MODIFIED CELLS FOR PROGRAMMING EXTRA ENVELOPED VIRUSES (EEVs) AND THEIR USE FOR PRODUCING THE EEVs, THE PROGRAMMED EEVs AND USES THEREOF

Abstract

Provided are modified cells and cell lines containing extracellular enveloped viruses (EEV), methods of producing serum-resistant EEVs, and programmed EEVs for systemic oncolytic viral therapy. The viruses are high EEV producing viruses that contain genome modifications that alter properties and / or activities of the viruses. The cells and cell lines are modified to encode polypeptides or peptides that are displayed on their membranes, which, when virus is propagated in the cells, encases the virus so that the polypeptides and peptides from the cell membrane are displayed on the virus. The viruses, thus contain modified outer envelopes that display polypeptides and / or peptides encoded by the virus and from the cell membranes. This combination provides viruses whose properties and activities can be programmed by virtue of the cells in which they are produced and viral genome modifications. The viruses can be used for treating cancers or, because the properties of the virus, including tropisms, other diseases, disorders, and conditions involving targeted cells.

Description

[0001] Attorney Docket No. 120276-2615PC

[0002] -1-

[0003] MODIFIED CELLS FOR PROGRAMMING EXTRA ENVELOPED VIRUSES

[0004] (EEVs) AND THEIR USE FOR PRODUCING THE EEVs, THE PROGRAMMED EEVs AND USES THEREOF

[0005] RELATED APPLICATIONS

[0006] Benefit of priority is claimed to U. S. provisional application Serial No.

[0007] 63 / 875,990, entitled “MODIFIED CELLS FOR PROGRAMMING EXTRA ENVELOPED VIRUSES (EEVs) AND THEIR USE FOR PRODUCING THE EEVs, THE PROGRAMMED EEVs AND USES THEREOF,” filed September 04, 2025, to inventors Antonio Fernandez Santidrian, Duong Hoang Nguyen, Yunyi Kang, Karolin Waterstraat, Thomas Herrmann, Lina Schulte, Sinje Tigges, and Applicant Calidi Biotherapeutics (Nevada), Inc.

[0008] Benefit of priority is claimed to U. S. provisional application Serial No.

[0009] 63 / 719,604, entitled “SERUM-RESISTANT EEV VIRUSES AND USES THEREOF,” filed November 12, 2024, to inventors Antonio Fernandez Santidrian, Duong Hoang Nguyen, and Yunyi Kang, and Applicant Calidi Biotherapeutics (Nevada), Inc.

[0010] Benefit of priority is claimed to International PCT application No.

[0011] PCT / US2025 / 017701, published as WO 2025 / 184411, on September 04, 2025, entitled SERUM-RESISTANT EEV VIRUSES AND USES THEREOF,” filed February 27, 2025, to inventors Antonio Fernandez Santidrian, Duong Hoang Nguyen, Yunyi Kang, Karolin Waterstraat, Thomas Herrmann, and Applicant Calidi Biotherapeutics (Nevada), Inc. Benefit of priority is claimed for subject matter having the priority date of November 12, 2024, or later.

[0012] Where permitted the subject matter of each of these applications is incorporated in its entirety.

[0013] INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

[0014] An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The electronic file was created on November 12, 2025, is 46,716,374 bytes bytes in size, and is titled 2615PCSEQ001.xml.

[0015] FIELD

[0016] The field is oncolytic viruses, manufacture, and uses thereof for therapy.

[0017] BACKGROUND Attorney Docket No. 120276-2615PC

[0018] -2-

[0019] The ability of administered viruses to infect a tumor and colonize and / or replicate within tumors is decreased by circulating neutralizing antibodies, innate and adaptive immune mechanisms, and other clearing mechanisms directed against the viruses. These mechanisms have impeded the goal of systemic oncolytic therapy. There is a need to address these problems to improve to the therapeutic efficacy of oncolytic viruses.

[0020] SUMMARY

[0021] Provided are enveloped viruses that derive a membrane from the host in which they are propagated. These include poxviruses, particularly the EEV form of vaccinia, and RNA viruses. The viruses are propagated in modified host cells that express a polypeptide or functional portion thereof on membranes that, when the viruses are propagated in the modified cells are displayed the virus on the host-derived membrane. The viruses also are genome-modified to express a polypeptide or portion thereof on the host-derived membrane. The displayed polypeptides encoded by the virus and on the host-derived membrane and also displayed on the virus are chosen to modify, alter, or confer a property or activity on the virus. The displayed polypeptides generally are displayed as transmembrane fusion proteins or chimeric proteins where the functional portion of the polypeptide that modifies, alters, and / or confers a property or activity is displayed and active. The viruses thus can be programmed to have advantageous properties, including increased serum resistance, altered tropism for targeting to cells, such as tumor cells or immune cells, in which the virus can deliver encoded therapeutic proteins or propagate and lyse ells in which they are delivered.

[0022] Provided are viruses that are high EEV viruses that are genome-modified to encode a fusion protein or chimeric between an EEV outer membrane transmembrane protein, and a polypeptide that alters a property or activity of the virus, where: the virus is a poxvirus, such as vaccinia virus; the transmembrane fusion or chimeric protein comprises B5R, A33R, A34R, A56R, F13L protein, which are virus-encoded transmembrane proteins, and the polypeptide that alters a property or activity of the virus, wherein the polypeptide is displayed on the outer membrane of the virus; the EEV outer membrane is a host cell-derived membrane that comprises a host cell transmembrane fusion protein or chimera with a polypeptide that alters a property or activity of the virus when displayed on the virus; and the host cell-derived membrane is the outer membrane that the virus acquires from the cell in which it is propagated. The Attorney Docket No. 120276-2615PC

[0023] -3-

[0024] virally-encoded A56 protein generally is intact. In some embodiments, it can be a chimeric or fusion protein to display a polypeptide, such as a protein that inhibits complement, on the surface of the virus. High EEV viruses can be prepared as known in the art and detailed herein; they can be produced from any known virus by mutation and / or selection, or a known strain with such property can be used and modified and propagated in a modified host cell as detailed herein. Host cells are modified to express a polypeptide that alters an activity or property of the virus by expressing such polypeptide or functional portion thereof on host cell membranes, which are deposited on EEV viruses and other enveloped viruses. In some embodiments, the viruses are any of the red tail (RT) viruses detailed herein or are derivatives thereof, or are derives of IHD, such as IHD-W or IHD-J strains of vaccinia virus modified or selected to be high EEV.

[0025] The viruses, such as vaccinia virus, can include knock-outs of genes or portions thereof so that the functional product encoded by the genes are not produced. The knockouts are selected to confer advantageous properties on the virus, such as by increasing serum resistance, as detailed herein. For example, the vaccinia viruses an include knockouts of the products encoded by A46R, VGF, and TK, and comprises an intact A56 gene. This is effected by deletion, insertions, and / or rearrangements. Generally, the gene is deleted all or in part and / or another coding sequence open reading frame or transgene is inserted, such as one the encodes a therapeutic product. Exemplary viruses are the RT viruses provided herein and derivatives thereof produced by modifications and / or culturing the viruses and selecting for a particular property or activity, or just changes that occur upon propagation without altering properties / activities of the virus. An exemplary virus is the virus detailed herein is the virus designated RT-134 or a virus having at least 95% nucleic acid sequence identity thereto (at least 95% sequence identity to SEQ ID NO: 901, 917 or 918) and retaining the high EEV phenotype and encoded therapeutic protein and the displayed virally-encoded fusion protein or chimeric protein. The vaccinia viruses have an intact A56 gene.

[0026] The host cell-derived chimeric or fusion protein that alters a property or activity of the virus can be a property or activity selected from one or more of:

[0027] (a) attenuation or blocking of anti-viral immunity, and / or

[0028] (b) reduction of neutralizing antibody binding, and / or

[0029] (c) reduction of antibody-dependent opsonization, and / or Attorney Docket No. 120276-2615PC

[0030] -4-

[0031] (d) reduction of phagocytic uptake, and / or

[0032] (e) attenuation of natural killer (NK) cell cytotoxicity, and / or

[0033] (f) enhancement of delivery of virus to tumors, and / or

[0034] (g) enhancement of anti-tumor immunity.

[0035] The viruses can comprise nucleic acid encoding a therapeutic protein for treatment of a disease, disorder, or condition, such as an anti-cancer therapeutic or an immune modulatory protein. The encoded product is inserted into or in place of a non-essential locus, such as the VGF or A46R locus. The nucleic acid encoding the therapeutic protein can be inserted into the VGF gene locus replacing all or a part of the encoded product to effect the knockout. Encoded therapeutics are user selected and depend upon the disease, disorder, or condition to be treated and the target cell. For example, for treating tumors encoded products include any that modify the tumor microenvironment to render it an anti-tumor environment. Products include cytokines, such a cytokine that comprises 11-15, such as an IL-15 superagonist or modified IL-15 superagonist.

[0036] The host cell-derived fusion or chimeric transmembrane protein can comprise, for example an anti-cancer antibody or antigen-binding portion thereof or a receptor or ligand for targeting the virus to a tumor or immune cell, or comprise a polypeptide that attenuates anti-viral immunity, such as, but not limited to, an anti-cancer antibody that comprises an scFv or antigen-binding portion thereof.

[0037] Thus, provided are enveloped viruses that comprise a host-derived membrane, wherein the host-derived membrane is modified to display a transmembrane chimeric or fusion protein comprising the host transmembrane protein and a second polypeptide or portion thereof that alters properties or activities of the virus, such as an extracellular envelope virus (EEV) that comprises a host-derived membrane and a virally produced membrane. Extra envelope viruses include the EEV form of vaccinia virus, such as high EEV vaccinia virus, a retrovirus or other RNA virus that has a single membrane derived from the host. Provided are enveloped viruses that comprise a host cell-derived membrane, where the viruses comprise a non-virally encoded polypeptide (a polypeptide encoded by the host cell in which the virus is produced) displayed on the host-derived membrane; the non-virally encoded membrane-displayed polypeptide alters properties and / or activities of the envelope virus, such as an EEV vaccinia virus; and the non-virally Attorney Docket No. 120276-2615PC

[0038] -5-

[0039] encoded membrane-displayed polypeptide is a fusion protein or chimeric protein that comprises all or a portion of a host transmembrane protein and a polypeptide that alters the properties or activities of the virus.

[0040] Provided are programmed extracellular envelope viruses, such as EEV vaccinia viruses. The programmed viruses comprise a virally-encoded modified transmembrane polypeptide, and a non-virally encoded polypeptide (a polypeptide encoded by the host cell in which the virus is produced) displayed on the outer second membrane; and the membrane-displayed polypeptide(s) alter properties and / or activities of the EEV virus. Provided are programmed extracellular envelope viruses, such as EEV vaccinia viruses, and other viruses, such as single membrane RNA viruses, that have a host cell-derived membrane. The host cell-derived membrane includes a host cell-encoded modified transmembrane polypeptide or the a membrane-displayed polypeptide where the membrane-displayed displayed polypeptide(s) alter properties and / or activities of the virus.

[0041] Enveloped viruses include viruses such as extracellular envelope vaccinia virus (EEV) and retroviruses and other RNA viruses. For example, the viruses can be selected from among species of Herpesviridae, Iridoviridae, Poxviridae, Hepadnaviridae, Togaviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Bunyaviridae, Coronaviridae, Arenaviridae, Retroviridae, and Flioviridae.

[0042] Also provided are modified cells that are modified to express a transmembrane protein that is a fusion protein or chimeric protein containing a polypeptide or functional portion thereof. The cell comprises a virus was propagated in the host cell, and has the host-derived membrane. The cell is modified to express one or more polypeptide(s) and / or peptide(s) in the cell membrane, whereby the polypeptide or peptide is displayed on the membrane; and the polypeptide(s) and / or or peptide(s) alters activity or properties of the virus when the membrane envelops the virus. The virus can be a high EEV vaccinia virus. Provided are modified cells or cell lines that comprise an EEV virus, such as an EEV vaccinia virus, that produces more than 1% EEV viruses when propagated in the cell or cell line, or a virus that acquires it membrane from the host cell; and the cell is genome-modified to encode and express on the cell membrane a polypeptide that, when displayed on the second membrane of the EEV virus or on the membrane of the virus that acquires its membrane from the cell, alters an activity or Attorney Docket No. 120276-2615PC

[0043] -6-

[0044] property of the virus. The host cell transmembrane protein can comprise a single- or multi-pass transmembrane protein, where the functional domain of the fusion or chimeric protein of the transmembrane protein is displayed on the surface of the host-derived membrane of the virus. Exemplary host cell membranes include, but are not limited to, CD8a, PDGFR, CD28, CD4, and CD3(^, which are host cell transmembrane proteins that are single-pass transmembrane protein.

[0045] Polypeptides and portions there of that that alters, confer, and / or hance the properties or activities of the virus on which they are displayed are known in the art, and a multitude of examples are described in the disclosure herein. Included are polypeptides that increase serum resistance of the immune system of the host to whom the virus is administered, and polypeptides that target the virus by virtue of interaction with a receptor or ligand displayed on the cell or virus to a cell, such as a tumor cell or an immune cell. Many receptors and ligands that interact with tumor-specific antigens and receptors and immune cell, such as T-cells, known to those of skill in the art

[0046] The host cell for propagating viruses can comprise fusion or chimeric protein, which is the encases a virus propagated in the cell so that the host-derived virus membrane that comprises all or an active portion of polypeptide that alters, enhances, or confers a property or activity of the virus. These include, complement inhibiting polypeptides and polypeptides modulates the immune response of a subject for treatment with the virus, and receptors, and ligands, including antibodies and antigen-binding portions thereof. Exemplary vaccinia viruses, such as EEV vaccinia viruses, that display such polypeptides are described and exemplified herein. These include high EEV-producing EEV vaccinia viruses, such as a high EEV-producing EEV virus that, when propagated, produces more than 1%, 5%, 10%, 15%, 20%, 25%, 30%, generally more than 30%, or more than IDH-W strain of vaccinia virus. As detailed throughout the disclosure herein, the viruses include EEV vaccinia viruses have higher anti-tumor activity and higher EEV production than the virus HTD-W, wherein the EEV is a clone of the polyclonal vaccinia IHD strain NR-52. Included are those where the EEV virus genome has an intact A56 locus, whereby the virus has increased serum stability compared to the polyclonal IHD strain, such as where: the extra enveloped vaccinia virus (EEV) that has higher anti-tumor activity and higher EEV production than the virus HTD-W; the EEV is a clone of the polyclonal vaccinia IHD strain NR-52 (BEI Resources Attorney Docket No. 120276-2615PC

[0047] -7-

[0048] catalog no. NR-52); and contains a deletion of at least one amino acid in the K7R gene, a TLR modulator receptor; and / or contains a gene identical to the gene encoding RPXV102, a cell surface-binding protein and carbonic anhydrase homolog, which does not occur in IHD-W, and which is in the IMV and binds to chondroitin sulfate on the cell surface, providing virion attachment to a target cell; and / or has 2 SNPs in the A30L gene compared with IHD-W; and / or when its sequence is compared with the sequences of each of A33R, A34R, A36R, A56R, B5R, F13L, A45R, A29L A31R A30L, A32L, and / or A13L in the strain IHD-J, the genome of the clonal EEV has 2 SNPs in A30L and 1 in A45R.

[0049] The viruses are manufactured in modified host cells for manufacture of the virus. Modified cells and cell lines, include stem cells and cell lines comprising a modified transmembrane protein, such as HEK293 cell, HeLa cells, or iPSC. The modified host-derived transmembrane proteins include, for example, TROP2, HER2, CD20, CD38, BCMA Fusion proteins and chimeric proteins containing these transmembrane proteins can comprise a receptor or ligand or functional portion thereof for targeting a virus to tumor cell or an immune cell, or altering the response of the immune system of the host to whom the virus is administered. As detailed herein, EEV vaccinia viruses are modified, and can include gene knock-outs that confer advantageous properties, particularly for increasing serum half-life and / or evading the immune system of the host. These include high EEV vaccinia viruses that comprise knock-outs (KOs) of the VGF, TK, and A46R loci. The viruses can further comprises nucleic acid encoding a therapeutic product that is an anti-cancer therapeutic or a product that promotes an antitumor response in the tumor microenvironment, such as a cytokine, such as an IL- 15 or a modified form thereof. Examples of IL- 15 cytokines include, but are not limited to, an IL-15 superagonist (IL-15 / IL-15R alpha chain complex), or conjugate thereof with an scFv, and / or comprising a mutation that increases activity. Examples include IL- 15 polypeptides where the cytokine is IL-15 / IL-15R alpha chain complex (IL-15 superagonist) or a modified IL-15 / IL-15R alpha chain complex that comprises a replacement, deletion or insertion of one or more amino acids, whereby activity in vivo is increased compared to IL-15 / IL-15R alpha chain complex without the modifications. Modified IL-15 polypeptides include IL-15 superagonist that comprises the replacement N72D or N72E. IL-15 superagonists, for example, comprise Sushi domain, a linker, and Attorney Docket No. 120276-2615PC

[0050] -8-

[0051] IL- 15, where: a) the Sushi domain has the sequence:

[0052] ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH WTTPS LKCIRDPALVHQRPAPPSTVTTAGV (SEQ ID NO:635); the sequence of the linker is SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO:636), and the IL-15 comprises N72D and comprises the sequence:

[0053] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS (SEQ ID NO:637); or b) a variant of the IL-15 superagonist that has at least 95%, 96%, 97%, 98%, 99% sequence identity to the IL- 15 superagonist of a) and retains antitumor activity.

[0054] Other exemplary virus-encoded therapeutics can be selected from among one or more of CCL21, IL-1, IL-2, IL-3, IL-7, IL-12, IL-15, IL-15 / IL15a receptor complex (referred to as IL 15 superagonist) and variants thereof that increase activity, IL- 18, IL-21, IFN-a, IFN-P, IFN-y, TNF-a, EPO, GM-CSF, G-CSF, Flt3L, FGF, EGF, IL-4, IL-6, IL-10, IL-11, IL-13, IL-17, IL-32, and IL-7-IL-21 fusion protein. The viral genome encodes an IL- 15 superagonist that comprises the IL- 15 sushi domain linked, via a peptide linker, to a modified IL-15 that has increased activity. Exemplary are viruses that are produced in the cells, and cell lines provide herein where the virus displays a virally-encoded transmembrane fusion or chimeric protein comprising A33 and CD55. Included for propagation to acquire a host cell-derived membrane from the cells and cell lines, modified as described herein and as apparent from the description herein are the RT viruses described above and throughout the disclosure herein. Exemplary thereof is the virus designated RT-134 whose genome comprises the sequence set forth in SEQ ID NO: 901, 917, 918 or a sequence having least 95%, 96%, 97%, 98%, 99% sequence identity thereto to the SEQ ID NO:901 without the ITRs. Such viruses include those where the genome of the virus comprises the knockouts of the VGF, TK, and A46 loci, and the genome encodes an IL-15 superagonist (an IL-15 / IL-15R alpha chain complex); the encoded IL-15 / IL-15R alpha chain complex comprises a replacement at residue N72 that is D or E; and the genome of the virus has at least 95% sequence identity to SEQ ID NO: 901 or a the portion that does not include the ITRs, and includes the knockouts of VGF, TK, A46, and the knockout of VGF is effected by encoding the IL-15 superagonist inserted therein or replacing all or a portion of the encoded VGF protein. The virus Attorney Docket No. 120276-2615PC

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[0056] further can display host cell-membrane derived proteins that further alter, enhance, or confer properties on the virus as detailed throughout the disclosure herein. The viruses, cells, cell lines, where the host cell membrane is modified display a modified transmembrane protein that alters one or more properties or activities of the virus that comprises the modified membrane.

[0057] As detailed throughout the disclosure herein a property and / or activity that is conferred, enhanced, and / or altered by a transmembrane protein displayed on the host cell-derived membrane is selected from among one or more of: (a) attenuation or blocking of anti-viral immunity, (b) reduction of neutralizing antibody binding, (c) reduction of antibody-dependent opsonization, (d) reduction of phagocytic uptake, (e) attenuation of natural killer (NK) cell cytotoxicity, (f) enhancement of delivery to tumors, (g) enhancement of anti-tumor immunity, and other such properties / activities as are apparent from the disclosure herein and the knowledge of the skilled artisan. Other such properties and / or activities include one or more of: a) enhancing delivery to tumors and / or target tissues, such as by expressing tissue and / or tumor homing proteins, such as, for example, chemokine receptors and integrins; b) targeting to and / or comprising receptor or ligand binding domains such as, for example, antibodies or antigen-binding portions thereof, scFvs, and fusion or chimeric proteins; and c) enhancing anti-tumor immunity such as by expressing on the membrane a transmembrane protein comprising all or an active portion of a co-stimulatory molecule; a TNF superfamily of ligands (TNFSF); a TGF-beta polypeptide antagonists; a checkpoint inhibitor; and a cytokine and / or chemokine.

[0058] The cell or virus or cell line can include modifications wherein the host cells membranes are modified by one or more of:

[0059] a) express on or more chemokine receptors (CCR1, CCR2, CCR4, CCR5, CCR7, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, XCR1, and atypical chemokine receptors (ACKRs), such as ACKR1 and ACKR1); integrins (a6pi, a6p4, a6p5, avP5, pi, a2pi, a2p3, avP3, a5pi, a6pi, avP3,a3pi, avP6, all, a6pi, a5, P3, a9pi, P4, ax, a9p5, a9, ITGB1); and / or other targeting molecules such as CD44, C-met, and CLA4 tissue-specific or tumor specific targeting or tropism; and / or b) target or include binding domains, wherein proteins for targeting include: single-chain variable fragments (scFvs), variable heavy domains of heavy chains (VHHs), Fab fragments, or Attorney Docket No. 120276-2615PC

[0060] -10-

[0061] engineered ligand mimetics, DARPin, ligands or receptors that recognize or are expressed on target cells in a host. As detailed herein and understood by a skilled artisan, binding domains can be selected from one or more of: TROP2, HER2, BCMA, CD38, EGFR, BCMA, CD19, CD20, Mesothelin, CD22, B7-H3, and PTK7. For example, the host-derived viral membrane can comprise chimeric antigen receptor (CAR). For example, the CAR can comprise an extracellular antigen-binding domain, a hinge or spacer region, a transmembrane domain, and one or more intracellular signaling domains. For example, the CAR can comprise an extracellular single-chain variable fragment (scFv) targeting an antigen expressed on a tumor cell or other targeted cell, such as, where the CAR that comprises an scFv or other single chain antibody that targets a tumor antigen, such as TROP2, HER2, CD20, CD38, and BCMA, a hinge region, and a transmembrane domain. The host-derived membrane on the virus can target a tumor marker and / or antigen, or a tumor malignancy marker, or an autoimmune / inflammatory disorder marker, such as where the host-derived membrane on the virus targets a tumor marker and / or antigen, or a tumor malignancy marker, or an autoimmune / inflammatory disorder marker, is one or more of: a) a tumor marker / antigen that is HER2, Trop-2, Nectin-4, Tissue Factor, Folate Receptor a, c-MET, HER3, CLDN18.2, EGFR, EGFRvIII, Mesothelin, FAP, CEA, B7-H3, PSMA, GPC3, MUC1, IL-13Ra2, GD2, or ROR1; b) a tumor hematologic malignancy markers that is BCMA, CD38, SLAMF7, GPRC5D, CD19, CD22, CD30, CD33, CD123, FLT3, CLL1 (CLEC12A), SLAMF7 (CS1), or CD79b; and c) an autoimmune inflammatory disorder marker include CD 19, BCMA, CD20, CD22, and CD52. As detailed herein, the host cell transmembrane protein that is modified is CD8a, PDGFR, CD28, CD4, and CD3Q or comprises a glycosylphosphatidylinositol (GPI) anchors with or without linkage to a signaling domain, such as where a transmembrane fusion or chimeric protein on the host-derived membrane on the virus is selected from among one or more of TROP2(PDGFR-TM), HER2(PDGFR-TM), BCMA(PDGFR-TM), CD38(PDGFR-TM), TROP2(CD8-TM), where the sequences are set forth in SEQ ID NOs: 881-885, respectively, or variants thereof that have at least 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NOs: 881-885, respectively and retaining the activity of the scFv or antigen binding portion of the fusion or chimeric protein. Attorney Docket No. 120276-2615PC

[0062] -11-

[0063] The host cell membrane or host cell-derived membrane can be modified, for example to contain one or more of a peptide or polypeptide that:

[0064] a) attenuates or block anti-viral immunity, such as by: increasing resistance to complement / attenuated complement deposition, optionally selected from one or more of: expressing complement regulator / inhibitor, such as CD46, CD55, CD59, and CD35 / CR1; and / or expressing a soluble complement regulator / inhibitor in a membrane tethered or pericellularly retained format, such as Factor H, C4b binding protein, and functional portions thereof; and / or expressing a protein to inhibit lectin-pathway initiation -Inhibitors of MASP proteases, such as Cl -inhibitor (Cl -inh), human astrovirus coat protein (CoPt), and Flavivirus non-structural protein 1 (NS1); and / or expressing a viral mimic of a complement regulator / inhibitor, such as CP, MOPICE, SPICE, CCPH, Kaposi -sarcoma associated herpesvirus Kaposica I KCP, Herpesvirus saimiri (HVS) HVS-CD59, Rhesus rhadinovirus RCP-H and RCP-1, murine gamma herpesvirus 68 (yHV-68) RCA, Influenzavirus Ml, EMICE, IMP, and functional portions thereof; and / or

[0065] b) attenuates neutralizing antibody binding / Attenuate antibody dependent opsonization, such as one or more of: expressing ST6GAL1, B4GALT1, FUT8, and MGAT5 to promote a dense, terminally sialylated and fucosylated glycocalyx such that recognition of the viral envelope by neutralizing antibodies is sterically hindered / reduce Fc clustering on envelope membrane; expressing an antibody decoy receptor to act as a neutralizing antibody sink, such as a membrane anchored Fc binding domain, such as Fc binding domains derived from Protein A or Protein G Fc binding domains; expressing an Fc Receptor, as an antibody decoy receptor, such as IgA (e.g., FcaRI (CD89)); IgM (e.g., plgR, and Fca / pR); IgE (e.g., FcaRI and FcsRII (CD23)); and IgG (e.g., FcyRI (CD64), FcyRII (CD32), FcγRIIII (CD16), and FcRn).

[0066] The cell or virus or cell line can include modifications wherein the host cells membranes are modified by one or more of:

[0067] a) removing host cell features that facilitate classical and lectin pathway activation on the envelope or host cell membrane such as by eliminating Fc gamma receptor expression by removing adhesion molecules that stabilize Fc driven lytic synapses, such as by - knockout LFA-1, ICAM-1, CD2, CD58, DNAM-1, Nectin and Nectin-like molecules, Cadherins, and Tetraspanins; Attorney Docket No. 120276-2615PC

[0068] -12-

[0069] b) attenuating phagocytic uptake, such as by overexpressing CD47;

[0070] c) attenuating natural killer cell cytotoxicity, such as by HLA-E and HLA-G overexpression to maintain inhibitory NK signaling;

[0071] d) expressing antagonists of NK cell receptors / ligands of NK inhibitory receptors - host cells can be engineered to express of one or more of: antagonists of MIC-A and MIC-B (NKG2D ligands) (e.g., kK5 (KHSV)); antagonists of the NKG2D receptor (e.g., Cowpox OMCP); antagonists of natural cytotoxicity receptors (NCRs) - targeting NKp30, NKp44, NKp46 receptors (e.g., HA (hemagglutinin - in vaccinia and other viruses)); ligands for the NK inhibitory receptors (KIR) (e.g., HLA-Bw4; HLA-C2); ligands for the NK inhibitory receptors (NKG2a / CD94) (e.g., HLA-E and derivatives alone or combined with 21M HLA-B ligands to generate HLA-E binding peptides and stabilize HLA-E surface expression);

[0072] e) eliminating stress induced NKG2D ligands Membrane-Bound MICA / B (NKG2D Ligands); Membrane-Bound PVR (DNAM-1 Ligand); Membrane-Bound Nectin-2 (DNAM-1 Ligand), MICA, MICB, and ULBP family members, suppression or knockout of NECTIN 2 and PVR;

[0073] f) silencing immunogenic and immune-activating determinants, such as by suppression or deletion of B2M; CIITA; MHC Class I molecules (HLA-A, B, C); MHC Class II molecules (HLA-DP, DQ, DR); MHC -like molecules (CDla / b / c / d); or regulators of transcription or expression of MHC Class I, MHC Class II, MHC-like molecules (e.g., TAP1 / 2, Tapasin, Beta-2 microglobulin, CIITA, RFXANK, RFX5 and RFXAP);

[0074] g) antagonizing immunogenic and immune-activating determinants - express of one or more of: B2M Antagonists of Viral Origin (e.g., ULI 8 (HCMV); and / or MHC Antagonists of Viral Origin (e.g., one or more of A40R MHCI (Vaccinia); Nef, TAT (HIV); E3-19K (Adenovirus); ICP47 (HSV-1 / 2); CPXV012, CPXV203 (Cowpox); EBNA1, BNLF2a, BGLF5, BILF1 (EBV); ORF66 (VZV); US2 / gp24, US3 / gp23, US6 / gp21, US10, USll / gp33 (hCMV); rhl78 / VIHCE (RhCMV); U21 (HHV-6 / 7); LANA1, ORF37 / SOX, kK3 / MIR1, kK5 / MIR2 (KHSV); mK3 (MHV-68); UL41 / vhs (a-herpesvirus, HSV, BHV-1, PRV); UL49.5 (Varicellovirus, BHV-1, EHV-1 / 4, PRV); and m4 / gp34, m6 / gp48, m27, ml52 / gp40 (mCMV)); Attorney Docket No. 120276-2615PC

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[0076] h) reducing immune co-stimulation and danger signaling in host cell by silencing or knockout of CD80, CD86, TLR3, TLR7, TLR9, IFI16, AIM2; and

[0077] i) expressing immunosuppressive factors of human or viral origin.

[0078] For example, the host cell-derived virus membrane can be modified to enhance anti-tumor immunity by expressing one or more of:

[0079] a co- stimulatory molecules, such as, for example, CD40, CD40L, 4- IBB, 4-1BBL, 4-1BBL with a deletion of the cytoplasmic domain (4-lBBLAcyt), 4-1BBL with a truncated cytoplasmic domain, 0X40 (CD 134), OX40L (CD252), other members of the TNFR superfamily (e.g., CD27, CD27 ligand, GITR, CD30, Fas receptor, TRAIL-R, TNF-R, HVEM, and RANK), B7, CD80, CD86, ICOS, ICOS ligand (B7RP1), and CD28;

[0080] a truncated co-stimulatory molecules (e.g., 4-1BBL, CD80, CD86, CD27L, B7RP1, OX40L), with a full or partial (complete, or truncated, or modified to ensure proper orientation when expressed in a cell) cytoplasmic domain deletion;

[0081] a TNF superfamily of ligands - CD30, Fas-L, TRAIL-R, and TNF-R, which induce apoptosis, and CD27, OX40L, CD40L, GITR-L, and 4-1 BBL;

[0082] a TGF-beta polypeptide antagonist;

[0083] an immune checkpoint scFv (tethered to a transmembrane protein - an scFv targeting CTLA-4, PD-L1 (B7-H1), PD-L2, PD-1, PD-2, IDO1, IDO2, SIRP alpha (CD47), VISTA (B7-H5), LIGHT, HVEM, CD28, LAG3, TIM3, TIGIT, Galectin-9, CEACAM1, CD155, CD112, CD226, CD244 (2B4), B7-H2, B7-H3, CD137, ICOS, GITR, B7-H4, B7-H6, CD137, CD27, CD40, CD40L, CD48, CD70, CD80, CD86, CD137 (4-1BB), 4-1BBL, CD200, CD272 (BTLA), CD160, A2a receptor, A2b receptor, HHLA2, ILT-2, ILT-4, gp49B, PIR-B, 0X40, OX-40L, HLA-G, ILT-2 / 4, KIR, TIM1, TIM4, and CLEVER- 1 / Stabilin-l;

[0084] a cytokine as a fusion / chimeric protein with a transmembrane protein - IL2, IL4, IL6, IL7, IL7-IL21 fusion proteins, IL9, IL10, IL11, IL12, IL15, IL18, or IL21 can be fused at the cytokine C-terminus, through a linker, to N- or C- terminus of a transmembrane protein or any other membrane bound protein.

[0085] As detailed throughout the disclosure herein, cells for propagating the virus can be selected from among: adult stem cells; embryonic stem cells; fetal stem cells; neural stem cells; mesenchymal stem cells (for example, isolated / derived from: adult bone Attorney Docket No. 120276-2615PC

[0086] -14-

[0087] marrow, adipose tissue, blood, dental pulp, neonatal umbilical cord, umbilical cord blood, placenta, placenta-derived adherent stromal cells, placenta-derived decidual stromal cells, endometrial regenerative cells, placental bipotent endothelial / mesenchymal progenitor cells, amniotic membrane or fluid mesenchymal stem cells, amniotic fluid derived progenitors, Wharton’s Jelly mesenchymal stem cells, pelvic girdle stem cells, Chorionic Villus Mesenchymal Stromal cells, subcutaneous white adipose mesenchymal stem cells, pericytes, adventitial reticular stem cells, hair follicle-derived stem cells, hematopoietic stem cells, periosteum-derived mesenchymal stem cells, lateral plate mesenchymal stem cells, exfoliated deciduous teeth stem cells, periodontal ligament stem cells, dental follicle progenitor cells, stem cells from apical papilla, muscle satellite cells, etc.); neural stem cells; totipotent stem cells; pluripotent stem cells; induced pluripotent stem cells (iPSCs); multipotent stem cells; oligopotent stem cells; unipotent stem cells; adipose stromal stem cells; endothelial stem cells (for example, endothelial progenitor cells, placental endothelial progenitor cells, angiogenic endothelial Cells, pericytes); adult peripheral blood stem cells; myoblasts; small juvenile stem cells; skin fibroblast stem cells; tissue / tumor-associated fibroblasts; epithelial stem cells; and embryonic epithelial stem cells. Exemplary of such cells and cell lines are iPSCs, stem cells, and cell lines, such as HEK293, HEK293T, A549, PerC6, Vero, Vero STAT1 KO, HEK293. STAT1 BAX KO AGE1.CR.pIX, CV1, HELA, HELA S3, CHO, VPCs, VPCs 2.0, FS293, MDCK, and MDCK. STAT1 KO cells. As discussed, the host cell-derived membrane or the host cell-derived membrane and ae virally encoded membrane protein are modified to display a fusion or chimeric polypeptides that:

[0088] a) attenuate or block anti-viral immunity, such as by providing:

[0089] enhanced resistance to complement and / or attenuated complement deposition and / or attenuating or eliminating neutralizing antibody binding and / or antibody dependent opsonization;

[0090] b) attenuate or modulate phagocytic uptake;

[0091] c) enhance, increase specificity, or increase delivery to tumors, such as by including tissue and tumor homing proteins;

[0092] d) comprise targeting and / or binding domains, such as those that target solid tumor markers, hematologic malignancy markers, or autoimmune or inflammatory disease markers selected from among: solid tumor markers, hematologic malignancy Attorney Docket No. 120276-2615PC

[0093] -15-

[0094] markers, and autoimmune or inflammatory disease markers, where the solid tumor markers, hematologic malignancy markers, and autoimmune or inflammatory disease markers are targeted by approved or investigational monoclonal antibodies, antibodydrug conjugates (ADCs), cell therapies, CAR T-cells, CAR-NK cells, and CAR-macrophages, and transgenic T-cell receptors;

[0095] e) increase or enhance anti-tumor immunity and / or the anti-tumor response of the immune system of the host, such expressing a co-stimulatory molecule or truncated costimulatory molecule (e.g., 4-1BBL, CD80, CD86, CD27L, B7RP1, and OX40L) and / or to display or contain a single-chain antibody or antigen binding portion thereof, such as an immune check point inhibitor; and / or display or contain a cytokine or chemokine. The host cell-derived membrane or the genome of the virus can be modified so that host-derived membrane comprises an immunomodulatory protein, a receptor, a ligand, an antigen-binding portion of an antibody, a chimeric receptor, or an immune modulator, such as, for example, a chimeric of fusion protein that comprises one or more of all or an active portion of CD55, CD47, and / or a CAR. The cell membrane and / or the virally encoded polypeptide comprises a target polypeptide or peptide selected from ALIX, KCNJ2, MICA, NECTIN2, or PVR. For example, the membrane can comprise a chimeric antigen receptor (CAR), such as, but are not limited to, one or more of a CAR selected from one or more of an epidermal growth factor receptor (EGFR) CAR, TROP2, CD38, a HER2 CAR, a CD 19 CAR, a mesothelin CAR, a GD2 CAR, and a BCMA CAR.

