complement control protein.

[0238] In some aspects and embodiments, chimeric gene constructs are designed to insert heterologous gene sequences into the corresponding envelope protein loci, such that a gene encoding an A13 fusion protein is inserted into and replaces the A13L wild-type gene, and a gene encoding an A27 fusion protein is inserted into and replaces the A27L wild-type gene. In such embodiments, the resulting engineered viral particles are devoid of all the respective wild-type proteins, but studies have demonstrated that such engineered viral particles are capable of replicating, assembling, and infecting cells regardless of whether they contain the A13 or A27 fusion protein. Furthermore, according to some aspects and embodiments, such engineered viruses may be beneficial in that they display a higher number / concentration of complement control proteins on their surface, and thus are more resistant to complement evasion / inactivation compared to engineered viruses that retain a constant percentage of expression of wild-type A13 or A27, respectively, on their surface. Thus, these two different approaches to generating engineered viral vectors provide alternative strategies for complement evasion and generation of engineered viruses optimized for specific therapeutic applications.

[0239] These studies demonstrate that, although the exact location of the fusion of the complement control protein sequence with the A13 or A27 protein sequence does not appear to be critical to the efficacy of the resulting protein and the corresponding engineered viral particles, fusing a complement control protein sequence to (or near) the C-terminus of the A13 protein can produce novel, viable complement control fusion proteins, and fusing a complement control protein sequence to (or near) the N-terminus of the A27 protein can also produce novel, viable complement control fusion proteins. This further demonstrates that the data show that it is possible to produce

[0240] Furthermore, it has been demonstrated that a variety of engineered viral particles can be produced to express a wide range of different fusion proteins on their surface, particularly on the surface of MV particles. The fusion proteins can include any one of several different complement control protein sequences (disclosed herein) fused to viral envelope protein sequences (particularly A13 and / or A27), and such fusion proteins can confer complement inhibition / evasion activity, respectively, to the engineered viral particles.

[0241] Thus, the present disclosure describes a series of novel engineered viruses that have potential as therapeutic agents, as well as the production of corresponding viral vectors, nucleic acid sequences and novel fusion proteins. In particular, the therapeutic agents of the present disclosure may be useful in the treatment of a variety of proliferative diseases and cancers (described above).

[0242] [Table 4] TIFF2025503515000008.tif221170TIFF2025503515000009.tif233170TIFF2025503515000010.tif232170TIFF202 5503515000011.tif232170TIFF2025503515000012.tif231170TIFF2025503515000013.tif230169TIFF2025503515 000014.tif232170TIFF2025503515000015.tif231170TIFF2025503515000016.tif231170TIFF2025503515000017. tif225170TIFF2025503515000018.tif232170TIFF2025503515000019.tif232170TIFF2025503515000020.tif22517 0TIFF2025503515000021.tif232170TIFF2025503515000022.tif226170TIFF2025503515000023.tif231169TIFF20 25503515000024.tif231169TIFF2025503515000025.tif231169TIFF2025503515000026.tif231170TIFF2025503515 000027.tif232170TIFF2025503515000028.tif231170TIFF2025503515000029.tif232170TIFF2025503515000030. tif232170TIFF2025503515000031.tif232170TIFF2025503515000032.tif233170TIFF2025503515000033.tif52170

[0243] Terms Further expressions of the inventive concepts are set forth in the following numbered clauses:

[0244] C1. An isolated nucleic acid encoding a fusion polypeptide, the fusion polypeptide comprising a vaccinia virus envelope protein or a portion thereof and at least one complement control protein or a functional fragment thereof.

[0245] C2. The isolated nucleic acid according to clause C1, wherein the vaccinia virus envelope protein is selected from A13 and A27 or a portion thereof.

[0246] C3. The isolated nucleic acid of clause C1 or C2, wherein the vaccinia virus envelope protein is A13.

[0247] C4. (i) the vaccinia virus envelope protein is not H3, D8 and / or A27 or a portion thereof; and / or (ii) the fusion polypeptide is not a fusion of the vaccinia virus envelope protein H3, D8 or A27 with a complement control protein selected from CD55, CD59, CD46, CD35, factor H and C4 binding protein; and / or (iii) The isolated nucleic acid of any one of clauses C1-C3, wherein the fusion polypeptide does not contain a complement control protein selected from CD55, CD59, CD46, CD35, Factor H and C4 binding protein attached to the N-terminus of A27.

[0248] C5. The isolated nucleic acid of any one of clauses C1-C4, wherein the vaccinia virus envelope protein is an envelope protein from a vaccinia mature virion (MV).