[0096] Provided are cells that are modified to express heterologous proteins on their membranes. The cells contain viruses that produce high levels of extracellular envelope viruses and other viruses that, when produced, are encased in the host cell membrane, and methods for producing extra enveloped viruses (EEVs), such as the EEV form of vaccinia virus. The cells also can be used for any virus, such as a lentivirus, that is encased in a membrane derived from the host in which it is propagated. The cells contain extra-enveloped viruses for propagation. EEVs acquire their second membrane from the membranes of cells in which they are produced. The cellular membranes are their second membrane, the outer membrane, which display products, such as proteins or polypeptides or peptides from the host cell membrane and also the products encoded by the virus. Hence by modifying cells to express products on their cell membranes, the EEVs Attorney Docket No. 120276-2615PC

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[0098] produced in the cells express the products on the second membranes. EEVs also encode proteins that are expressed on the outer membrane. Hence modification of the cells and the modification of the viral genome results in EEVs that can display heterologous products, proteins, polypeptides, or peptides encoded in the viral genome and also from the host cell membrane. These products are chosen to modulate or alter or enhance or inhibit a property or activity of the virus. Provided are compositions, including pharmaceutical compositions, that contain the viruses, cells, and cell lines. Also provided are uses of the viruses, cells, cell lines, compositions, and pharmaceutical compositions for treating diseases, disorders, and conditions, such as but are not limited to, cancer and autoimmune diseases, disorders, and conditions. Provided are methods of treatment by administering the viruses and compositions containing the viruses to a subject.

[0099] Modified cells and cell lines provided herein are modified to express heterologous proteins, such immune modulators, ligands, receptors, and / or antigens expressed on tumor cells or immune cells and / or that target the virus to receptors on tumor cells or immune cells or other cells involved in effecting an anti-cancer response or cells involved in other diseases, disorders, and conditions, such as autoimmune diseases in which B cells or other are overexpressed. The EEVs display products, generally peptides and polypeptides, on the second membrane that are ligands or receptors antigens or antibodies that target the virus to receptors on tumor cells or immune cells or other cells involved in effecting an anti-cancer response and deliver the virus to cells and / or products that increase serum resistance and / or resistant to the immune system of a host to which the virus is administered. The oncolytic viruses, including those described herein and known in the art, can amplify in the cells in the host. The virus also can encode a therapeutic product or products. The membrane is derived from the cells in which the EEVs are produced; such cells are modified to express the ligand, receptor, antibody, antigen or other protein / polypeptide on their membranes, which are then are deposited on the virus in the cells. Cells include any in which the viruses that have an extracellular membrane, such as poxviruses can be produced. These cells and cell lines, include for example cell lines such as Hela cells, induced pluripotent stem cells, and others. The viruses generally are those that produce high levels of EEV viruses. These include any known in the art, any modified or selected to be high EEV. They include the RT-00 virus and derivatives of the viruses provided herein, and also, those known in the art, such as Attorney Docket No. 120276-2615PC

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[0101] the virus of SEQ ID NO: 1 of US Publication No. US 2024 / 0033347) and others described herein and known to those of skill in the art. Virus is introduced into the modified cells and cultured to produce EEVs, which are released into the culture medium. The amount of EEVs can be increased by culturing and producing virus according to methods herein in which the EEV second membrane is retained to a greater extent than prior methods. In general, such methods are designed to be sufficiently gentle so that the second membrane, which is fragile, is retained.

[0102] Provided are programmed extracellular envelope viruses (EEVs) or viruses with a host cell-derived membrane, comprising a virally-encoded modified transmembrane polypeptide, and a non-virally encoded polypeptide displayed on the second membrane, wherein the membrane-displayed polypeptide(s) alter properties and / or activities of the EEV virus. Also provided are modified cells that comprise modified cell, comprising a virus that is an extracellular envelope virus (EEV) or virus with a host cell-derived membrane, where the cell is modified to express a polypeptide or peptide in the cell membrane; and the polypeptide or peptide is alters activity or properties of the virus when the membrane envelops the virus.

[0103] A modified cell or cell line, comprising an EEV virus that produces more than 1% EEV viruses when propagated in the cell or line, or a virus that acquires its membrane from the host cell, wherein the cell is genome-modified to encode and express on the cell membrane a polypeptide that, when displayed on the second membrane of the EEV virus or on the membrane of the virus that acquires its membrane from the cell, alters an activity or property of the virus are provided.

[0104] The cells are used in propagating the viruses. EEVs, which contain the host cell membrane and also include virally-encoded proteins in the membrane are released into the cell culture medium and isolated from the medium.

[0105] Compositions containing cells are provided as are the compositions, including pharmaceutical compositions containing the viruses are provided.

[0106] Various embodiments are detailed in the claims and herein incorporated by reference.

[0107] The viruses that are propagated in the cells include those described herein and also any known to the those of skill in the art. The viruses detailed below can be propagated in the modified host cells and the resulting viruses. Attorney Docket No. 120276-2615PC

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[0109] Detailed herein and provided herein are viruses and virus preparations and compositions that have a high percentage of extra enveloped vaccinia (EEV) forms of vaccinia virus and other poxviruses. These viruses can be introduced into the cells and propagated to acquire the modified cell membranes.

[0110] Also provided are methods of manufacturing such viruses, whereby compositions with high levels of the EEV form are produced. These methods can be used for production of any virus, particularly enveloped viruses, such as other poxviruses, herpesvirus, and lentiviruses that acquire a membrane for the host cell in which they are propagated. By virtue of acquisition of the membrane from the host, tropisms and other properties and activities, such as targeting to particular cells, and resistance to the immune system of the host, can be introduced onto the virus particles. With respect to vaccinia and other poxviruses that encode proteins that are expressed on the host-derived envelope the various properties and / or activities can be introduced combined with the ability to express virally-encoded polypeptides on the outer membrane, the viruses can be programmed to manifest the various properties and / or activities and combinations thereof.

[0111] In particular, provided are cells and cell lines modified to express proteins of interest on cell membranes so that EEV viruses produced in such cells and cell lines include proteins on the second membrane. The cells and cell lines also can be modified to express complement inhibiting proteins, such as CD55 or other modulators, and also polypeptides and peptides that alter a property or activity of virus encased in membrane derived from the cell, when the membrane is including those described herein, of the immune system of the host so that that the viruses that include such membranes exhibit increased serum resistance. Exemplary of proteins and polypeptides for expression / display in cell membranes and / or encoded in the virus for display on the second membrane are those for example that:

[0112] a) attenuate or block anti-viral immunity, such as by providing:

[0113] enhanced resistance to complement and / or attenuated complement deposition and / or attenuating or eliminating neutralizing antibody binding and / or antibody dependent opsonization;

[0114] b) attenuate or modulate phagocytic uptake; Attorney Docket No. 120276-2615PC

[0115] -19-

[0116] c) enhance, increase specificity, or increase delivery to tumors, such as by including tissue and tumor homing proteins;

[0117] d) comprise targeting and / or binding domains, such as those that target solid tumor markers, hematologic malignancy markers, or autoimmune or inflammatory disease markers. Exemplary solid tumor markers, hematologic malignancy markers, and autoimmune or inflammatory disease markers include those that are targeted by approved or investigational monoclonal antibodies, antibody-drug conjugates (ADCs), cell therapies, such as CAR T-cells, CAR-NK cells, and CAR-macrophages, and transgenic T-cell receptors; and / pr

[0118] e) increase or enhance anti-tumor immunity and / or the anti-tumor response of the immune system of the host, such expressing a co-stimulatory molecule or truncated costimulatory molecule (e.g., 4-1BBL, CD80, CD86, CD27L, B7RP1, and OX40L) and / or to display or contain a single-chain antibody or antigen binding portion thereof, such as an immune check point inhibitor; and / or a cytokine or chemokine.

[0119] For expression on the host-derived membrane, virally encoded peptides are expressed as fusion proteins or chimeric proteins with a virally-encoded extracellular envelope transmembrane protein. For vaccinia virus, the virally encoded outer membrane protein is A33R, A34R, B5R, and F13L, generally B5R or A33R, and the peptide / polypeptide is produced as a fusion (chimeric) protein, generally B5R or A33R. It is shown herein that an intact A56 membrane protein contributes to or enhances serum stability of the virus, he vaccinia virus is a high EEV-producing virus, generally higher than 1%, particularly greater than 30% of progeny viruses and / or viruses produced by methods that do not disrupt the EEV membrane to result in compositions that contain very high concentrations and / or amounts of EEV, such as 80% or more of the viruses as manufactured. Thus, the viruses encode fusion or chimeric proteins between of a virally-encoded transmembrane protein, such as A33 or B5R, and a peptide or polypeptide that increases serum resistance. As detailed herein, the viruses are produced in cells that natively express serum resistance proteins, such as CD55, and / or that are modified to express a peptide or polypeptide as a fusion or chimera with a host cell transmembrane protein, to alter a property or activity of the virus, such as to further increase serum resistance and / or to target the virus to a tumor cell or other cells, such as an immune cell. Attorney Docket No. 120276-2615PC

[0120] -20-

[0121] Provided are viruses that produce a high level of EEV particles, such as greater than 1% of the virus particles, or more, when propagated, and modified forms thereof. Also provided are methods for purifying the EEV viruses so that the EEV membrane is retained during propagation and isolation to produce preparations that have a high percentage, generally at least 30%, up to 80% and more EEV virus. In accord with the cells are cultured for a time sufficient for virus to propagate and produce progeny virus such that EEV virus is released by cells into the culture medium, which is harvested before the cells are lysed by virus. The purification methods from the harvested medium are sufficiently gentle under low shear conditions, such as using low shear pumps for filtration, to preserve the fragile second membrane. As a result, provided are preparations that have a very high percentage of intact EEVs. These viruses can be systemically administered. Methods of treatment of cancers by systemic administration of the resulting virus preparations are provided.

[0122] Viruses that produce a high level of EEV (generally greater than 1%, generally at least about 5% to 10%, EEV) are provided or employed in methods herein and / or used for modification as detailed herein. The viruses can include additional genome modifications that increase advantageous properties including serum resistance and tumor selectivity to further render them effective for systemic administration. Provided are viruses that are modified to have increased serum resistance and so that the serum resistance is retained when the viruses propagate in vitro and in vivo. The resistance is not a function of the cells in which the viruses propagate. The viruses include modifications to virally-encoded second membrane (EEV membrane) proteins so that the viruses encode complement resistance proteins (or other such humoral immunity modulating proteins) that are displayed on the surface of the second membrane of the EEV. This is effected by producing transmembrane fusion proteins (chimeric proteins) between a viral EEV membrane protein and the humoral immunity modulating protein, which increases or effects serum resistance.

[0123] Among the viruses provided are tumor-selective vaccinia strain designated RT (for the red tails that form when the virus is cultured) and derivatives thereof that produce a high amount (generally 30% or more of the virions) of enveloped vaccinia viruses (generically referred to as envRTs), which exhibit resistance to humoral immunity. Derivatives of these viruses and other high EEV producing viruses also are Attorney Docket No. 120276-2615PC

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[0125] provided that are engineered to encode and display proteins on the outer membrane (the second membrane) that confer resistance to humoral immunity. These displayed proteins also provide for independence from the cells in which the virus propagates in vivo and in vitro. In general, serum resistance is a function of the cells in which the viruses are produced, which provide the second membrane; the methods and viruses herein can be cultured or propagated in any cell line and retain the high serum resistance because the resistance proteins, such as complement resistance proteins, are virally encoded as fusion proteins with a viral outer membrane protein, such as A33R or B5R or other such viral protein. A fusion or chimera with the viral protein results in display of the fused portion on the outer membrane. The viruses and methods provided herein provide for systemic administration, high levels of killing of tumor cells, and systemic dissemination of virus to distal tumor sites and metastases. The viruses can deliver therapeutic payloads and can be modified to target particular cells. Also provided is a manufacturing process that enriches the EEVs, and maintains integrity of EEV for long-term storage. An exemplary high EEV-producing virus and derivatives thereof modified as detailed herein are provided.

[0126] Among the modifications or modification are modified virally-encoded EEV membrane proteins whereby the virus displays protein that reduce or inhibit humoral immunity, such as complement resistance proteins and other proteins that inhibit humoral immunity particularly anti-viral immunity. The EEV viruses that are modified to display a protein (or portion thereof) that reduces or inhibits humoral immunity also are referred to herein as IV-EEV viruses to emphasize that they exhibit increased survival in serum, generally human serum, compared to the same virus that does not express or display such protein, because of the resistance to humoral immunity, such as complement. These modifications increase the serum stability of the resulting EEV virus particles. The protein that reduces or inhibits humoral immunity or portion of such protein can be provided as a chimeric protein, such as, as a fusion protein with an EEV envelope protein, or portion thereof. Because the protein that reduces or inhibits humoral immunity is encoded on the viral genome as part of the proteins specific to the EEV particle, the resulting viruses, upon propagation in vivo and in vitro, display the protein that reduces or inhibits humoral immunity. Thus, upon propagation, the resulting viruses retain the resistance to the immune system of the host; retention of the resistance to the Attorney Docket No. 120276-2615PC

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[0128] immune system of EEV viruses heretofore has not been achieved; because most cells do not confer the immunity resistance on the virus. Such resistance is a function of the cells in which the virus replicates. As demonstrated and described herein, such resistance is not specific to tumor type. The resistance of the modified EEV viruses provided herein do not depend upon the cells in which the virus replicates or is amplified in vivo or in vitro. It is understood herein that the modifications to vaccinia viruses exemplified herein can be applied to other poxviruses and any vaccinia virus strain. Thus, methods for improving serum resistance of any EEV are provided.

[0129] The viruses also include knockouts of genes, such as virally-encoded TK, A46, and VGF, in the viral genome that increase serum stability, such as by increasing resistance to complement and other host anti-viral immune responses. Numerous such strains are provided herein. Exemplary of the strains are those that include the three knockouts and encode a complement resistance protein, such as CD55, in a virally encoded outer membrane protein, such as A33R, A34R, A56R, B5R, and F13L, as a fusion (chimeric) protein, generally B5R or A33R. It is shown herein that an intact A56R gene improves or contributes to increased serum stability. The viruses also can encode payloads inserted into a non-essential gene (inserted into or in place of the non-essential viral gene). Payloads include anti -tumor therapeutics, such as a cytokine, such as IL- 15, particularly IL-15 / IL-15R alpha chain complex (also referred to as IL-15 superagonist). Exemplary of such strains provided herein are:

[0130] (TK -, A46-, VGF-), a high EEV - producing strain RT-01 with the knockouts;

[0131] (TK -, A46-, VGF-) - A33+CD55 - the strain encoding a fusion protein;

[0132] (TK -, A46-, VGF-) + Payload IL-15 (cytokine form) - the strain encoding a cytokine inserted into the VGF locus;

[0133] (TK -, A46-, VGF-) - A33+CD55+ Payload IL-15 (cytokine form) - the strain encoding the fusion protein and the cytokine;

[0134] (TK -, A46-, VGF-) + Payload IL-15 superagonist (IL-15 / IL-15R alpha chain complex)-the strain encoding the IL-15 / IL-15R alpha chain complex; and

[0135] TK -, A46-, VGF-) - A33+CD55+ Payload IL-15 superagonist - the strain encoding the fusion protein and the IL- 15 superagonist. Sequences and description of such strains are provided herein. Attorney Docket No. 120276-2615PC

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[0137] Provided are extra enveloped vaccinia virus (EEV) particles that also have higher anti-tumor activity and EEV production than the virus IHD-W, where the EEV is a clone of the polyclonal vaccinia IHD strain NR-52. For example, provide is an EEV parti cle derived from an IHD parental strain, but that differs from other IHD virus. For example, provided are EEV particles differ from IHD-W as shown in Figure 26. The EEV viruses can include further modifications, such as knockouts of one or more genes selected from among A46R, B8R, J2R, A52R, F1L, VGF, TK, and B19R. Knockouts can be achieved by deletion of all or portion of the gene, insertion into the gene, transposition of nucleotides in the gene, combinations thereof, and any other modification that results in elimination of an active product encoded by the gene. These EEV particles can include additional modifications, including those whereby an EEV outer membrane transmembrane protein comprises a protein or portion thereof that, when administered to a host, reduces or inhibits humoral immunity, wherein the portion is sufficient to inhibit or reduce humoral immunity; and the protein or portion thereof is display on the outer membrane of the EEV. As shown herein, RT-00 (SEQ ID NO:1) is derived from a the IHD-W strain, but has a number of distinguishing features detailed herein. Among them is that in RT-00 the A56 protein is intact; whereas IHD-W contains a truncated form of the A56R protein. RT-00 was selected for increased anti-tumor activity.

[0138] The A56R protein has several functions, including regulating the presence of viral-encoded complement regulatory proteins (VCP). The vaccinia virus A56 protein: a multifunctional transmembrane glycoprotein that can anchor two different secreted viral proteins. The A56R protein is expressed in the host membrane (the second membrane in EEV). The protein, VCP, which is secreted, can form a complex, via a cysteine bond with a free cysteine in the ectodomain of the A56R protein on the surface of the enveloped viral particle (EEV), which provides some protection from complement neutralization in vivo, the effect does not provide for systemic administration. The complex is not encoded as a transmembrane fusion or chimeric protein but forms a complex between A56 on the membrane and secreted VCP on EEV particles.

[0139] US publication US 2005 / 0208074 describes prior art attempts to exploit interaction of the A56 for targeting virus to tumors. This publication states that controlled targeting of poxviral particles has been hampered by the intrinsic complexity of the poxviruses and the existence of the two different infectious forms, and describes the Attorney Docket No. 120276-2615PC

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[0141] difficulties, citing Galmiche et al. ((1997) J. Gen. Virol. 78, 3019-3027), which reports fusion of the tumor-associated antigen ErbB-2 (an EGFR overexpressed in certain tumors) to A56 (then referred to as viral hemagglutinin (HA)) to express the anti-EGFR scFv on the EEV surface. No preferential infection towards ErbB-2 expressing cells of the EEV having the antibody-HA fusion was observed. US publication US 2005 / 0208074 describes a subsequent attempt to achieve viral targeting by localizing a ligand on the surface of a poxviral particle targeted to a tumor cell by forming fusions with IMV surface proteins. Others have employed such constructs for display for screening for binders (see, e.g., US patent application publications.

[0142] 2021 / 0348158, 2019 / 0112388, 2013 / 0288927, and 2013 / 0288927). Another group prepared a modified WR strain of vaccinia virus to target immune cells infected with HIV; the vaccinia virus was modified to encode a fusion protein of the HIV receptor CD4 and the B5R envelope glycoprotein Katz et al. (1997) Nature Biotechnology 15: 1374-1378) for treating HIV infection. WR is a toxic strain of virus, and is not a high EEV vaccinia virus (greater than 1%, generally 5% or more), and more specifically not in virus preparations produced as described herein that contain at least 30% and more EEV Katz et al. nor other references describes modifying high EEV viruses, particularly any that have an intact A56 locus, which contributes to serum resistance or in viruses that have genes in the ITRs that are deleted, which deletions can contribute to tumor selectivity.

[0143] The RT viruses provided herein, which were derived from IHD-W, have deletions in the ITRS relative to IHD-W and other viruses that have been isolated or derived therefrom. These deletions confer advantageous properties on the RT viruses. The RT-00 virus and derivatives thereof contain deletions in the ITRs that encode ankyrin-like proteins. Ankyrin-like proteins generally act as modulators of host- virus interactions. Deletion of these genes can have advantageous effects, such as reduction in viral pathogenicity and facilitating clearance in non-tumor tissues to thereby increase the increased tumor selectivity observed in with the RT viruses.

[0144] RT-00 contains a 3-nucleotide deletion in the K7R gene, a TLR modulator receptor. This deletion, which does not occur in other orthopox viruses, produces a protein that is one amino acid shorter than the K7R protein found in other vaccinia viruses. RT-00 virus contains a gene identical to RPXV102 (a cell surface-binding Attorney Docket No. 120276-2615PC

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[0146] protein and carbonic anhydrase homolog), which is not in IHD-W but is present with an identical amino acid sequence in the Tashkent clone TKT4 and Rabbitpox virus.

[0147] RPXV102 is a protein present in the IMV that binds to chondroitin sulfate on the cell surface, providing virion attachment to a target cell. RT-00 has 2 SNPs in the A30L gene compared with IHD-W. When compared with the available IHD-J sequences (A33R, A34R, A36R, A56R, B5R, F13L, A45R, A29L partial, A31R A30L, A32L partial and A13L). The RT-00 has 2 SNPs in A30L and 1 in A45R relative to the HTD-W strain. It has an intact A56 locus. RT-00 contains a 1,839 base pair deletion in the left ITR and a 1,841 base pair deletion in the right ITR, where the left ITR is from base pairs 1- 9,037 and the right ITR is from base pairs 185,372 - 194,269.

[0148] Also provided are methods for producing virus preparations with high levels of EEV viruses. Prior art methods of virus result in a loss of the fragile outer membrane; the methods provided herein have been developed to preserve this membrane. Hence, use of this method for any double-enveloped virus, particularly poxviruses, such as vaccinia virus, results in higher levels of EEVs per preparation. The method is particularly advantageous for purification of viruses, such as those provided herein that provide high amounts (greater than 1%, generally greater than 10%) as detailed herein of EEVs. The methods provided herein preserve the second membrane during purification so that higher levels of EEV viruses result. The methods can be applied to purification of any vaccinia virus to increase the amount of EEV viruses, and are particularly useful for viruses that produce higher EEV levels, including the viruses provided herein.

[0149] Provided are viruses that are selected to and identified as producing high levels of EEVs, where high levels are generally more 30% of the viral particles produced or more. Provided are EEV particles whose genome comprises an intact A56 gene. A virus that produces high level of EEVs are those where the EEV particles comprise more than 1%, 5%, 10%, 15%, 20%, 25%, 30%, generally at least 30% or more of the virus population. Provided are virus preparations that contain a high level of EEVs. These high EEV producing viruses are further modified as detailed herein by optional knock-outs as detailed herein, and / or by producing viruses that display proteins that reduce or inhibit complement activity or other such proteins so that the viruses are not recognized or have a reduction in recognition by the immune system of the host and also produce high levels of EEV virus, similar to the immunizing strain, independent of the type of tumor cell in Attorney Docket No. 120276-2615PC

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[0151] which the EEV virus is propagated in vivo or in vitro. The viruses can be further modified to encode various payloads, which are detailed and exemplified below.

[0152] Payloads include immunostimulatory proteins and therapeutic proteins including cytokines, chemokines, antibodies and antigen-binding portions thereof, and antigens or epitopes to express and deliver into tumors and the tumor microenvironment and / or to provide targets for other therapies. In some instances, these payloads are expressed on the outer membrane of the virus, generally as fusion proteins. The displayed proteins can possess activities that improve properties of the viruses, including resistance to the immune system of the host, such as is manifested by increased serum stability, and / or therapeutic properties or provide targets for therapeutics.

[0153] Provided are vaccinia viruses that are derivatives or derived from the virus designated RT-00 (SEQ ID NO: 1) and other viruses having the same identifying characteristics, particularly production of high levels (greater than 1% of the viral particles, such as at least 5% or at least 10%, particularly when isolated by the methods herein that minimize shear forced during purification) and / or propagated from RT-00 or derived therefrom or produced based on the sequence of the genome. The derivatives produce the high levels of EEV particles, and / or are modified to have high serum resistance as detailed herein.

[0154] Viruses with knock-outs of certain genes also are provided; these viruses include derivatives of RT-00 and variants thereof, as well as other high EEV-producing viruses known in the art or produced as described herein. Knock-outs of certain genes can improve anti-tumor activity of the virus and / or increase serum stability, and / or other such properties.

[0155] The derivatives retain (within at least 5% or about 10%) the high EEV production of RT-00 or have increased EEV production compared to the virus without the knockouts. Derivatives are produced by culturing the virus or modifying so that it includes detectable markers and / or encode therapeutic proteins or diagnostic or detectable proteins.

[0156] Also included are derivatives that encode complement resistance proteins (also referred to as complement inhibiting or immune modulating proteins herein) whereby the virus has increased serum resistance compared to RT-00. Exemplary of derivatives of RT-00 are the viruses designated RT-01, RT-02, RT-03, RT-04, RT-05, RT-06, RT-07, Attorney Docket No. 120276-2615PC

[0157] -27-

[0158] RT-08, RT-09, RT-10, RT-11, RT-12, RT-13, RT-14, RT-15, RT-16, RT-17, RT-18, RT-19, RT-20, RT-21, RT-22, RT-23, RT-24, RT-25, RT-26, RT-27, RT-28, RT-29, RT-30, RT-31, RT-32, RT-33, RT-34, RT-35, RT-36, RT-37, RT-38, RT-39, RT-40, RT-41, RT-42, RT-43, RT-45, RT-51, RT-52, RT-58, RT-61, RT-62, RT-63, RT-64, RT-65, RT-72, RT-73, RT-74, RT-75, RT-76, RT-77, RT-82, RT-83, RT-84, RT-85, RT-86, RT-87, RT-88, RT-89, RT-90, RT-91, RT-92, RT-93, RT-94, RT-95, RT-96, RT-97, RT-98, RT-99, RT-100, RT-101, RT-102, RT-103, RT-104, RT-105, RT-106, RT-107, RT-108, RT-109, RT-110, RT-111, RT-112, RT-113, RT-114, RT-114b, RT-115, RT-116, RT-117, RT-118, RT-118b, RT-118c, RT-119, RT-120, RT-121, RT-122, RT-123, RT-124, RT-125, RT-126, RT-127, RT-128, RT-129, and RT-134 (see table and description below), and variants thereof produced by culturing or amplifying or otherwise propagating these viruses, and / or modifying or replacing or containing variations in one or more ITRs and viruses that have the same properties and / or identifying characteristics thereof, or viruses encoding additional or different payloads. As detailed herein, viruses are modified to encode fusion or chimeric proteins with a virally-encoded EEV transmembrane protein or a membrane protein that can display a protein on the EEV surface.

[0159] Provided is a vaccinia virus genome or a vaccinia virus comprising the genome, wherein the virus genome comprises the sequence of the viruses or is prepared from a transfer vectors whose sequence is set forth in any of SEQ ID NOs: 1-21, and 518-524, 628-634, and 782-793 or a variants of any of SEQ ID NOs: 1-21 and 518-524, 628-634, 782-800, 886-898, and 901, having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, excluding the ITRs, or are degenerate sequences thereof, whereby the resulting viruses, produce greater than 1% EEV and have the same or greater anti-tumor activity than RT-00 or the same or greater serum stability than RT-00. Exemplary thereof is a vaccinia virus genome or vaccinia virus that is the vaccinia virus designated RT-00 and variants thereof or the genome thereof, and viruses and genomes derived therefrom. These include viruses described, for example, in Figure 24 and variants thereof. Provided are vaccinia viruses that comprise a genome or wherein the virus genome comprises the sequence set forth in any of SEQ ID NOs:l, 782-790, 887-898, and 901 or a variant of any of SEQ ID NOs: 1, 782-790, 887-898, and 901 or a virus or genome thereof as set forth in Figure 24 and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith, or variants lack all or a Attorney Docket No. 120276-2615PC

[0160] -28-

[0161] portion of the ITRs or comprise heterologous ITRs, or degenerate sequences of any of the preceding sequences.

[0162] Also included are knock-outs of these viruses that have increased EEV production or other advantageous property as detailed herein. Provided are high EEV viruses encoding a fusion protein of the EEV transmembrane protein with a complement regulatory protein, wherein: the genome of the unmodified virus comprises a genome is selected from among SEQ ID NOs: 1, 22-165, 251, 485, 615-627, 899-901, or a genome having at least 95% sequence identity thereto excluding the ITRs; and the modified virus retains the high EEV phenotype and encoded fusion protein whereby the virus has increased serum resistance compared to a virus comprising the unmodified genome.

[0163] Modified viruses and genomes include vaccinia virus genomes, comprising nucleic acid encoding a chimeric protein, whereby the genome is modified, wherein: the chimeric protein comprises all or a functional portion of an EEV outer membrane transmembrane protein and all or a functional portion of a protein that reduces or inhibits humoral immunity in a host upon expression of the protein in the host; the functional portion of the transmembrane protein is a sufficient portion to display the protein or portion thereof that reduces or inhibits humoral immunity on the surface of an EEV particle comprising the genome; and the functional portion of the protein that reduces or inhibits humoral immunity is a sufficient portion to reduce or inhibit humoral immunity in the host. These include vaccinia viruses and genomes wherein: a vaccinia virus comprising the genome, upon propagation, produces a high level of EEV; and a high level is higher than that produced by the Western Reserve (WR) strain virus. For example, high producers of EEV viruses can be prepared by introducing a mutation or mutations that render(s) the virus a high EEV producer; and / or by propagating the virus and selecting a clone that is a high EEV producer. A high EEV producer is one that, upon propagation, the EEV particles comprise more than 1%, 5%, 10%, 15%, 20%, 25%, 30% (at least 30%) or more of the virus population. More than 10% is a desirable target for viruses intended for systemic administration.

[0164] Provided are EEV viruses designated IV-EEV because they are highly resistant to inactivation by the host immune system. An IV-EEV is an EEV that comprises nucleic encoding a chimeric transmembrane protein; the transmembrane protein when transcribed and translated is expressed in the second membrane; and the chimeric Attorney Docket No. 120276-2615PC

[0165] -29-

[0166] transmembrane protein comprises a polypeptide that confers humoral immunity or comprises sufficient portion thereof to confer humoral immunity when expressed. The virally encoded EEV transmembrane protein can be selected from among EEV transmembrane proteins, which include A33R, A34R, A56R, B5R, and F13L, such as A33R and B5R. Unlike the HTD strains, viruses exemplified herein have an intact A56 locus. Included are IV-EEV, wherein: chimeric polypeptide comprises a polypeptide of portion thereof that confers humoral immunity; and, when expressed, the chimeric polypeptide is displayed on the surface of the second membrane. Proteins that confer humoral immunity include complement regulatory proteins and portions thereof, wherein the protein or portion thereof is a complement regulatory protein. Complement regulatory proteins inhibit complement activation, and include inhibition of any point in any of the complement pathways, whereby complement activation, and hence complement is reduced or eliminated. Complement inhibiting proteins and other proteins that reduces or inhibits humoral immunity can be species specific. Hence, generally the displayed protein is from the species to which the virus is administered. Complement pathways, which are well-known to those of skill in the art, are depicted in Figure 34. Complement regulatory proteins include, but are not limited to, CD35 / CR1, CD55, CD59, CD46, Factor H, VCP, MOPICE, SPICE, CCPH, C4- binding protein, Kaposi-sarcoma associated herpesvirus Kaposica I KCP, Herpesvirus saimiri (HVS) HVS-CD59, Rhesus rhadinovirus RCP-H and RCP-1, murine gamma herpesvirus 68 (yHV-68) RCA, Influenzavirus Ml, EMICE, IMP, and functional portions thereof, and variants thereof that have at least 95% amino acid sequence identity with any of the preceding and have complement regulatory activity, whereby complement is inhibited.

[0167] Provided are EEV particles, vaccinia virus genomes, and vaccinia viruses that the virus produces a high level of EEV virus, where a high level is more than 5%, 10%, or 20%, or is 30% or more of the total virus particles produced. The protein that reduces or inhibits humoral immunity thus, includes a complement regulating protein that inhibits complement. The EEV transmembrane protein can be selected from among A33R, A34R, A56R, B5R, and F13L, particularly A33R and / or B5R, and variants of the transmembrane proteins having at least 95% sequence identity thereto, whereby the protein displays the protein that reduces or inhibits humoral immunity or portion thereof. It is shown herein it is advantageous for the A56 locus to be intact, since the A56 protein Attorney Docket No. 120276-2615PC

[0168] -30-

[0169] contributes to increased serum resistance. For example, the transmembrane domain comprises a protein or DNA sequence set forth in any of, SEQ ID NOs: 168-174, 182-188, 196-202, 210-216, and 224 and variants thereof having at least 95% sequence identity and retaining the ability to display the protein on the EEV particle. Unmodified viruses from which the modified viruses can be produced include, but are not limited to, Western Reserve (WR), Copenhagen (Cop), Bern, Paris, Tashkent, Tian Tan, Lister, Wyeth, H4D-J, H4D-W, Brighton, Ankara, modified vaccinia Ankara (MV A), CVA382, Dairen I, LIPV, LC16M8, LC16M0, AC AM, WR 65-16, Connaught, JX-594 (pexastimogene devacirepvec), GL-ONC1, vvDD TK mutant, New York City Board of Health (NYCBH), EM-63, and NYVAC vaccinia virus strains, and variants thereof that produce virus particles that produce EEV particles that display the protein that reduces or inhibits humoral immunity or portion thereof. The unmodified viruses are modified, if not already high EEV producers, can be made to be high producers, such as by mutation or selection. Other unmodified viruses, include but are not limited to, KCTC 15195B, JX-594 (Pexastimogene Devacirepvec, Pexa-Vec); LIVP GLV-lh68 (GLV-ONC1 or GL-ONC1); vvDD; TG6002; VG9-GM-CSF; CVV; deVV5; CF33; Guang9; IN rVV; T601; vA34R; aCEA TCE; a modified WR. TK-GMCSF vaccinia virus;

[0170] WR. B5Rmut. TK-; mCCR5 / TK- virus; mCXCR4 / TK- virus; TK- PH20 DCK virus and KLS-3010 and those described in: 8,980,246; US 2019 / 0218522; WO 2022 / 182206; WO 2023 / 118603. Any vaccinia virus (or poxvirus) can be modified to be a high EEV producer and then further modified as described herein to be an IV-EEV.

[0171] Exemplary of the viruses provided herein are the RT (for red tail) viruses and derivatives thereof that retain substantially the level of EEV production as RT-00 (SEQ ID NO: 1). Exemplary of such viruses are EEV particles or vaccinia virus genomes or vaccinia viruses where the unmodified or modified genome comprises the inserts set forth in any of SEQ ID NOs: 2-21, 518-524, 628-634, and 791-800 into the virus of SEQ ID NO: 1 or a variant thereof that retains the level of EEV production of RT-00 and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% nucleotide sequence identity or degenerates thereof that comprise one or more degenerate codons in protein-encoding sequences. These viruses can be further modified by one or more knockouts of a gene, wherein: the knockout increases the resistance of a virus comprising the genome to the humoral immunity of a host or increases tumor selectivity accumulation of the virus or Attorney Docket No. 120276-2615PC

[0172] -31-

[0173] increases anti-tumor activity of the virus; and a knockout comprises an insertion or deletion or rearrangement of the knocked-out gene, whereby a native encoded product is not produced. Knockouts can include, but are not limited to, one or more knockouts that inactivate or more genes selected from among in one or more vaccinia virus genes selected from among: A46R, B8R, J2R, A52R, F1L, VGF, TK, and B19R, such as viruses that comprise two or three knockouts. Exemplary thereof are EEV particles or vaccinia virus genomes or vaccinia virus that are double or triple knockouts, wherein: double knockouts comprise a) TK, A46R; b) TK, A52R; c) TK, B8R; d) TK, VGF; e) TK, F1L; or f) TK, B19R; and three / triple knockouts comprise g) TK, A46R, VGF; h) TK, A52R, VGF; i) TK, B8R, VGF; j) TK, F1L, VGF; k) TK, B8R, B19R; 1) TK, A46R, B19R; m) TK, A52R, B19R; or n) TK, F1L, B19R.

[0174] The EEV particles, genomes and viruses provided herein can encode heterologous products, which include therapeutic products, reporters, and detectable products. The nucleic acid encoding the heterologous nucleic acid, for example, can be inserted into or in place of nucleic acid in a non-essential gene locus, or is inserted to effect a knockout of one or more of: A46R, B8R, J2R, A52R, F1L, VGF, and B19R. Exemplary encoded products include one or more of EGFP, EmGFP, mNeonGreen, EBFP, TagBFP, EYFP, TPet, GFP, BFP or TurboFP635. The RT-00 virus and viruses derived therefrom also can encode therapeutic or diagnostic payloads. For example, therapeutic proteins include, but are not limited to, cytokines (GM-CSF, IL-2, IL- 10, IL-12, IL-15, IL-15 / IL-15R alpha chain complex, IL-17, IL-18, IL-21, TNF, MIPla, FLt3L, IFN-b, IFN-g), chemokines (CC15, CC12, CC119, CXC111, RANTES), co-stimulators (OX40L, 4-1BBL, CD40L, B7.1 / CD80, GITRL, LIGHT, CD70), bi-specific t-cell engagers (BITEs), therapeutic antibodies, immune checkpoint inhibitors, single-chain antibodies such as single chain antibodies against VEGF, VEGFA, VEGFB, PGF, VEGFR2, PDGFR, Ang-1, Ang-2, ANGPT1, ANGPT2, HGF, TGF-P and immune checkpoint inhibitors, such as inhibitors of PD-1, PD-L1, CTLA4, or TIM-3, prodrug activators, such as lacZ, cytosine deaminase enzymes, human sodium iodide symporter, hNIS, and Aquaporin 1-AQP1. For example, the heterologous nucleic acid encodes one or more modulators of angiogenesis, immune system co-stimulators, or checkpoints inhibitors, such as, for example, anti -VEGF A and VEGFB and PGF; anti-VEGF and Attorney Docket No. 120276-2615PC

[0175] -32-

[0176] anti-ANGPT2; anti-VEGF, anti-ANGPT-2 and anti-CTL4; anti-VEGF and OX40L; anti-VEGF, anti-ANGPT2 and anti-PD-1 products.

[0177] Provided are isolated EEV virus particles that comprises the virus genome of any provided and described herein, and also EEV viruses from viruses and genomes modified as described herein to be IV-EEV. Any known vaccinia virus can be modified as described herein so that it is an IV-EEV, such as by modifying the genome to produce a chimeric (or fusion protein) of a protein that reduces or inhibits humoral immunity or functional portion thereof with a virus-encoded EEV membrane protein or portion thereof whereby the protein that reduces or inhibits humoral immunity is displayed on the virus particle. The genome of any high EEV producing virus, described herein, can be modified or further modified to comprise knockouts of at least two of A46R, B8R, J2R, A52R, F1L, VGF, and B19R or of A33R, A34R, A36R, A56R, B5R, F13L, A45R, A29L. Viruses exemplified herein include an intact A56R locus.

[0178] Compositions comprising virus particles that are high EEV producers and comprise a modification described herein, are provided. When EEVs are produced and / or isolated, the EEVs comprise at least 50%, 60%, 70%, 85%, 90%, 95%, or more of the virions in the composition. This can be following production in vitro or following isolation or purification, such as by methods provided herein, or formulation for administration. Provided are compositions comprising the EEV virus particles formulated for systemic administration. The compositions can comprise or consist essentially of EEV virus particles. Exemplary of compositions are those formulated for multiple dosage administration and those formulated for single dosage administration. Exemplary thereof are compositions, comprising the EEV virion particles in an amount that is: (i) between about IxlO3and about IxlO15pfu per ml; (ii) between about IxlO4and about IxlO14pfu per ml; or (iii) between about IxlO6and about IxlO12pfu per ml.

[0179] Uses of the viruses, and EEVs provided herein for treating cancer are provided. Methods for treating cancer comprising administering, such as systemically administering, an EEV particle or vaccinia virus genome or vaccinia virus composition provided here to a subject who has a cancer. Provided are EEV particles, vaccinia virus genomes, vaccinia viruses, and compositions for use for treating cancer. The compositions and viruses and particles can be formulated for systemic administration. Cancers comprise a solid tumor, or metastases, or is a homological malignancy, Attorney Docket No. 120276-2615PC

[0180] -33-

[0181] including for example, a malignant tumor or hematological malignancy, including metastatic cancers, lymphatic tumors, and blood cancers. For example, the cancers can include any type of malignant tumor or hematological malignancy, including metastatic cancers, lymphatic tumors, and blood cancers. Exemplary cancers include, but are not limited to, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoma, adrenal cancer, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brainstem glioma, brain cancer, carcinoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway or hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, Burkitt’s lymphoma, carcinoid tumor, carcinoma, central nervous system lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, epidermoid carcinoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer / intraocular melanoma, eye cancer / retinoblastoma, gallbladder cancer, gallstone tumor, gastric / stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, giant cell tumor, glioblastoma multiforme, glioma, hairy-cell tumor, head and neck cancer, heart cancer, hepatocellular / liver cancer, Hodgkin’s lymphoma, hyperplasia, hyperplastic corneal nerve tumor, in situ carcinoma, hypopharyngeal cancer, intestinal ganglioneuroma, islet cell tumor, Kaposi's sarcoma, kidney / renal cell cancer, laryngeal cancer, leiomyoma tumor, lip and oral cavity cancer, liposarcoma, liver cancer, non-small cell lung cancer, small cell lung cancer, lymphomas, macroglobulinemia, malignant carcinoid, malignant fibrous histiocytoma of bone, malignant hypercalcemia, malignant melanomas, marfanoid habitus tumor, medullary carcinoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic skin carcinoma, metastatic squamous neck cancer, mouth cancer, mucosal neuromas, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disorder, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neck cancer, neural tissue cancer, neuroblastoma, Attorney Docket No. 120276-2615PC

[0182] -34-

[0183] oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial tumor, ovarian germ cell tumor, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma, pituitary adenoma, pleuropulmonary blastoma, polycythemia vera, primary brain tumor, prostate cancer, rectal cancer, renal cell tumor, reticulum cell sarcoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, seminoma, Sezary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck carcinoma, stomach cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, throat cancer, thymoma, thyroid cancer, topical skin lesion, trophoblastic tumor, urethral cancer, uterine / endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia or Wilms’ tumor. Exemplary cancers commonly diagnosed in humans include, but are not limited to, cancers of the bladder, brain, breast, bone marrow, cervix, colon / rectum, kidney, liver, lung / bronchus, ovary, pancreas, prostate, skin, stomach, thyroid, or uterus.

[0184] Exemplary cancers commonly diagnosed in dogs, cats, and other pets include, but are not limited to, lymphosarcoma, osteosarcoma, mammary tumors, mastocytoma, brain tumor, melanoma, adenosquamous carcinoma, carcinoid lung tumor, bronchial gland tumor, bronchiolar adenocarcinoma, fibroma, myxochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma, Wilms’ tumor, Burkitt's lymphoma, microglioma, neuroblastoma, osteoclastoma, oral neoplasia, fibrosarcoma, osteosarcoma and rhabdomyosarcoma, genital squamous cell carcinoma, transmissible venereal tumor, testicular tumor, seminoma, Sertoli cell tumor, hemangiopericytoma, histiocytoma, chloroma (e.g., granulocytic sarcoma), corneal papilloma, corneal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, stomach tumor, adrenal gland carcinoma, oral papillomatosis, hemangioendothelioma and cystadenoma, follicular lymphoma, intestinal lymphosarcoma, fibrosarcoma and pulmonary squamous cell carcinoma. Exemplary cancers diagnosed in rodents, such as a ferret, include, but are not limited to, insulinoma, lymphoma, sarcoma, neuroma, pancreatic islet cell tumor, gastric MALT lymphoma and gastric adenocarcinoma. The viruses, EEVs, compositions, methods and uses can be used for treating an animal, including humans. Non-human subjects include animals, such as livestock and pets. Animal cancers include among leukemia, hemangiopericytoma and Attorney Docket No. 120276-2615PC

[0185] -35-

[0186] bovine ocular neoplasia (in cattle); preputial fibrosarcoma, ulcerative squamous cell carcinoma, preputial carcinoma, connective tissue neoplasia and mastocytoma (in horses); hepatocellular carcinoma (in swine); lymphoma and pulmonary adenomatosis (in sheep); pulmonary sarcoma, lymphoma, Rous sarcoma, reticulo-endotheliosis, fibrosarcoma, nephroblastoma, B-cell lymphoma and lymphoid leukosis (in avian species); retinoblastoma, hepatic neoplasia, lymphosarcoma (lymphoblastic lymphoma), plasmacytoid leukemia and swimbladder sarcoma (in fish), caseous lymphadenitis (CLA): chronic, infectious, contagious disease of sheep and goats caused by the bacterium Cory neb acterium pseudotuberculosis, and contagious lung tumor of sheep caused by jaagsiekte.

[0187] Provide are nucleic acid molecules and constructs encoding fusion or chimeric polypeptides, comprising an EEV or poxvirus vaccinia virus-encoded outer envelope protein or membrane spanning portion thereof, and at least one protein that reduces or inhibits humoral immunity or humoral immunity inhibiting portion thereof. The nucleic acid molecules encoding a fusion polypeptide or chimeric polypeptide, comprising a complement regulatory protein (CRP; also referred to herein as a regulator of complement activation (RCA) or a complement resistance protein) or sufficient portion thereof for activity, and an extracellular enveloped vaccinia virus (EEV) transmembrane protein or suffi cient portion thereof for display of the CRP on the surface of an EEV. Envelope proteins include for example, vaccinia vims, and the envelope protein is B5R, A33R, A34R, A56R or F13L from EEV viruses. The proteins that reduce or inhibit humoral immunity or portion thereof is selected among one or more of: CD35 / CR1, CD55, CD59, CD46, Factor H, VCP, MOPICE, SPICE, ORF4, CCPH, C4- binding protein, CD35, Kaposi -sarcoma associated herpesvirus Kaposica I KCP, Herpesvirus saimiri (HVS) -CD59, Rhesus rhadinovirus RCP-H and RCP-1, murine gamma herpesvirus 68 (yHV-68) RCA, Influenzavirus Ml, EMICE, CPXV034, CRASP-2, and IMP, as well as modified sequences thereof, or functional portions thereof. For example, the protein that reduces or inhibits humoral immunity is selected from among one or more of: CD35, CD55, VCP, mutated VCP, SPICE, CCPH and ORF4 or functional portions thereof. The protein that reduces or inhibits humoral immunity or portion thereof is fused, generally via peptide bond, to a transmembrane region of the EEV Attorney Docket No. 120276-2615PC

[0188] -36-

[0189] envelope protein, whereby the protein or portion thereof that reduces or inhibits humoral immunity is displayed on the EEV outer membrane.

[0190] Viruses for modification and from which the envelope proteins are derived include, but are not limited to, Vaccinia Copenhagen virus, Camelpox virus, Variola virus, Cowpox virus, Taterapox virus, Monkeypox virus Zaire-96-1-16, Vol epox virus, Akhmeta vims, Ectromelia vims, Orthopoxvirus Abatino vims, Skunkpox vims, 87 Raccoonpox virus, Yokapox vims, Murmansk poxvims, NY 014 poxvirus, and Yaba monkey tumor vims, and any discussed herein and / or known in the art. The protein that reduces or inhibits humoral immunity or portion thereof is linked to the N-terminus or into the stalk region of the envelope protein or is covalently linked to the C -terminus of the envelope protein, or inserted such that the protein that reduces or inhibits humoral immunity is displayed on the surface of the virus.

[0191] Vaccinia vims that comprise the nucleic acid molecules provided herein are provided. These include vaccinia viruses and virus genomes, where the nucleic acid encoding the chimeric or fusion protein replaces the respective envelope proteinencoding nucleic acid or is inserted into a gene locus to knockout the activity of the protein encoded at the locus. As discussed, the vimses can include further modifications as described herein and known in the art. For example, the vims can comprise a deletion in or of or insertion in the thymidine kinase (TK) gene. Vimses for used herein include, but are not limited to, a Western Reserve (WR), Copenhagen (Cop), Bern, Paris, Tashkent, Tian Tan, Lister, Wyeth, H4D-J, IHD-W, Brighton, Ankara, modified vaccinia Ankara (MVA), CVA382, Dairen I, LIPV, LC16M8, LC16M0, AC AM, WR 65-16, Connaught, JX-594 (pexastimogene devacirepvec), GL-ONC1, vvDD TK mutant, New York City Board of Health (NYCBH), EM-63, KCTC 15195B, and NYVAC vaccinia vims strains, and variants thereof that produce vims particles that produce a high level of EEV particles, or that produce a high level of EEV particles and that display the protein that reduces or inhibits humoral immunity or portion thereof; and a high level of EEV vims is at greater than 1%, 5%, 10%, 15%, 20%, 25%, 30% or more of the vims population.

[0192] Provided are compositions comprising the viruses and EEV particles provided herein. They are formulated in a pharmaceutically acceptable vehicle, particularly one suited for systemic administration. Exemplary compositions contain a have a unit dose Attorney Docket No. 120276-2615PC

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[0194] of: (i) between about IxlO3and about IxlO15pfu per ml; (ii) between about IxlO4and about IxlO14pfu per ml; or (iii) between about IxlO6and about IxlO12pfu per ml.

[0195] Methods of treating cancer and / or other proliferative diseases, disorders, and / or conditions, comprising administering the pharmaceutical composition or viruses provided herein. The viruses provided herein a designed for systemic administration. The viruses and EEV particles, and compositions provided herein are for use for treating cancer and / or proliferative diseases, disorders, and / or conditions. Provided are the EEV viruses, viruses, and compositions for use for treating cancer in combination with a second anti-cancer agent or treatment. Provided are method of treating cancer, comprising: a) systemically administering an EEV, virus, or composition; and b) administering a second agent or treatment, wherein: a) and b) are effected serially, simultaneously, or intermittently, or a) is effected before b), or b) is effected before a). The second anti-cancer agent or treatment can be, for example, chemotherapy, or immunotherapy, or cell therapy, or an anti-biotic, or radiation therapy, or surgery, or combinations of two or more. The second agent can be, for example, selected from among ceftazidime, cefepime, imipenem, aminoglycoside, vancomycin and antipseudomonal P-lactam. Exemplary antifungal agents which can be included in a combination with a virus provided herein include, but are not limited to, amphotericin B, dapsone, fluconazole, flucytosine, griseofulvin, itraconazole, ketoconazole, miconazole, clotrimazole, nystatin, and combinations thereof. Exemplary antiviral agents which can be included in a combination with a virus provided herein include, but are not limited to, cidofovir, alkoxyalkyl esters of cidofovir (CDV), cyclic CDV, and (S)-9-(3 -hydroxy -2 phosphonylmethoxypropyl)adenine, 5-(dimethoxymethyl)-2'-deoxyuridine, isatin-beta-thiosemicarbazone, N-methanocarbathymidine, brivudine, 7-deazaneplanocin A, ST-246, Gleevec, 2'-beta-fluoro-2', 3 '-dideoxyadenosine, indinavir, nelfinavir, ritonavir, nevirapine, AZT, ddl, ddC, and combinations thereof. Typically, combinations with an antiviral agent contain an antiviral agent known to be effective against the virus of the combination. For example, combinations can contain a vaccinia virus with an antiviral compound, such as cidofovir, alkoxyalkyl esters of cidofovir, ganciclovir, acyclovir, ST-246, Gleevec, and derivatives thereof.

[0196] The methods and uses can further include a step of administering an anti-viral agent or an anti-viral antibody to modulate the level of virus or to eliminate the virus. Attorney Docket No. 120276-2615PC

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[0198] Exemplary antivirals include an anti-viral agent or antibody, such as one or more selected from among cidofovir, alkoxyalkyl esters of cidofovir, ganciclovir, acyclovir, ST-246, Gleevec, and derivatives thereof. The methods and regimens and uses can include administering an immunomodulator to modulate the response of the immune system of the host. Immunomodulators include, for example, chemotherapeutic agents at dose sufficient to achieve an immunomodulatory effect but not lymphodepletion.

[0199] Administration regimens are provided. The treatment or use can comprise a regimen of systemic viral administration and an immunomodulatory agent, wherein the regiment comprises: a) treatment with an immunomodulatory agent; then treatment with virus; and then clear virus with ST-246 or other anti-viral; or b) regimen a) further comprising administration of an immunomodulatory agent after treatment with the virus, or after viral treatment, not before. Anti-viral agents are known in the art. Exemplary anti-viral agents, but are not limited to, ST-246, cidofovir, Gleevec® (Imatinib), ganciclovir, acyclovir, and other chemotherapeutic agents.

[0200] The viruses, virus genomes, EEVs, compositions, methods, and uses can be further modified whereby the genome of the virus is modified to encode a target antigen that, upon expression, is expressed on the surface of a cell infected with the virus. The target antigen can be a therapeutic target, such as a tumor-specific antigen or neoantigen. Therapies include, for example, immunotherapy, cell therapy, antibodies, and antibodydrug conjugates for treating cancer. Exemplary thereof is checkpoint inhibitor therapies, CAR-T cell therapy, NK cell therapy, gene-editing therapy, TIL cell therapy, and other such therapies that can include targeting a cell surface antigen. Exemplary of cell surface antigens that can be encoded by the virus and expressed on the surface of an infected tumor cell are CD20 and HER2.

[0201] Provided are the methods for manufacturing EEV virus, wherein the resulting product comprises at least 60% EEV virus. The methods comprise: culturing cells infected with vaccinia virus for a time sufficient for virus to replicate and to be released into the medium without lysing the cells; collecting the culture medium and filtering, under low shear force, through a filter that captures particulates; and purifying the virus from the culture medium with low shear force filtration. Following purification, the virus can be re-buffered into a formulation buffer for administration and / or storage at low temperature, such as at -20 °C to -80 °C, wherein the formulation buffer is suitable for Attorney Docket No. 120276-2615PC

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[0203] storage and for systemic injection. The cells for infection with the virus is cultured in a suitable format, such as a suspension reactor, a spinner flask, a wave bioreactor, or such format that permits culturing under conditions that will not disrupt the outer membrane of EEVs released from the cells. Exemplary cells include cell lines and cells for culturing vaccinia virus, such as, but not limited to, iPSCs (induced pluripotent stem cells), stem cells, and cell lines, such as HEK293, HEK293T, A549, PerC6, Vero, Vero STAT1 KO, HEK293. STAT1 BAX KO AGE1.CR.pIX, CV1, HELA, HELA S3, CHO, VPCs, VPCs 2.0, FS293, MDCK, and MDCK. STAT1 KO cells. Exemplary of cells for production are iPSCs and HeLa cells. In particular, the cells for manufacturing can be modified to express on the cell membranes polypeptides or peptides that modulate or program activities / properties of EEV vaccinia viruses produced in such cells. As detailed herein, the cells can be modified or obtained from other sources to express a polypeptide or peptide portion thereof to display on the cell membrane a ligand, receptor, modified receptor, immune modulator, and other such polypeptide / proteins that then will be displayed on EEV vaccinia virus, or any enveloped virus that acquires an outer membrane or its only membrane from the cell in which the virus is protein. The display polypeptide / protein will modify or program the virus to have a property or activity of interest, such as expression of a chimeric receptor, or a ligand or antibody to target the virus to particular cells, or an immune modulator to alter the response of the immune system of the host.

[0204] The cells are used to produce enveloped viruses, such as EEV vaccinia virus, and particularly the high EEV-producing vaccinia virus provided herein, including the EEV-viruses that encoded modified transmembrane EEV second membrane proteins to display immune modifiers, such as CD55, and / or to add other targeting proteins and ligands and receptors. The combination of producing EEV viruses in modified host cells and also modifying the viruses provides for programming EEV viruses to have modified properties that result in altered interactions, generally increased serum resistance, and targeting to cells of interest or adding activities. The EEVs also can be modified to encode payloads, such as anti-cancer products for treatment of cancer, or immunosuppressors for treatment of autoimmune diseases, where the EEVs are modified to infect activated B-cells, and other such modifications. Attorney Docket No. 120276-2615PC

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[0206] The methods of purification from such host cells of virus modified to produce high levels of EEVs can be isolated / purified by methods provided herein or other methods that are designed to isolate a high percentage of EEVs from cell culture medium. All steps of the methods detailed herein are performed under low shear force to avoid damaging the fragile outer membrane from the host cells on the viruses. For example, the tubing and pumps are selected so that the cells and medium are exposed to low or no shear force. For example, low shear force is less than 100 shear / seconds, such as from about 10 shear / seconds to less than about 100 shear / seconds. In some embodiments, the method comprises: a) infecting cultured cells with an IMV crude lysate and culturing the cells for a time sufficient for production of EEV particles and release thereof into the cell culture medium without lysing the cells, wherein the conditions are low shear force conditions; b) harvesting the culture medium; adding 5-10% sucrose; and filtering the resulting mixture under low shear force to remove particulates; c) treating the mixture with a DNAase to digest any host cell DNA in the mixture; d) low or shear force free concentration of viruses by a tangential flow filtration (TFF), wherein the pore size is about 0.05pm to about 0.1 m, and collecting the resulting virus composition; e) re-buffering the virus into a storage and injectable formulation buffer; and f) optionally filling a vial or vials for low temperature storage. For example, the method comprises: a) culturing cells in suspension spinner flasks to achieve S cell densities of 2xl0e6 cells per mL, wherein the culture conditions are 37°C and 5% CO2; b) directly infecting the cells with IMV crude lysates with at a multiplicity of infection (MOI) of about 0.1 to 1 virus particles per cell, such as at about 0.5 virus particles per cell, and culturing for about 35-50 hours, wherein the culturing is sufficient for release of EEV into the medium without lysing the cells to avoid release of IMV into the cell culture medium; c) harvesting the culture medium, adding 5-10% sucrose, and pre-filtering with a filter to remove cells and cell material from viruses to produce filtered medium; d) adding a DNAase, such as benzonase enzyme, to digest any host cell DNA in the filtered medium; e) concentrating the viruses by TFF under low shear force or force free conditions; f) shear force free or low shear force re-buffering of the viruses into a storage and IV injectable formulation buffer. An exemplary formulation buffer comprises lOmM Tris / HCl, 1% sucrose, 2% trehalose, 5% mannitol, 300 mM glycine, and 0.1% recombinant human albumin. The rebuffered virus composition can be stored in a container or containers, such as a vial or Attorney Docket No. 120276-2615PC

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[0208] vials, for storage and / or systemic injection. Viruses for manufacture for infecting the cells include any vaccinia virus or poxvirus that has high EEV as detailed herein, including any of the Red Tail (RT) viruses and derivatives thereof. These include an of the viruses described below or any EEV known to those of skill in the art. Exemplary of the viruses are high EEV producing viruses that include knockouts of genes TK, A46 and VGF and / or the EEV virus whose genome comprises the sequence set forth in SEQ ID NOs:782-790, 887-898, and 901.

[0209] BRIEF DESCRIPTION OF DRAWINGS FIGURE 1 depicts a strategy for selection of a vaccinia virus with high EEV production and resistances to humoral immunity.

[0210] FIGURE 2 shows spreading patterns of the CALI virus (low producer of EEV) and the EEV clone designated RT-01 (high producer of EEV).

[0211] FIGURE 3 shows that EEV, but not IMV are resistant to inhibition by humoral immunity.

[0212] FIGURE 4 show spread of the RT-01 and CAL2 viruses to neighboring cells at 24 and 48 hours.

[0213] FIGURE 5 shows that the EEV virus RT-01 survives systemic administration and reaches all tumors; the corresponding IMV virus does not.

[0214] FIGURE 6 presents a gene map of a representative vaccinia virus genome. Genome fragments are alphabetically labeled from A to O based on fragment size obtained by digestion of the viral genome using the restriction enzyme Hindlll. For example, the largest fragment is named A and the smallest as O. The vaccinia virus genome is bidirectional; genes are labeled R (right) and L (left) to indicate the genomic orientation of open reading frames (ORFs). Exemplary ORFs include, but are not limited to, VGF, F1L, J2R, A46R, A52R, B8R, and B19R. There can be variation in the ORF nomenclature among vaccinia viruses, such as, for example, between Western Reserve and Copenhagen. Alternative nomenclature is based on sequencing data of the full vaccinia virus genome; coding sequences are numbered according to their first appearance starting from the left to the right end of the genome. The first ORF present 5’ in the genome is termed 001 and labeled R or L to indicate the genomic orientation. Adapted from Ali etal. Viruses 2016, 8(5), 134. Attorney Docket No. 120276-2615PC

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[0216] FIGURE 7 shows the distribution of virus in the tumors, where the virus is injected into the right tumor and amplification and distribution in the left tumor is shown. The N2 EEV (RT-02) virus has greater amplification and distribution to the right tumor than the CAL2 IM V virus.

[0217] FIGURE 8 shows fluorescence in the left and right tumors following injection into the right tumor with CAL2 or RT-02 virus in an A549 rodent model.

[0218] FIGURE 9 shows the distribution of virus in the tumors, where the virus is injected into the right tumor and amplification and distribution in the left tumor is shown.

[0219] FIGURE 10 shows that the N2 virus can amplify and kill mouse cells.

[0220] FIGURES 11A and 11B illustrate the amplification potential of RT-01 in B16-F10 melanoma cancer cells and CT26 prostate cancer cells at various multiplicities of infection (MOIs).

[0221] FIGURE 12 shows the expression of RNA in various tumors demonstrating that expression is not tumor-specific. Figure reproduced from Expression of CD55 in cancer -Summary - The Human Protein Atlas (proteinatlas.org).

[0222] FIGURES 13A and 13B show the levels of RNA encoding CD55 among various cancer cell lines. FIGURES 13A and 13B show the levels of RNA encoding CD55 in cervical cancers lines and in breast cancers (TPM= transcripts per million of protein encoding genes), respectively demonstrating that CD55 levels are not specific to a type of tumor.

[0223] FIGURE 14 depicts the display of hCD55 on the EEV second membrane; the resulting virus is referred to herein as an IV-EEV.

[0224] FIGURE 15 show the structure of A46R-hCD55-B5R as constructed and inserted into the parental virus to produce RT-02.

[0225] FIGURE 16 (from Example 13) shows that RT-05 virus (modified N2 virus encoding a CD55 / B5R fusion protein) infection leads to an increase in CD55 expression, especially in the CD55-negative cell line, BT549. MDA-MB-231 uninfected cells (dashed line) show basal CD55 expression when compared with BT549, which also was increased after infection with B5R-CD55 armed vaccinia virus (solid color), especially in the intracellular stain. Surface and intracellular staining confirm that BT549 uninfected cells are CD55-negative (dashed line), while BT549 cells infected with RT-05 (solid color) show significant expression of CD55;

[0226] infected Attorney Docket No. 120276-2615PC

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[0228] Cells + RT-05.

[0229] FIGURE 17 shows the amounts of virus in the injected right flank tumors and in the uninjected left flank tumors on days 3 and 6. Controls show no virus in the tumors. FIGURE 18 shows the types of immune cells in the tumors, indicating a conversion to an anti-tumor phenotype.

[0230] FIGURE 19 shows and describes the resistance of the N2 (RT-01) virus to humoral immunity but not to immune cell-mediated clearance. The virus lyses the cells and by a cascade of cellular immune responses converts the tumor microenvironment into an antitumor phenotype, which also decreases viral replication and persistence. This can be achieved by treatment with an agent that reduces immune suppression in the tumor, by treatment with an immune modulator to target only subsets of immune cells, such as achieved by lower dose chemotherapy (see, e.g., Sistigu et al. (2011) Semin Immunopathol 33:369-383, DOI 10.1007 / s00281-011-0245-0). Figure 19 shows the resistance of the virus RT-01 to humoral immunity but not to immune cell-mediated clearance. RT-01 infects and amplifies in tumor cells and transforms all tumor microenvironments. Immune cell activation leads to clearance of the virus, making the virus safe, but also eliminating cancer cells.

[0231] FIGURE 20 shows that administration of RT-N2 (RT-01) virus and chemotherapy increased the anti-tumor response showing increased tumor regression, persistence of virus, and anti-tumor phenotype in the tumor microenvironment.

[0232] FIGURES 21A, 21B and 21C show that pre-treatment by administration of with (RT-N2) vaccinia virus enhances NK infiltration in a solid. Figure 21A depicts the protocol;

[0233] Figure 21B shows a graph of virus presence in the tumors; Figure 21C shows the amount of infiltrated NK cells in the tumors measured by fluorescence signals, the images and a graphical depiction of the results.

[0234] FIGURE 22 depicts a tumor cell infected with a vaccinia virus that encodes a target antigen whereby the antigen is expressed on the surface of the tumor cell.

[0235] FIGURES 23A and 23B show that target antigens are expressed on the surface of the infected tumor cell. Figure 23A shows that virally-encoded CD20 is expressed, and FIGURE 23B shows that virally encoded HER2 is expressed. Such tumor cells express the targeted antigens for treatment with therapeutic products that target the antigens. Attorney Docket No. 120276-2615PC

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[0237] FIGURE 24 summarizes EEV viruses provided herein, the transfer vectors for generating the viruses, and inserts and loci of the inserts, and nomenclature.

[0238] FIGURE 25 shows the potency of EEV viruses following various manufacturing procedures. EEVs are produced by methods with increasing improvements, particularly in reducing shear forces. Bar one (#1) shows percentage of EEVs produced using a standard manufacturing process (pellet purification) for the primary virus fraction released from infected host cells. This initial process exhibits less than 20% survival of active particles. EEVs produced with improvements in the manufacturing process are shown in bars #2 - #4, each improvement is described in Example 9.

[0239] FIGURE 26 presents a graphic representation of major differences in sequence between the virus designated RT-00 (N2 or H4D-RT) herein, and the virus designated IHD-W1; functionalities that are not present in IHD-W1 are in gray; SNPS that lead to changes in the amino acid sequence are in white.

[0240] FIGURE 27 depicts the orientation of the various EEV transmembrane proteins in or EEV membrane or between the IMV and EEV membranes. The A33R, A34R, A56R, and B5R proteins are exposed, and F13L is located between the EEV outer envelope and the IMV surface. The N-terminus of BR, A56R and the C-term of A33R and A34R are exposed to the outside of the EEV. (Adapted from Smith et al. Journal of General Virology (2002), 83, 2915-2931).

[0241] FIGURE 28A depicts the structure and configuration of an exemplary CCP (complement control protein, also referred to as a complement inhibitory protein (CIP) or a complement regulatory protein (CRP)). FIGURE 28B provides a schematic representation of CD55 with other enveloped membrane vaccinia virus proteins, such as A33R, with similar structure to the CD55-B5R construct. The extracellular portion of CD55 (SCR1-4) is fused with intracellular and transmembrane portions of A33R protein under control of 3 different promoters, pSE, pSEL, and pSL. The CD55 portion is inserted in other EEV envelope proteins, including A34, A56, F13. Modified from Riccardo etal. Viruses. 2023, Dec (8); 15(12).

[0242] FIGURE 29 shows that incorporation of all or a portion of CD55 into the second membrane of an EEV virus confers increased resi stance to human serum of enveloped CD55 virus. Attorney Docket No. 120276-2615PC

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[0244] FIGURES 30A and 30B show that systemic administration of enveloped virus targets lungs and metastasized Tumors. As depicted in Figure 30A, the virus specifically targeted lung tumors. As shown in the Figure 30B, the virus significantly reduced the metastatic burden in the liver compared to the control (left panel).

[0245] FIGURES 31A, 31B, and 31C show heat maps of fluorescence signal intensity for single, double, and tripe knockout viruses. FIGURES 31A and 31B depict heat maps representing the intensity of fluorescence signal by organ (n=5), showing that single and double knockout viruses result in significant viral amplification within tumor tissue. FIGURE 31C shows that triple-knockout viruses exhibited significantly reduced off-target amplification in non-tumor tissues while maintaining potent tumor targeting and amplification of the payload.

[0246] FIGURES 32A, 32B and 32C show the survival of EEVs produced in human ipSCs compared to EEVs produced in Hela cells and IMVs produced in Hela cells when exposed to serum from three different human donors.

[0247] FIGURE 33 shows that treatment with RT-52 (see Figure 24) and cyclophosphamide as detailed in Example 24 has remarkable therapeutic efficacy after systemic administration.

[0248] FIGURE 34 depicts complement pathways and components.

[0249] FIGURES 35A, 35B, 35C, and 35D summarize an exemplary method of manufacturing EEV. Figure 35A depicts an upstream culture method / process in a spinner flask; Figure 35B depicts the process in a perfusion reactor; Figure 35C shows the downstream process for isolation of virus from the culture medium harvested from spinner culture and suspension; and Figure 35D shows the process in a wave bioreactor.

[0250] FIGURES 36A, 36B, and 36C examine the effects of CPA treatment. Figure 36A shows that CPA treatment prolonged virus presence at the tumor sites. Figure 36B shows that virus treatment led to reduced tumor volumes. RT-65 showed significantly higher tumor regression even when used as monotherapy. When combined with 150 mg / kg CPA, RT-64 and RT-65 showed significantly improved efficacy. Figure 36C confirms the enhanced efficacy was also confirmed by a mouse survival curve where two mice achieved complete remission with the RT-65 and CPA combination treatment. FIGURES 37A and 37B show that EEVs released from RT-77-infected Hela or hiPSCs were highly resistant to human serum complement-mediated inactivation compared to Attorney Docket No. 120276-2615PC

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[0252] EEVs from RT-65. Figure 37A shows results from CBD27 human serum; Figure 37B shows results from AB2391 human serum.

[0253] FIGURES 38A and 38B show that canine cell lines are permissive to RT viruses. Figure 38A shows that the D-17 cell line exhibited high cytolysis in a dose-dependent manner, with most of the cells being killed by RT-01 and RT-05 within 24 hours postinfection. In contrast, Figure 38B shows that the CMT-U27 cell line showed resistance at lower MOI, with cytolysis only observed at the highest MOI (MOI 10).

[0254] FIGURES 39A, 39B, and 39C show results of administration of RT-65 (TK-, A46R-, VGF-) or RT-96 (RT-65 with IL-15 superagonist payload) to C57 / BL6 immunocompetent mice (6-8 weeks old, n=16) implanted with one million (le6) LL2 lung cancer cells were implanted subcutaneously into both flanks 4 days post implantation. At day 6 after RT treatment, 6 tumors were collected, dissociated, stained, and analyzed with Cytoflex flow cytometer for the TIL analysis. FIGURE 39A shows the quantification by ELISA of IL15 superagonist in the tumors from the RT-96-treated mice at 6 days post treatment; FIGURE 39B shows changes in exemplary cellular composition of the tumor microenvironment upon RT-65 and RT-96 administration, FIGURE 39C shows reductions in the growth of LL2 lung cancer tumors upon RT-65 and RT-96 administration.

[0255] FIGURES 40A and 40B show, using that EEVs (infecting virus RT-65 (TK-, A46R-, VGF- RT virus) produced in and harvested from specifically activated HER2 CAR-T, that HER2 CAR was readily detectable on the membrane of HER2-CAR RT-65 EEVs, but not on HER2-CAR RT-65 IMV or the parental RT-65 virus. These results demonstrate generating EEVs that display functional CARs by employing genetically engineered host cells.

[0256] FIGURE 41 show that host cells that are modified to express a TROP2-PDGFR CAR construct transfer the TROP2-PDGFR fusion / chimeric protein to an EEV envelope. FIGURE 42 depicts a genome map corresponding to the virus RT-134.

[0257] DETAILED DESCRIPTION

[0258] A. Definitions

[0259] B. Overview

[0260] C. Extra Enveloped Viruses (EEVs) as exemplified by EEV Vaccinia Viruses (EEVs) Attorney Docket No. 120276-2615PC

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[0262] 1. Vaccinia viruses

[0263] 2. Vaccinia Virus EEV and Their Production

[0264] 3. IMV

[0265] 4. Insufficient EEV for System Administration

[0266] 5. Selection of high producing EEV

[0267] 6. Exemplary high EEV-producing viruses

[0268] 7. Modifications of EEV to increase resistance to humoral immunity D. Methods of Production of High Amounts of EEVs

[0269] 1. Existing methods result in low production of EEVs

[0270] 2. Methods of manufacturing / producing virus resulting in higher EEV Viruses

[0271] 3. iPSCs

[0272] 4. Manufacturing the high EEV viruses, such as the RT viruses and other high EEV viruses detailed herein in HeLa Cells 5. Use of iPSCs to Manufacture Enveloped Viruses

[0273] 6. iPSCs and their use for manufacturing EEVs from poxviruses and other viruses

[0274] 7. Exemplary Advantages of iPSCs

[0275] E. Programmed EEV Viruses

[0276] 1. Serum-resistance of EEV viruses is limited by short-term protection from host immune system, such as protection from complement 2. EEV that display or express a CRP (or CRA) or other protein that inhibits or reduces the humoral immune response or sufficient of such proteins on the EEV outer membrane

[0277] 3. Advantages of the S-R EEVs and IV EEVs

[0278] 4. Enveloped Viruses in General

[0279] 6. Complement Regulating Proteins

[0280] 7. Properties of complement regulatory proteins that inhibit complement activation (complement inhibitory proteins) 8. EEV Viruses That Are Modified To Display Immune Modulating Proteins On The Outer Membrane Of The EEV

[0281] 9. Viruses modified to encode Complement Regulating Proteins — Functional or active or sufficient portions of the complement regulating proteins or other such proteins

[0282] 10. Assays for measuring or assessing the complement regulating activity of portions Attorney Docket No. 120276-2615PC

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[0284] 11. Exemplary of immune system regulating proteins are the complement regulatory proteins

[0285] 12. EEV outer membrane proteins

[0286] F. Any Poxviruses Can Be Modified As Described Herein

[0287] 1. Methods for modifying vaccinia viruses

[0288] 2. Therapeutic, transgenic, and attenuated vaccinia viruses

[0289] 3. Exemplary Therapeutic Vaccinia Virus

[0290] 4. Virally Encoded Immunomodulators and Effects thereof

[0291] G. EEV Vaccinia Viruses Can be Modified to Express Heterologous Genes and / or Payloads

[0292] H. Chimeras and Fusion of Virally Encoded Membrane Proteins and Proteins That Inhibit or Modulate the Humoral Immunity of The Host

[0293] 1. Fusion proteins and Chimeric proteins

[0294] 2. Identification of regions for EEV membrane proteins to affix the complement inhibitory protein (also referred to as complement regulatory protein or complement control protein (CCP)) and region(s) for insertion. I. Modified Viral Envelops and Methods of Modifying Viral Envelopes

[0295] 1. Modifications host and / or manufacturing cells

[0296] 2. Modifications of host cell membranes to program viral envelopes 3. Selection of Components for Programmable Enveloped Viruses J. Viruses Containing Modified Viral Envelopes

[0297] K. Pharmaceutical Compositions, Combinations, and Kits

[0298] L. Methods of Treating Cancer and Other Proliferative Diseases, Disorders, and Conditions

[0299] M. Examples

[0300] A. DEFINITIONS

[0301] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and information on the internet can come and go, but equivalent information can be Attorney Docket No. 120276-2615PC

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[0303] found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0304] As used herein, a “virus” refers to any of group of infectious entities that cannot grow or replicate without a host cell. Viruses typically contain a protein coat and RNA or DNA as genetic material; they have no semipermeable membrane, and are capable of growth and multiplication only in living cells. Examples include influenza virus, mumps virus, poliovirus, Seneca Valley Virus, and semliki forest virus.

[0305] As used herein, “oncolytic viruses” refer to viruses that replicate selectively in tumor cells in tumorous subjects. These include viruses that naturally preferentially replicate and accumulate in tumor cells, such as poxviruses, and viruses that have been engineered to do so. Some oncolytic viruses can kill a tumor cell following infection of the tumor cell. For example, an oncolytic virus can cause death of the tumor cell by lysing the tumor cell or inducing cell death of the tumor cell. Exemplary oncolytic viruses include, but are not limited to, poxviruses, herpesviruses, adenoviruses, adeno-associated viruses, lentiviruses, retroviruses, rhabdoviruses, papillomaviruses, vesicular stomatitis virus, measles virus, Newcastle disease virus, picornavirus, Sindbis virus, papillomavirus, parvovirus, reovirus, and coxsackievirus.