[0249] C6. At least one complement regulatory protein is (i) CD35, CD55, CD59, CD46, CR1, factor H, VCP, MOPICE, SPICE, CCPH, C4 binding protein, CD35, Kaposi's sarcoma-associated herpesvirus Kaposica / KCP, herpesvirus saimiri (HVS) and HVS-CD59, rhesus monkey rhadinovirus RCP-H and RCP-1, murine gamma herpesvirus 68 (γHV-68) RCA, influenza virus M1, EMICE and IMP, and modified sequences thereof or functional fragments thereof. The isolated nucleic acid according to any one of clauses C1 to C5, selected from one or more of the group consisting of:

[0250] C7. The isolated nucleic acid of any one of clauses C1-C6, wherein the at least one complement control protein is selected from one or more of the group consisting of CD35, CD55, VCP, mutated VCP, SPICE, CCPH and ORF4 or a functional fragment thereof.

[0251] C8. The isolated nucleic acid of any one of clauses C1-C7, wherein at least one complement control protein or functional fragment thereof is fused to a transmembrane domain of a vaccinia virus MV envelope protein.

[0252] C9. The isolated nucleic acid of clause 8, wherein the transmembrane region of the vaccinia virus envelope protein is not H3 or D8.

[0253] C10. The isolated nucleic acid of any one of clauses C1-C9, wherein the vaccinia virus envelope protein or portion thereof is fused to at least one complement control protein or functional fragment thereof via a peptide bond.

[0254] C11. The isolated nucleic acid of any one of clauses C1-C10, wherein the peptide bond comprises one or more repeats of the sequence (Gly4Ser); optionally, the peptide bond comprises (Gly4Ser), (Gly4Ser)2 or (Gly4Ser)3.

[0255] C12. Vaccinia virus envelope protein A13 is (i) an A13 protein sequence selected from Vaccinia Copenhagen virus, Camelpox virus, Smallpox virus, Cowpox virus, Taterapox virus, Monkeypox virus Zaire-96-I-16, Volepox virus, Aqumeta virus, Ectromelia virus, Orthopoxvirus Abatino virus, Skunkpox virus, Raccoonpox virus, Yokapox virus, Murmansk pox virus, NY_014 pox virus, and Yabasa monkey tumor virus, or a portion thereof; or (ii) The isolated nucleic acid of any one of clauses C1-C11, comprising at least amino acids 2-21 of a sequence selected from one of SEQ ID NOs: 1-16, or a sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto.

[0256] C13. The vaccinia virus envelope protein A13L is (i) amino acids 2 to 23 of a sequence selected from any one of SEQ ID NOs: 1 to 16; (ii) amino acids 2 to 25 of a sequence selected from any one of SEQ ID NOs: 1 to 16; (iii) amino acids 2 to 30 of a sequence selected from any one of SEQ ID NOs: 1 to 16; (iv) amino acids 2 to 40 of a sequence selected from any one of SEQ ID NOs: 1 to 16; (v) amino acids 2 to 50 of a sequence selected from any one of SEQ ID NOs: 1 to 16; (vi) amino acids 2 to 60 of a sequence selected from any one of SEQ ID NOs: 1 to 16; (vii) amino acids 2 to 70 of a sequence selected from any one of SEQ ID NOs: 1 to 16; (viii) amino acids 1 to 21 of a sequence selected from any of SEQ ID NOs: 1 to 16; or (ix) any one of SEQ ID NOs: 1 to 16; or a sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto. The isolated nucleic acid of any one of clauses C1 to C12, comprising a sequence selected from:

[0257] C14.(i) at least one complement control protein or functional fragment thereof is linked to or toward the C-terminus of a vaccinia virus envelope protein or portion thereof, optionally, the vaccinia virus envelope protein is A13; or (ii) The isolated nucleic acid of any one of clauses C1 to C13, wherein at least one complement control protein or functional fragment thereof is linked to or towards the N-terminus of a vaccinia virus envelope protein or portion thereof, and optionally the vaccinia virus envelope protein is A27.

[0258] C15. The isolated nucleic acid of any one of clauses C1-C14, wherein the at least one complement control protein comprises two, three, or four complement control proteins or functional fragments thereof arranged in sequence; optionally, the two, three, or four complement control proteins are linked to each other via a flexible linker peptide, such as GGGGS or multiple repeats thereof.

[0259] C16. The isolated nucleic acid of clause C15, wherein the at least one complement control protein comprises two VCP or four VCP proteins arranged in series or a functional fragment thereof.

[0260] C17. The fusion polypeptide is selected from A13 fused to VCP, A13 fused to two VCP proteins arranged in tandem, A13 fused to four VCP proteins arranged consecutively, A13 fused to a mutant VCP, A13 fused to CD55, A13 fused to CD35, A13 fused to CCPH, or A13 fused to ORF4, or a functional fragment thereof; Optionally, the mutated VCP comprises SEQ ID NO:31, CD55 comprises amino acids 35-284 of CD55 (e.g., SEQ ID NO:71), CD35 comprises amino acids 42-1584 of CD35 (e.g., SEQ ID NO:72), CCPH comprises amino acids 21-266 of CCPH (e.g., SEQ ID NO:73), and ORF4 comprises amino acids 22-268 of ORF4 (e.g., SEQ ID NO:74); Optionally, the VCP protein is a poxvirus complement control protein or a modified poxvirus complement control protein selected from SPICE (SEQ ID NO: 32), MOPICE (SEQ ID NO: 33), EMICE (SEQ ID NO: 75, 76, particularly SEQ ID NO: 76) or IMP (SEQ ID NO: 77, 78, particularly SEQ ID NO: 78).