[0306] As used herein, the term “therapeutic virus” refers to a virus that is administered for the treatment of a disease or disorder, such as a neoplastic disease, such as cancer, a tumor and / or a metastasis or inflammation or wound or diagnosis thereof and / or both. Generally, a therapeutic virus herein is one that exhibits anti-tumor activity and minimal toxicity. The viruses also can be modified to target immune cells, such as for treatment of autoimmune disorders.

[0307] As used herein the term “vaccinia virus” or “VACV” or “ VV” denotes a large, complex, enveloped virus belonging to the poxvirus family. It has a linear, doublestranded DNA genome approximately 190 kbp in length, which encodes approximately 200 proteins. Vaccinia virus strains include, but are not limited to, strains of, derived from, or modified forms of Western Reserve (WR), Copenhagen (Cop), Bern, Paris, Tashkent, Tian Tan, Lister, Wyeth, H4D-J, IHD-W, Brighton, Ankara, modified vaccinia Ankara (MVA), CVA382, Dairen I, LIPV, LC16M8, LC16M0, AC AM, WR 65-16, Connaught, JX-594 (pexastimogene devacirepvec), GL-ONC1, vvDD TK mutant, New York City Board of Health (NYCBH), EM-63, and NYVAC vaccinia virus strains. Attorney Docket No. 120276-2615PC

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[0309] As used herein, “marker” or “selection marker” in reference to engineered viruses refer to a compound, such as a protein, whose expression and / or presence within and / or on the surface of the virus permits selection of a virus with desired engineered properties, such as viruses that express a recombinantly expressed therapeutic gene or other protein, including a marker protein.

[0310] As used herein, Lister Strain of the Institute of Viral Preparations (LIVP) or LIVP virus strain refers to a virus strain that is the attenuated Lister strain (ATCC Catalog No. VR-1549) that was produced by adaption to calf skin at the Institute of Viral Preparations, Moscow, Russia (Al’tshtein et al. (1985) Dokl. Akad. Nauk USSR 285:696-699). The LIVP strain can be obtained, for example, from the Institute of Viral Preparations, Moscow, Russia (see, e.g., Kutinova etal. (1995) Vaccine 13:487-493); the Microorganism Collection of FSRI SRC VB Vector (Kozlova etal. (2010) Environ. Sci. Technol. 44:5121-5126); or can be obtained from the Moscow Ivanovsky Institute of Virology (C0355 K0602; Agranovski et al. (2006) Atmospheric Environment 40:3924-3929). It also is well-known to those of skill in the art; as it was the vaccine strain used for vaccination in the USSR and throughout Asia and India. The strain now is used by researchers and is well-known (see e.g., Altshteyn et al. (1985) Dokl. Akad. Nauk USSR 285:696-699,' Kutinova et al. (1994) Arch. Virol. 134:1-9; Kutinova etal. (1995) Vaccine 73:487-493; Shchelkunov et al. (1993) Virus Research 28:213-283; Sroller et al. (1998) Archives Virology 743:1311-1320; Zinoviev et al, (1994) Gene 747:209-214; and Chkheidze et al. (1993) FEBS 336:340-342). An LIVP virus strain encompasses any virus strain or virus preparation that is obtained by propagation of LIVP through repeat passage in cell lines. As used herein, polypeptide refers to two or more amino acids covalently joined. The terms polypeptide and protein are used interchangeably herein.

[0311] As used herein, a polypeptide is a linear chain of amino acids linked by peptide bonds. A peptide generally is used with reference to a smaller polypeptide that is from 2 to about or 40 amino acids in length; and a polypeptide refers to a longer amino acid chain. A protein is a functional biological molecule made up of one or more polypeptides that are folded into a specific 3D structure.

[0312] As used herein, reference to proteins, unless otherwise specified, includes all forms of peptides, polypeptides, small peptides, and proteins. In general, the polypeptides displayed on a viral membrane or host cell membrane are polypeptides that Attorney Docket No. 120276-2615PC

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[0314] exhibit or manifest an activity or confer a property they can comprise a functional domain of protein or polypeptide. Recitation of peptide or polypeptide or protein is not to be interpreted as limiting to a particular form or size. The cells and / or viruses include genome modifications whereby a functional portion or all of polypeptide are displayed on the viral membrane surface to confer a property, such as serum resistance, or activity targeting to particular cells of a treated subject. The size and / or configuration of the particular displayed product depends on the product.

[0315] As used herein, a functional portion of a polypeptide or portion is a sufficient portion that is of the protein or polypeptide that exhibits an activity or property.

[0316] As used herein, a polypeptide or peptide that alters a property or activity of the virus is a polypeptide or peptide that confers, enhances, inhibits, reduces, or otherwise changes a property and / or an activity of the virus.

[0317] As used herein, EE Vs refer to viruses encased in a membrane-derived from a host cell, and include poxviruses, such as vaccinia virus, herpesviruses, and lentiviruses (for purposes herein). Vaccinia virus is exemplary of such viruses and many high EEV-producing vaccinia viruses are provided herein and / can be prepared as described herein, and / or are known in the art. For purposes herein EEVs refer to the viruses generically, and EEV vaccinia virus refers vaccinia virus EEVs or high EEV-producing vaccinia virus. In some instances, where they context is vaccinia virus, the viruses are referred to as EEVs.

[0318] A used herein, an enveloped virus is a virus that obtains its outer membrane from the cell in which it is produced. This host-derived membrane comprises a viral envelope. The virus acquires this envelope when it exits the host cell through a process called budding, often from the plasma membrane, and also from internal membranes, such as the endoplasmic reticulum, Golgi apparatus, or nuclear membrane. Some viruses have a single membrane that is derives from the host; others, such as poxviruses, including the EEV form of vaccinia virus, include two membranes — a host cell-derived membrane and a virally-derived membrane.

[0319] As used herein, reference to host-derived membranes that express or display a modified transmembrane protein refers to membranes on virus that was propagated in host cells that are genome-modified to encode and express the modified transmembrane protein. Envelope virus propagated in such host cells acquire the membrane single Attorney Docket No. 120276-2615PC

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[0321] membrane in enveloped viruses, such as lentiviruses and other RNA viruses, and outer membrane in viruses, such as vaccinia virus and other poxviruses, that produce their own membrane as well.

[0322] As used herein, a chimeric protein used interchangeably with a fusion protein refers to a protein that comprises portions from at least two different proteins. The chimeric protein can comprise two full length proteins or portions of one or both proteins (or all of the component proteins). The proteins are covalently linked via peptide bonds and can be linked end-to-end ( / .<?., N-terminus to C-terminus, N-terminus to N-terminus, C -terminus to C-terminus, and combinations thereof). Alternatively, one protein can be inserted into another, such as in or in place of domain of the second protein. Other combinations of two or more proteins are contemplated as long as the proteins or portions thereof are linked via peptide bonds to comprise a continuous protein. The proteins can subsequently be processed, such as for activation.

[0323] As used herein, a unit dose can be measure or provided as plaque forming units (pfu) of a virus particle or particles, A single dose, measured as PFU / ml / kg, for example, can vary between IxlO3and IxlO15PFUs; for example, about IxlO4, about IxlO6, about IxlO8, about IxlO10, about IxlO12or about IxlO14or higher, per mL, per kg.

[0324] As used herein, the term "carrier cell," used interchangeably with "cell," "cell vehicle," "carrier vehicle," cell-based delivery vehicle" and "cell-based vehicle" refers to any cell that can be or is infected with virus or otherwise associated with virus, such as through chemical or physical interaction between the virus and a surface protein, or by infection of the cytoplasm or nucleus of the cell with the virus. As used herein, a carrier cell refers to a cell that can be infected with a virus, such as an oncolytic virus, and in which a virus / oncolytic virus can replicate. The resulting carrier cell contains or is in association with an oncolytic virus.

[0325] As used herein, extra-enveloped viruses are a distinct type of virus that possess an additional lipid bilayer outside of the primary envelope. The extra bilipid layer is acquired from the host cell in which the virus replicates during budding process, giving them multiple protective membranes. Poxviruses and herpesvirus are exemplary of such viruses, and vaccinia virus exemplified herein. Viruses, such as lentivirus, while not having a second membrane, acquire the lipid membrane from the host cell. Vaccinia virus, as described herein and known in the art, produces an intracellular mature virus Attorney Docket No. 120276-2615PC

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[0327] (IMV) particle, which contains a single membrane, assembled within the cell. A portion, of the IMV particles get wrapped in an additional double-membrane sac derived from the Golgi apparatus or endosomes. This creates an intracellular enveloped virus (IEV), which is then transported to the cell surface, where it fuses with the plasma membrane, releasing the virion, which is now enclosed by an extra layer, forming the extracellular enveloped virus (EEV). The second membrane, thus, is derived from the host cell membrane and also includes virally encoded proteins. For purposes herein, any virus that acquires a membrane from the host cell membrane can be cultured in modified host cells as detailed herein to program properties of the resulting virus.

[0328] As used herein, an extracellular enveloped vaccinia virus (EEV vaccinia virus) is a vaccinia virus that contains a second membrane. Any vaccinia virus can be modified, such as by modifying a gene, such as A33R, A34R, A56R, B5R, and F13L (see, SEQ ID NOs: 168-174, 182-188, 196-202, 210-216, 224, and 225 for exemplary sequences from various vaccinia virus strains) or any virally encoded second membrane protein, by propagating the virus and selecting for a virus that produces a high percentage (greater than at least, for example 5%) EEV clones or a greater percentage than the parental virus from which the clone is derived. The EEV virus, when cultured in a cell line that has a high level of a protein, such as complement inhibiting proteins, such as CD46, CD55, CD59, CD71, CD81, (or sufficient portion thereof to confer serum resistance) or other protein or portion thereof that results in serum -resistant EEV (SR-EEV) clones by virtue of the CD46, CD55, CD59, CD71, CD81 or other such protein (or sufficient portion thereof to confer serum resistance) in the second membrane. Upon propagation in a tumor or cell line that does not express high levels at least one of CD46, CD55, CD59, CD71, CD81, the resulting viruses are not serum resistant. Amino acid sequences of exemplary human complement regulatory proteins are set forth in SEQ ID NOs:237-248.

[0329] As used herein, in general a virus that produces high levels of EEVs when propagated results in EEV particles in an amount greater than about 1% of the virus particles. The level of EEV is a function of the virus that is propagated, the cells in which the virus is propagated, and the method by which the virus is propagated and / or isolated. For example, provided herein are methods for manufacturing virus that result in virus preparation in which at least about 60% or more of the viruses in any preparation are EEVs. Attorney Docket No. 120276-2615PC

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[0331] As used herein, the “modified virus” refers to a virus that is altered compared to a parental strain of the virus. Typically, modified viruses have genome modifications, such as one or more truncations, mutations, insertions or deletions in the genome of virus. A modified virus can have one or more endogenous viral genes modified and / or one or more intergenic regions modified. Exemplary modified viruses can have one or more heterologous nucleic acid sequences inserted into the genome of the virus. Modified viruses can contain one or more heterologous nucleic acid sequences in the form of a gene expression cassette for the expression of a heterologous gene.

[0332] Typically, the genome of the virus is modified by substitution (replacement), insertion (addition) or deletion (truncation) of nucleotides. Modifications can be made using any method known to one of skill in the art, including as provided herein, such as genetic engineering and recombinant DNA methods. Hence, a modified virus is a virus that is altered in its genome compared to the genome of a parental virus. Exemplary modified viruses have one or more heterologous nucleic acid sequences inserted into the genome of the virus. The heterologous nucleic acid can contain an open reading frame encoding a heterologous protein, which can be inserted under control of a viral promoter or a heterologous non-viral promoter. For example, modified viruses herein can contain one or more heterologous nucleic acid sequences in the form of a gene expression cassette for the expression of a heterologous gene.

[0333] Also included are EEV viruses or viruses encased in a host cell-derived membrane deposited when the virus has been propagated in modified host cells that are modified to express proteins / polypeptides on cell membranes for deposition on the virus, where proteins / polypeptides (or active portions thereof) alter, enhance, or confer a property on the virus. Such viruses also are referred to herein as programmed EEVs.

[0334] As used herein, complement regulatory protein or complement regulator protein (also referred to herein as a complement resistance protein or a regulator of complement activity (RCA)) is a protein that plays a regulatory role in humans and animals to ensure that the complement system of (innate) immunity does not become over-activated, thus causing harm to self-tissues. Included are regulatory proteins, such as, but not limited to, as Cl inhibitor, C4b binding protein, and factors H, B, D, and I. Membrane bound complement regulatory proteins (mCRPs) provide another complement control mechanism; these include, for example, CD35 (Complement receptor 1, CR1), CD46 Attorney Docket No. 120276-2615PC

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[0336] (membrane cofactor protein, MCP), CD55 (decay acceleration factor, DAF), and CD59 (protectin). Complement regulatory proteins (CRPs) are expressed on every cell in the human body, though the expression of these mCRPs varies across tissue type. Since different tissues face different immune interactions within the body, mCRP expression across tissue types also can be variable (Qin et al., (2001), Mamm. Genome, 12:582-589).

[0337] As used herein, complement inhibitor protein refers to any protein, polypeptide, and or portion thereof that can inhibit complement.

[0338] As used herein, humoral immunity regulatory proteins refer to proteins that interact with or modulate immunity. Particularly, humoral immunity resistance proteins, are proteins that when all or a portion is expressed on the surface of an EEV, reduce or eliminate serum inactivation of the virus. For purposes herein, these include CRPs or other such proteins that inhibit the anti-viral response of the host.

[0339] As used herein, a protein the reduces or inhibits humoral immunity in a host, and variations of such language, refers to proteins, such as complement regulating proteins and complement inhibiting proteins, that, when such proteins are displayed on the surface of a virus, such as an EEV in systemic circulation, immune response or system of the host is modulated to reduce the humoral response of the host against the virus. Such proteins include, but are not limited to, CD55, DAF-2, CD46, CD59, and CD35, and immune modulating portions thereof.

[0340] As used herein, humoral immunity is a type of immunity that is mediated by macromolecules found in extracellular fluids such as secreted antibodies, complement proteins, and certain antimicrobial peptides. It is one of the two primary branches of the adaptive immune system; the other is cell-mediated immunity. Humoral immunity is so named because it involves substances found in the humors, or body fluids. Proteins that confer such immunity include any that inhibit or reduce complement or expression thereof.

[0341] As used herein, immunomodulatory with respect to combination treatments reduce or modulate the immune response so that the anti-viral response is reduced.

[0342] As used herein, serum resistant EEVs (SR-EEVs) are EEVs that are resistant to serum. These include EEVs produced in cells lines that express a high level of CD55 or other such complement inhibitory protein. Attorney Docket No. 120276-2615PC

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[0344] As used herein, IV-EEVs are EEVs that are resistant to complement and / or other anti-viral mediators in vitro and in vivo independent of the cell in which they are propagated or amplified in vivo or in vitro. The IV-EEVs exhibit humoral immunity and can be administered systemically and disseminate to distal tumors in vivo. IV-EEVs are SR-EEVs that have been genetically modified to encode a protein that protects against complement inactivation and / or humoral immunity. The IV-EEVs encoding such protein (or a sufficient portion thereof to confer humoral immunity) as a fusion protein in a transmembrane protein that, when the virally-encoded fusion protein is expressed, it is displayed in the second membrane.

[0345] As used herein, a fusion protein, used interchangeably with a chimeric protein, refers to a protein that comprises portions of at least 10 contiguous amino acids from two or more different proteins. The contiguous portions are linked via peptide bonds. This is distinct from a protein in which two are more distinct portions comprise two or more chains, such as chains linked by cysteine bonds.

[0346] As used herein, therapeutically effective with reference to a virus refers to parameters, such as reduction in tumor size, increased survival, increased progression free survival, durable response rate (DRR; objective response lasting continuously > 6 months) and overall survival (OS) as defined by the US FDA for approval of a treatment. Objective response refers to the percentage of patients on whom a therapy has a defined effect. Such parameters can vary by type of cancer, where effective treatments for intractable cancers, such as pancreatic cancer are more modest than for cancers have been treated. For pancreatic cancer, for example, an extension of life by two months can be considered effective. As another example, the oncolytic virus, T-VEC was the first oncolytic immunotherapy to demonstrate therapeutic benefit against melanoma in a phase III clinical trial in melanoma. T-VEC was considered well-tolerated and resulted in a statistically significant higher durable response rate (16.3% of patients, P <.001) and longer median OS (23.3 months; P =.051) compared to the control with GM-CSF.

[0347] As used herein, serum-resistant EEVs (or S-R EEVs) provided herein are modified EEVs that produce second membranes that display a polypeptide or protein that confers resistance to serum, generally by inactivation of resistance to complement.

[0348] Native serum-resistance of EEVs is a function of the cell in which they are propagated; as described herein some cell lines, such as HELA cells produce high levels of EEVs that Attorney Docket No. 120276-2615PC

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[0350] exhibit serum resistance because the HELA cells produce high levels of CD55 that get incorporated in to the second membrane of the EEV. Provided herein are modified viruses that produce S-R EEVs that can be propagated in any cell line or in any tumor and they retain the serum resistance. Such EEVs can be designated IV-EEVs; IV-EEVs are modified so that they express a humoral immunity resistance protein or portion thereof so that the EEVs are not inactivated by humoral immunity. As exemplified herein, this is achieved by virally encoding a chimeric EEV membrane protein that includes all or a portion of a protein that resists humoral immunity, such as a complement inactivating protein, such as CD46, CD55, and CD59. They are expressed as a chimera with an EEV membrane protein, such as A33R, A34R, A56R and / or B5R to display the complement inactivating protein (CIP; also referred to as complement resistance protein (CRA) also referred to as a complement regulatory protein (CRP) herein) the external EEV membrane. CRP and CRA and CIP are used interchangeably herein. The IV-EEVs also can be modified to express other humoral tumor modulators whose expression increase serum stability.

[0351] As used herein, “multiplicity of infection (MOI)” refers to the number of virions that are added per cell during infection ( / .<?., one million virions added to one million cells is an MOI of one).

[0352] As used herein, "sensitized" or "sensitizing a cell" to alter a property of the cell, refers to treating the cell by treatment, generally before use, with an agent to modify a property of the cell, such as by inducing expression of a gene.

[0353] As used herein, amplification of a virus in a carrier cell means that the virus replicates in the cell to sustain the virus or increase the amount of virus in the cell.

[0354] As used herein, a “host cell” or “target cell” are used interchangeably to mean a cell that can be infected by a virus.

[0355] As used herein, the term “tissue” refers to a group, collection or aggregate of similar cells generally acting to perform a specific function within an organism.

[0356] As used herein, the term “immunomodulatory protein” or “immunomodulator” refers to a protein that is expressed by a virus that can protect the virus from attack by innate and / or acquired immune systems of the target cell, such as, for example, cells of the tumor. Viral immunomodulatory products have evolved to withstand the selective evolutionary pressure imposed by the host immune system. These products can modulate Attorney Docket No. 120276-2615PC

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[0358] innate and adaptive host immune responses. Exemplary immunomodulatory products encoded by vaccinia, for example, include, but are not limited to, VCP (C3L), B5R, HA (A56R), B18R / B19R, B8R, CmrC and CmrE.

[0359] As used herein, the term, “therapeutic gene product” or “therapeutic polypeptide” refers to any heterologous protein expressed by a therapeutic gene encoded by a virus, such as an oncolytic virus, that ameliorates the symptoms of a disease or disorder or ameliorates the disease or disorder. Therapeutic gene products include, but are not limited to, moieties that inhibit cell growth or promote cell death that can be activated to inhibit cell growth or promote cell death, or that activate another agent to inhibit cell growth or promote cell death. Optionally, the therapeutic agent can exhibit or manifest additional properties, such as, properties that permit its use as an imaging agent, as described elsewhere herein. Exemplary therapeutic gene products include, for example, immune checkpoint inhibitors, cytokines, growth factors, photosensitizing agents, radionuclides, toxins, anti-metabolites, signaling modulators, anti-cancer antibodies, angiogenesis inhibitors or a combination thereof.

[0360] As used herein, recitation of “antibody” (e.g., antibody directed to an antigen expressed on an immune cell population such as, for example, T cells, 76 (gd) T cells, NK cells, and NKT cells to be depleted or inhibited for suppression of an immune response) includes full-length antibodies and portions thereof including antibody fragments. Antibody fragments, include, but are not limited to, Fab fragments, Fab' fragments, F(ab’)2 fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fd’ fragments, single-chain Fvs (scFv), single-chain Fabs (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above.

[0361] Antibody also includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, and intrabodies. Antibodies provided herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA and IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass (e.g., IgG2a and IgG2b).

[0362] Antibodies, such as monoclonal antibodies, can be prepared using standard methods known to those with skill in the art (see, e.g., Kohler et al., Nature 256:495-497 (1975); Kohler et al., Eur. J. Immunol. 6:511-519 (1976); and WO 02 / 46455). For Attorney Docket No. 120276-2615PC

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[0364] example, an animal is immunized by standard methods to produce antibody-secreting somatic cells. These cells then are removed from the immunized animal for fusion to myeloma cells. Somatic cells that can produce antibodies, such as B cells, can be used for fusion with a myeloma cell line. These somatic cells can be derived from the lymph nodes, spleens, and peripheral blood of primed animals. Specialized myeloma cell lines have been developed from lymphocytic tumors for use in hybridoma-producing fusion procedures (Kohler and Milstein, Eur. J. Immunol. 6:511-519 (1976); Shulman et aL, Nature, 276:269-282 (1978); Volk etal., J. Virol., 42:220-227 (1982)). These cell lines have three useful properties. The first is they facilitate the selection of fused hybridomas from unfused and similarly indefinitely self-propagating myeloma cells by having enzyme deficiencies that render them incapable of growing in selective medium that support the growth of hybridomas. The second is they have the ability to produce antibodies and are incapable of producing endogenous light or heavy immunoglobulin chains. A third property is they efficiently fuse with other cells. Other methods for producing hybridomas and monoclonal antibodies are well known to those of skill in the art. It is routine to produce antibodies against any polypeptide, e.g., antigenic marker on an immune cell population, or an immune checkpoint.

[0365] As used herein, therapeutic agents are agents that ameliorate the symptoms of a disease or disorder or ameliorate the disease or disorder. Therapeutic agent, therapeutic compound, or therapeutic regimens include conventional drugs and drug therapies, including vaccines for treatment or prevention (i.e., reducing the risk of getting a particular disease or disorder), which are known to those skilled in the art and described elsewhere herein. Therapeutic agents for the treatment of neoplastic disease include, but are not limited to, moieties that inhibit cell growth or promote cell death that can be activated to inhibit cell growth or promote cell death, or that activate another agent to inhibit cell growth or promote cell death. Therapeutic agents for use in the methods provided herein can be, for example, an anticancer agent. Exemplary therapeutic agents include, for example, therapeutic microorganisms, such as therapeutic viruses and bacteria, cytokines, growth factors, photosensitizing agents, radionuclides, toxins, antimetabolites, signaling modulators, anticancer antibiotics, anticancer antibodies, angiogenesis inhibitors, radiation therapy, chemotherapeutic compounds or a combination thereof. Attorney Docket No. 120276-2615PC

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[0367] As used herein, a tumor cell or cancer cell refers to a cell that divides and reproduces abnormally because growth and division are not regulated or controlled, i.e., cells that are susceptible to uncontrolled growth. A tumor cell can be a benign or malignant cell. Typically, the tumor cell is a malignant cell that can spread to other parts of the body, a process known as metastasis.

[0368] As used herein, a virus preparation or virus composition, refers to a virus composition obtained by propagation of a virus strain, for example a vaccinia virus strain, a vaccinia virus clonal strain or a modified or recombinant virus strain, in vivo or in vitro in a culture system. For example, a vaccinia virus preparation refers to a viral composition obtained by propagation of a virus strain in host cells, typically upon purification from the culture system using standard methods known in the art. A virus preparation generally is made up of a number of virus particles or virions. If desired, the number of virus particles in the sample or preparation can be determined using a plaque assay to calculate the number of plaque forming units per sample unit volume (pfu / mL), assuming that each plaque formed is representative of one infective virus particle. Each virus particle or virion in a preparation can have the same genomic sequence compared to other virus particles (i.e., the preparation is homogenous in sequence) or can have different genomic sequences i.e., the preparation is heterogenous in sequence). It is understood to those of skill in the art that, in the absence of clonal isolation, heterogeneity or diversity in the genome of a virus can occur as the virus reproduces, such as by homologous recombination events that occur in the natural selection processes of virus strains (Plotkin & Orenstein (eds) “Recombinant Vaccinia Virus Vaccines” in Vaccines, 3rdedition (1999)).

[0369] As used herein, plaque forming unit (pfu) or infectious unit (IU) refers to the number of infectious or live viruses. It thus reflects the amount of active virus in the preparation. The pfu can be determined using a virus plaque assay (plaque formation assay) or an end-point dilution assay, which are standard assays known to one of skill in the art.

[0370] As used herein, “targeting molecule” or “targeting ligand” refers to any molecular signal directing localization to specific cells, tissues or organs. Examples of targeting ligands include, but are not limited to, proteins, polypeptides or portions thereof that bind to cell surface molecules, including, but not limited to, proteins, carbohydrates, lipids or Attorney Docket No. 120276-2615PC

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[0372] other such moieties. For example, targeting ligands include proteins or portions thereof that bind to cell surface receptors or antibodies directed to antigens expressed selectively on a target cell. Targeting ligands include, but are not limited to growth factors, cytokines, adhesion molecules, neuropeptides, protein hormones and single-chain antibodies (scFv).

[0373] As used herein, accumulation of a virus in a particular tissue refers to the distribution or colonization of the virus in particular tissues of a host organism after a time period following administration of the virus to the host, long enough for the virus to infect the host’s organs or tissues. One skilled in the art recognizes that the time period for infection of a virus varies depending on the virus, the organ(s) or tissue(s) to be infected, the immunocompetence of the host, and the dosage of the virus. Generally, accumulation can be determined at time points from about less than 1 day, about 1 day to about 2, 3, 4, 5, 6 or 7 days, about 1 week to about 2, 3 or 4 weeks, about 1 month to about 2, 3, 4, 5, 6 months or longer after infection with the virus. Oncolytic viruses preferentially accumulate in immunoprivileged tissue, such as inflamed tissue or tumor tissue, but are cleared from other tissues and organs, such as non-tumor tissues, in the host to the extent that toxicity of the virus is mild or tolerable and at most, not fatal.

[0374] As used herein, “preferential accumulation” refers to accumulation of a virus at a first location at a higher level than accumulation at a second location (i.e., the concentration of viral particles, or titer, at the first location is higher than the concentration of viral particles at the second location). Thus, a virus that preferentially accumulates in immunoprivileged tissue (tissue that is sheltered from the immune system), such as inflamed tissue, and tumor tissue, relative to normal tissues or organs, refers to a virus that accumulates in immunoprivileged tissue, such as tumor, at a higher level (i.e., concentration or viral titer) than the virus accumulates in normal tissues or organs.

[0375] As used herein, activity refers to the in vitro or in vivo activities of a compound or virus provided herein. For example, in vivo activities refer to physiological responses that result following in vivo administration of a compound or virus provided herein (or of a composition or other mixture thereof). Activity, thus, encompasses resulting therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures. Attorney Docket No. 120276-2615PC

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[0377] Activities can be observed in in vitro and / or in vivo systems designed to test or use such activities.

[0378] As used herein, “anti-tumor activity” or “anti-tumorigenic” refers to virus strains that prevent or inhibit the formation or growth of tumors in vitro or in vivo in a subject. Anti-tumor activity can be determined by assessing a parameter or parameters indicative of anti-tumor activity.

[0379] As used herein, “greater” or “improved” activity with reference to anti-tumor activity or anti-tumorigenicity means that a virus strain is capable of preventing or inhibiting the formation or growth of tumors in vitro or in vivo in a subject to a greater extent than a reference or control virus or to a greater extent than absence of treatment with the virus. Whether anti-tumor activity is “greater” or “improved” can be determined by assessing the effect of a virus and, if necessary, a control or reference virus, on a parameter indicative of anti-tumor activity. It is understood that when comparing the activity of two or more different viruses, the amount of virus (e.g., pfu) used in an in vitro assay or administered in vivo is the same or similar, and the conditions (e.g., in vivo dosage regime) of the in vitro assay or in vivo assessment are the same or similar.

[0380] As used herein, “toxicity” (also referred to as virulence or pathogenicity herein) with reference to a virus refers to the deleterious or toxic effects to a host upon administration of the virus. For an oncolytic virus, such as vaccinia virus, the toxicity of a virus is associated with its accumulation in non-tumorous organs or tissues, which can impact the survival of the host or result in deleterious or toxic effects. Toxicity can be measured by assessing one or more parameters indicative of toxicity. These include accumulation in non-tumorous tissues and effects on viability or health of the subject to whom it has been administered, such as effects on body weight.

[0381] As used herein, “reduced toxicity” means that the toxic or deleterious effects upon administration of the virus to a host are attenuated or lessened compared to a host not treated with the virus or compared to a host that is administered with another reference or control virus. Whether toxicity is reduced or lessened can be determined by assessing the effect of a virus and, if necessary, a control or reference virus, on a parameter indicative of toxicity. It is understood that when comparing the activity of two or more different viruses, the amount of virus e.g., pfu) used in an in vitro assay or administered in vivo is the same or similar and the conditions (e.g., in vivo dosage Attorney Docket No. 120276-2615PC

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[0383] regime) of the in vitro assay or in vivo assessment are the same or similar. For example, when comparing effects upon in vivo administration of a virus and a control or reference virus the subjects are the same species, size, gender and the virus is administered in the same or similar amount under the same or similar dosage regime. In particular, a virus with reduced toxicity can mean that upon administration of the virus to a host, such as for the treatment of a disease, the virus does not accumulate in non-tumorous organs and tissues in the host to an extent that results in damage or harm to the host, or that impacts survival of the host to a greater extent than the disease being treated does or to a greater extent than a control or reference virus does. For example, a virus with reduced toxicity includes a virus that does not result in death of the subject over the course of treatment.

[0384] As used herein, a “control” or “standard” refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a plasma sample, it can be from a normal volunteer not affected with the condition of interest. A control also can be an internal control. For example, a control can be a sample, such as a virus, that has a known property or activity.

[0385] As used herein, dosing regimen refers to the amount of agent, for example, a carrier cell or virus or other agent, administered, and the frequency of administration over the course of a cycle of administration. The dosing regime is a function of the disease or condition to be treated, and thus can vary.

[0386] As used herein, frequency of administration refers to the number of times an agent is administered during the cycle of administration. For example, frequency can be days, weeks or months. For example, frequency can be administration once during a cycle of administration, two times, three times, four times, five times, six times or seven times. The frequency can refer to consecutive days during the cycle of administration. The frequency is a function of the disease or condition treated.

[0387] As used herein, a “cycle of administration” refers to the repeated schedule of the dosing regimen of administration of a virus that is repeated over successive administrations. For example, an exemplary cycle of administration is a 28-day cycle.

[0388] As used herein, immunoprivileged cells and immunoprivileged tissues refer to cells and tissues, such as solid tumors, which are sequestered from the immune system. An immunoprivileged cell or tissue tolerates the introduction of antigens without eliciting an inflammatory immune response. For example, administration of a virus to a Attorney Docket No. 120276-2615PC

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[0390] subject elicits an immune response that clears the virus from the subject.

[0391] Immunoprivileged sites, however, are shielded or sequestered from the immune response, permitting the virus to survive and generally to replicate. Immunoprivileged tissues include proliferating tissues, such as tumor tissues and other tissues and cells involved in other proliferative disorders, wounds and other tissues involved in inflammatory responses.

[0392] As used herein, a tumor, also known as a neoplasm, is an abnormal mass of tissue that results when cells proliferate at an abnormally high rate. Tumor encompasses hematopoietic tumors as well as solid tumors. Tumors can show partial or total lack of structural organization and functional coordination with normal tissue. Tumors can be benign (not cancerous), or malignant (cancerous).

[0393] As used herein, malignant, as applied to tumors, refers to primary tumors that have the capacity of metastasis with loss of growth control and positional control.

[0394] As used herein, metastasis refers to a growth of abnormal or neoplastic cells distant from the site primarily involved by the morbid process.

[0395] As used herein, malignant tumors can be classified into three major types.

[0396] Carcinomas are malignant tumors arising from epithelial structures, such as, but not limited to, breast, prostate, lung, colon, and pancreas. Sarcomas are malignant tumors that originate from connective tissues, or mesenchymal cells, such as muscle, cartilage, fat or bone. Leukemias and lymphomas are malignant tumors affecting hematopoietic structures (structures pertaining to the formation of blood cells), including components of the immune system. Other malignant tumors include, but are not limited to, tumors of the nervous system (e.g., neurofibromatomas), germ cell tumors, and blastic tumors.

[0397] As used herein, a disease or disorder or condition refers to a pathological condition in an organism resulting from, for example, infection or genetic defect, and characterized by identifiable symptoms. An exemplary disease as described herein is a neoplastic disease, such as cancer.

[0398] As used herein, neoplastic disease refers to any disorder involving cancer, including tumor development, growth, metastasis and progression.

[0399] As used herein, cancer is a term for diseases caused by or characterized by any type of malignant tumor or hematological malignancy, including metastatic cancers, lymphatic tumors, and blood cancers. Exemplary cancers include, but are not limited to, Attorney Docket No. 120276-2615PC

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[0401] acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoma, adrenal cancer, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brainstem glioma, brain cancer, carcinoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway or hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, Burkitt’s lymphoma, carcinoid tumor, carcinoma, central nervous system lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, epidermoid carcinoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer / intraocular melanoma, eye cancer / retinoblastoma, gallbladder cancer, gallstone tumor, gastric / stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, giant cell tumor, glioblastoma multiforme, glioma, hairy-cell tumor, head and neck cancer, heart cancer, hepatocellular / liver cancer, Hodgkin’s lymphoma, hyperplasia, hyperplastic corneal nerve tumor, in situ carcinoma, hypopharyngeal cancer, intestinal ganglioneuroma, islet cell tumor, Kaposi's sarcoma, kidney / renal cell cancer, laryngeal cancer, leiomyoma tumor, lip and oral cavity cancer, liposarcoma, liver cancer, non-small cell lung cancer, small cell lung cancer, lymphomas, macroglobulinemia, malignant carcinoid, malignant fibrous histiocytoma of bone, malignant hypercalcemia, malignant melanomas, marfanoid habitus tumor, medullary carcinoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic skin carcinoma, metastatic squamous neck cancer, mouth cancer, mucosal neuromas, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disorder, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neck cancer, neural tissue cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial tumor, ovarian germ cell tumor, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma, pituitary adenoma, pleuropulmonary blastoma, polycythemia vera, Attorney Docket No. 120276-2615PC

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[0403] primary brain tumor, prostate cancer, rectal cancer, renal cell tumor, reticulum cell sarcoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, seminoma, Sezary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck carcinoma, stomach cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, throat cancer, thymoma, thyroid cancer, topical skin lesion, trophoblastic tumor, urethral cancer, uterine / endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia or Wilms’ tumor. Exemplary cancers commonly diagnosed in humans include, but are not limited to, cancers of the bladder, brain, breast, bone marrow, cervix, colon / rectum, kidney, liver, lung / bronchus, ovary, pancreas, prostate, skin, stomach, thyroid, or uterus.

[0404] Exemplary cancers commonly diagnosed in dogs, cats, and other pets include, but are not limited to, lymphosarcoma, osteosarcoma, mammary tumors, mastocytoma, brain tumor, melanoma, adenosquamous carcinoma, carcinoid lung tumor, bronchial gland tumor, bronchiolar adenocarcinoma, fibroma, myxochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma, Wilms’ tumor, Burkitt's lymphoma, microglioma, neuroblastoma, osteoclastoma, oral neoplasia, fibrosarcoma, osteosarcoma and rhabdomyosarcoma, genital squamous cell carcinoma, transmissible venereal tumor, testicular tumor, seminoma, Sertoli cell tumor, hemangiopericytoma, histiocytoma, chloroma (e.g., granulocytic sarcoma), corneal papilloma, corneal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, stomach tumor, adrenal gland carcinoma, oral papillomatosis, hemangioendothelioma and cystadenoma, follicular lymphoma, intestinal lymphosarcoma, fibrosarcoma and pulmonary squamous cell carcinoma. Exemplary cancers diagnosed in rodents, such as a ferret, include, but are not limited to, insulinoma, lymphoma, sarcoma, neuroma, pancreatic islet cell tumor, gastric MALT lymphoma and gastric adenocarcinoma. Exemplary neoplasias affecting agricultural livestock include, but are not limited to, leukemia, hemangiopericytoma and bovine ocular neoplasia (in cattle); preputial fibrosarcoma, ulcerative squamous cell carcinoma, preputial carcinoma, connective tissue neoplasia and mastocytoma (in horses); hepatocellular carcinoma (in swine); lymphoma and pulmonary adenomatosis (in sheep); pulmonary sarcoma, lymphoma, Rous sarcoma, reticulo-endotheliosis, fibrosarcoma, nephroblastoma, B-cell lymphoma and lymphoid leukosis (in avian species); retinoblastoma, hepatic neoplasia, Attorney Docket No. 120276-2615PC

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[0406] lymphosarcoma (lymphoblastic lymphoma), plasmacytoid leukemia and swimbladder sarcoma (in fish), caseous lymphadenitis (CLA): chronic, infectious, contagious disease of sheep and goats caused by the bacterium Corynebacterium pseudotuberculosis, and contagious lung tumor of sheep caused by jaagsiekte.

[0407] As used herein, a cell involved in a disease or disease process refers to cells whose presence contributes to, exacerbates, causes or otherwise is involved in the etiology of a disease or disease process. Inhibition or killing of such cells can ameliorate the symptoms of the disease or can ameliorate the disease. Examples of such cells are tumor cells. Killing or inhibiting the growth or proliferation of tumor cells effects treatment of tumors. Other examples are immune effector cells, which participate in inflammatory responses that contribute to the pathology of a variety of diseases.

[0408] Inhibiting or killing immune effector cells can treat diseases that have an inflammatory component.

[0409] As used herein, “killing or inhibiting growth or proliferation of cells” means that the cells die or are eliminated. Inhibiting growth or proliferation means that the number of such cells does not increase, and can decrease.

[0410] As used herein, a “tumor cell” is any cell that is part of a tumor. Typically, carrier cells provided herein preferentially home to tumor cells and the viruses provided herein preferentially infect tumor cells in a subject compared to normal cells.

[0411] As used herein, a “metastatic cell” is a cell that has the potential for metastasis. Metastatic cells have the ability to metastasize from a first tumor in a subject and can colonize tissue at a different site in the subject to form a second tumor at the site.

[0412] As used herein, “tumorigenic cell,” is a cell that, when introduced into a suitable site in a subject, can form a tumor. The cell can be non-metastatic or metastatic.

[0413] As used herein, a “normal cell” is a cell that is not derived from a tumor, but is derived from healthy non-diseased tissue.

[0414] As used herein, a “metastasis” refers to the spread of cancer from one part of the body to another. For example, in the metastatic process, malignant cells can spread from the site of the primary tumor in which the malignant cells arose and move into lymphatic and blood vessels, which transport the cells to normal tissues elsewhere in an organism where the cells continue to proliferate. A tumor formed by cells that have spread by metastasis is called a “metastatic tumor,” a “secondary tumor” or a “metastasis.” Attorney Docket No. 120276-2615PC

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[0416] As used herein, an anti-cancer agent or compound (used interchangeably with "anti-tumor or anti -neoplastic agent") refers to any agents or compounds used in anticancer treatment. These include any agents, when used alone or in combination with other compounds, that can alleviate, reduce, ameliorate, prevent, or place or maintain in a state of remission of clinical symptoms or diagnostic markers associated with neoplastic disease, tumors and cancer, and can be used in methods, combinations and compositions provided herein. Anticancer agents include antimetastatic agents. Exemplary anticancer agents include, but are not limited to, chemotherapeutic compounds, such as, but not limited to toxins, alkylating agents, nitrosoureas, anticancer antibiotics, antimetabolites, antimitotics, and topoisomerase inhibitors, cytokines, growth factors, hormones, photosensitizing agents, radionuclides, signaling modulators, immunotherapeutic agents, CAR-T cells, checkpoint inhibitors, CRISPR therapies, anticancer antibodies, anticancer oligopeptides, anticancer oligonucleotides (e.g., antisense RNA and RNAi, such as siRNA and shRNA), angiogenesis inhibitors, radiation therapy, or a combination thereof. Exemplary chemotherapeutic compounds include, but are not limited to, Ara-C, cisplatin, carboplatin, paclitaxel, doxorubicin, gemcitabine, camptothecin, irinotecan, cyclophosphamide, 6-mercaptopurine, vincristine, 5-fluorouracil, and methotrexate.

[0417] As used herein, reference to an anticancer or chemotherapeutic agent includes combinations or a plurality of anticancer or chemotherapeutic agents unless otherwise indicated.

[0418] As used herein, a subject includes any organism, including an animal for whom diagnosis, screening, monitoring or treatment is contemplated. Animals include mammals such as primates and domesticated animals. An exemplary primate is a human. A patient refers to a subject, such as a mammal, primate, human, or livestock subject afflicted with a disease condition or for which a disease condition is to be determined or risk of a disease condition is to be determined.

[0419] As used herein, a patient refers to a human subject exhibiting symptoms of a disease or disorder.

[0420] As used herein, treatment of a subject that has a condition, disorder or disease means any manner of treatment in which the symptoms of the condition, disorder or disease are ameliorated or otherwise beneficially altered. Treatment encompasses any Attorney Docket No. 120276-2615PC

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[0422] pharmaceutical use of the cell-assisted viral expression systems described and provided herein.

[0423] As used herein, treatment refers to amelioration of the symptoms of a disease or disorder.

[0424] As used herein, prevention refers to prophylactic treatment to reduce the risk of getting a disease or condition or reducing the severity thereof.

[0425] As used herein, a subject refers to any mammal that can be treated by the methods and uses herein. Mammals include humans, other primates, such as chimpanzees, bonobos, and gorillas, dogs, cats, cows, pigs, goats and other farm animals and pets. Patients refer to human subjects.

[0426] As used herein, treatment of a subject that has a neoplastic disease, including a tumor or metastasis, means any manner of treatment in which the symptoms of having the neoplastic disease are ameliorated or otherwise beneficially altered. Typically, treatment of a tumor or metastasis in a subject encompasses any manner of treatment that results in slowing of tumor growth, lysis of tumor cells, reduction in the size of the tumor, prevention of new tumor growth, or prevention of metastasis of a primary tumor, including inhibition of vascularization of the tumor, tumor cell division, tumor cell migration or degradation of the basement membrane or extracellular matrix.

[0427] As used herein, therapeutic effect means an effect resulting from treatment of a subject that alters, typically improves or ameliorates the symptoms of a disease or condition or that cures a disease or condition. A therapeutically effective amount refers to the amount of a composition, molecule or compound which results in a therapeutic effect following administration to a subject.

[0428] As used herein, amelioration or alleviation of the symptoms of a particular disease, disorder, or condition, such as by administration of a pharmaceutical composition, refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.

[0429] As used herein, efficacy means that upon administration of a virus or virus composition, the virus will colonize proliferating or immunoprivileged cells, such as tumor cells, and replicate. Colonization and replication in tumor cells is indicative that the treatment is or will be an effective treatment. Attorney Docket No. 120276-2615PC

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[0431] As used herein, effective treatment with a cell carrier / virus is one that can increase survival compared to the absence of treatment therewith. For example, a virus is an effective treatment if it stabilizes disease, causes tumor regression, decreases severity of disease or slows down or reduces metastasizing of the tumor.

[0432] As used herein, an effective amount, or therapeutically effective amount, of a virus or compound for treating a disease, disorder, or condition is an amount to ameliorate, or in some manner reduce the symptoms associated with the disease. The amount will vary from one individual to another and will depend upon one or several factors, including, but not limited to, age, weight, the overall physical condition of the patient, and the severity of the disease. A therapeutically effective amount can be administered as a single dosage or can be administered in multiple dosages according to a regimen, whereby it is effective. The amount can cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease. Repeated administration can be required to achieve the desired amelioration of symptoms.

[0433] As used herein, an effective amount, or therapeutically effective amount, of a virus or compound for treating a neoplastic disease, including a tumor or metastasis is an amount to ameliorate, or in some manner reduce the symptoms associated with the neoplastic disease, including, but not limited to slowing of tumor growth, lysis of tumor cells, reduction in the size of the tumor, prevention of new tumor growth, or prevention of metastasis of a primary tumor.

[0434] As used herein, prevent a disease or condition means reduce the probability or rise of getting the disease or condition.

[0435] As used herein, a "composition" refers to any mixture of two or more products or compounds. It can be a solution, a suspension, liquid, powder, a paste, aqueous, nonaqueous, or any combination thereof.

[0436] As used herein, a formulation refers to a composition containing at least one active pharmaceutical or therapeutic agent and one or more excipients.

[0437] As used herein, a co-formulation refers to a composition containing two or more active or pharmaceutical or therapeutic agents and one or more excipients.

[0438] As used herein, a combination refers to any association between or among two or more items. The combination can be two or more separate items, such as two compositions or two collections, can be a mixture thereof, such as a single mixture of the Attorney Docket No. 120276-2615PC

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[0440] two or more items, or any variation thereof. The elements of a combination are generally functionally associated or related. Exemplary combinations include, but are not limited to, two or more pharmaceutical compositions, a composition containing two or more active ingredients, such as two viruses, or a virus and an anticancer agent, such as a chemotherapeutic compound, two or more viruses, a virus and a therapeutic agent, a virus and an imaging agent, a virus and a plurality of therapeutic and / or imaging agents, or any association thereof. Such combinations can be packaged as kits.

[0441] As used herein, a composition refers to a mixture of two or more components, such as a therapeutic agent in or mixed with a pharmaceutically acceptable vehicle.

[0442] As used herein, direct administration refers to administration of a composition without dilution.

[0443] As used herein, a kit is a packaged combination, optionally, including instructions for use of the combination and / or other reactions and components for such use.

[0444] As used herein, an “article of manufacture” is a product that is made and sold. As used throughout this application, the term is intended to encompass articles containing a carrier cell and vaccinia virus alone or in combination with a second therapy or a therapeutic energy source contained in the same or separate articles of packaging.

[0445] As used herein, a device refers to a thing made or adapted for a particular task. Exemplary devices herein are devices that cover or coat or are capable of contacting the epidermis or surface of the skin. Examples of such devices include, but are not limited to, a wrap, bandage, bind, dress, suture, patch, gauze or dressing.

[0446] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0447] As used herein, ranges and amounts can be expressed as “about” or “approximately” a particular value or range. “About” or “approximately” also includes the exact amount. Hence, “about 5 milliliters” means “about 5 milliliters” and also “5 milliliters.” Generally, “about” includes an amount that is expected to be within experimental error for the parameter.

[0448] As used herein, “about the same” means within an amount that one of skill in the art considers to be the same or to be within an acceptable range of error. For example, typically, for pharmaceutical compositions, within at least 1%, 2%, 3%, 4%, 5% or 10% Attorney Docket No. 120276-2615PC

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[0450] is considered about the same. Such amounts can vary depending upon the tolerance for variation in the composition by subjects.

[0451] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0452] As used herein, "allogeneic cells" are cells that are genetically different with respect to a particular subject because they are derived from genetically different individual, generally of the same species. For example, allogeneic stem cells are stem cells that are derived from a donor other than the patient (or identical twin).

[0453] As used herein, "autologous cells" are cells obtained from the individual to be treated with the cells. For example, autologous cells are obtained from the subject to be treated (i.e., the patient). For example, autologous stem cells are stem cells that are derived from the patient.

[0454] As used herein, induced pluripotent stem cells (iPSCs) are cells that can be reprogrammed from skin or blood cells to have the same properties as embryonic stem cells. iPSCs can differentiate into any cell type in the body, except for cells in the placenta. The iPSCs can be generated from the subject to be treated, or can be allogeneic, including from commercial or academic sources.

[0455] As used herein, induced pluripotent stem cells (iPSCs) are cells that can be reprogrammed from skin or blood cells to have the same properties as embryonic stem cells. iPSCs can differentiate into any cell type in the body, except for cells in the placenta. The iPSCs can be generated from the subject to be treated, or can be allogeneic, including from commercial or academic sources.

[0456] As used herein, the term "engineered," with respect to cell vehicles or carrier cells, denotes the genetic modification of the cells, such that they express proteins that can improve or enhance the performance of the cells. For example, cells can be engineered for improved viral amplification and / or improved immunomodulation.

[0457] As used herein, "immunomodulation" refers to any process in which an immune response is modified to a desired level, for example by inducing, enhancing or suppressing an immune response.

[0458] As used herein, '"immune suppression" or "immunosuppression" refers to the suppression or reduction of the immune response. Attorney Docket No. 120276-2615PC

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[0460] As used herein, "immune privileged" or "immunoprivileged" refers to cells or tissues that do not elicit an immune response and can evade the immune system.

[0461] Immunoprivileged cells and tissues refer to cells and tissues, such as solid tumors and the tumor microenvironment, which are sequestered from the immune system by virtue of immunosuppressive properties of tumors. As a result, oncolytic viruses preferentially accumulate in tumors in the tumor microenvironment because they are shielded from the immune system. Immunoprivileged tissues and cells, however, are shielded or sequestered from the immune response, permitting the viruses to survive and generally to replicate.

[0462] As used herein, "resistant" with respect to viral infection refers to a cell that is not infected, or is infected to a very low degree, with a virus upon exposure to the virus.

[0463] As used herein, "permissive" with respect to viral infection refers to a cell that is readily infected upon exposure to the virus.

[0464] As used herein, immunologically compatible refers to a cell or virus that is sufficiently compatible with the immune system of the subject / host, to evade the subject's immune system for a sufficient time to deliver virus to a tumor or cancerous cell in the subject.

[0465] As used herein, "co-culture" refers to a cell culture in which two or more different populations of cells are grown.

[0466] As used herein the term "loading," with respect to cells, can refer to the association of a cell with an agent, such as, for example, a virus, small molecule, therapeutic agent, and antibody or antigen binding fragment of thereof, through a chemical or physical interaction between the cell and the agent on the surface of the cell or inside the cell.

[0467] As used herein, " ACAM2000" (AC AMI 000 and ACAM2000, which have the same genomic sequence, deposited as ATCC Deposit No. PTA-3321; see, U. S. Patent Nos. 6,723,325, 6,723,325, 7,115,270 and 7,645,456) is a wild type thymidine kinase (TK)-positive Wyeth strain of vaccinia virus. It is a smallpox vaccine strain that is available from the CDC. ACAM1000 is the designation of the virus when propagated in MRC5 cells; ACAM2000 is the designation of the virus when propagated in Vero cells. In embodiments, the ACAM2000 virus has the sequence set forth in SEQ ID NO:25. Attorney Docket No. 120276-2615PC

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[0469] As used herein, “CAL-01” or “CALI” used interchangeably herein, designates a virus that is amplified or cultured from ACAM2000 or ACAM1000. In exemplary embodiments, the CAL1 virus has the sequence set forth in SEQ ID NO:251. CAL2 is CAL1 encoding TurboFP635 at the TK locus.

[0470] As used herein, “inactivation” of a gene or genetic locus means that the expression of one or more products encoded by the gene or locus is partially or completely inhibited, e.g., by 10% or more, generally by 50% or more, e.g., about or at 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. The inactivation can be effected, e.g., by partial or complete truncation of a locus and / or by insertion of an exogenous gene, such as a therapeutic gene. A knockout means that the gene is not expressed.

[0471] As used herein, a human protein is one encoded by a nucleic acid molecule, such as DNA, present in the genome of a human, including all allelic variants and conservative variations thereof. A variant or modification of a protein is a human protein if the modification is based on the wild type or prominent sequence of a human protein.

[0472] As used herein, the residues of naturally occurring a-amino acids are the residues of those 20 a-amino acids found in nature which are incorporated into protein by the specific recognition of the charged tRNA molecule with its cognate mRNA codon in humans.

[0473] As used herein, non-naturally occurring amino acids refer to amino acids that are not genetically encoded.

[0474] As used herein, “nucleic acid” refers to at least two linked nucleotides or nucleotide derivatives, including a deoxyribonucleic acid (DNA) and a ribonucleic acid (RNA) and analogs thereof, joined together, typically by phosphodiester linkages. Also included in the term “nucleic acid” are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives or combinations thereof. Nucleic acids also include DNA and RNA derivatives containing, for example, a nucleotide analog or a "backbone" bond other than a phosphodiester bond, for example, a phosphotriester bond, a phosphoramidate bond, a phosphorothioate bond, a thioester bond, or a peptide bond (peptide nucleic acid). The term also includes, as equivalents, derivatives, variants and analogs of either RNA or DNA made from Attorney Docket No. 120276-2615PC

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[0476] nucleotide analogs, single (sense or antisense) and double-stranded nucleic acids.

[0477] Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the uracil base is uridine. Nucleic acids can be single or double-stranded. When referring to probes or primers, which are optionally labeled, such as with a detectable label, such as a fluorescent or radiolabel, single-stranded molecules are contemplated. Such molecules are typically of a length such that their target is statistically unique or of low copy number (typically less than 5, generally less than 3) for probing or priming a library. Generally, a probe or primer contains at least 14, 16 or 30 contiguous nucleotides of sequence complementary to or identical to a gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleotides long.

[0478] As used herein, an isolated nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0479] As used herein, “synthetic,” with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and / or by chemical synthesis methods.

[0480] As used herein, “polypeptide” refers to two or more amino acids covalently joined. The terms “polypeptide” and “protein” are used interchangeably herein.

[0481] As used herein, a “peptide” refers to a polypeptide that is from 2 to about or 40 amino acids in length.

[0482] As used herein, the amino acids which occur in the various sequences of amino acids provided herein are identified according to their known, three-letter or one-letter abbreviations (Table 2). The nucleotides which occur in the various nucleic acid fragments are designated with the standard single-letter designations used routinely in the art.

[0483] As used herein, an “amino acid” is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes herein, amino acids include the twenty naturally-occurring amino acids (Table Attorney Docket No. 120276-2615PC

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[0485] 2), non-natural amino acids and amino acid analogs ( / .<?., amino acids wherein the a-carbon has a side chain).

[0486] As used herein, the amino acids, which occur in the various amino acid sequences of polypeptides herein, are identified according to their well-known, three-letter or one-letter abbreviations (see Table 2). The nucleotides, which occur in the various nucleic acid molecules and fragments, are designated with the standard singleletter designations used routinely in the art.

[0487] As used herein, “amino acid residue” refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are presumed to be in the “L” isomeric form. Residues in the “D” isomeric form, which are so designated, can be substituted for any L-amino acid residue as long as the desired functional property is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described in J. Biol. Chem., 243: 3557-3559 (1968), and adopted in 37 C. F. R. §§ 1.821-1.822, abbreviations for amino acid residues are shown in Table 2:

[0488] Table 2 - Table of Correspondence

[0489] SYMBOL

[0490] 1-Letter 3-Letter AMINO ACID

[0491] Y Tyr Tyrosine

[0492] G Gly Glycine

[0493] F Phe Phenylalanine

[0494] M Met Methionine

[0495] A Ala Alanine

[0496] S Ser Serine

[0497] I He Isoleucine

[0498] L Leu Leucine

[0499] T Thr Threonine

[0500] V Vai Valine

[0501] P Pro Proline

[0502] K Lys Lysine

[0503] H His Histidine

[0504] Q Gin Glutamine

[0505] E Glu Glutamic acid

[0506] Z Glx Glu and / or Gin

[0507] W Trp Tryptophan

[0508] R Arg Arginine

[0509]

[0510] D Asp Aspartic acid Attorney Docket No. 120276-2615PC

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[0512] SYMBOL

[0513] 1-Letter 3-Letter AMINO ACID

[0514] N Asn Asparagine

[0515] B Asx Asn and / or Asp

[0516] C Cys Cysteine

[0517]

[0518] X Xaa Unknown or other

[0519] All sequences of amino acid residues represented herein by a formula have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus. In addition, the phrase “amino acid residue” is defined to include the amino acids listed in the Table of Correspondence (Table 2), modified, non-natural and unusual amino acids. Furthermore, a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to an amino-terminal group such as NH2 or to a carboxyl-terminal group such as COOH.

[0520] As used herein, “naturally occurring amino acids” refer to the 20 L-amino acids that occur in polypeptides. As used herein, the residues of naturally occurring a-amino acids are the residues of those 20 a-amino acids found in nature which are incorporated into protein by the specific recognition of the charged tRNA molecule with its cognate mRNA codon in humans.

[0521] As used herein, “non-natural amino acid” refers to an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid. Non-naturally occurring amino acids, thus, include, for example, amino acids or analogs of amino acids other than the 20 naturally occurring amino acids and include, but are not limited to, the D-stereoisomers of amino acids. Exemplary non-natural amino acids are known to those of skill in the art, and include, but are not limited to, para-acetyl Phenylalanine, para-azido Phenylalanine, 2 -Aminoadipic acid (Aad), 3 -Aminoadipic acid (bAad), P-alanine / p -Amino-propionic acid (Bala), 2-Aminobutyric acid (Abu), 4-Aminobutyric acid / piperidinic acid (4Abu), 6-Aminocaproic acid (Acp), 2-Aminoheptanoic acid (Ahe), 2-Aminoisobutyric acid (Aib), 3-Aminoisobutyric acid (Baib), 2-Aminopimelic acid (Apm), 2,4-Diaminobutyric acid (Dbu), Desmosine (Des), 2,2'-Diaminopimelic acid (Dpm), 2,3 -Diaminopropionic acid (Dpr), N-Ethylglycine (EtGly), N-Ethylasparagine (EtAsn), Hydroxylysine (Hyl), allo-Hydroxylysine (Ahyl), 3-Hydroxyproline (3Hyp), 4-Hydroxyproline (4Hyp), Isodesmosine (Ide), allo-Isoleucine (Aile), N-Methylglycine, sarcosine (MeGly), N- Attorney Docket No. 120276-2615PC

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[0523] Methylisoleucine (Melle), 6-N-Methyllysine (MeLys), N-Methylvaline (MeVal), Norvaline (Nva), Norleucine (Nle), and Ornithine (Orn). Exemplary non-natural amino acids are described herein and are known to those of skill in the art.

[0524] As used herein, an isokinetic mixture is one in which the molar ratios of amino acids has been adjusted based on their reported reaction rates (see, e.g., Ostresh et al. (1994) Biopolymers 34:1681).

[0525] As used herein, a DNA construct is a single or double stranded, linear or circular DNA molecule that contains segments of DNA combined and juxtaposed in a manner not found in nature. DNA constructs exist as a result of human manipulation, and include clones and other copies of manipulated molecules.

[0526] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in the art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0527] Such substitutions can be made in accordance with the exemplary substitutions set forth in the following Table:

[0528] Exemplary Conservative Amino Acid Substitutions

[0529] Exemplary

[0530] Original Residue Conservative

[0531] Substitution(s)

[0532] Ala (A) Gly; Ser

[0533] Arg (R) Lys

[0534] Asn (N) Gin; His

[0535] Cys (C) Ser

[0536] Gln (Q) Asn

[0537] Glu (E) Asp

[0538] Gly (G) Ala; Pro

[0539] His (H) Asn; Gin

[0540] lie (I) Leu; Vai

[0541] Leu (L) He; Vai

[0542] Lys (K) Arg; Gin; Glu

[0543] Met (M) Leu; Tyr; lie

[0544] Phe (F) Met; Leu; Tyr

[0545] Ser (S) Thr

[0546] Thr(T) Ser

[0547] Trp (W) Tyr

[0548]

[0549] Tyr(Y) Trp; Phe Attorney Docket No. 120276-2615PC

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[0551] Exemplary

[0552] Original Residue Conservative

[0553] Substitution(s)

[0554]

[0555] Vai (V) He; Leu

[0556] Other substitutions also are permissible and can be determined empirically or in accord with other known conservative or non-conservative substitutions.

[0557] As used herein, “naturally occurring amino acids” refer to the 20 L-amino acids that occur in polypeptides.

[0558] As used herein, the abbreviations for any protective groups, amino acids and other compounds are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726).

[0559] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.

[0560] B. OVERVIEW

[0561] Provided are enveloped viruses, such as vaccinia viruses, for systemic administration, and methods and cells for manufacturing viruses. The viruses can be programmed to have or exhibit modified or new properties and / or activities. Vaccinia viruses, which are exemplary of poxviruses, are modified or selected to produce a high level of EEV form of virus. The EEV form contains a host cell-derived second membrane which contains a non-virally encoded polypeptide displayed on the host-derived membrane. This is achieved by modifying the host cells to express non-virally encoded polypeptide or modifying the genome of the virus to encode heterologous proteins, and particularly by modifying virally-encoded transmembrane protein that are expressed on the surface of the second (also referred to as the outer) membrane. These proteins and modified EEV viruses are detailed herein and in co-pending application International PCT application No. PCT / US2025 / 017701, published as WO2025 / 184411 on September 04, 2025. These modified proteins, which are virally encoded, are expressed independently of the cell, in vivo or in vitro, in which the virus propagated. The viruses also are modified and new properties / activities are conferred by virtue of the host cell in which the viruses are propagated. The host cells can be modified to encode proteins for expression in the host cell membranes and are thereby incorporated on the second membrane in the viruses. The host cell can be modified so that membranes Attorney Docket No. 120276-2615PC

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[0563] express or display polypeptides or peptides that confer properties or activities on the EEV virus. These properties / activities, however, are not retained in progeny virus that are produced in different cells, since the second membrane is derived from the host. Modified host cells that contain the EEV virus are provided herein.

[0564] Provided are compositions of viruses that contain high percentages, such as more the 1%, 2%, 3%, 4%, 5% and more, of EEV forms of poxviruses, such as vaccinia virus. As described herein, provided for example are high EEV vaccinia viruses and derivatives thereof. These viruses are selected or designed or modified to produce a high level of EEVs and, optionally, for increased anti-tumor activity. Also provided are derivatives of these viruses that have particular knock-outs that increase the amount of EEVs that are produced, and also have other modifications. Also provided are derivatives of these viruses and any vaccinia viruses that are modified to have chimeric second membrane proteins outer membrane encoded by the virus, such that the chimeric portion is displayed on the membrane. Generally, the chimeric protein is a chimera or fusion protein between a virally-encoded outer membrane protein and an immunomodulatory protein that, when expressed on the surface of the virus increases serum resistance. Also provided are methods of manufacturing EEVs; the method results in compositions that contain a high percentage of EEVs, such as at least 60%, 70%, 80% or more of the virus is an EEV virus. Modified cell lines for producing EEV viruses also are provided. Since the second envelope is derived from the cell in which the virus is produced, cells can be modified so that the membranes on the EEV display functional polypeptides for increasing serum resistance, and / or for targeting tumors, or for interacting with therapeutic molecules or other cells.

[0565] Provided herein are enveloped viruses that are encased in a host cell-derived membrane. The host cell derived membraned encases the virus and includes virally encoded transmembrane proteins, which in a fusion protein or chimeric protein that contains a virally-encoded transmembrane protein and a heterologous protein or polypeptide or functional portion thereof that confers or alters property or activity of the virus. The host-derived membrane also comprises host cell-encoded modified host transmembrane proteins that are fusion proteins or chimeric proteins between the host cell transmembrane protein and a heterologous polypeptide r functional portion thereof that confers or alters a viral activity or property. The viruses are viruses that are encased Attorney Docket No. 120276-2615PC

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[0567] in a host-derived membrane, such as RNA viruses, and also viruses that are encased in viral membrane and in an outer membrane that is derived from the host in which the viruses are propagated. Poxviruses, such as vaccinia virus EEV forms, are exemplary of the latter viruses. These viruses are produced in vitro in host cells from which the membranes are derived, and such host cells, are modified to express on their membranes, generally as fusion or chimera with a host cell transmembrane protein.

[0568] Provided herein are modified host cells, cell lines and compositions containing the host cells or cell lines for propagating the viruses, the modified host cells containing the viruses, and methods for propagating and purifying EEV viruses, particularly EEV vaccinia viruses. The methods include steps to minimize the loss of the second membrane that occurs during purification. Also provided are exemplary host cells, such as iPSCs, that as detailed herein are advantageous for modification and for propagating EEV viruses. iPSCs can be readily obtained and / or modified to express polypeptides or peptides on the membranes to provide for the EEV vaccinia viruses that are propagated in the host cells. As shown herein, iPSCs are effective host cells for propagating EEV viruses. Even without modification they express complement regulatory proteins and other such proteins that confer serum resistance on the results EEV viruses. The iPSCs can be modified to increase their expression of the complement regulatory proteins and other immune modulatory proteins, and to express other proteins / polypeptides / peptides on the cell membranes for deposition on viruses that acquire a cell membrane from the host. This is particularly advantageous for culturing / propagating vaccinia viruses that produce high levels of EEV forms of the virus.

[0569] As described herein, the modified host cells can provide additional programming to EEV viruses, such as EEV vaccinia viruses that have been selected or modified to produce high levels of EEV forms of the viruses. The resulting viruses that express virally encoded proteins in the second membrane and also host-derived proteins that can confer properties and activities are provided. These properties / activities can be chosen based on the intended use of the viruses. They can alter tropism of the virus, increase serum stability, target the virus to particular cells, for treatment of different diseases. The viruses also can be genome-modified to encode therapeutic or active products for treating diseases, disorders, and conditions in which the target cell type is involved, and / or for diagnosis of a disease, disorder, and / or condition, and / for monitoring treatment. Attorney Docket No. 120276-2615PC

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[0571] All of these aspects of the cells, viruses, methods, uses, and others are detailed in the sections and Examples that follow throughout this section, the Detailed Description, Examples, and claims.

[0572] Vaccinia Viruses and other Poxviruses for Systemic Administration Oncolytic viral therapy uses naturally occurring or genetically engineered viruses to selectively lyse tumor cells. Oncolytic viruses preferentially accumulate in and then replicate in tumor tissue and in cells and tissues other immunoprivileged and immunosuppressed environments. Oncolytic viruses, including vaccinia viruses, exert anti-tumor effects via oncolysis and activation of anti-tumor immune responses.

[0573] Upon infection of tumors with virus, the tumor microenvironment is remodeled to stimulate an anti-tumor immune response, the viruses amplify, and lyse tumor cells. Thereby releasing virus and tumor antigens; the viruses, if they can disseminate and are not destroyed by the immune system of the host can infect other tumors, and released tumor antigens can result in an anti -turn or response. For successful therapy, the viruses must be able to disseminate and must survive attack by the immune system of the host.

[0574] Oncolytic therapy has had limited success. The successful oncolytic virotherapies require administration by intratumoral or local injection. Approved oncolytic viruses for cancer therapy require local or intratumorally administration. These viruses include Rigvir® (enteric cytopathic human orphan virus approved in Latvia for melanoma in 2004), Oncorine® (adenovirus approved in China for head and neck cancer in 2005), T-VEC® (HSV approved in the United States for melanoma in 2015) and DEL YT ACT® (HSV approved in Japan for glioblastoma in 2021). For example, T-VEC is approved in patient without visceral metastases.

[0575] Effective systemic administration of virotherapy has not been successful for several reasons, including the presence of complement and neutralizing antibodies in the bloodstream, and dilution in the bloodstream when administrated. For cancers that metastasize or tumors that cannot be reached by intratumoral administration, oncolytic therapy has not been effective. Systemic delivery of therapeutic levels of oncolytic viruses, such as vaccinia virus, has not been achieved.

[0576] Systemic delivery of virotherapy has been an elusive goal. Systemic delivery provide advantages for treatment of many cancers, including late-stage and metastatic cancers that are not effectively treated by intratumoral administration. Systemic delivery Attorney Docket No. 120276-2615PC

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[0578] of oncolytic viruses can target cancer cells that are inaccessible by localized therapies and provides the ability to target multiple tumor sites upon administration.

[0579] Provided herein are viruses for systemic administration. Effective systemic administration of virotherapy has not been effected for several reasons, including the presence of complement and neutralizing antibodies in the bloodstream, and the dilution of the oncolytic virus in the bloodstream when systemically administrated. The neutralization systemically administered viruses by the host immune system limits the amount of virus that can reach and amplify in tumors and tumor metastasis to effect therapy.

[0580] The success of treatment using oncolytic viruses is limited by host (adaptive and innate) immune responses. The presence of pre-existing antiviral neutralizing antibodies and the development of anti-oncolytic viral neutralizing antibodies during therapy has limited the therapeutic potential of oncolytic viral therapy. For example, neutralizing antibodies bind viruses, block their attachment to cell surface receptors and inhibit viral infection (Jennings etal. (2014) Int. J Cancer 134: 1091-1101). Previous exposure can result in adaptive immunity, leading to more specific and potent anti-viral immunity. Older subjects have been vaccinated against smallpox, resulting in pre-existing antiviral immunity against Ortho poxviruses, including vaccinia virus. Even if a subject does not already possess pre-existing immunity to a specific virus, the initial dose of an oncolytic virus results in a robust anti-viral immune response, limiting the effectiveness of repeated doses, which are often required to achieve a potent anti-tumor response.

[0581] Provided herein are vaccinia viruses for systemic therapy and methods for modifying any vaccinia virus so that the virus can be systemically administered and be therapeutically effective for treating cancers. Similar modifications can be applied to other poxviruses, such as, for example, entomopox, monkeypox, swinepox and pinguin pox, to produce viruses that can be systemically administered. The methods, modifications, and virus products are exemplified with vaccinia; it is understood that the methods and modifications can be practiced with other poxviruses to produce systemically administrable virus products for use as therapeutics and vaccines. The viruses can be further modified / programmed by propagating them in particular host cells, including modified host cells as detailed herein. Attorney Docket No. 120276-2615PC

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[0583] Vaccinia viruses are large DNA viruses that have been used as smallpox vaccines. Vaccinia viruses have a broad host and cell type range, they are not limited by receptors during infection, and the virus exhibits high infectivity in various host species and a large range of tissues. Vaccinia virus produces four different types of virion from each infected cell: intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), and extracellular enveloped virus (EEV). These virions occur in different abundance, structure, and location, and have different roles in the virus life-cycle. The IEV and CEV are EEV precursors. IMV is the most abundant form of virus and is retained in cells until lysis; it is a robust, stable virion and is well suited to transmit infection between hosts. IEV is formed by wrapping of IMV with intracellular membranes, and is an intermediate between IMV and CEV / EEV that provides virus dissemination to the cell surface on microtubules. CEV induces the formation of actin tails that drive CEV particles away from the cell and is facilitates for cell-to-cell spread. EEV mediates dissemination of virus. Seven virus-encoded proteins have been identified that are components of IEV, and five of them are present in CEV or EEV.

[0584] Intracellular mature virus (IMV) particles are formed in the from non-infectious precursors called crescents and immature virus (IV). IMV generally constitute the majority of infectious progeny and most remain within the cell until lysis. Some IMV leave the cytoplasm on microtubules and become wrapped by a double layer of intracellular membrane derived from the early endosomes or trans-Golgi network (TGN) to form intracellular enveloped virus (IEV). IEV move on microtubules to the cell surface where the outer membrane fuses with the plasma membrane to thereby present an enveloped virion on the cell surface. Particles retained on the cell surface are cell-associated enveloped virus (CEV) and those released are called extracellular enveloped virus (EEV). CEV and EEV are physically indistinguishable and contain one fewer membrane than IEV and one more membrane than IMV. CEV induce the formation of actin tails that drive the virions away from the cell and are important for cell-to-cell spread. EEV mediate long-range dissemination of virus in vivo.

[0585] Because EEV viruses disseminate in vivo, they are candidates for viruses for systemic administration. Vaccinia viruses that produce relatively high levels or EEV have been sought or produced for systemic administration. It is known that a mutation in Attorney Docket No. 120276-2615PC

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[0587] the A34 gene results in viruses that produce high levels of EEVs. Thus, far, however, systemic administration has not been successful. EEVs that are propagated in cells that express complement resistant proteins, such as CD55, by virtue of inclusion of the host cell membrane in the EEV, for the first generation, exhibit resistance to host innate humoral immunity. Since such resistance is a function of the host cell in which they are propagated, once they infect tumor cells in vivo, unless the tumor is one that produces proteins in the membrane that resist the host cell immune system, the progeny viruses lose the resistance when they amplify in a tumor.

[0588] Provided herein are vaccinia virus that have advantageous properties so that they can be therapeutically effective upon systemic administration. As detailed herein, a known high EEV producing vaccinia virus isolate was propagated and a clone that produces high levels of EEV and that has high anti -tumor activity was selected. This virus designated RedTail-00 (RT-00; N2 referencing the source of the virus) was selected for its high level of EEVs and its selectivity for tumors and high anti-tumor activity. Differences between this virus and other known EEV producers are described herein. As described herein, knockouts of various genes and combinations thereof can be introduced to increase the resistance of the virus to the innate humoral immunity of human hosts. The virus and its derivatives (see, e.g., Figure 24) are suitable for systemic delivery. The combination of the high EEV production, resistance to the immune system of the host, the accumulation in tumors, and high anti-tumor activity, render this virus, and its derivatives and viruses similarly produced suitable for systemic administration. The knockouts detailed herein can be introduced into EEV viruses known in the art to improve their resistance to the immunity system of the host.

[0589] Additionally, and significantly, provided herein is a solution to the problem that the resistance of the EEV to the immune system of the host is a function of the host cell in which the EEV are produced, and is eliminated when they propagate in the tumor in the host, unless the tumor is a rare tumor that happens to produce high levels of a complement resistance protein. As detailed in sections that follow, a complement resistance protein or sufficient portion thereof or other such protein to confer resistance, is introduced into a virally-encoded transmembrane protein to produce a chimeric protein that is expressed on the EEV membrane, thereby introducing resistance to humoral immunity into the membrane such that when the virus propagates, the protein is Attorney Docket No. 120276-2615PC

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[0591] expressed on the EEV membrane. Exemplary of this, the RT-00 virus has been modified by introducing a CD 55 protein (or domain thereof that confers resistance, into a virally-encoded transmembrane protein, such as B5R. The resulting virus is a high EEV producer and also has resistance to the host humoral immune system. The chimeric protein also can be expressed in derivatives of RT-00 (or other EEV viruses) that have additional modifications that increase resistance to the host immune system or otherwise increases the amount of virus produced, particularly in tumors.

[0592] EEV viruses are difficult to manufacture because the second membrane is fragile and is lost during the manufacturing process, thereby lowering the percentage of EEVs produced. Provided herein a manufacturing process that preserves this membrane so that the yield of EEVs is high. The process, described herein, gently isolates the EEVs so that the membranes remain intact.