[0261] C18. The isolated nucleic acid according to clause C18, wherein one or more complement control proteins or functional fragments thereof are fused to the C-terminus of A13.

[0262] C19. The isolated nucleic acid according to clause C17 or clause C18, having a sequence selected from any one of SEQ ID NOs: 37-46, or a sequence with at least about 80%, at least about 90%, at least about 95%, at least about 98% or at least about 99% sequence identity thereto; or encoding a fusion polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 47-56, or a sequence with at least about 90%, at least about 95%, at least about 98% or at least about 99% sequence identity thereto.

[0263] C20. Vaccinia virus envelope protein A27 or a part thereof (i) an A27 protein sequence selected from Vaccinia Copenhagen virus, Vaccinia virus, Cowpox virus, Camelpox virus, Smallpox virus, Orthopox Abatino virus, Aquameta virus, Ectromelia virus, Taterapox virus, Monkeypox virus, Volepox virus, Raccoonpox virus, Skunkpox virus, or a portion thereof; (ii) An isolated nucleic acid according to clause C1 or clause C2 or, when subject to clause C1 or C2, any one of clauses C5 to C16, comprising at least 90 contiguous amino acids of a sequence selected from any one of SEQ ID NOs: 17 to 29 or a sequence having at least about 90%, at least about 95%, at least about 98% or at least about 99% sequence identity thereto.

[0264] C21. Vaccinia virus envelope protein A27 is selected from the group consisting of SEQ ID NOs: 17 to 29. The isolated nucleic acid according to clause C20, comprising a sequence selected from any one of the above, or a sequence having at least about 90%, at least about 95%, at least about 98% or at least about 99% sequence identity thereto.

[0265] C22. the fusion polypeptide is selected from A27 fused to VCP, A27 fused to a mutated VCP, A27 fused to CD55, A27 fused to CD35, A27 fused to CCPH, or A27 fused to ORF4, or a functional fragment thereof; Optionally, the mutated VCP comprises SEQ ID NO:31; CD55 comprises amino acids 35-284 of CD55 (e.g., SEQ ID NO:71), CD35 comprises amino acids 42-1584 of CD35 (e.g., SEQ ID NO:72), CCPH comprises amino acids 21-266 of CCPH (e.g., SEQ ID NO:73), or ORF4 comprises amino acids 22-268 of ORF4 (e.g., SEQ ID NO:74); Optionally, the isolated nucleic acid according to clause C20 or clause C21, wherein the VCP protein is a poxvirus complement control protein or a modified poxvirus complement control protein selected from SPICE (sequence number 32), MOPICE (sequence number 33), EMICE (sequence numbers 75, 76, particularly SEQ ID NO: 76) or IMP (sequence numbers 77, 78, particularly SEQ ID NO: 78).

[0266] C23. The isolated nucleic acid according to clause C22, wherein one or more complement control proteins or functional fragments thereof are fused to the N-terminus of A27.

[0267] C24. The isolated nucleic acid according to clause C22 or clause C23, encoding a fusion polypeptide having a sequence selected from any one of SEQ ID NOs: 57-62, or a sequence with at least about 80%, at least about 90%, at least about 95%, at least about 98% or at least about 99% sequence identity thereto; or having an amino acid sequence selected from any one of SEQ ID NOs: 63-68, or a sequence with at least about 90%, at least about 95%, at least about 98% or at least about 99% sequence identity thereto.

[0268] C25. The isolated nucleic acid of any one of clauses C1-C24, comprising an engineered vaccinia virus genome or vector.

[0269] C26. An engineered vaccinia virus vector comprising a nucleic acid according to any of clauses C1-C25.

[0270] C27. The engineered vaccinia virus vector of clause C26, wherein a nucleic acid encoding a fusion polypeptide comprising a vaccinia virus envelope protein or a portion thereof and at least one complement control protein or a functional fragment thereof is inserted into the vector at a locus outside of the corresponding wild-type vaccinia virus envelope protein locus.

[0271] C28. The engineered vaccinia virus vector of clause C26 or clause C27, comprising a deleted or inactive thymidine kinase (TK) gene.

[0272] C29. The engineered vaccinia virus vector of clause 28, wherein a nucleic acid encoding a fusion polypeptide is inserted into the locus of the TK gene; optionally, the TK gene is deleted or inactivated.

[0273] C30. The nucleic acid encoding the fusion polypeptide is inserted into a corresponding envelope protein gene locus; optionally, the corresponding envelope protein gene is a deleted 28. The engineered vaccinia virus vector of clause 26 or clause 27, which has been deleted or inactivated.

[0274] C31.(i) the fusion polypeptide comprises A13 or a portion thereof, and the corresponding envelope protein locus is the A13L locus; or (ii) The engineered vaccinia virus vector according to clause C30, wherein the fusion polypeptide comprises A27 or a portion thereof and the corresponding envelope protein locus is the A27L locus.

[0275] C32. The engineered vaccinia virus vector of any one of clauses C26 to C31 or the isolated nucleic acid of any one of clauses C1 to C25, wherein the nucleic acid encoding the fusion polypeptide comprises a vaccinia virus early or late transcription promoter sequence, and the early or late transcription promoter sequence is operably linked to the nucleic acid encoding the fusion polypeptide.

[0276] C33. The engineered vaccinia virus vector of any one of clauses C26 to C31 or the isolated nucleic acid of any one of clauses C1 to C25, wherein the nucleic acid encoding the fusion polypeptide is operably linked to a native promoter of a corresponding vaccinia virus envelope protein.

[0277] C34. The engineered vaccinia virus vector according to any one of clauses C26 to C33 or the isolated nucleic acid according to clause C25, comprising a deletion or an inactivated vaccinia virus gene of one or more vaccinia virus genes selected from the group of C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, J2R, F1L, F2L, F3L, B14R, B15R, B16R, B17L, B18R, B19R, B20R, K ORF A, K ORF B, B ORF E, B ORF F, B ORF G, B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R and B29R.

[0278] C35. Engineered vaccinia virus vectors (i) a cytokine, optionally GM-CSF; (ii) a prodrug converting enzyme, optionally a cytosine deaminase (CD); (iii) a theranostic payload, optionally a sodium iodide symporter (NIS); (iv) an affinity reagent, optionally a bispecific antibody; (v) an imaging or reporter agent, optionally being luciferase, Renilla luciferase-GFP fusion protein, β-galactosidase, β-glucuronidase or green fluorescent protein; The engineered vaccinia virus vector according to any one of clauses C26 to C34 or the isolated nucleic acid according to clause C25, comprising a nucleic acid sequence encoding

[0279] C36. The engineered vaccinia virus vector of any one of clauses C26 to C35 or the isolated nucleic acid of clause C25, wherein the engineered vaccinia virus vector comprises a nucleic acid sequence encoding a cytokine; optionally, the cytokine is GM-CSF.

[0280] C37.Abatino macacapox virus, Acmeta virus, Camelpox virus 903, Camelpox virus CMG, Camelpox virus CMS, Camelpox virus CP1, Camelpox virus CP5, Camelpox virus M-96, Cowpox virus (Brig hton Red), Cowpox virus (GRI-90 strain), Cowpox virus (Hamburg-1985 strain), Cowpox virus (Turkmenia-1974 strain), Elephantpox virus, Belo Horizonte virus, Ectromeliavirus ERPV, Ectromeliavirus Moscow, Ectromeliavirus Naval, Ectromeliavirus WH, Common marmoset (Callithrix jacchus) orthopoxvirus, Monkeypox virus (Sierra Leone 70-0266 strain), Monkeypox virus (Zaire 77-0666 strain), Monkeypox virus Zaire-96-I-16, Raccoonpox virus, Skunkpox virus, Taterapox virus, Aracatuba virus, Buffalopox virus, Cantagalo virus, Guarani P1 virus, Guarani P2 virus, Horsepox virus, Modified vaccinia virus Ankara, Rabbitpox virus, Rabbitpox virus Utrecht, SPAN 232 virus, Vaccinia virus Acambis 3000 MVA, Vaccinia virus Ankara, Vaccinia virus Copenhagen, Vaccinia virus Dalian I, Vaccinia virus GLV-1h68, Vaccinia virus IHD-J, Vaccinia virus L-IPV, Vaccinia virus LC16M8, Vaccinia virus LC16MO, Vaccinia virus Lister, Vaccinia virus LIVP, Vaccinia virus Mariana, Vaccinia virus Tashkent, Vaccinia virus Tian Tan, Vaccinia virus WAU86 / 88-1, Vaccinia virus Western Reserve, Vaccinia virus WR, Vaccinia virus WR 65-16, Vaccinia virus Wyeth, Variola major virus, Variola5. The engineered vaccinia virus vector of any one of clauses C26 to C36 or the isolated nucleic acid of clause C25, comprising an engineered vaccinia virus genome selected from Orthopoxvirus GCP2010, Orthopoxvirus GCP2013, Orthopoxvirus NY99014 / 1999, Orthopoxvirus OH08 / 2008, Orthopoxvirus Tena Dona, Orthopoxvirus VPXV_CA85, Orthopoxvirus WA01960 / 2001, Todopoxvirus, Orthopoxvirus species.

[0281] C38.(i) Copenhagen, Western Reserve, Wyeth, Lister or modified vaccinia Ankara strain; (ii) Copenhagen or Western Reserve strains; or (iii) Copenhagen strain The engineered vaccinia virus vector according to any one of clauses C26 to C37 or the isolated nucleic acid according to clause C25, comprising an engineered vaccinia virus genome selected from:

[0282] C39. A modified vaccinia virus virion obtained from the engineered vaccinia virus vector or isolated nucleic acid of any one of clauses C25 to C38.