[0593] Hence, provided are methods to produce vaccinia viruses for systemic administration. The methods can be applied to any known vaccinia virus, particularly any developed as anti-tumor therapeutics. Any such virus can be modified by mutation or selection to 1) produce high levels of EEVs, and 2) encode a transmembrane chimeric protein on the EEV membrane to confer heritable resistance to the host immune system. Optionally additional modifications as described herein can be introduced into the resulting viruses to confer further resistance to the host cell immune system or other advantageous properties. The viruses also can be modified to encode therapeutic products, proteins or nucleic acids, and other such payloads, such as gene editing systems for modification the tumor cell genome. The viruses can be further modified to encode antigens expressed on tumor cell surfaces for combination therapy with targeted cell immunotherapies or other therapies.

[0594] As provided herein are viruses that produce high levels, 1% or greater, such as 5%- 10% EEV particles. Also provided are methods for producing or manufacturing EEV preparations. In general, the methods herein result in compositions in which at least about 60%, 70%, 80%, 90%, and more of the virus particles are EEVs. Also provided herein, are methods for modifying the viruses, so that they are resistant or have increased serum resistance or stability compared to the unmodified virus, to the immune system of the host. The modified viruses can be propagated by the methods herein, and result in Attorney Docket No. 120276-2615PC

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[0596] viruses that exhibit 80% and greater serum resistance. These and other properties are described and exemplified in the sections and Examples below.

[0597] It is understood that the methods and uses and viruses are detailed with respect to vaccinia virus, but the same modifications, methods and uses can be applied to other enveloped viruses, such as any poxvirus or herpes virus or other virus that acquires a second membrane from a host cell, and also to viruses, such as retroviruses, such as lentiviruses, that have single membranes, where the membrane is acquired from the host.

[0598] C. EXTRA ENVELOPED VIRUSES (EEV) AS EXEMPLIFIED BY EEV VACCINIA VIRUSES (EEVs)

[0599] Certain virus families are known to poses an outer membrane or envelope which typically is derived from host cell membranes. In general, viral envelops contain a lipid bilayer membrane which can contain virus-encoded proteins, transmembrane proteins, and host-derived proteins. Viruses that acquire their envelope by budding through the plasma membrane of the host cell where the lipid composition of the viral envelope can reflect that of the host membrane. Enveloped viruses can carry host cell proteins as components of the viral envelope. In addition to virus-specified envelope proteins, a lipid bilayer envelope from the host cell membrane, the viral envelop can be embedded with glycoproteins which aid in host cell entry, which can aid in the efficient delivery of genetic material, making them ideal for gene therapy (Gelderblom, H. R., Medical Microbiology. 4th edition. Ch 41.).

[0600] Families of exemplary enveloped viruses include Herpesviridae, Iridoviridae. Poxviridae, Hepadnaviridae, Togaviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Bunyaviridae, Coronaviridae, Arenaviridae.

[0601] Retroviridae, and Flioviridae. Vaccinia virus, a notable enveloped virus, is particularly valuable in genetic medicine due to its large genome, accommodating significant genetic insertions and the incorporation of transgenes, direct tumor targeting, and selective replication within cancer cells while sparing normal tissues. These and other oncolytic properties allow vaccinia virus to deliver therapeutic genes and induce tumor cell lysis and thereby enhance the anti-tumor effects. By targeting tumors specifically, vaccinia virus-based therapies improve the precision and effectiveness of cancer treatments, reducing off-target effects and minimizing damage to healthy tissues. Attorney Docket No. 120276-2615PC

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[0603] Provided herein are EEV viruses, methods for producing EEV viruses, and methods and modifications for increasing resistance of EEV viruses to humoral immunity.

[0604] 1. Vaccinia viruses

[0605] Vaccinia Virus

[0606] Examples of vaccinia viruses include, but are not limited to, Lister (also known as Elstree), New York City Board of Health (NYCBH), Dairen, Ikeda, LC16M8, Western Reserve (WR), Copenhagen (Cop), Tashkent, Tian Tan, Wyeth, Dryvax, IHD-J, IHD-W, Brighton, Ankara, Modified Vaccinia Ankara (MV A), Dairen I, LIPV, LC16M0, LIVP, WR 65-16, EM63, Bern, Paris, CVA382, NYVAC, ACAM2000, ACAM1000 and Connaught strains. Vaccinia viruses are oncolytic viruses that possess a variety of features that make them particularly suitable for use in wound and cancer gene therapy. For example, vaccinia is a cytoplasmic virus, thus, it does not insert its genome into the host genome during its life cycle. Unlike many other viruses that require the host’s transcription machinery, vaccinia virus can support its own gene expression in the host cell cytoplasm using enzymes encoded in the viral genome. Vaccinia viruses also have a broad host and cell type range. In particular, vaccinia viruses can accumulate in immunoprivileged cells or immunoprivileged tissues, including tumors and / or metastases, and also including wounded tissues and cells. Yet, unlike other oncolytic viruses, vaccinia virus can typically be cleared from the subject to whom the viruses are administered by activity of the subject’s immune system, and hence are less toxic than other viruses such as adenoviruses. Thus, while the viruses can typically be cleared from the subject to whom the viruses are administered by activity of the subject’s immune system, viruses can nevertheless accumulate, survive and proliferate in immunoprivileged cells and tissues such as tumors, because such immunoprivileged areas are isolated from the host’s immune system.

[0607] Vaccinia viruses also can be modified by insertion of heterologous genes. This can result in the attenuation of the virus and / or permit delivery of therapeutic proteins. For example, the vaccinia virus genome has a large carrying capacity for foreign genes, where up to 25 kb of exogenous DNA fragments (approximately 12% of the vaccinia genome size) can be inserted. The genomes of several of the vaccinia strains have been completely sequenced, and many essential and nonessential genes identified. Due to high sequence homology among different strains, genomic information from one vaccinia Attorney Docket No. 120276-2615PC

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[0609] strain can be used for designing and generating modified viruses in other strains. Finally, the techniques for production of modified vaccinia strains by genetic engineering are well established (Moss (1993) Curr. Opin. Genet. Dev. 3:86-90; Broder and Earl, (1999) Mol. Biotechnol. 13:223-245; Timiryasova et al. (2001) Biotechniques 31:534-540).

[0610] Various vaccinia viruses have been demonstrated to exhibit antitumor activities. In one study, for example, nude mice bearing non-metastatic colon adenocarcinoma cells were systemically injected with a WR strain of vaccinia virus modified by having a vaccinia growth factor deletion and an enhanced green fluorescence protein inserted into the thymidine kinase locus. The virus was observed to have antitumor effects, including one complete response, despite a lack of exogenous therapeutic genes in the modified virus (McCart et al. (2001) Cancer Res. 1:8751-8757). In another study, vaccinia melanoma oncolysate (VMO) was injected into sites near melanoma positive lymph nodes in a Phase III clinical trial of melanoma patients. As a control, a New York City Board of Health strain vaccinia virus (VV) was administered to melanoma patients. The melanoma patients treated with VMO had a survival rate better than that for untreated patients, but similar to patients treated with the VV control (Kim et al. (2001) Surgical Oncol. 10:53-59).

[0611] LIVP strains of vaccinia virus also have been used for the diagnosis and therapy of tumors, and for the treatment of wounded and inflamed tissues and cells (see e.g., Zhang etal. (2007) Surgery 142:976-983; Lin et al. (2008) J. Clin. Endocrinol. Metab.

[0612] 93:4403-7; Kelly etal. (2008) Hum. Gene Ther. 19:774-782; Yu etal. (2009) Mol.

[0613] Cancer Ther. 8:141-151; Nu etal. (2009) Mol. Cancer 8:45; U. S. Patent No. 7,588,767; U. S. Patent No. 8,052,968; and U. S. Publication No. 2004 / 0234455). For example, when intravenously administered, LIVP strains have been demonstrated to accumulate in internal tumors at various loci in vivo, and have been demonstrated to effectively treat human tumors of various tissue origin, including, but not limited to, breast tumors, thyroid tumors, pancreatic tumors, metastatic tumors of pleural mesothelioma, squamous cell carcinoma, lung carcinoma and ovarian tumors. LIVP strains of vaccinia, including attenuated forms thereof, exhibit less toxicity than WR strains of vaccinia virus, and result in increased and longer survival of treated tumor-bearing animal models (see, e.g., U. S. Publication No. 2011 / 0293527). Wyeth strains of vaccinia virus, such as JX-594, also exhibit lower toxicity, and have been used for the treatment of cancers. Attorney Docket No. 120276-2615PC

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[0615] Vaccinia is a cytoplasmic virus; thus, it does not insert its genome into the host genome during its life cycle. Vaccinia virus has a linear, double-stranded DNA genome of approximately 180,000 base pairs in length that is made up of a single continuous polynucleotide chain (Baroudy et al. (1982) Cell 28:315-324). The structure is due to the presence of 10,000 base pair inverted terminal repeats (ITRs). The ITRs are involved in genome replication. Genome replication involves self-priming, leading to the formation of high molecular weight concatemers (isolated from infected cells) which are subsequently cleaved and repaired to make virus genomes (see, e.g., Traktman, P., Chapter 27, Poxvirus DNA Replication, pp. 775-798, in DNA Replication in Eukaryotic Cells, Cold Spring Harbor Laboratory Press (1996)). The genome contains approximately 250 genes. In general, the non-segmented, non-infectious genome is arranged such that centrally located genes are essential for virus replication (and are thus conserved), while genes near the two termini effect more peripheral functions such as host range and virulence. Vaccinia viruses practice differential gene expression by using open reading frames (ORFs) arranged in sets that, as a general principle, do not overlap.

[0616] Vaccinia virus possesses a variety of features for use in cancer gene therapy and vaccination including broad host and cell type range, and low toxicity. For example, while most oncolytic viruses are natural pathogens, vaccinia virus has a unique history in its widespread application as a smallpox vaccine that has resulted in an established track record of safety in humans. Toxicities related to vaccinia administration occur in less than 0.1% of cases, and can be effectively addressed with immunoglobulin administration. In addition, vaccinia virus possesses a large carrying capacity for foreign genes (up to 25 kb of exogenous DNA fragments, approximately 12% of the vaccinia genome size, can be inserted into the vaccinia genome) and high sequence homology among different strains for designing and generating modified viruses in other strains. Techniques for production of modified vaccinia strains by genetic engineering are well established (Moss (1993) Curr. Opin. Genet. Dev. 3: 86-90; Broder and Earl (1999) Mol. Biotechnol. 13: 223-245; Timiryasova et al. (2001) Biotechniques 31: 534-540). Vaccinia virus strains have been shown to specifically colonize solid tumors, while not infecting other organs (see, e.g., Zhang et al. (2007) Cancer Res. 67:10038-10046; Yu et al.

[0617] (2004) Nat. Biotech. 22:313-320; Heo et al. (2011) Mol. Ther. 19:1170-1179; Liu et al. (2008)Afo / . Ther. 16:1637-1642; Park c / . (2008) Lancet Oncol. 9:533-542). Attorney Docket No. 120276-2615PC

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[0619] 2. Vaccinia Virus EEV and Their Production

[0620] Vaccinia virus, exemplary of poxviruses, has two infectious forms, which differ in their structure and number of membranes. One form, the Intracellular Mature Virus (IMV), generally is the most abundant form and has a single, very stable, envelope. This single envelope is derived from the virus's own assembly process within the cytoplasm (known as "viral factories"). The other form is the Extracellular Enveloped Virus (EEV): A fraction of the IMV particles acquire an additional, second, lipid membrane derived from the host cell's trans-Golgi network (TGN) or early endosomal membranes to become an intracellular enveloped virus (IEV). This IEV is transported to the cell surface, where the outermost membrane can fuse with the plasma membrane to release the EEV, which is surrounded by two membranes. The EEV form is provides for long-range dissemination within the host. The outer membrane, thus, acquires membrane surface proteins form the host cell in which it is propagated, and also includes virally-encoded proteins. Both of these are exploited herein. The virus can be manufacture in host cells, such as HeLa cells, that express proteins, such as CD55, that improve serum resistance. As detailed herein, the cells in which the virus is propagated can be modified to express proteins on cell membranes so that the membrane a virus acquires from the cell displays such proteins. The proteins can be any that alter, particularly enhance, a property or activity of the virus. Any host cell can be modified to express a protein or polypeptide or peptide of interest, particularly by encoding it in the host cell as a transmembrane protein. As detailed throughout the disclosure herein, viruses also can be genome-modified to encode a protein, polypeptide, or peptide, on the membrane to also alter properties or activities of the virus. Provided herein are viruses, exemplified as high EEV vaccinia viruses, that comprise an outer or second membrane that displays host-derived proteins, particularly as fusion or chimeric protein with host cell transmembrane proteins, and virally-encoded transmembrane proteins. The virally-encoded transmembrane proteins are expressed and retained when the viruses propagate in vivo in target cells, such as tumors, thereby retaining the properties / activities conferred by the virally encoded fusion proteins.

[0621] The presence of the extra, host-derived envelope in the EEV form provides distinct biological and immunological properties compared to the IMV, such as resistance to complement-mediated neutralization. Attorney Docket No. 120276-2615PC

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[0623] As described above, vaccinia virus (VV), as well as other dual enveloped poxviruses, produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). When EEV is challenged with complement from the same species as the cells used to grow the virus, EEV is resistant to neutralization by complement, whereas IMV is not. EEV complement resistance does not result from an endogenous EEV protein. Complement resistance of vaccinia virus EEV is mediated by host regulators of complement activation (RCA) that is expressed by a host cell and passively incorporated into the EEV outer envelope along with host cell membrane proteins that are naturally expressed by the host cell. The EEV outer membrane is extremely fragile and is damaged by virus purification. Once the EEV outer membrane is ruptured, the particle retains infectivity as an IMV. Methods provided herein retain the fragile outer membrane during purification.

[0624] Complement activation on host cells is prevented by several membrane RCA, the activity of which is restricted predominantly to complement of the same species, a phenomenon called homologous restriction. These proteins down-regulate complement activity at two steps in the classical and the alternative pathways: complement receptor 1 (CD35) and decay-accelerating factor (CD55) inhibit the formation and accelerate the decay of the classical pathway and alternative pathway C3 -activating enzymes (C3 convertases); complement receptor 1 and membrane cofactor protein (CD46) act as cofactors for Factor I (a serum protease), which catabolizes C4b and C3b, thereby inhibiting formation of the C3 convertases C4b2a and C3bBb; and, finally, at the end of the complement cascade, CD59 and possibly also homologous restriction factor (C8-binding protein) prevent the formation of the membrane attack restrict complement deposition and amplification on their surfaces.

[0625] Vaccinia virus, the prototype of the poxvirus family, produces two morphologically distinct infectious forms of virions, termed intracellular mature virus (IMV) and extracellular enveloped virus (EEV). IMV represents the majority of infectious progeny and remains within the cytoplasm until cell lysis. A fraction of IMV acquire a double membrane derived from the trans-Golgi network or early tubular endosomes of the host cell to form intracellular enveloped virus (IEV). EEV is formed when the outer IEV membrane fuses with the plasma membrane. EEV also arise by IMV budding through the plasma membrane. EEV are disseminated in vitro and in vivo. Attorney Docket No. 120276-2615PC

[0626] -93-

[0627] At least 10 proteins are associated with the outer envelope of EEV and 6 vaccinia virus encode EEV membrane proteins, which as detailed below, can be modified as described herein to include all or an active portion of a heterologous protein, such as an RCA, to modulate properties or activities, including biological and immunological properties, of the EEV. The protein include: A56R, encoding the virus hemagglutinin (HA) gp86; F13L, encoding a 37-kDa protein (37K protein), p37; A34R, encoding a triplet of glycoproteins, gp22-24; B5R, encoding a 42-kDa glycoprotein, gp42; A36R, encoding a 45- to 50-kDa protein, p45-50; and A33R, encoding a 23- to 28-kDa glycoprotein, gp23-28.

[0628] Unlike the passive incorporation of naturally occurring host cell proteins, it is shown herein that the properties of an EEV can be programed or controlled by selecting host cells and engineering the membrane of such host cells so that any number of modified host cell membrane proteins, including for example polypeptides such as immunomodulatory proteins, receptors, antigens, and other polypeptides that can alter a property or activity of the virus, can incorporated into the viral envelop to modify the properties of the virus. Provided here in are modified cells that contain high EEV, modified cells, and methods of culturing modified cells to amplify the virus to produce EEV that contain the cell membrane-displayed proteins. For example, the properties of an EEV can be altered to enhance anti-tumor immunity, enhance immune evasion, and enhance tumor-targeting by the incorporation of modified host cell membrane proteins into the viral envelope. As shown herein, the envelope can be modified to improve targeting specificity and reduce anti-viral immunogenicity to thereby increase the safety and efficacy of enveloped viral therapies compared to the same virus with an unmodified envelope.

[0629] Such modified EEVs, modified cells, and methods exploit: 1) the fact that cell membranes end up on the virus as a second membrane; 2) high EEV-producing virus can be amplified in such cells to result in EEVs that display the host cell derived cell membrane proteins; and 3) this is further combined with the viruses provided herein in which one or more viral envelope proteins are fused with polypeptides, particularly where a viral envelope protein is fused to one or more immune modulating proteins as detailed herein and in the priority applications. Result are high levels of EEV viruses that Attorney Docket No. 120276-2615PC

[0630] -94-

[0631] are serum resistant, which serum resistance is propagated with the virus, and that express host cell proteins.

[0632] Vaccinia virus (VV), thus, produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). When EEV is challenged with complement from the same species as the cells in which the virus is grown, EEV is resistant to neutralization by complement, whereas IMV is not. Complement resistance is mediated by host resistance to complement proteins incorporated into the EEV outer envelope.

[0633] The vaccinia virus EEV, thus, has resistance to anti-viral immunity, such as complement, and has the ability to infect and amplify in tumors and spread to tumor metastases. Generally, vaccinia virus follows a complex morphogenic pathway that results in the formation of intracellular virus (IMV, intracellular mature virus; IEV, intracellular enveloped virus) and extracellular virus (EEV, extracellular enveloped virus; CEV, cell-associated extracellular virus).

[0634] Upon amplification, the first virion produced, intracellular mature virus (IMV), is surrounded by a single membrane, and remains within the infected host cell until cell lysis. The IMV form comprises the majority of infectious progeny; the IMV particles are susceptible to elimination by host immunity through complement and antibody neutralization, and, thus, when administered they do not disseminate. Systemic delivery of IMV vaccinia viruses for oncolytic viral therapy is limited by the relative sensitivity of IMV to neutralization by the immune system and its inefficient cell to adjacent cell spread within solid tumors following host cell death. IMV spread to distant tumors, such as metastatic tumors, through the blood stream or lymphatic system is limited compared to EEV, which disseminate.

[0635] A fraction of IMV acquires a double membrane derived from the trans-Golgi network or early tubular endosomes to form intracellular enveloped virus (IEV). IEV is an intermediate between IMV and CEV / EEV that provides virus dissemination to the cell surface on microtubules. IEV is formed by wrapping of IMV with intracellular membranes. IEV traffics to the cell surface via microtubules where the IEV membrane fuses with the host cell plasma membrane exposing an enveloped virion on the host cell surface. Viral particles retained on the host cell surfaces are CEV; viral particles released from the host cell membrane are EEV. CEV induces the formation of actin tails that Attorney Docket No. 120276-2615PC

[0636] -95-

[0637] drive CEV particles away from the cell and is important for cell-to-cell spread. EEV thus is formed when the outer IEV membrane fuses with the host plasma membrane and is released from the host cell membrane (see, e.g., Smith etal. J. Gen. Virology. (2002), 83, 2915-2931).

[0638] Much of the EEV remains attached to the host cell surface and is retained as CEV. EEV that is released into the extracellular milieu (generally less than about 1% of virions) is responsible for viral spread within the infected host. EEVs are unstable outside of the host and are difficult to manufacture. The co-existence of IMV and EEV forms has been described for vaccinia strains and other poxviruses, including the fowl poxvirus, entomopox, monkeypox, swinepox and pinguin pox. EEV virions exhibit long-range spread in cell culture and systemic dissemination in vivo. The EEV form of poxviruses such as vaccinia virus have been developed for possible systemic delivery of viruses to tumors and tumor metastasis.

[0639] EEV exhibit rapid and efficient spread through local and systemic tumor sites within an infected host. Whereas IMV form is relatively stable in the environment and is primarily responsible for spread between individuals, EEV is responsible for viral spread within the infected host and is relatively easily degraded outside of the host. The EEV form is unstable outside of the body, which can reduce the risk of transmission to individuals in the public.

[0640] EEV has several mechanisms to inhibit its neutralization within the bloodstream. First, EEV is has resistance to complement due to the incorporation of host cell regulators / inhibitors of complement into its outer membrane coat plus secretion of vaccinia virus complement control protein (VCP) into local extracellular environment. Second, EEV exhibits resistance to neutralizing antibody effects compared to IMV, although smallpox vaccines can generate antibody responses to the EEV form in immunized humans. EEV also is released at earlier time points following infection (e.g., 4-6 hours) than IMV (which is only released during / after infected cell death). The host spread of the EEV virion is faster than IMV (Blasco et al. J. Virology, 67(6):3319-3325, 1993).

[0641] When propagated in a cell from the same species of the cell to be infected, the EEV can exhibit resistance to complement. Complement resistance occurs for virus propagated in cells that express high levels of Complement regulatory proteins (CRPs) or Attorney Docket No. 120276-2615PC

[0642] -96-

[0643] regulators host complement activation (RCA). EEV is relatively resistant to complement and antibody-mediated neutralization relative to standard preparations of vaccinia virus containing exclusively IMV when administered intravascularly; the EEV form shows enhanced stability and retain activity longer in the blood over IMV (Vanderplasschen et al., (1998) Proc Natl Acad Sci USA. (13):7544-9; Smith etal. (1998) Adv Exp Med Biol.

[0644] 440: 395-414). This plays a role for repeat administration once neutralizing antibody levels have increased and when anti -cancer therapies require repeat administration. EEV have been developed for systemic administration, but none have been successfully developed for this purpose. The EEV form is a low percentage of any virus produced, serum resistance derives from the cells in which the EEV is propagated; as shown in the Examples, such serum resistance is not specific to cell type or to tumor type. Hence, one cannot develop an EEV for treatment of a particular disease, since serum resistance is not specific to tumor type. Upon administration, the production of EEV is low, and serum resistance is lost upon replication in most cells.

[0645] 3. IMV

[0646] Upon propagation of vaccinia viruses, IMVs comprise the majority of infectious progeny. In strains that are not modified or selected to produce high levels of -99% of virions or IMV. The IMVs remain within the cytoplasm until host cell lysis. The cell-to-cell spread of IMV within an infected host is inefficient; IMV are susceptible to elimination by host immunity through complement and antibody neutralization. For example, the IMV proteins A27L, H3L, L1R, and D8L are immunogenic proteins and are targets for anti-viral neutralizing antibodies. To overcome these limitations and to capitalize upon the natural abundance of IMVs, recombinant vaccinia viruses have been modified or engineered to contain one or more viral glycoproteins with mutations in neutralizing antibodies epitopes, resulting in viral escape from neutralization see, e.g., US Publication No. US 2021 / 0388388; Published International PCT application No. WO 2020 / 086423 Al. IMV viruses have been modified to encode and express complement regulatory proteins covalently linked to an IMV protein to provide resistance to complement (see e.g., Published International PCT applications Nos. WO 2021 / 071534 Al; WO 2022 / 182206 Al; and WO 2023 / 118603 Al; see, also SongK. etal.

[0647] Biomedicines. 20208(11), 491). Vaccinia virus IMV have been modified to encode one or more of these proteins, such as CD35, CD55, CD59, CD46, CR1, Factor H, VCP, Attorney Docket No. 120276-2615PC

[0648] -97-

[0649] MOPICE, SPICE and CCPH, as a chimeric protein with an IMV membrane protein (see, Internationa] PCT application No. WO 2023 / 118603). The IMV is said to have increased resistance to complement. IMV, however, do not disseminate but move from cell-to-cell, so that inactivation by complement is not a substantial problem. A problem, however, is that IMV cannot be systemically administered and do not disseminate systemically. Hence the problems of host immunity to the virus and problems with systemic administration have not been solved.

[0650] IMVs are distinct virus particles from EEVs. IMV formation occurs in cytoplasmic sites called “virus factories” from which cellular organelles largely are excluded, by contrast the EEV is formed when the outer IEV membrane fuses with the host plasma membrane and is released from the host cell membrane. In this way, EEV, but not IMV, incorporates an outer membrane or outer envelope that covers the IMV membrane.

[0651] The EEV encodes and expresses proteins on the outer membrane, and incorporates host cell proteins into the outer membrane or outer envelope. Cells infected by IMV or modified / engineered IMV produce little EEV, unless they have been further modified or engineered to contain mutations in the viral genome that increase EEV production. EEV progeny of any IMV viruses do not contain IMV membrane modifications on the EEV outer membrane. The EEV outer membrane or outer envelope covers the IMV membrane. Any IMV membrane protein, expressed or naturally occurring, is enveloped by the EEV membrane.

[0652] The spread of the IMV viral form to distant tumors, such as metastatic tumors, through the blood stream or lymphatic system is inefficient compared to EEV. IMV remains inside the host cell until host cell lysis, EEV is released from the infected host cell membrane. EEV can colonize distant tumor sites which is needed for systemic oncolytic viral therapy.

[0653] 4. Insufficient EEV for Systemic Administration

[0654] The systemic capabilities of oncolytic vaccinia viruses are limited by the relatively small amount of EEV generated by an infected cell, further the need to propagate the EEV in cell that produces high levels of a complement resistant protein, and because the complement resistance is not passed on to progeny viruses upon replication. Additionally, much of the EEV remains attached to the host cell surface and Attorney Docket No. 120276-2615PC

[0655] -98-

[0656] is retained as CEV. EEV that is released into the extracellular milieu (~1% of virions) is responsible for viral spread within the infected host and is unstable outside of the host (Smith etal., 1998 Adv Exp Med Biol. 1998:440:395-414).

[0657] To address these limitations, virus with increased EEV production can be produced. This was effected by selecting for EEV viruses with enhanced or improved EEV production and enhanced or improved resistance to host immunity. Mutations to can be introduced to increase the production of EEV. Methods for increasing the amount of EEV isolated from any strain are provided, and are particularly useful for strains that are high EEV producers (generally more than 1%, such as 5% to 10%). Methods herein increase the amount of EEV isolated from any strain.

[0658] 5. Selection of high producing EEV

[0659] Viruses can be screened for high EEV production and / or known mutations can be introduced to increase EEV production. Gene knockouts or mutations can be introduced to increase resistance to humoral immunity, such as complement, to increase serum stability. Viruses can be screened for tumor selectivity and for other properties of interest.

[0660] Methods for Selection of high EEV viruses

[0661] Viruses are selected for high production, generally greater than 1%, 5%, 10%, or more EEV production. As described in the Examples, a virus was selected and the further modified.

[0662] Exemplary methods describing the production and isolation of EEV forms of vaccinia virus are well known (see, e.g., Blasco et al. J. Virology, 67(6):3319-3325, 1993; U. S. Patent No. 8,329,164; Published International PCT application No.

[0663] W02013038066A1; Published International PCT application No. WO2023128672A1; U. S. Publication Nos. US 2022 / 0049228A1 US 2023 / 0002740A1). High EEV producing viruses (>1% of virions, or more, such as at least 2, 3, 4, or 5% or more) are known in the art. Such viruses can be modified as detailed herein and / or manufactured by the methods herein. These include viruses known to those of skill in the art, such as those described, for example, in any of the following: Blasco etal. J. Virology, 67(6):3319-3325, 1993; U. S. Patent No. 8,329,164; Published International PCT application No.

[0664] W02013038066A1; Published International PCT application No. WO2023128672A1; and U. S. Publication Nos. US 2022 / 0049228A1 and US 2023 / 0002740A1. For example, Attorney Docket No. 120276-2615PC

[0665] -99-

[0666] US 22023002740 describes viruses that produce high levels of EEV, such as a vaccinia virus (OVV), comprising: a) a nucleotide sequence encoding a variant A33 polypeptide; b) a nucleotide sequence encoding a variant A34 polypeptide; or c) a nucleotide sequence encoding a variant A33 polypeptide and a nucleotide sequence encoding a variant A34 polypeptide, where the variant A33 polypeptide and variant A34 polypeptide each provides for enhanced viral spreading or enhanced production of extracellular enveloped virion (EEV), compared to the corresponding wild-type A33 polypeptide and wild-type A34 polypeptide, respectively; and or a nucleotide sequence encoding a variant B5 polypeptide, where the variant B5 polypeptide provides for enhanced viral spreading or enhanced production of EEV, compared to the corresponding wild-type B5 polypeptide. Any poxvirus, such as a vaccinia virus, can be modified as detailed herein and / or manufactured by the methods herein and combinations thereof to produce high concentrations of EEVs that are serum stable or resistant. Among these are EEVs who serum resistance is independent of the cells, including tumor cells in which they are produced.

[0667] Cells are infected and the EEV particles are released into the culture supernatant before lysis of the infected cell; an exemplary protocol that was employed herein is depicted in Figure 1 and exemplified in Example 1. After a sufficient time, postinfection, the culture supernatant can be collected, and number of infectious virus particles produced in the supernatant (EEV) can be determined by a plaque assay. High EEV production capacity of selected viruses can be assessed by performing the Comet assay, where the head of the comet represent the primary plaque and the comet tails represent the secondary plaques caused by spread of EEV particles and indicates high EEV production. A virus with high EEV production forms a distinct tail when performing the Comet assay. A long “tail” in a Comet assay indicates that the virus can produce high levels of EEV leading to further spread. By contrast, short and round plaques signifies that the virus mainly spreads from cell to cell such as with IMV. As another example, the vaccinia strain Western Reserve (WR) strain is a low EEV producing strain, in which less than 1% of infectious progeny are EEV and forms round well-defined plaques. By contrast, International Health Department (IHD)-J is a high EEV producing strain, where up to 30% of infectious progeny are EEV. IHD-J forms large plaques, with diffuse elongated comet shape caused by the distribution of EEV- Attorney Docket No. 120276-2615PC

[0668] -100-

[0669] derived secondary plaques (Blasco etal. J. Virology, 67(6):3319-3325, 1993). A target herein EEV produced greater than 10%, 20%, 30% of progeny. The selected exemplary virus has EEV production of about 30%.

[0670] Virus replication (amplification) in cells can be determined by infecting a monolayer of cells with virus at a sufficient multiplicity of infection (MOI) generally less than 10, such as, for example, an MOI of 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10, and that incubation of virus with cells should proceed sufficiently long for viral replication to commence. A sufficient time for viral replication / amplification can be, for example, about 1 hour or longer, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or longer. The amount of time can vary, for example, from more than 2 hours, e.g., 3 or more hours, more than 4 hours, e.g., 6-48 hours to e.g., 72 or more hours. Following infection, the viral inoculum can be washed and replaced with fresh medium.

[0671] Virus produced in the supernatant and cells can be harvested separately, e.g., 24 hours post-infection. The virus in the supernatant is typically tittered by plaque assay and EEV assessed by performing a Comet assay. Virus contained in the infected cell also can be pelleted, frozen and stored, for example, at -80° C.

[0672] IMV or EEV virions originating from a virus are incubated with human serum (e.g, at least 20% human serum) followed by infection of a cell, such as a cancer cell line for a sufficient time (e.g, 24-hours incubation). IMV can be enriched from the pellet of an infected cell, while EEV particles can be enriched from the supernatants from infected cells. IMV and EEV can be quantified by plaque assay, where high EEV producing viruses are expected to form more plaques that IMV when challenged by human serum containing neutralizing antibodies and active complement or can be exposed to neutralizing antibodies (such as an anti-Ll NR-45114 antibody or the Vaccinia Immune Globulin (VIG) antibody). Viral plaque formation (e.g., in PFU / mL) can be compared with appropriate control viruses (e.g., an unmodified poxvirus or vaccinia virus that does not possess A34R / K151E or other mutations that are known to enhance EEV production). Increased resistance to neutralizing antibodies can be measured by quantifying the number of plaques in a viral plaque assay, following treatment with an anti-Ll NR-45114 antibody or an anti-Vaccinia Immune Globulin Attorney Docket No. 120276-2615PC

[0673] -101-

[0674] (VIG) antibody. Higher plaque formation indicates greater EEV production and resistance to neutralizing antibodies.

[0675] Virus samples containing enrichment of IMV or enrichment of EEV can be separated and purified using a CsCl density gradient ultracentrifugation method. Virus can be overlaid on a CsCl gradient and centrifuged overnight. Bands containing the enriched fractions can be extracted at defined locations within the density gradient, and CsCl is then removed.

[0676] The number of EEV virions can be quantified can be treated by antibody staining for a EEV membrane protein, such as, A33R, A34R, A56R, B5R or F13L or any protein expressed or present on the EEV outer membrane. For example, the amount of EEV can be quantified by incubating an EEV containing sample with a fluorescent molecule conjugated anti-B5 antibody. For example, the number of EEV can be quantified by measuring virus sized particles (VSP) and VSP containing the B5 antigen can then be quantified by flow cytometry.

[0677] 6. Exemplary high EEV-producing viruses

[0678] Provided herein are RT-00 vaccinia virus (or N2, also referred to as RT-00) derivatives thereof. Details of production of RT-00 and derivative are described in the Examples, and its sequence is set forth in SEQ ID NO:01 and the viruses are summarized in Figure 24.

[0679] RT-00 was selected for high EEV production and high tumor selectivity. It can be used as a systemically administered extracellular enveloped virus (EEV) for oncolytic virus therapy. As detailed herein, the RT viruses can be further modified, such as by gene knockouts and other modifications to reduce inactivation by host immune responses, for example, by serum inactivation, complement and neutralizing antibodies. Exemplary knock-outs and their effects are detailed in the Examples. These knockouts can be introduced into the RT viruses and any virus provided herein, including modified RT viruses, and can be introduced into any therapeutic vaccinia virus.

[0680] The RT vaccinia viruses were identified by production comet-shaped plaques indicating the production high levels of EEV and enhanced viral spreading, compared to IMV producing viruses.

[0681] It is shown herein, that EEV purified from RT viruses, can be systemically administered, and that upon systemic administration, the EEV resists inactivation by the Attorney Docket No. 120276-2615PC

[0682] -102-

[0683] host immune system, including complement and / or neutralizing antibodies, to thereby reach tumors and metastases, if present. EEV purified from RT viruses (viruses derived from RT-00, where RT refers to the red tail seen on assays to detect dissemination) can reach multiple tumors, amplify in multiple tumor sites and in metastases and, upon administration, such as by systemic administration. Because of the high level of EEV virus produced, upon administration by any route, such as systemic or intratumor administration or local administration, the virus can disseminate because of the high level of EEV and also because of the serum resistance. Unlike IMV, it is shown herein that purified RT EEV particles, colonize tumors following exposure to human serum.

[0684] RT-00 possesses A34R SNP from the amino acid replacement KI 5 IE. It is this mutation, along with other variations in the genome compared to the parental virus, that confers the high level of EEV produced and the serum resistance. The A34R gene encodes an EEV-specific glycoprotein with homology to C-type animal lectins which is expressed in the outer membrane of extracellular enveloped virus (EEV). The A34R lectin homology / carbohydrate recognition domain binds EEV virions to the host cell membrane and inhibits the release of the EEV from the host cell membrane. A substitution at codon 151 of A34R from a lysine to glutamic acid (KI 5 IE mutation) renders the A34R protein less able to tether the EEV to the host cell membrane, resulting in release of EEV (see, e.g., U. S. Patent No. 8,329,164; US2022 / 0049228A1). The vaccinia virus strains International Health Department (HTD)-J and W strains (IHD-J and HTD-W) and rabbit poxvirus strains possess A34R / K151E substitution are known to produce more EEV, while most vaccinia virus strains have the Western Reserve genotype (McIntosh & Smith, J Virol. 1996 Jan; 70(1): 272-281). The K151E mutation in the A34R gene results in increased EEV production.

[0685] The genome of RT-00 is related IHD-W but possesses key distinguishing features. HTD-W contains a truncated form of the A56R protein, whereas in RT-00, the A56R protein is intact. The A56R protein has several functions, including regulating the presence of viral-encoded complement regulatory proteins (VCP) on the cell surface. The expression of A56R on tumor cells can protect the cell from complement neutralization leading to better spread of oncolytic virotherapy. A56R protein is expressed in in the host membrane of the EEV, facilitating the location of VCP on the surface of the enveloped viral particle. Attorney Docket No. 120276-2615PC

[0686] -103-

[0687] RT-00 contains a 3-nucleotide deletion in the K7R gene, a TLR modulator receptor. This deletion, which is not found in any other orthopox virus, produces a protein that is one amino acid shorter than the K7R protein found in other vaccinia viruses. RT-00 virus contains a gene identical to RPXV102 (a cell surface-binding protein and carbonic anhydrase homolog), which is not found in H D-W but is present with an identical amino acid sequence in the Tashkent clone TKT4 and Rabbitpox virus. RPXV102 is a protein present in the IMV that binds to chondroitin sulfate on the cell surface, providing virion attachment to a target cell. RT-00 has 2 SNPs in the A30L gene compared with IHD-W. RT-00 contains a 1,839 base pair deletion in the left ITR and a 1,841 base pair deletion in the right ITR, where the left ITR is from base pairs 1- 9,037 and the right ITR is from base pairs 185,372 - 194,269.