[0283] C40. A modified vaccinia virus virion comprising a nucleic acid according to any one of clauses C1-C38.

[0284] C41. A pharmaceutical composition comprising an engineered vaccinia virus vector according to any one of clauses C26 to C38, an isolated nucleic acid according to any one of clauses C1 to C25 and C32 to C38, an engineered vaccinia virus vector according to any one of clauses C26 to C38, an engineered vaccinia virus virion according to clause C39 or a modified vaccinia virus virion according to clause C40, and a pharma- ceutically acceptable carrier.

[0285] C42. The pharmaceutical composition according to clause C41, formulated for systemic or local or topical administration.

[0286] C43. Modified or engineered vaccinia virus virions; and (i) Approximately 1 × 10 per ml 3 ~Approx. 1×10 15 pfu; (ii) Approximately 1 × 10 per ml 4 ~Approx. 1×10 14 pfu; or (iii) Approximately 1 × 10 per ml 6 ~Approx. 1×10 12 p.f.u. A pharmaceutical composition according to clause C41 or clause C42, formulated to have a unit dose of

[0287] C44. An engineered vaccinia virus vector according to any one of clauses C26 to C38, an isolated nucleic acid according to any one of clauses C1 to C25 and C32 to C38, an engineered or modified vaccinia virus virion according to clause C39 or clause C40 or a pharmaceutical composition according to any one of clauses C41 to C43 for use in a method of treating cancer and / or a proliferative disease or disorder in a subject.

[0288] C45. A method of treating cancer and / or a proliferative disease or disorder in a mammalian subject, comprising administering to the subject a therapeutically effective amount of an engineered vaccinia virus vector according to any one of clauses C26 to C38, an isolated nucleic acid according to any one of clauses C1 to C25 and C32 to C38, an engineered or modified vaccinia virus virion according to clause C39 or clause C40 or a pharmaceutical composition according to any one of clauses C41 to C43.

[0289] The engineered vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus virion or pharmaceutical composition for use according to clause C44 or the method according to clause C45, wherein the cancer and / or proliferative disease or disorder is selected from lung cancer (e.g., lung adenocarcinoma), cervical cancer, breast cancer, heart cancer, colon cancer, prostate cancer, brain glioblastoma, pancreatic cancer, leukemia (e.g., acute monocytic leukemia), lymphoma, kidney cancer, colorectal cancer, bladder cancer, testicular cancer, gastrointestinal cancer, liver cancer (e.g., hepatocellular carcinoma) and / or glioblastoma. The present invention may also be useful for treating one or more of skin cancer (e.g., melanoma), head and / or neck cancer, gallbladder cancer, uterine cancer, stomach cancer, thyroid cancer, laryngeal cancer, lip and / or oral cavity cancer, pharyngeal cancer, eye cancer and bone cancer.

[0290] C47. Cancer and / or proliferative disease or disorder is liver cancer (HC), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratocarcinoma / rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, extrahepatic cancer, Ewing's sarcoma family Lee, osteosarcoma and malignant fibrous histiocytoma, central nervous system embryonal tumor, central nervous system germ cell tumor, craniopharyngioma, ependymoma, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasm, colon cancer, extrahepatic bile duct carcinoma, ductal carcinoma in situ (DCIS), endometrial carcinoma, ependymoma, esophageal cancer, olfactory neuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube carcinoma, fibrous tissue of the bone Myeloma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brain stem glioma, hairy cell leukemia, hepatocellular carcinoma, Langerhans cell histiocytosis, Hodgkin's lymphoma, hypopharyngeal carcinoma, pancreatic islet cell tumor, pancreatic neuroendocrine tumor, Wilms' tumor and other childhood renal tumors, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastasis Squamous cell neck cancer, midline carcinoma, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, myelodysplastic syndrome, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low-grade ovarian cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, brown cell or the method according to clause C45, wherein the engineered vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus virion or pharmaceutical composition for use according to clause C44 or the method according to clause C45 is selected from any one or more of the group consisting of: pulmonary tumour, pituitary tumour, pleuropulmonary blastoma, primary peritoneal carcinoma, rectal carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, Kaposi's sarcoma, rhabdomyosarcoma, Sezary syndrome, small intestine cancer, soft tissue sarcoma, pharyngeal carcinoma, thymoma and thymic carcinoma, thyroid carcinoma, transitional cell carcinoma of the renal pelvis and ureter, urethral carcinoma, endometrial carcinoma, uterine sarcoma, vaginal carcinoma, vulvar carcinoma and Waldenstrom's macroglobulinemia.

[0291] C48. The engineered vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus virion or pharmaceutical composition for use according to any one of clauses C44, C46 or C47 or the method according to any one of clauses C45 to C47, wherein the subject is selected from a human, a non-human primate, a cow, a sheep, a pig, a dog, a cat, a rabbit, a bat, a mouse or a rat; optionally, the subject is a human, a non-human primate, a rabbit or a mouse; optionally, the subject is a human.

[0292] C49. The engineered vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus virion or pharmaceutical composition for use according to any one of clauses C44 or C46 to C48 or the method according to any one of clauses C45 to C48, wherein the vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus virion or pharmaceutical composition is administered systemically or locally to the subject; optionally the administration is by a route selected from intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, local (e.g. near a tumor, particularly the vasculature of a tumor or adjacent vasculature), percutaneous, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage or oral.

[0293] C50. The engineered vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus virion or pharmaceutical composition for use according to any one of clauses C44 or C46 to C49 or the method according to any one of clauses C45 to C49, wherein the vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus virion or pharmaceutical composition is administered in combination with one or more additional therapeutic agents or therapies, and the administration of the one or more additional therapeutic agents or therapies is simultaneous, separate or sequential to the administration of the vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus virion or pharmaceutical composition.

[0294] C51. The engineered vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus virion or pharmaceutical composition for use according to clause C50 or the method according to clause C50, wherein the one or more additional therapeutic agents or therapies comprise chemotherapy, radiation therapy, oncolytic virus therapy, immunomodulatory proteins, anti-cancer agents or any combination thereof.

[0295] C52. The engineered vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus virion or pharmaceutical composition or method for use according to clause C51, wherein the anti-cancer agent is selected from one or more chemotherapeutic agents, radiotherapeutic agents, cytokines, immune checkpoint inhibitors, anti-angiogenic agents, apoptosis inducers, anti-cancer antibodies and / or anti-cyclin dependent kinase agents.

[0296] C53. The engineered vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus for use according to clause C51 or C52, wherein the therapy is selected from one or more of chemotherapy, biological therapy, radiation therapy, immunotherapy, hormonal therapy, antivascular therapy, cryotherapy, toxin therapy and / or surgery or a combination thereof. Subirion or pharmaceutical composition or method.

[0297] C54. The engineered vaccinia virus vector, the isolated nucleic acid, the engineered or modified vaccinia virus virion or the pharmaceutical composition for use according to any one of clauses C44 or C46 to C53 or the method according to any one of clauses C45 to C53, wherein the cancer is a primary cancer, a secondary cancer or a metastasis.

[0298] C55. The engineered vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus virion or pharmaceutical composition for use according to any one of clauses C44 or C46 to C54, or the method according to any one of clauses C45 to C54, which reduces the size of a tumor or the proportion or proliferation of tumor cells in a subject by about 10% to about 100%, about 20% to about 90%, about 30% to about 80%, or about 40% to about 70%.

[0299] C56. Engineered or modified vaccinia virus virions are (i) Approximately 1 × 10 per kg 3 ~Approx. 1×10 15 pfu; (ii) Approximately 1 × 10 per kg 4 ~Approx. 1×10 14 pfu; or (iii) Approximately 1 × 10 per kg 6 ~Approx. 1×10 12 p.f.u. 5. A method comprising the engineered or modified vaccinia virus virion for use according to any one of clauses C44 or C46 to C55 or the engineered or modified vaccinia virus virion according to any one of clauses C45 to C55 administered to a subject at a dose of

[0300] C57. One or more first active agents selected from an engineered vaccinia virus vector according to any one of clauses C26 to C38, an isolated nucleic acid according to any one of clauses C1 to C25 and C32 to C38, an engineered or modified vaccinia virus virion according to clause C39 or clause C40, or a pharmaceutical composition according to any one of clauses C41 to C43, and a container containing the first active agent; and optionally one or more second active agents selected from the group consisting of anti-cancer agents, immunomodulatory agents, or any combination thereof, which may be administered to the subject in combination with the one or more first active agents; one or more devices or apparatus for administering at least one first active agent and an optional second active agent; and Kit Instructions and a kit comprising:

[0301] C58. An isolated polypeptide encoded by a nucleic acid according to any one of clauses C1-C25.

[0302] C59. The isolated polypeptide of clause A58, having a sequence selected from any one of SEQ ID NOs: 47-56 or 63-68, or a sequence having at least about 90%, at least about 95%, at least about 98% or at least about 99% sequence identity thereto.

Claims

1. An isolated nucleic acid encoding a fusion polypeptide, said fusion polypeptide comprising a vaccinia virus envelope protein or a portion thereof and at least one complement control protein or functional fragment thereof.

2. 2. The isolated nucleic acid of claim 1, wherein the vaccinia virus envelope protein is selected from A13 and A27 or a portion thereof.

3. 3. The isolated nucleic acid of claim 1 or 2, wherein the vaccinia virus envelope protein is A13.

4. 3. The isolated nucleic acid of claim 1 or 2, wherein the vaccinia virus envelope protein is an envelope protein from a vaccinia mature virion (MV).