[0688] When compared with the available H D-J sequences (A33R, A34R, A36R, A56R, B5R, F13L, A45R, A29L partial A31R, A30L, A32L partial and A13L) RT-00 has 2 SNPs in A30L and 1 in A45R.

[0689] Other high EEV viruses

[0690] Numerous high EEV viruses are known in the art. Those viruses can be produced by the methods herein to increase EEV levels and / or can be modified as detailed herein to improve serum stability and / or other properties, such as by modifying the virus to display humoral immunity modulating (generally increasing) on the outer membrane. Knock-outs also can be introduced to improve anti-tumor and resistance to humoral immunity. Examples of prior art viruses for modification and production as detailed herein include the following.

[0691] No prior art contemplates modifying an EEV virus by producing a fusion with an outer membrane transmembrane protein, such one or more of A33R, A34R, A56R, B5R, and F13L, to display a humoral immunity modulating protein, such as a complement inhibiting protein, on the outer membrane. If any transmembrane proteins are contemplated, they are displayed on the mature virus (IMV, also referred to as an MV); none describe or appreciate the advantages of display on the outer membrane described herein. All can be modified as detailed herein.

[0692] SillaJen / Jennerex viruses

[0693] Another virus derived from an H D-J virus preparation (parent H D-J obtained from ATCC® Catalog No. VR-156) is detailed in International PCT publication No. Attorney Docket No. 120276-2615PC

[0694] -104-

[0695] W02024 / 011250 (see, also US publication No. US2024 / 0033347), which provides an IHD clone (see SEQ ID NO:616 herein) and modified forms thereof (see SEQ ID NOs: 617-627 herein) that has high EEV production. These viruses can be modified as detailed herein (see below) so that the encode chimeric outer transmembrane fusion proteins, such as A33R, A34R, A56R, B5R, and F13L, with humoral immunity modulating proteins, such as complement inhibiting or modulating proteins, as exemplified herein for RT-00. As detailed below, fusion with an outer membrane protein confers additional properties on the virus whereby the serum resistance of the virus is retained independent of the tumor in which the virus propagates (in vivo or in vitro). These viruses provided herein and known in the art can be modified so that they are serum resistant and maintain this phenotype when they disseminated after administration.

[0696] 7. Modifications of EEV to increase resistance to humoral immunity

[0697] The genome of a vaccinia virus can be modified to increase resistance to humoral immunity by knocking out genes that contribute to such resistance or that reduce or limit infection of host cells, particularly tumor cells. Exemplary loci for knockouts are detailed and exemplified in the Examples. Exemplary of such viruses is the virus designated RT-00 is a vaccinia virus having the sequence set forth in SEQ ID NO: 1 or variants thereof having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity in the genome. As described herein, RT-00 is an exemplary viral clone that produces a high level (about or greater than 30% of progeny) that are EEV, and that has other properties, including high tumor selectivity that render it suitable for systemic administration for treatment of cancers, including metastatic cancers.

[0698] In some embodiments, RT-00 or a virus derived therefrom, or a vaccinia virus with high levels of EEV production, can be modified or engineered to enhance tumor selectivity. RT-00 or a virus derived therefrom or any high EEV producing virus or S-R virus or EEV virus known in the art can be modified or engineered by knockout (e.g., by complete or partial deletions or insertions or transpositions so that functional product is not produced) in one or more vaccinia virus genes selected from among: A46R, B8R, J2R, A52R, F1L, VGF, and / or B19R. In some embodiments, RT-00 or a virus derived therefrom can comprise two or three knockouts in the A46R, B8R, J2R, A52R, F1L, VGF, or B19R genes or gene loci. In some embodiments, the two or double knockouts Attorney Docket No. 120276-2615PC

[0699] -105-

[0700] can be produced and include any two of from among A46R, B8R, J2R, A52R, F1L, VGF, and B19R, or other such genes / loci. For example, double knockouts include, but are not limited to knockouts of: a) TK, A46R; b) TK, A52R; c) TK, B8R; d) TK, VGF; e) TK, F1L; and f) TK, B19R. Similarly, three / triple knockouts can be produced by combining knockouts of A46R, B8R, J2R, A52R, F1L, VGF, and B19R. For example, exemplary triple knockouts can be selected from among: g) TK, A46R, VGF; f) TK, A52R, VGF; g) TK, B8R, VGF; h) TK, F1L, VGF; i) TK, B8R, B19R; f) TK, A46R, B19R; g) TK, A52R, B19R; and h) TK, F1L, B19R.

[0701] In some embodiments, RT-00 or a virus derived therefrom can be modified or engineered to encoded and express heterologous nucleic, such as, for example, by replacement of or insertion into any one of A46R, B8R, J2R, A52R, F1L, VGF, and / or B19R genes, or in place or inserted into any non-essential gene in vaccinia virus. Those of skill in the art are familiar with non-essential genes in vaccinia virus and many vaccinia viruses that encode heterologous nucleic acid, such as heterologous proteins, such as a therapeutic protein, are known and available to those of skill in the art.

[0702] Any such known vaccinia virus can be modified or selected to be an EEV high producer, and, modified as described herein, to encode and express in the EEV membrane a chimeric polypeptide for display of a complement resistance protein or a complement regulating protein or other modulator of humoral immunity in the host on the EEV, such that the protein is displayed in EEV progeny thereof.

[0703] The table below and Figure 24, which includes viruses RT-32 to RT43, summarizes viruses derived from RT-00 / N2, their nomenclature and modifications. SEQ ID NO: 1 sets forth the sequence of the RT-00 virus. Inserts are added to the vectors in the noted sites and are added either by recombination with a transfer vector, or by insertion with a Cre-Lox system. The sequences of the transfer vectors are set forth in the listed SEQ ID NOs. They also are set forth in the Examples below. The transfer vectors are pUC-derived vectors that include the nucleic acid encoding the inserted sequence, which includes the nucleic acid encoding the noted polypeptide and promoter, and includes flanking vaccinia virus DNA to insert into the noted loci (i.e., TK, A46R, VGF, and others as noted or known to those of skill in the art) SEQ ID NOs: 2-21, 518-524, 628-634, and 791-800 refer to the sequences of the transfer vectors or inserts, which Attorney Docket No. 120276-2615PC

[0704] -106-

[0705] sequences also are set forth in the Examples. The subsequent Table summarizes the genotype of each virus. The complete table is presented in Figure 24 as a single table.

[0706] SEQ NAME PARENT TRANSFER TRANSFER ID ID

[0707] Modification / insertion VIRUS Vector (NAME) Vector

[0708] NO:

[0709] IHD RT-00 N2 N. A. 1

[0710] (NR-52) Wild Type

[0711] RT-01

[0712] Turbo (TK) RT-00 pUC-Turbo (TK) Seq: RT-01 2

[0713] T pUC-eGFP

[0714] RT-02 urbo (TK) / RT-01 Se 3

[0715] eGFP (A46R) (A46R) q: RT-02

[0716] RT-03 Turbo (TK) / RT-01 pUC-eGFP (B8R) Seq: RT-03 4

[0717] eGFP (B8R)

[0718] RT-04 Turbo (TK) / pUC-eGFP

[0719] RT-01

[0720] eGFP (A52R) (A52R) Seq: RT-04 5 Turbo (TK) / pUC-CD55-B5R

[0721] RT-05 pSEL-CD55- RT-02 6

[0722] (A46R) Seq: RT-05 B5R(A46R)

[0723] RT-06 Turbo (TK) / pUC-CD55-B5R

[0724] RT-02 Seq: RT-06 7 pSE-CD55-B5R(A46R) (A46R)

[0725] pUC-CD55-B5R

[0726] RT-07 Turbo (TK) / RT-02 S 8

[0727] pSL-CD55-B5R(A46R) (A46R) eq: RT-07

[0728] eGFP(TK) /

[0729] pUC-lox-eGFP

[0730] RT-08 pSEL-CD55- RT-05 9

[0731] (TK) Seq: RT-08 B5R(A46R)

[0732] Turbo (TK) /

[0733] RT-09 pSEL-CD55(sol)- RT-02 pUC-CD55(sol)- B5R (A46R) Seq: RT-09 - B5R(A46R)

[0734] pSL-antiVEGF(TK) /

[0735] pUC-Turbo- RT-10 pSEL-CD55- RT-08 Seq: RT-10 10 B5R(A46R) antiVEGF (TK)

[0736] eGFP(TK) /

[0737] pSEL-CD55- RT-11 RT-08 Seq: RT-11 11 B5R(A46R) / pUC-Turbo (VGF)

[0738] Turbo(VGF)

[0739] eGFP(TK) /

[0740] RT-12 RT-14 12 Turbo(VGF) pUC-Turbo (VGF) Seq: RT-12

[0741] Tur pUC-eGFP- RT-13 bo (TK) / RT-01 Seq 13

[0742] pSL-antiVEGF(FlL): RT-13

[0743] antiVEGF (F1L)

[0744] RT-14 eGFP(TK) RT-01 pUC-eGFP (TK) Seq: RT-14 14 eGFP(TK) /

[0745] RT-15 RT-14 pUC-Turbo

[0746] T Seq: RT-15 15 urbo(B19R) (A19R)

[0747] non

[0748] RT-16 (TK) / (B19R) RT-15 (cre-lox syste Seq: RT-16 -m)

[0749] non

[0750] RT-17 (TK) / Turbo(B19R) RT-15 - (cre-lox system) Seq: RT-17

[0751] non

[0752] RT-18 eGFP(TK) / (B19R) RT-15 -

[0753]

[0754] (cre-lox system) Seq: RT-18 Attorney Docket No. 120276-2615PC

[0755] -107-

[0756] SEQ NAME PARENT TRANSFER TRANSFER ID Modification / insertion I VIRUS Vector (NAME) Vector D NO: non

[0757] RT-19 (TK) / (VGF) RT-12 (cre-lox system) Seq: RT-19 - RT-20 eGFP(TK) / (VGF) RT-12 none - - (cre-lox system)

[0758] RT-21 (TK) / Turbo(VGF) RT-12 none - - (cre-lox system)

[0759] (TK) / (VGF) /

[0760] RT-22 RT-15 pUC-Turbo

[0761] Tur Seq: RT-22 16 bo(B19R) (B19R)

[0762] RT-23 Turbo (TK) / (A46R) RT-02 none - - (cre-lox system)

[0763] RT-24 Turbo (TK) / (B8R) RT-03 none - - (cre-lox system)

[0764] RT-25 Turbo (TK) / (A52R) RT-04 none - - (cre-lox system)

[0765] (TK) / eGFP(A52R) / pUC-eGFP

[0766] RT-26 RT-21 17 Turbo(VGF) (A52R) Seq: RT-26 (TK) / eGFP(B8R) /

[0767] RT-27 RT-21 pUC-eGFP (B8R) 18 Turbo(VGF) Seq: RT-27 (TK) / eGFP(B8R) /

[0768] RT-28 RT-17 pUC-eGFP (B8R) 19 Turbo(B19R) Seq: RT-28 (TK) / eGFP(A52R) / pUC-eGFP

[0769] RT-29 RT-17 20 Turbo(B19R) (A52R) Seq: RT-29 (TK) / pSL- antiVEGF eGFP(FlL) pUC-eGFP- RT-30 RT-17 21 / antiVEGF (F1L) Seq: RT-30 Turbo(B19R)

[0770] pSEL-CD55- none (cre-lox

[0771] RT-31 RT-08 - - B5R(A46R) system)

[0772] none (cre-lox

[0773] RT-32 (TK) RT-14 - - system)

[0774] pUC-Nluc-eGFP

[0775] RT-33 Nluc-eGFP (TK) RT-01 51

[0776] (TK) Seq: RT-33 8 (TK) / (B19R) / pUC-eGFP

[0777] RT-34 RT-16 3 eGFP(A46R) (A46R) Seq: RT-02 (TK) / (VGF) / pUC-eGFP

[0778] RT-35 RT-19 3 eGFP(A46R) (A46R) Seq: RT-02 (TK) / (B19R) /

[0779] RT-36 RT-34 none - - (A46R) (cre-lox system)

[0780] RT-37 (TK) / (VGF) / A46R) RT-35 none

[0781] (cre-lox system) - - RT-38 (TK) / (VGF) / (B19R) RT-22 none - - (cre-lox system)

[0782] CD55-B5R(TK) / pUC-lox-eGFP- RT-39 RT-36 519 (B19R) / (A46R) CD55-B5R (TK) Seq: RT-39 CD55-B5R(TK) / pUC-lox-eGFP- RT-40 520 (VGF) / (A46R) RT-38 CD55-B5R (TK) Seq: RT-40 CD55-B5R(TK) / pUC-lox-eGFP- RT-41 RT-17 521 (VGF) / (B19R) CD55-B5R (TK) Seq: RT-41 CD55-A33R(TK) / pUC-lox-eGFP- RT-42 RT-38 522 (VGF) / (B19R) CD55-A33R (TK) Seq: RT-42

[0783]

[0784] RT-43 CD55-B5R(TK) / RT-38 pUC-lox-Turbo- Seq: RT-43 523 Attorney Docket No. 120276-2615PC

[0785] -108-

[0786] SEQ NAME PARENT TRANSFER TRANSFER ID Modification / insertion Vector (NAME) Vector ID VIRUS NO:

[0787] (VGF) / (B19R) / Nluc (Ig)

[0788] Luc(Ig)

[0789] CD55-B5R(TK) / pUC-lox-eGFP- RT-45 RT-23 628 (A46R) CD55-B5R (TK) Seq: RT-45 eGFP(A46R) / (TK) / pUC-eGFP

[0790] RT-51 RT-17 3 Turbo(B19R) (A46R) Seq: RT-02 eGFP(A46R) / (TK) / pUC-eGFP

[0791] RT-52 RT-21 3 Turbo(VGF) (A46R) Seq: RT-02 CD55-A33R(TK) / pUC-lox-eGFP- RT-58 RT-21 629 Turbo(VGF) CD55-A33R (TK) Seq: RT-58 CD55-B5R(TK) / pUC-lox-eGFP- RT-61 RT-17 630 Turbo(B19R) CD55-B5R(TK) Seq: RT-61 (TK) / Turbo(VGF) / pUC-lox-TagBFP- RT-62 RT-21 631 FLT3L(B19R) FLT3L (B19R) Seq: RT-62 (TK) / Turbo(VGF) / pUC-lox-TagBFP- RT-63 RT-21 632 IL15(B19R) IL15 (B19R) Seq: RT-63 (A46R) / (TK) none (cre-lox

[0792] RT-64 / Turbo(B19R) RT-51 system) - - (A46R) / (TK) none (cre-lox

[0793] RT-65 RT-52 - - / Turbo(VGF) system)

[0794] Turbo(TK) / FLT3L pUC-lox-TagBFP- RT-72 631 (B19R) RT-01 FLT3L (B19R) Seq: RT-62 Turbo(TK) / IL- pUC-lox-TagBFP- RT-73 632 15(B19R) RT-01 IL15(B19R) Seq: RT-63 (A46R pUC-lox-eGFP- RT-74 ) / Turbo (TK) / RT-02 633 FLT3L(VGF) FLT3L (VGF) Seq: RT-74 (A46R) / Turbo(TK) / pUC-lox-eGFP- RT-75 RT-02 634 IL-15(VGF) IL15(VGF) Seq: RT-75 (A46R) / CD55- pUC-lox-eGFP- RT-76 A33R(TK) RT-64 629

[0795] CD55-A33R(TK) Seq: RT-58 / Turbo(B19R)

[0796] (A46R) / CD55- pUC-lox-eGFP- RT-77 A33R(TK) / RT-65 629

[0797] CD55-A33R(TK) Seq: RT-58 Turbo(VGF)

[0798] (A46R) / CD-55- RT-82 A33R(TK) / FLT3L RT-78 none - - (B19R) (cre-lox system)

[0799] (A46R) / CD-55- RT-83 A33R(TK) / IL 15 RT-79 none - - (cre-lox system)

[0800] (B19R)

[0801] (A46R) / CD-55- none (cre-lox

[0802] RT-84 A33R(TK) / FLT3L RT-80 - - system)

[0803] (VGF)

[0804] (A46R) / CD-55- none (cre-lox

[0805] RT-85 A33R(TK) / IL 15 RT-81 - - system)

[0806] (VGF)

[0807] RT-86 (A46R) / (TK) / FLT3L RT-64 pUC-lox-TagBFP- (B1 Seq: RT-62 631 9R) FLT3L (B19R)

[0808] (A46R) / (TK) / IL 15 pUC-lox-TagBFP- RT-87 RT-64 632 (B19R) IL15(B19R) Seq: RT-63

[0809]

[0810] Attorney Docket No. 120276-2615PC

[0811] -109-

[0812] SEQ NAME PARENT TRANSFER TRANSFER ID Modification / insertion I VIRUS Vector (NAME) Vector D NO:

[0813] (A46R) / (TK) / FLT3L pUC-lox-eGFP- RT-88 RT-65 633 (VGF) FLT3L (VGF) Seq: RT-74 (A46R) / (TK) / IL 15 pUC-lox-eGFP- RT-89 RT-65 634 (VGF) IL15 (VGF) Seq: RT-75 (A46R) / (TK) / non

[0814] - - RT-90 Flt3L(B19R) RT-86 (cre-lox system)

[0815] (A46R) / (TK) / non

[0816] - - RT-91 IL15(B19R) RT-87 (cre-lox system)

[0817] (A46R) / (TK) / non

[0818] - - RT-92 Flt3L(VGF) RT-88 (cre-lox system)

[0819] (A46R) / (TK) / non

[0820] - - RT-93 IL15(VGF) RT-89 (cre-lox system)

[0821] (A46R) / (TK) / pUC-lox-eGFP

[0822] - - RT-94 eGFP(VGF) RT-65 (VGF)

[0823] non

[0824] - - RT-95 (A46R) / (TK) / (VGF) RT-94 (cre-lox system)

[0825] (A46R) / (TK) / IL 15 pUC-lox-eGFP- 793, RT-96 super(VGF) RT-65 IL15 super (VGF) Seq: RT-96 794 anti-VEGF(A46R) / pUC-eGFP- - - RT-97 (TK) / Turbo(VGF) RT-65 antiVEGF (A46R)

[0826] (A46R) / (TK) / pUC-lox-eGFP- 795 RT-98 eGFP(VGF) RT-65 (VGF) Seq: RT-98

[0827] non

[0828] - - RT-99 (A46R) / (TK) / (VGF) RT-98 (cre-lox system)

[0829] RT- anti-VEGF(A46R) / pUC-lox-eGFP- - - 100 (TK) / eGFP(VGF) RT-97 (VGF)

[0830] (A46R) / CD55- RT- A33R(TK) / pUC-lox-eGFP- - - 101 eGFP(VGF) RT-77 (VGF)

[0831] RT- anti-VEGF(A46R) / non

[0832] - - 102 (TK) / (VGF) RT-100 (cre-lox system)

[0833] RT- (A46R) / CD55- non

[0834] - - 103 A33R(TK) / (VGF) RT-101 (cre-lox system)

[0835] RT- (A46R) / (TK) / IL-15 non

[0836] - 793 104 super(VGF) RT-96 (cre-lox system)

[0837] anti-VEGF(A46R) / pUC-Turbo- RT- CD55-A33R(TK) / antiVEGF (A46R) - - 105 (VGF) RT-77 - noFlag

[0838] anti-VEGF(A46R) /

[0839] RT- CD55-A33R(TK) / non - - 106 (VGF) RT-105 (cre-lox system)

[0840] (A46R) / CD55- pU C-lox-TurboFP- RT- A33R(TK) / IL-15 IL15 superagonist - 793 107 super(VGF) RT-77 (VGF) - noFlag

[0841] (A46R) / CD55- RT- A33R(TK) / IL-15 non - 793 108 super(VGF) RT-107 (cre-lox system)

[0842] (A46R) / CD55- pU C-lox-TurboFP- RT- A33R(TK) / IL- IL15 (VGF) - - 791 109 15(VGF) RT-103 noFlag

[0843] RT- (A46R) / CD55- non

[0844] - 791

[0845]

[0846] 110 A33R(TK) / IL- RT-109 (cre-lox system) Attorney Docket No. 120276-2615PC

[0847] -110-

[0848] SEQ NAME PARENT TRANSFER TRANSFER ID Modification / insertion Vector (NAME) Vector ID VIRUS NO:

[0849] 15(VGF)

[0850] pU C-lox-TurboFP- IL15

[0851] - 792 RT- (A46R) / (TK) / IL-15 superagonist ori

[0852] 111 super ori(VGF) RT-99 (VGF) - noFlag

[0853] RT- (A46R) / (TK) / IL- non

[0854] - 792 112 15super ori(VGF) RT-111 (cre-lox system)

[0855] (A46R) / CD55- pU C-lox-TurboFP- RT- A33R(TK) / IL- IL15 super ori - 792 113 15super ori(VGF) RT-103 (VGF) - noFlag

[0856] (A46R) / CD55- RT- A33R(TK) / IL- non - 792 114 15super ori(VGF) RT-113 (cre-lox system)

[0857] (A46R) /

[0858] Turbo_pE_CD55- pU C-lox-TurboFP- Seq: RT-120 799 RT- A33R(TK) / GFP IL- pE CD55-A33R

[0859] 114b 15super noFlag(VGF) RT-113 (TK)

[0860] (A46R) / pE_CD55- - - RT- A33R(TK) / IL- non

[0861] 115 15super noFlag(VGF) RT-114 (cre-lox system)

[0862] pUC-lox-GFP- - - RT- (A46R) / (TK) / IL- IL15_ori_noFlag

[0863] 116 15 ori noFlag(VGF) RT-65 (VGF)

[0864] pUC-lox-GFP- - - IL15

[0865] RT- (A46R) / (TK) / IL-15 super ori noFlag

[0866] 117 super ori noFlag(VGF) RT-65 (VGF)

[0867] (A46R) / CD55- pUC-lox-Turbo- - - RT- A33R(TK) / IL- hIgK-IL15_super

[0868] 118 15* super (VGF) RT-103 (VGF) - noFlag

[0869] (A46R) / CD55- pUC-lox-GFP- RT- A33R(TK) / IL- IL15*_super Seq: RT-118 797 118b 15* super (VGF) RT-98 (VGF) - noFlag

[0870] (A46R) / CD55- pUC-lox-GFP- Seq: RT-119 798 RT- A33R(TK) / IL- IL15*_super

[0871] 118c 15* super (VGF) RT-101 (VGF) - noFlag

[0872] (A46R) / CD55- pUC-lox-GFP- - - RT- A33R(TK) / Turbo-IL- IL 15 *_super_TM

[0873] 119 15* super TM (VGF) RT-103 (VGF) - noFlag

[0874] (A46R) / CD55- - - RT- A33R(TK) / IL- non

[0875] 120 15* super (VGF) RT-118 (cre-lox system)

[0876] (A46R) / Turbo-pE- pU C-lox-TurboFP- RT- CD55-A33R(TK) / IL- pE CD55-A33R Seq: RT-120 799 121 15 super (VGF) RT-104 (TK)

[0877] (A46R) / pE-CD55- - - RT- A33R(TK) / IL- non

[0878] 122 15 super (VGF) RT-121 (cre-lox system)

[0879] RT- (A46R) / (TK) / IL- non - - 123 15* super (VGF) RT-118 b (cre-lox system)

[0880] RT- (A46R) / Turbo-pE- pU C-lox-TurboFP- Seq: RT-120 799

[0881]

[0882] 124 CD55-A33(TK) / IL- RT-123 pE CD55-A33R Attorney Docket No. 120276-2615PC

[0883] -111-

[0884] SEQ NAME PARENT TRANSFER TRANSFER ID Modification / insertion Vector (NAME) Vector ID

[0885] VIRUS NO:

[0886] 15* super (VGF) (TK)

[0887] (A46R) / pE-CD55- RT- A33(TK) / IL- non - - 125 15* super (VGF) RT-124 (cre-lox system)

[0888] (A46R) / eGFP-pE- CD55-A33(TK) /

[0889] 796

[0890] RT- Turbo-IL- 15 *_super pUC-lox-eGFP- Seq: RT-58

[0891] 126 (VGF) RT-118-b CD55-A33R (TK)

[0892] (A46R) / pE-CD55- RT- A33(TK) / IL- non - - 127 15* super (VGF) RT-126 (cre-lox system)

[0893] Turbo-IL- 15 *_super pUC-lox-Turbo- RT- (A46R) / CD55- IL15*_super Seq: RT-128 800

[0894] 128 A33R(TK) / (VGF) RT-103 (A46R) - noFlag

[0895] IL-15*_super (A46R) /

[0896] RT- CD55-A33R(TK) / non - - 129 (VGF) RT-128 (cre-lox system)

[0897] (A46R) / CD55- RT- A33R(TK) / IL-15 non - 793

[0898] 134 super(VGF) no-flag RT-107 (cre-lox system)

[0899]

[0900] The table below summarizes structures of the above viruses, which are derived from RT-00 / N2, their nomenclature, genotype with reference to the vaccinia virus locus and insert, if present, and heterologous nucleic acid encoded by the virus, (see also Figure 24 for insertion sites for RT-31 through RT-43).

[0901] Genotype

[0902] Name Transfer ID (*: single K. O, **: Vector double K. O, ***: triple A46R B8R J2R A52R F1L VGF B19R Ig SEQ ID K. O) NO. RT-00

[0903] (SEQ

[0904] ID N2 n.a. NO:1)

[0905] pSEL- RT- 01* Turbo (TK) Turbo SEQ ID FP635 NO: 2 RT- Turbo (TK) / eGFP pSEL- pSEL- 02** (A4 R Turbo SEQ ID 6 ) eGFP FP635 NO: 3 RT- Turbo (TK) / eGFP pSEL- pSEL- SEQ ID 03** (B8R) eGFP Turbo

[0906] FP635 NO: 4 RT- Turbo (TK) / eGFP pSEL- Turbo pSEL- SEQ ID 04** (A52R) FP635 eGFP NO: 5 RT- Turbo (TK) / pSEL- pSEL- pSEL- 05** CD55 CD55- Turbo SEQ ID -B5R (A46R) NO: 6 B5R FP635

[0907] pSE- pSEL- RT- Turbo (TK) / pSE- SEQ ID CD55- Turbo 06** CD55-B5R (A46R) NO: 7 B5R FP635

[0908] pSL- pSEL- RT- Turbo (TK) / pSL - SEQ ID CD55- Turbo 07** CD55- B5R (A46R) NO: 8 B5R FP635

[0909]

[0910] Attorney Docket No. 120276-2615PC

[0911] -112-

[0912] Genotype

[0913] Name Transfer ID (*: single K. O, **: A46R B8R J2R A52R F1L VGF B19R Vector double K. O, ***: triple Ig SEQ ID K. O) NO. RT- eGFP (TK) / pSEL- pSEL- pSEL- SEQ ID 08** CD55-B5R(A46R) CD55- B5R eGFP NO: 9 RT- Turbo (TK) / pSEL- pSL- pSEL- Q9** CD55(sol)-B5R(A46R) CD55 Turbo n.a.

[0914] (sol)-B5R FP635

[0915] pSL- anti- RT- pSL-anti-VEGF(TK) / pSEL- VEGF SEQ ID 10** pSEL-CD55- CD55- B5R(A46R) B5R pSEL- NO: 10

[0916] Turbo

[0917] FP635

[0918] RT- eGFP(TK) / pSEL- pSEL- pSEL- 11** CD55-B5R(A46R) / CD55- pSEL- Turbo SEQ ID * eGFP NO: 11 Turbo(VGF) B5R FP635

[0919] RT- eGFP(TK) / pSEL- pSEL- SEQ ID 12** Turbo

[0920] Turbo(VGF) eGFP FP635 NO: 12 pSL- RT- Turbo (TK) / pSL-anti- pSEL- anti- 1 Turbo VEGF SEQ ID 3** VEGF(FIL) FP635 pSEL- NO: 13 eGFP

[0921] RT- pSEL - SEQ ID 14* eGFP (TK) eGFP NO: 14 RT- eGFP (TK) / pSEL - pSEL- Turbo SEQ ID 15** Turbo(B19R) eGFP FP635 NO: 15 RT- 16** (TK) / (B19R) (TK-) (B19R-) n.a. RT- pSEL- (TK) / Turbo(B19R) (TK-) Turbo n.a.17**

[0922] FP635

[0923] RT- eGFP(TK) / (B19R) pSEL - 18** eGFP (B19R-) n.a. RT- (TK) / (VGF) (TK-) (VGF ) n.a.19**

[0924] RT- pSEL - eGFP(TK) / (VGF) (VGF ) n.a.20** eGFP

[0925] pSEL- RT- (TK) / Turbo(VGF) (TK-) Turbo n.a.2i**

[0926] FP635

[0927] pSEL- RT- (TK) / (VGF) / Turbo SEQ ID (TK-) (VGF ) Turbo 22 * * * (B19R) NO: 16 FP635

[0928] pSEL- RT- Turbo (TK) / (A46R) (A46R-) Turbo n.a.23**

[0929] FP635

[0930] pSEL- RT- (B8R- Turbo (TK) / (B8R) Turbo n.a.24** ) FP635

[0931] pSEL RT- (A52R- Turbo (TK) / (A52R) Turbo n.a.

[0932] 25** ) FP635

[0933] (TK) / eGFP(A52R) / pSEL- RT- pSEL- SEQ ID Turbo (TK-) Turbo 26*** eGFP NO: 17 (VGF) FP635

[0934] (TK) / eGFP(B8R) / pSEL- RT- pSEL- SEQ ID Turbo (TK-) Turbo 27 * * * eGFP NO: 18 (VGF) FP635

[0935]

[0936] Attorney Docket No. 120276-2615PC

[0937] -113-

[0938] Genotype

[0939] Name Transfer ID (*: single K. O, **: A46R B8R J2R A52R F1L VGF B19R Vector double K. O, ***: triple Ig SEQ ID K. O) NO. (TK) / eGFP(B8R) / pSEL- RT- Turbo pSEL- 28*** eGFP (TK-) Turbo SEQ ID NO: 19 (B19R) FP635

[0940] (TK) / eGFP(A52R) / pSEL- RT- pSEL- SEQ ID Turbo (TK-) Turbo 29*** eGFP NO: 20 (B19R) FP636

[0941] pSL- (TK) / pSL-anti-VEGF anti- pSEL- RT- SEQ ID eGFP(FlL) / Turbo (TK-) VEGF Turbo

[0942] 30*** NO: 21 (B19R) pSEL- FP635

[0943] eGFP

[0944] pSEL- RT- pSEL-CD55- CD55- (TK-) n.a.31** B5R(A46R) B5R

[0945] RT- (TK) (TK-) n.a.

[0946] 32*

[0947] pSEL- RT- Nluc SEQ ID Nluc-eGFP (TK) 33* pSEL- NO: 518 eGFP

[0948] RT- (TK) / (B19R) / eGFP pSEL- SEQ ID (TK-) (B19R-) 34*** (A46R) eGFP NO: 3 RT- (TK) / (VGF) / eGFP pSEL- SEQ ID (TK-) (VGF ) 35*** (A46R) eGFP NO: 3 RT- (TK) / (B19R) / (A46R) (A46R-) (TK-) (B19R-) n.a.36***

[0949] RT- (TK) / (VGF) / A46R) (A46R-) (TK-) (VGF ) n.a.37***

[0950] RT- (TK) / (VGF) / (B19R) (TK-) (VGF ) (B19R-) n.a.38***

[0951] pSEL - eGFP RT- CD55-B5R(TK) / SEQ ID (A46R-) pSL- (B19R-) 39*** (B19R) / (A46R) NO: 519 CD55- B5R

[0952] pSEL - eGFP RT- CD55-B5R(TK) / (VGF) SEQ ID (A46R-) pSL- (VGF ) 4Q*** / (A46R) NO: 520 CD55- B5R

[0953] pSEL - eGFP

[0954] RT- CD55-B5R(TK) / SEQ ID pSL- (VGF ) (B19R-) 41*** (VGF) / (B19R) NO: 521 CD55- B5R

[0955] pSEL - eGFP RT- CD55-A33R(TK) / SEQ ID pSL- (VGF ) (B19R-) 42*** (VGF) / (B19R) NO: 522 CD55- A33R

[0956] pSEL - pSEL- CD55-B5R(TK) / eGFP Nlue RT- SEQ ID (VGF) / (B19R) / pSL- (VGF ) (B19R-) pSEL- 43*** NO: 523 Luc(Ig) CD55- Turbo B5R FP635 pSEL- eGFP RT- CD55-B5R(TK) / SEQ ID (A46R-) pSE- 45** (A46R) NO: 628 CD55- B5R

[0957] pSEL- RT- eGFP(A46R) / (TK) / pSEL- SEQ ID (TK-) Turbo 51*** Turbo(B19R) eGFP NO: 3 FP635

[0958]

[0959] Attorney Docket No. 120276-2615PC

[0960] -114-

[0961] Genotype

[0962] Name Transfer ID (*: single K. O, **: A46R B8R J2R A52R F1L VGF B19R Vector double K. O, ***: triple Ig SEQ ID K. O) NO. RT- eGFP(A46R) / (TK) / pSEL- pSEL- SEQ ID 52*** Turbo(VGF) eGFP (TK-) Turbo

[0963] FP635 NO: 3 pSEL- RT- CD55-A33R(TK) / eGFP pSEL- pSL- Turbo SEQ ID 58** Turbo(VGF) CD55- FP635 NO: 629

[0964] A33R

[0965] pSEL- RT- CD55-B5R(TK) / eGFP pSEL- 61** Tur pSL- Turbo SEQ ID bo(B19R) CD55- FP635 NO: 630

[0966] B5R

[0967] RT- (TK) / u pSEL- pSEL- T rbo(VGF) / TagBFP SEQ ID 62*** FLT3L(B19R) (TK-) Turbo

[0968] FP635 pSL- NO: 631

[0969] FLT3L

[0970] R - pSEL- pSEL- T (TK) / Turbo(VGF) / TagBFP SEQ ID 63*** IL15(B19R) (TK-) Turbo

[0971] FP635 pSL- NO: 632

[0972] IL15

[0973] RT- (A46R) / (TK) pSEL- 54*** / Turbo(B19R) (A46R- (TK-) Turbo n.a.

[0974] FP635

[0975] RT- (A46R) / (TK) pSEL- (A46R-) (TK-) Turbo n.a.

[0976] 65*** / Turbo(VGF) FP635

[0977] pSEL- RT- Turbo(TK) / FLT3L pSEL- TagBFP SEQ ID 72** (B19R) Turbo

[0978] FP635 pSL- NO: 631

[0979] FLT3L

[0980] pSE

[0981] RT- Turbo(TK) / IL- pSEL- L- Turbo TagBFP SEQ ID 73** 15(B19R) FP635 pSL- NO: 632

[0982] IL15

[0983] RT- (A46R) / Turbo (TK) / pSEL- (A46R-) Turb pSEL-eGFP SEQ ID 74*** FLT3L(VGF) o pSL-FLT3L NO: 633

[0984] FP635

[0985] RT- (A46R) / Turbo(TK) / pSEL- pSEL-eGFP SEQ ID 75*** IL-15(VGF) (A46R-) Turbo pSL-IL15 NO: 634

[0986] FP635

[0987] pSEL- (A46R) / CD55- eGFP pSEL- RT- SEQ ID 75*** A33R(TK) (A46R-) pSL- Turbo

[0988] / Turbo(B19R) CD55- FP635 NO: 629

[0989] A33R

[0990] pSEL- RT- (A46R) / CD55- eGFP pSEL- 77 * * * A33R(TK) / (A46R-) pSL- Turbo SEQ ID Turbo(VGF) CD55- FP635 NO: 629

[0991] A33R

[0992] RT- (A46R) / CD-55- pSL- 82*** A33R(TK) / FLT3L (A46R-) CD55- pSL- ( FLT3L n.a. B19R) A33R

[0993] RT- (A46R) / CD-55- pSL- 83*** A33R(TK) / IL15 (A46R-) CD55- pSL- (B1 IL15 n.a.

[0994] 9R) A33R

[0995] RT- (A46R) / CD-55- pSL- 84*AA A33R(TK) / (A46R-) CD55- pSL-FLT3L n.a.

[0996] FLT3L(VGF) A33R

[0997] RT- (A46R) / CD-55- pSL- 85*AA A33R(TK) / IL15 (VGF) (A46R-) CD55- pSL-IL15 n.a.

[0998] A33R

[0999] RT- (A46R) / (TK) / FLT3L pSEL- SEQ ID

[1000]

[1001] 86*** (B19R) (A46R-) (TK-) TagBFP NO: 631 Attorney Docket No. 120276-2615PC

[1002] -115-

[1003] Genotype

[1004] Name Transfer ID (*: single K. O, **: A46R B8R J2R A52R F1L VGF B19R Vector double K. O, ***: triple Ig SEQ ID K. O) NO.

[1005] pSL- FLT3L

[1006] pSEL- RT- (A46R) / (TK) / IL15 TagBFP SEQ ID g7*** (B19R) (A46R-) (TK-) pSL- NO: 632

[1007] IL15

[1008] RT- (A46R) / (TK) / FLT3L pSEL-eGFP SEQ ID gg*** (VGF) (A46R-) (TK-) pSL-FLT3L NO: 633 RT- (A46R) / (TK) / IL15 pSEL-eGFP SEQ ID g2*** (VGF) (A46R-) (TK-) pSL-IL15 NO: 634 RT- (A46R) / (TK) / n.a 20*** Fl (A46R-) (TK-) pSL-.