5. The at least one complement regulatory protein is (i) CD35, CD55, CD59, CD46, CR1, Factor H, VCP, MOPICE, SPICE, CCPH, C4-binding protein, CD35, Kaposi's sarcoma-associated herpesvirus Kaposica / KCP, herpesvirus saimiri (HVS) and HVS-CD59, rhesus monkey rhadinovirus RCP-H and RCP-1, murine gammaherpesvirus 68 (γHV-68) RCA, influenza virus M1, EMICE and IMP, and modified sequences thereof or functional fragments thereof.

3. The isolated nucleic acid of claim 1 or 2, selected from one or more of the group consisting of:

6. 3. The isolated nucleic acid of claim 1, wherein the at least one complement control protein is selected from one or more of the group consisting of CD35, CD55, VCP, mutated VCP, SPICE, CCPH, and ORF4, or functional fragments thereof.

7. 3. The isolated nucleic acid of claim 1, wherein the at least one complement regulatory protein or functional fragment thereof is fused to the transmembrane domain of a vaccinia virus MV envelope protein.

8. 8. The isolated nucleic acid of claim 7, wherein the transmembrane domain of the vaccinia virus envelope protein is not H3 or D8.

9. The vaccinia virus envelope protein A13 is (i) an A13 protein sequence selected from vaccinia Copenhagen virus, camelpox virus, smallpox virus, cowpox virus, taterapox virus, monkeypox virus Zaire-96-I-16, volepox virus, aquametavirus, ectromelia virus, orthopoxvirus Abatino virus, skunkpox virus, raccoonpox virus, yokapox virus, murmansk pox virus, NY_014 pox virus, and yaba monkey tumor virus, or a portion thereof; or (ii) The isolated nucleic acid of claim 1 or 2, comprising at least amino acids 2 to 21 of a sequence selected from one of SEQ ID NOs: 1 to 16, or a sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto.

10. the fusion polypeptide is selected from A13 fused to VCP, A13 fused to two VCP proteins arranged in tandem, A13 fused to four VCP proteins arranged consecutively, A13 fused to a mutant VCP, A13 fused to CD55, A13 fused to CD35, A13 fused to CCPH, or A13 fused to ORF4, or a functional fragment thereof; Optionally, the mutant VCP comprises SEQ ID NO:31; the CD55 comprises amino acids 35-284 of CD55 (e.g., SEQ ID NO:71), the CD35 comprises amino acids 42-1584 of CD35 (e.g., SEQ ID NO:72), the CCPH comprises amino acids 21-266 of CCPH (e.g., SEQ ID NO:73), or the ORF4 comprises amino acids 22-268 of ORF4 (e.g., SEQ ID NO:74); Optionally, the VCP protein is a poxvirus complement control protein or a modified poxvirus complement control protein selected from SPICE (SEQ ID NO: 32), MOPICE (SEQ ID NO: 33), EMICE (SEQ ID NOs: 75, 76, particularly SEQ ID NO: 76) or IMP (SEQ ID NOs: 77, 78, particularly SEQ ID NO: 78).

11. The isolated nucleic acid of claim 2, wherein the one or more complement regulatory proteins or functional fragments thereof are fused to the C-terminus of A13.

12. 12. The isolated nucleic acid of claim 10 or 11, encoding a fusion polypeptide having a sequence selected from any one of SEQ ID NOs: 37-46, or a sequence with at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto; or an amino acid sequence selected from any one of SEQ ID NOs: 47-56, or a sequence with at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto.

13. the fusion polypeptide is selected from A27 fused to VCP, A27 fused to a mutant VCP, A27 fused to CD55, A27 fused to CD35, A27 fused to CCPH, or A27 fused to ORF4, or a functional fragment thereof; Optionally, the mutant VCP comprises SEQ ID NO:31; the CD55 comprises amino acids 35-284 of CD55 (e.g., SEQ ID NO:71), the CD35 comprises amino acids 42-1584 of CD35 (e.g., SEQ ID NO:72), the CCPH comprises amino acids 21-266 of CCPH (e.g., SEQ ID NO:73), or the ORF4 comprises amino acids 22-268 of ORF4 (e.g., SEQ ID NO:74); 3. The isolated nucleic acid of claim 1, wherein the VCP protein is a poxvirus complement control protein or a modified poxvirus complement control protein selected from SPICE (SEQ ID NO: 32), MOPICE (SEQ ID NO: 33), EMICE (SEQ ID NOs: 75, 76, particularly SEQ ID NO: 76) or IMP (SEQ ID NOs: 77, 78, particularly SEQ ID NO: 78).

14. 14. The isolated nucleic acid of claim 13, wherein the one or more complement regulatory proteins or functional fragments thereof are fused to the N-terminus of A27.

15. 14. The isolated nucleic acid of claim 13, encoding a fusion polypeptide having a sequence selected from any one of SEQ ID NOs: 57-62, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto; or an amino acid sequence selected from any one of SEQ ID NOs: 63-68, or a sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto.

16. 3. An engineered vaccinia virus vector comprising the nucleic acid of claim 1 or 2.

17. 17. The engineered vaccinia virus vector of claim 16, wherein the nucleic acid encoding a fusion polypeptide comprising a vaccinia virus envelope protein or portion thereof and at least one complement control protein or functional fragment thereof is inserted into the vector at a locus outside the corresponding wild-type vaccinia virus envelope protein locus.

18. 17. The engineered vaccinia virus vector of claim 16, comprising a deleted or inactive thymidine kinase (TK) gene.

19. 19. The engineered vaccinia virus vector of claim 18, wherein the nucleic acid encoding the fusion polypeptide is inserted into the locus of the TK gene; and optionally, the TK gene is deleted or inactivated.

20. 17. The engineered vaccinia virus vector of claim 16 or the isolated nucleic acid of claim 1, wherein the nucleic acid encoding the fusion polypeptide is operably linked to the native promoter of the corresponding vaccinia virus envelope protein.

21. (i) Copenhagen, Western Reserve, Wyeth, Lister or modified vaccinia Ankara strain; (ii) Copenhagen or Western Reserve strains; or (iii) Copenhagen strain 17. The engineered vaccinia virus vector of claim 16 or the isolated nucleic acid of claim 1, comprising an engineered vaccinia virus genome selected from:

22. 20. A modified vaccinia virus virion comprising the nucleic acid of claim 1 or 16.

23. 23. A pharmaceutical composition comprising the engineered vaccinia virus vector of claim 16, the isolated nucleic acid of claim 1, or the modified vaccinia virus virion of claim 22, and a pharmaceutically acceptable carrier.

24. 24. The pharmaceutical composition of claim 23, formulated for systemic or local or topical administration.

25. comprising a modified vaccinia virus virion, and (i) Approximately 1 x 10 per ml ３ ~Approx. 1×10 １５ pfu; (ii) about 1 x 10 per ml ４ ~Approx. 1×10 １４ pfu; or (iii) about 1 x 10 per ml ６ ~Approx. 1×10 １２ pfu 24. The pharmaceutical composition of claim 23, formulated to have a unit dose of

26. 24. An engineered vaccinia virus vector according to claim 16, an isolated nucleic acid according to claim 1, a modified vaccinia virus virion according to claim 22 or a pharmaceutical composition according to claim 23 for use in a method for treating cancer and / or a proliferative disease or disorder in a subject.

27. A method for treating cancer and / or a proliferative disease or disorder in a mammalian subject, comprising administering to the subject a therapeutically effective amount of an engineered vaccinia virus vector described in claim 16, an isolated nucleic acid described in claim 1, a modified vaccinia virus virion described in claim 22, or a pharmaceutical composition described in claim 23.

28. 28. The engineered vaccinia virus vector, isolated nucleic acid, modified vaccinia virus virion or pharmaceutical composition for use according to claim 26 or the method according to claim 27, wherein the cancer and / or proliferative disease or disorder is selected from lung cancer (e.g. lung adenocarcinoma), cervical cancer, breast cancer, heart cancer, colon cancer, prostate cancer, brain glioblastoma, pancreatic cancer, leukemia (e.g. acute monocytic leukemia), lymphoma, kidney cancer, colorectal cancer, bladder cancer, testicular cancer, gastrointestinal cancer, liver cancer (e.g. hepatocellular carcinoma) and / or glioblastoma. The present invention may also be useful in treating one or more of skin cancer (e.g., melanoma), head and / or neck cancer, gallbladder cancer, uterine cancer, stomach cancer, thyroid cancer, laryngeal cancer, lip and / or oral cavity cancer, pharyngeal cancer, eye cancer, and bone cancer.

29. 28. The engineered vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus virion or pharmaceutical composition for use according to claim 26 or the method according to claim 27, wherein the vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus virion or pharmaceutical composition is administered to the subject systemically or locally; optionally, administration is by a route selected from intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, local (e.g., near a tumor, particularly the vasculature of a tumor or adjacent vasculature), transdermal, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage or oral.

30. 28. The engineered vaccinia virus vector, isolated nucleic acid, modified vaccinia virus virion or pharmaceutical composition for use according to claim 26 or the method of claim 27, wherein the vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus virion or pharmaceutical composition is administered in combination with one or more additional therapeutic agents or therapies, the administration of which is simultaneous, separate or sequential to the administration of the vaccinia virus vector, isolated nucleic acid, engineered or modified vaccinia virus virion or pharmaceutical composition.

31. The engineered vaccinia virus vector, isolated nucleic acid, modified vaccinia virus virion or pharmaceutical composition for use according to claim 26 or the method according to claim 27, wherein the cancer is a primary cancer, a secondary cancer or a metastasis.

32. The modified vaccinia virus virion comprises: (i) Approximately 1 x 10 per kg ３ ~Approx. 1×10 １５ pfu; (ii) about 1 x 10 per kg ４ ~Approx. 1×10 １４ pfu; or (iii) about 1 x 10 per kg ６ ~Approx. 1×10 １２ pfu 28. A method comprising the modified vaccinia virus virion for use according to claim 26 or the modified vaccinia virus virion according to claim 27, administered to the subject at a dose of

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