[1009] t3L(B19R) FLT3L RT- (A46R) / (TK) / n.a 21* (A46R-) (TK-) pSL-. ** IL15(B19R) IL15

[1010] RT- (A46R) / (TK) / n.a.

[1011] 22*** Flt3L(VGF) (A46R-) (TK-) pSL-Flt3L

[1012] RT- (A46R) / (TK) / n.a.

[1013] 2j*** IL15(VGF) (A46R-) (TK-) pSL-IL15

[1014] RT- (A46R) / (TK) / pSEL - n.a.

[1015] 24*** eGFP(VGF) (A46R-) (TK-) eGFP

[1016] RT- n.a.

[1017] 25*** (A46R) / (TK) / (VGF) (A46R-) (TK-) (VGF )

[1018] pSEL - SEQ ID RT- (A46R) / (TK) / IL15 eGFP NO: 793 26*** super(VGF) (A46R-) (TK-) pSL-IL15

[1019] super

[1020] pSL- n.a. RT- anti-VEGF(A46R) / antiVEGF pSEL- 27*** (TK-)

[1021] (TK) / Turbo(VGF) pSEL- TurboFP635

[1022] eGFP

[1023] RT- (A46R) / (TK) / n.a.

[1024] 2g*** (A46R-) (TK-) pSEL - eGFP(VGF) eGFP

[1025] RT- n.a.

[1026] 22*** (A46R) / (TK) / (VGF) (A46R-) (TK-) (VGF )

[1027] pSL- n.a. RT- anti-VEGF(A46R) / antiVEGF pSEL - 100*** (TK) / eGFP(VGF) pSEL- (TK-) eGFP

[1028] eGFP

[1029] pSEL - n.a. RT- (A46R) / CD55- eGFP

[1030] A33R(TK) / (A46R-) pS pSEL - 101*** L- eGFP(VGF) CD55- eGFP

[1031] A33R

[1032] RT- anti-VEGF(A46R) / pSL- n.a.

[1033] 102*** (TK) / (TK-) (VGF ) (VGF) antiVEGF

[1034] RT- (A46R) / CD55- pSL- n.a.

[1035] 103*** A33R(TK) / (VGF) (A46R-) CD55- (VGF )

[1036] A33R

[1037] RT- (A46R) / (TK) / IL-15 pSL-IL15 SEQ ID 104*** super(VGF) (A46R-) (TK-) super NO: 793 RT- anti-VEGF(A46R) / pSL- n.a.

[1038] antiVEGF pSL- 105*** CD55-A33R(TK) /

[1039] (VGF) pSEL- CD55- (VGF )

[1040] A33R

[1041] eGFP

[1042] anti RT- -VEGF(A46R) / pSL pSL- n.a.

[1043] 1 CD55-A33R(TK) / - 06*** antiVEGF CD55- (VGF )

[1044] (VGF) A33R

[1045] pSEL - SEQ ID RT- (A46R) / CD55- pSL- eGFP NO: 793

[1046] A33R(TK) / IL-15 (A46R-) CD55- 107* * * pSL-IL15 super(VGF) A33R super

[1047] (A46R) / CD55- pSL- SEQ ID RT- pSL-IL15 A33R(TK) / IL-15 (A46R-) CD55- NO: 793108*** super-flag super(VGF) A33R

[1048]

[1049] Attorney Docket No. 120276-2615PC

[1050] -116-

[1051] Genotype

[1052] Name Transfer ID (*: single K. O, **: A46R B8R J2R A52R F1L VGF B19R Vector double K. O, ***: triple Ig SEQ ID K. O) NO. RT- (A46R) / CD55- pSL- pSEL - SEQ ID 109* * * A33R(TK) / IL- (A46R-) CD55- eGFP NO: 791

[1053] 15(VGF) A33R pSL-IL15

[1054] RT- (A46R) / CD55- pSL- SEQ ID no*** A33R(TK) / IL- (A46R-) CD55- pSL-IL15 NO: 791

[1055] 15(VGF) A33R

[1056] pSEL - SEQ ID RT- (A46R) / (TK) / eGFP NO: 792 111*** IL-15 super_ori(V GF) (A46R-) (TK-) pSL-IL15

[1057] super ori

[1058] RT- (A46R) / (TK) / IL- SEQ ID ( pSL-IL15

[1059] H2*** 15super ori(VGF) A46R-) (TK-) super ori NO: 792 pSEL - SEQ ID RT- (A46R) / CD55- pSL- eGFP NO: 7 H A33R(TK) / IL- (A46R-) CD55- 92 3***

[1060] 15super ori(VGF) A33R pSL-IL15

[1061] super ori

[1062] RT- (A46R) / CD55- pSL- SEQ ID A33R(TK) / IL- (A46 pSL-IL15R-) CD55- NO: 79211^*** super ori 15super ori(VGF) A33R

[1063] (A46R) / pSEL - pSEL

[1064] RT- T urbo jE_CD55- TurboFP -eGFP

[1065] SEQ ID 114b A33R(TK) / GFP IL- (A46R-) pE- pSL NO: 799 * * * 15super_ CD55- -IL15 super

[1066] noFlag(VGF) A33R noFLAG

[1067] (A46R) / pE CD55- n.a. pE- pSL- RT- A33R(TK) / IL- (A46R-) CD55- IL15 super_

[1068] 115*** 15super_ A33R noFLAG noFlag(VGF)

[1069] pSEL - n.a. (A46R) / (TK) / IL- RT- eGFP 15 ori (A46R-) (TK-) 116*** pSL-IL15 noFlag(VGF) ori noFlag

[1070] pSEL - n.a. RT- eGFP (A46R) / (TK) / IL-15 117 (A46R-) (TK-) pSL-IL15

[1071] super_ori_noFlag(y GF) * * * super ori

[1072] noFlag

[1073] pSEL - n.a. RT- (A46R) / CD55- pSL- Turbo

[1074] 118 A33R(TK) / IL- (A46R-) CD55- pSL-hIgK_

[1075] * * * 15*_super (VGF) A33R IL15_super

[1076] noFlag

[1077] pSEL - SEQ ID RT- Turbo NO: 797 (A46R) / (TK) / Turbo- 118b (A46R-) (TK-) pSL- IL-15* super (VGF) * * * IL15*_super

[1078] noFlag

[1079] pSEL - pSEL - n.a. RT- (A46R) / CD55- eGFP Turbo

[1080] 118c A33R(TK) / IL- (A46R-) pSL- pSL- * * * 15*_super (VGF) CD55- IL15*_super

[1081] A33R noFlag

[1082] pSEL - SEQ ID pSEL - Turbo NO: 798 (A46R) / CD55- eGFP RT- pSL- A33R(TK) / Turbo-IL- (A46R-) pSL- H9*** IL15*_s 15* super TM (VGF) CD55- uper TM A33R noFlag

[1083] (A46R) / CD55- pSL- pSL- n.a. RT- A33R(TK) / IL- (A46R-) CD55- IL15*_super 120***

[1084] 15* super (VGF) A33R noFlag

[1085] (A46R) / Turbo-pE- SEQ ID RT- pSEL- pSL-IL15 CD55-A33R(TK) / IL- (A46R-) NO: 799121*** Turbo super 15 super (VGF)

[1086]

[1087] Attorney Docket No. 120276-2615PC

[1088] -117-

[1089] Genotype

[1090] Name Transfer ID (*: single K. O, **: A46R B8R J2R A52R F1L VGF B19R Vector double K. O, ***: triple Ig SEQ ID K. O) NO.

[1091] pSE- CD55- A33R

[1092] RT- (A46R) / pE-CD55- pSE- n.a.

[1093] 122 A33R(TK) / IL- (A46R-) CD55- pSL-IL15

[1094] AAA 15 super (VGF) A33R super

[1095] RT- 12 (A46R) / (TK) / IL- pSL- n.a.

[1096] 3

[1097] AAA 15*_super (VGF) (A46R-) (TK-) IL15*_super

[1098] noFlag

[1099] pSEL- SEQ ID RT- (A46R) / Turbo-pE- Turbo pSL- NO: 799 124 CD55-A33(TK) / IL- (A46R-) pSE- IL15*_super

[1100] AAA 15*_super (VGF) CD55- noFlag

[1101] A33R

[1102] RT- (A46R) / pE-CD55- pSE- pSL- n.a.

[1103] 125 A33(TK) / IL- (A46R-) CD55- IL15*_super

[1104] AAA 15* super (VGF) A33R noFlag

[1105] pSEL - pSEL - SEQ ID RT- (A46R) / eGFP-pE- eGFP Turbo NO 126 CD55-A33(TK) / : 796

[1106] (A46R-) pS

[1107] AAA Turbo-IL-15*_super L- pSL- (VGF) CD55- IL15*_super

[1108] A33R noFlag

[1109] pSEL - n.a. RT- (A46R) / pE-CD55- eGFP pSL- 127 A33(TK) / IL- (A46R-) pSL- IL15*_super

[1110] AAA 15*_super (VGF) CD55- noFlag

[1111] A33R

[1112] pSEL - SEQ ID Turbo-IL-15*_super Turbo NO: 800 RT- pSL- 128 (A46R) / CD55- pSL- CD55- (VGF )

[1113] AAA A33R(TK) / IL15*_s

[1114] (VGF) uper A33R

[1115] noFlag

[1116] RT- IL-15* super (A46R) / pSL- pSL- n.a.

[1117] 129 CD55-A33R(TK) / IL15*_

[1118] AAA (VGF super CD55- (VGF )

[1119] ) noFlag A33R

[1120] RT- (A46R) / CD55- pSL- pSL-IL15 SEQ ID 134 A33R(TK) / IL-15 (A46R-) CD55- super-no NO: 793 AAA

[1121]

[1122] super(VGF) A33R flag

[1123] RT-00 or a virus derived therefrom can be and has been modified or engineered for recombinant expression of selection markers or detectable and encoded payloads.

[1124] Selection markers include, but are not limited to, EGFP, EmGFP, mNeonGreen, EBFP, TagBFP, EYFP, TPet, GFP, BFP or TurboFP635. The RT-00 virus and viruses derived therefrom also are modified to encode therapeutic and / or diagnostic payloads. For example, therapeutic proteins include, but are not limited to, cytokines (GM-CSF, IL-2, IL-7, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, IL-7-IL-21 fusion proteins, TNF, MIPla, FLt3L, IFN-b, IFN-g), chemokines (CC15, CC12, CC119, CXC111, RANTES), costimulators (OX40L, 4-1BBL, CD40L, B7.1 / CD80, GITRL, LIGHT, CD70), bi-specific t-cell engagers (BITEs), therapeutic antibodies, immune checkpoint inhibitors, single chain antibodies such as single chain antibodies against e.g., VEGF, e.g., VEGFA, Attorney Docket No. 120276-2615PC

[1125] -118-

[1126] VEGFB, PGF, VEGFR2, PDGFR, Ang-1, Ang-2, ANGPT1, ANGPT2, HGF, TGF-P and immune checkpoint inhibitors, such as inhibitors of PD-1, PD-L1, CTLA4, or TIM-3, prodrug activators, such as lacZ, cytosine deaminase enzymes, human sodium iodide symporter, hNIS, Aquaporin 1-AQP1, and any other payload of interest, particularly any that have anti-cancer activity, or promote an anti-cancer phenotype or activity.

[1127] RT-00 or a virus derived therefrom can be modified and / or or engineered to encode and express detectable and or selection markers including, to express 1, 2 or more of the recombinant proteins described above under control of heterologous or different viral promoters (e.g., Pel, pL, CMV). The viruses can be engineered to encode and express combinations of therapeutic proteins, e.g, against modulators of angiogenesis and immune system co-stimulators or checkpoints, e.g, Anti-VEGFA and VEGFB and PGF; anti-VEGF and anti-ANGPT2; anti-VEGF, anti-ANGPT-2 and anti-CTL4; anti-VEGF and OX40L; Anti-VEGF, Anti-ANGPT2 and anti-PD-1.

[1128] RT-00 or a virus derived therefrom, can be modified, or engineered to encode and express an anti-complement protein or a complement regulatory protein (CRP or RCA) or other regulator of humor immunity such that the immune system of the host has reduced or eliminated recognition of the virus.

[1129] RT-00 or a virus derived therefrom, can be modified, or engineered for expression or overexpression of an anti-complement protein or a regulator of complement activation or other such protein, or active or functional portion thereof, on the EEV outer membrane, such as, part of a chimeric or fusion protein with an EEV membrane protein or portion thereof, whereby the CRP. In some embodiments, embodiments, RT-00 or a virus derived therefrom, can be modified, or engineered to overexpress an CRP, or other such protein, or a portion thereof on the EEV outer membrane as a fusion protein such that it is covalently linked to a protein expressed or present on the EEV outer membrane. Thus, provided herein are EEV viruses designated RT-00 - RT-45, RT-51 - RT65, RT-72 - RT-89, RT-90 -RT-114, RT-115 - RT-129, and RT-134 summarized in the above tables and variants thereof encoding different payloads or including minor variations resulting upon culturing the viruses and / or degeneracy of the genetic code.

[1130] As detailed herein, above and below, and in the Examples below, the RT viruses were selected for improved tumor selectivity and higher production of EEV and then Attorney Docket No. 120276-2615PC

[1131] -119-

[1132] further modified as detailed herein. Any vaccinia virus can be modified as detailed herein to alter properties, such as serum stability, anti-tumor activity and other such properties. These modifications include, but are not limited to, knocking out particular genes, and / or by encoding fusion proteins (chimeric proteins) of a protein that inhibits complement or other anti-viral serum product with a viral EEV second envelope protein. The ability of the RT-00 virus, as shown in the Examples, to kill cancer cells was tested using an NCI-60 panel was assessed as was the resistance of this vaccinia virus against human humoral immunity and its rapid spread were assessed ex vivo. Targeting, biodistribution, therapeutic efficacy, and safety profile of the virus was evaluated in multiple animal models. Among the RT vaccinia viruses identified, the exemplified virus designated RT-00 (SEQ ID NO:1) was sequenced. It has three knockouts (3KO): TK (Thymidine kinase), A46R (immunomodulator), and VGF (Vaccinia virus growth factor). These genetic modifications significantly improved tumor-selective amplification and the safety profile while maintaining therapeutic efficacy. The 3KO RT virus demonstrates strong oncolytic activity against more than 60 different human cancer cell lines (NCI-60) at low multiplicity of infection (MOI). This 3KO RT virus was further engineered with to encode a CD55-domain fusion protein with the viral envelope A33R to display the CD55 portion. This chimeric (fusion) protein is expressed in the extracellular enveloped viral particle and protects the manufactured particle and viral progeny from inactivation by the human complement system. Targeting and biodistribution studies revealed that RT virus accumulated in all tumors on day one, with tumor-selective amplification observed on day seven following intravenous administration. In various immunocompetent mouse models, including metastatic lung cancer, this RT virus demonstrated excellent tumor targeting, killing, and expression of selected therapeutic payload.

[1133] Generation of the viruses

[1134] The viruses can be generated from RT-00 (SEQ ID NO: 1), or a virus having degenerate codons or other sequence variants thereof, by insertion of any of the transfer vectors (or similar vectors) set forth in the table by homologous combination or any other method by which modifications can be introduced into a vaccinia virus backbone.

[1135] Production of the RT recombinant viruses can be achieved through known techniques and protocols for producing recombinant vaccinia viruses (see e.g., Earl P. L. etal. Curr Protoc Protein Sci.,' 89: 5.13.1-5.13.18; Broder and Earl, (1999)Afo / . Attorney Docket No. 120276-2615PC

[1136] -120-

[1137] Biotechnol. 13:223-245; Falkner, F. G.; Moss, B. J. Virol. 1990, 64, 3108-3111). In general, cell monolayers are infected with vaccinia virus and transfected with a plasmid transfer vector that contains a transgene of interest, driven by a viral promoter, that is flanked by vaccinia virus DNA segments. Homologous recombination occurs during the replication cycle of virus between the vaccinia virus sequences in the transfer vector and the viral genome. The resulting recombinant vaccinia virus genome is packaged within the infected cells to form progeny vaccinia virus.

[1138] To obtain a transfer vector, a transgene of interest is cloned into any suitable plasmid, such as a pUC or pUC-19 vector, that contains: 1) a vaccinia virus promoter, 2) a multiple cloning site adjacent to the promoter for the addition of a transgene, 3) flanking sequences derived from vaccinia virus insertion site, and 4) necessary elements for replication and selection of the plasmid transfer vector in bacteria.

[1139] Viral promoters are selected to affect the time and level of expression of transgenes, exemplary of such include natural viral promoters (p), heterologous non-viral promoters, and synthetic promoters such as, synthetic early (pSE), synthetic early late (pSEL), and synthetic late (pSL), and inducible promoters (e.g. Azad et. al. Nat Commun. 2023 May 26; 14: 3035). Other exemplary promoters for use in the production of recombinant vaccinia viruses include:

[1140] SEQ ID NO: Promoter Sequence

[1141] 166 synthetic early late (pSEL) aaaaattgaaattttattttttttttttggaatataaata

[1142] 167 / 249 synthetic late (pSL) ttttttttttttttttttttggcatataaata

[1143] 525 synthetic early (pSE) aaaaattgaaaaactagcgtctttttttgctcgaagt

[1144] aaaaattgaa attttatttt ttttttttgg aatataaata

[1145] 526

[1146] earl...

Claims

1. Attorney Docket No. 120276-2615PC2.-516-3.WHAT IS CLAIMED:

1. A virus that is a high EEV vaccinia virus that is genome-modified to encode a fusion or chimera between an EEV outer membrane transmembrane protein, and a polypeptide that alters a property and / or activity of the virus, wherein:5.the transmembrane fusion or chimeric protein comprises an A33R, B5R, A34R, A56R, or F13L protein and the polypeptide that alters a property and / or activity of the virus, wherein the polypeptide is displayed on the outer membrane of the virus; and the EEV outer membrane is a host cell-derived membrane that comprises a host cell transmembrane fusion protein or chimera with a polypeptide that alters a property and / or activity of the virus when displayed on the virus; and6.the host cell-derived membrane is the outer membrane that the virus acquires from the cell in which it is propagated.

2. The virus of claim 1 that comprises at least three knockouts of the products encoded by A46R, VGF, and TK, and comprises an intact A56 gene.

3. The virus of claim 1 or claim 2 that is derived from an RT virus, optionally, the virus is the virus designated RT-134 (SEQ ID NO:901 or 917), or a virus whose genome comprises the sequence set forth in SEQ ID NO:918, or a virus that has least 95%, 96%, 97%, 98%, 99% or more sequence identify to the nucleotides 9,038 -185,371 set forth SEQ ID NO: 901 or 917 or to SEQ ID NO: 918.

4. The virus of any of claims 1-3, wherein the host cell-derived chimeric or fusion protein alters a property and / or activity of the virus selected from one or more of:10.(a) attenuation or blocking of anti-viral immunity,11.(b) reduction of neutralizing antibody binding,12.(c) reduction of antibody-dependent opsonization,13.(d) reduction of phagocytic uptake,14.(e) attenuation of natural killer (NK) cell cytotoxicity,15.(f) enhancement of delivery of virus to tumors,16.(g) enhancement of anti-tumor immunity.

5. The virus of any of claims 1-4, comprising nucleic acid encoding a therapeutic protein for treatment of a disease, disorder, or condition, such as an anticancer therapeutic or an immune modulatory protein.

6. The virus of any of claims 1-5, wherein the virus encodes a cytokine.Attorney Docket No. 120276-2615PC19.-517-7. The virus of claim 3 or claim 4, wherein the nucleic acid encoding the therapeutic protein is inserted into the VGF gene locus replacing all or a part of the encoded product to effect the knockout.

8. The virus of claim 6 or claim 7, wherein the cytokine comprises IL-15, such as an IL-15 superagonist or modified IL-15 superagonist.

9. The virus of any of claims 1-8, wherein host cell-derived fusion or chimeric transmembrane protein comprises an anti-cancer antibody or antigen-binding portion thereof or a receptor or ligand for targeting the virus to a tumor or immune cell, or comprises a polypeptide that attenuates anti-viral immunity.

10. The virus of claim 9, wherein the anti-cancer antibody comprises an scFv or antigen-binding portion thereof.

11. An enveloped virus, comprising a host-derived membrane, wherein the host-derived membrane is modified to display a transmembrane chimeric or fusion protein comprising the host transmembrane protein and a second polypeptide or functional portion thereof that alters properties or activities of the virus.

12. The enveloped virus of claim 11 that is an extracellular envelope virus (EEV) that comprises a host-derived membrane and a virally produced membrane.

13. The virus of claim 11 or claim 12 that is an extra envelope virus (EEV) form of vaccinia virus.

14. The enveloped virus of claim 13 that is a high EEV vaccinia virus.

15. The enveloped virus of claim 11 that is a retrovirus or other RNA virus that has a single membrane derived from the host.

16. An enveloped virus with a host cell-derived membrane, comprising a non-virally encoded polypeptide displayed on the host-derived membrane, wherein:30.the non-virally encoded membrane-displayed polypeptide alters properties and / or activities of the enveloped virus, such as the EEV vaccinia virus; and31.the non-virally encoded membrane-displayed polypeptide is a fusion protein or chimeric protein that comprises all or a portion of a host transmembrane protein and a polypeptide that alters the properties or activities of the virus.

17. The virus of any of claims 1-16 that is selected from among species of Herpesviridae, Iridoviridae, Poxviridae, Hepadnaviridae, Togaviridae, Flaviviridae,Attorney Docket No. 120276-2615PC33.-518-34.Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Bunyaviridae, Coronaviridae, Arenaviridae, Retroviridae, and Flioviridae.

18. The virus of claim 17 that is an extracellular envelope vaccinia virus (EEV) or a retrovirus or other RNA virus.

19. A programmed extracellular envelope virus (EEV), comprising a virally-encoded modified transmembrane polypeptide, and a non-virally encoded polypeptide displayed on the outer membrane, wherein the membrane-displayed polypeptide(s) alter properties and / or activities of the EEV virus.

20. A programmed extracellular envelope virus (EEV) or enveloped virus with a host cell-derived membrane, comprising a host cell-encoded modified transmembrane polypeptide on the second membrane, wherein the membrane-displayed polypeptide(s) alter properties and / or activities of the virus.

21. A modified cell, comprising a virus that is an extracellular envelope virus (EEV) or an enveloped virus with a host cell-derived membrane, wherein:39.the cell is modified to express one or more polypeptide(s) and / or peptide(s) in the cell membrane, whereby the polypeptide or peptide is displayed on the membrane and;40.the displayed polypeptide(s) and / or or peptide(s) alters a property and / or activity of the virus when the membrane envelops the virus; and41.the virus is a high EEV virus.

22. A modified cell or cell line, comprising an EEV virus that produces more than 1% EEV viruses when propagated in the cell or line, or a virus that acquires it membrane from the host cell, wherein the cell is genome-modified to encode and express on the cell membrane a polypeptide that, when displayed on the second or outer membrane of the EEV virus or on the membrane of the virus that acquires from the cell, alters an activity and / or property of the virus.

23. The virus or cell or cell line of any of claims 1-22, wherein the host cell transmembrane protein comprises a single- or multi-pass transmembrane protein, wherein the functional domain of the fusion or chimeric protein of the transmembrane protein is displayed on the surface of the host-derived membrane of the virus.Attorney Docket No. 120276-2615PC44.-519-24. The virus or cell or cell line of any of claims 1-23, wherein the host cell transmembrane protein comprises a single-pass transmembrane protein selected from CD8a, PDGFR, CD28, CD4, and CD3<25. The virus or cell or cell line of any of claims 1-23 wherein the polypeptide that alters the properties or activities of the virus is interacts with a receptor on a tumor cell or an immune cell.

26. The virus or cell or cell line of any of claims 1-25, wherein the cell comprises a fusion or chimeric protein on the host-derived virus membrane that comprises all or an active portion of complement inhibiting protein or a protein that modulates the immune response of a subject for treatment with the virus.

27. The virus or cell or cell line of claim 26, wherein the protein is chimeric protein or fusion protein that comprises a receptor or ligand.

28. The virus or cell or cell line of any of claims 1-27, wherein the virus is an EEV vaccinia virus.

29. The virus or cell or cell line of any of claims 1-28, wherein the virus is a high EEV-producing EEV virus.

30. The virus or cell or cell line of any of claims 1-28, wherein:52.the virus is a high EEV-producing EEV virus; and53.a high EEV-producing virus is a virus that, when propagated, produces more than 1%, 5%, 10%, 15%, 20%, 25%, 30%, generally more than 30%, or more than H D-W strain of vaccinia virus.

31. The virus or cell or cell line of any of claims 1-27 wherein the virus is a lentivirus.

32. The virus or cell or cell line of any of claims 1-31, wherein the virus or host cell also displays a transmembrane virus-encoded chimeric or fusion protein.

33. The virus or cell or cell line of any of claims 1-32, wherein the modified host cells for manufacture of the virus are modified cell lines or modified stem cells comprising a modified transmembrane protein.

34. The virus or cell or cell line of any of claims 1-33, wherein the cells are HEK293 cell, HeLa cells, or iPSCs.Attorney Docket No. 120276-2615PC58.-520-35. The virus or cell or cell line of any of claims 1-34, wherein the virus or cell or cell line comprises a modified host-derived transmembrane protein that comprises a receptor or ligand for targeting a tumor cell or an immune cell.

36. The virus or cell or cell line of any of claims 1-35, wherein the modified host-derived transmembrane protein comprises an anti-cancer antibody or antigenbinding portion thereof or a receptor or ligand for targeting TROP2, HER2, BCMA, CD38, EGFR, BCMA, BCMA, CD19, CD20, mesothelin, CD22, B7-H3, or PTK7.

37. The virus or cell or cell line of any of claims 1-36, wherein the virus is a high EEV vaccinia virus, comprising a knock-outs (KOs) of the VGF, TK, and A46R loci.

38. The virus or cell or cell line of any of claims 1-37, wherein the genome of the virus comprises nucleic acid encoding a therapeutic product that is an anti-cancer therapeutic or a product that promotes an anti-tumor response in the tumor microenvironment.

39. The virus or cell or cell line of claim 38, wherein the encoded product is a cytokine.

40. The virus or cell or cell line of claim 39, wherein the cytokine is an IL-15 or a modified form thereof.

41. The virus or cell or cell line of claim 40, wherein the IL-15 is an IL-15 superagonist (IL-15 / IL-15R alpha chain complex), or conjugate thereof with an scFv, and / or comprising a mutation that increases activity.

42. The virus or cell or cell line of any of claims 38-41, wherein the cytokine is IL-15 / IL-15R alpha chain complex (IL-15 superagonist) or a modified IL-15 / IL-15R alpha chain complex that comprises a replacement, deletion or insertion of one or more amino acids, whereby activity in vivo is increased compared to IL-15 / IL-15R alpha chain complex without the modifications.

43. The virus or cell or cell line of claim 42, wherein the IL-15 superagonist comprises the replacement N72D or N72E.

44. The virus or cell or cell line of any of claims 1-43, wherein the virus encodes a therapeutic selected from among one or more of CCL21, IL-1, IL-2, IL-3, IL-7, IL-12, IL-15, IL-15 / IL15a receptor complex (referred to as IL15 superagonist) and variants thereof that increase activity, IL- 18, IL-21, IFN-a, IFN-P, IFN-y, TNF-a, EPO,Attorney Docket No. 120276-2615PC69.-521-70.GM-CSF, G-CSF, Flt3L, FGF, EGF, IL-4, IL-6, IL-10, IL-11, IL-13, IL-17, IL-32, and IL-7-IL-21 fusion protein.

45. The virus or cell or cell line of any of claims 38-44, wherein the viral genome encodes an IL- 15 superagonist that comprises the IL- 15 sushi domain linked, via a peptide linker, to a modified IL-15 that has increased activity.

46. The virus or cell or cell line of claim 45, wherein sequence the IL- 15 superagonist comprises a Sushi domain, a linker, and IL- 15;73.a) the Sushi domain has the sequence:74.ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH WTTPS LKCIRDPALVHQRPAPPSTVTTAGV (SEQ ID NO:635)75.the sequence of the linker is SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 636), and76.the IL- 15 comprises N72D and comprises the sequence:77.NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS (SEQ ID NO:637); or78.b) a variant of the IL-15 superagonist that has at least 95%, 96%, 97%, 98%, 99% sequence identity to the IL-15 superagonist of a) and retains anti-tumor activity.

47. The virus or cell or cell line of any of claims 38-46, wherein a virus displays a virally-encoded transmembrane fusion or chimeric protein comprising A33 and CD55.

48. The virus or cell or cell line of any of claims 1-47, designated RT-134 whose genome comprises the sequence set forth in SEQ ID NO:901, 917, 918 or a sequence having least 95%, 96%, 97%, 98%, 99% or more sequence identity thereto to the SEQ ID NO:901, 917 or 918.

49. The virus or cell or cell line of claim 48, wherein:82.the genome of the virus comprises the knockouts of the VGF, TK, and A46 loci, and the genome encodes an IL-15 superagonist (an IL-15 / IL-15R alpha chain complex);83.the encoded IL-15 / IL-15R alpha chain complex comprises a replacement at residue N72 that is D or E; and84.the genome of the virus has at least 95% sequence identity to SEQ ID NO: 901, and includes the knockouts of VGF, TK, A46, and the knockout of VGF is effected by Attorney Docket No. 120276-2615PC85.-522-86.encoding the IL-15 superagonist inserted therein or replacing all or a portion of the encoded VGF protein.

50. The virus or cell or cell line of any of claims 1-49, wherein the host cell membrane is modified display a modified transmembrane protein that alters one or more properties or activities of the virus that comprises the modified membrane.

51. The virus or cell or cell line of any of claims 1-50, wherein the property and / or activity that is altered by a transmembrane protein displayed on the host cell-derived membrane is selected from among wherein the property / activity is selected from one or more of:89.(a) attenuation or blocking of anti-viral immunity,90.(b) reduction of neutralizing antibody binding,91.(c) reduction of antibody-dependent opsonization,92.(d) reduction of phagocytic uptake,93.(e) attenuation of natural killer (NK) cell cytotoxicity,94.(f) enhancement of delivery to tumors,95.(g) enhancement of anti-tumor immunity.

52. The virus or cell or cell line of any of claim 1-51, wherein the property and / or activity of the virus that is altered is selected from among one or more of:97.a) enhancing delivery to tumors and / or target tissues, such as by expressing tissue and / or tumor homing proteins, such as, for example, chemokine receptors and integrins;98.b) targeting to and / or comprising receptor or ligand binding domains such as, for example antibodies or antigen-binding portions thereof, scFvs and fusion or chimeric proteins thereof;99.c) enhancing anti-tumor immunity such as by expressing on the membrane a transmembrane protein comprising all or an active portion of a co-stimulatory molecule; a TNF superfamily of ligands (TNFSF); a TGF-beta polypeptide antagonists; a checkpoint inhibitor; and a cytokine and / or chemokine.

53. The virus or cell of any of claims 1-52, wherein the host cell membrane or host cell-derived membrane is modified to:101.a) express one or more chemokine receptors (CCR1, CCR2, CCR4, CCR5, CCR7, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, XCR1, and atypical chemokine receptors (ACKRs), such as ACKR1 and ACKR1); integrins (a6pi, a6p4, a6p5, avP5, pi, a2pi, a2p3, avP3, a5pi, a6pi, avP3,a3pi, avP6, all, a6pi, a5, Attorney Docket No. 120276-2615PC102.-523-103.P3, a9pi, P4, ax, a9p5, a9, ITGB1); and / or other targeting molecules such as CD44, C-met, and CLA4 tissue-specific for tumor specific targeting or tropism; and / or104.b) target or include binding domains, wherein proteins for targeting include: single-chain variable fragments (scFvs), variable heavy domains of heavy chains (VHHs), Fab fragments, or engineered ligand mimetics, DARPin, ligands or receptors that recognize or are expressed on target cells in a host.

54. The virus or cell or cell line of claim 53, wherein binding domains are selected from one or more of TROP2, HER2, BCMA, CD38, EGFR, BCMA, CD 19, CD20, Mesothelin, CD22, B7-H3, and PTK7.

55. The virus or cell or cell line of any of claims 1-54, wherein the host-derived viral membrane comprises a chimeric antigen receptor (CAR).

56. The virus or cell of claim 55, wherein the CAR comprises an extracellular antigen-binding domain, a hinge or spacer region, a transmembrane domain, and one or more intracellular signaling domains.

57. The virus or cell or cell line of claim 55, wherein the CAR comprises an extracellular single-chain variable fragment (scFv) targeting an antigen expressed on a tumor cell or other targeted cell.

58. The virus or cell or cell line of any of claims 55-57, comprising a CAR that comprises an scFv or other single chain antibody that targets a tumor antigen, such as TROP2, HER2, CD20, CD38, and BCMA, a hinge region, and a transmembrane domain.

59. The virus or cell or cell line of any of claims 1-58, wherein the host-derived membrane on the virus targets a tumor marker and / or antigen, or a tumor malignancy marker, or an autoimmune / inflammatory disorder marker.

60. The virus or cell or cell line of claim 59, wherein the host-derived membrane on the virus targets a tumor marker and / or antigen, or a tumor malignancy marker, or an autoimmune / inflammatory disorder marker, is one or more of112.a) a tumor marker / antigen that is HER2, Trop-2, Nectin-4, Tissue Factor, Folate Receptor a, c-MET, HER3, CLDN18.2, EGFR, EGFRvIII, Mesothelin, FAP, CEA, B7-H3, PSMA, GPC3, MUC1, IL-13Ra2, GD2, orRORl; Attorney Docket No. 120276-2615PC113.-524-114.b) a tumor hematologic malignancy markers that is BCMA, CD38, SLAMF7, GPRC5D, CD19, CD22, CD30, CD33, CD123, FLT3, CLL1 (CLEC12A), SLAMF7 (CS1), or CD79b; and115.c) an autoimmune inflammatory disorder marker include CD 19, BCMA, CD20, CD22, and CD52.

61. The virus or cell or cell line of any of claims 1-60, wherein the host cell transmembrane protein that is modified is CD8a, PDGFR, CD28, CD4, and CD3Q or comprises a glycosylphosphatidylinositol (GPI) anchors with or without linkage to a signaling domain.

62. The virus or cell or cell line of any of claims 1-61, wherein transmembrane fusion or chimeric protein on the host derived membrane on the virus are selected from among one or more of TROP2(PDGFR-TM), HER2(PDGFR-TM), BCMA(PDGFR-TM), CD38(PDGFR-TM), TROP2(CD8-TM), where the sequences are set forth in SEQ ID NOs: 881-885, respectively, or variants thereof that have at least 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NOs:881-885, respectively and retaining the activity of the scFv or antigen binding portion of the fusion or chimeric protein.

63. The virus or cell or cell line of any of claims 1-62, wherein the host-derived cell membrane comprises a peptide or polypeptide that119.a) attenuates or block anti-viral immunity, such as by:120.increasing resistance to complement / attenuated complement deposition, optionally selected from one or more of: expressing complement regulator / inhibitor, such as CD46, CD55, CD59, and CD35 / CR1; and / or expressing a soluble complement regulator / inhibitor in a membrane tethered or pericellularly retained format, such as Factor H, C4b binding protein, and functional portions thereof; and / or expressing a protein to inhibit lectin-pathway initiation - Inhibitors of MASP proteases, such as Cl-inhibitor (Cl -inh), human astrovirus coat protein (CoPt), and Flavivirus non-structural protein 1 (NS1); and / or expressing a viral mimic of a complement regulator / inhibitor, such as CP, MOPICE, SPICE, CCPH, Kaposi-sarcoma associated herpesvirus Kaposi ca I KCP, Herpesvirus saimiri (HVS) HVS-CD59, Rhesus rhadinovirus RCP-H and RCP-1, murine gamma herpesvirus 68 (yHV-68) RCA, Influenzavirus Ml, EMICE, IMP, and functional portions thereof; and / or Attorney Docket No. 120276-2615PC121.-525-122.b) attenuates neutralizing antibody binding / Attenuate antibody dependent opsonization, such as one or more of: expressing ST6GAL1, B4GALT1, FUT8, and MGAT5 to promote a dense, terminally sialylated and fucosylated glycocalyx such that recognition of the viral envelope by neutralizing antibodies is sterically hindered / reduce Fc clustering on envelope membrane; expressing an antibody decoy receptor to act as a neutralizing antibody sink, such as a membrane anchored Fc binding domain, such as Fc binding domains derived from Protein A or Protein G Fc binding domains; expressing an Fc Receptor, as an antibody decoy receptor, such as IgA (e.g., FcaRI (CD89)); IgM (e.g., plgR, and Fca / pR); IgE (e.g., FcaRI and FcsRII (CD23)); and IgG (e.g., FcyRI (CD64), FcyRII (CD32), FcγRIIII (CD16), and FcRn).

64. The cell or virus or cell line of any of claims 1-63, wherein the host cells membranes are modified by one or more of:124.a) removing host cell features that facilitate classical and lectin pathway activation on the envelope or host cell membrane such as by eliminating Fc gamma receptor expression by removing adhesion molecules that stabilize Fc driven lytic synapses, such as by knockout LFA-1, ICAM-1, CD2, CD58, DNAM-1, Nectin and Nectin-like molecules, Cadherins, and Tetraspanins;125.b) attenuating phagocytic uptake, such as by overexpressing CD47;126.c) attenuating natural killer cell cytotoxicity, such as by HLA-E and HLA-G overexpression to maintain inhibitory NK signaling;127.d) expressing antagonists of NK cell receptors / ligands of NK inhibitory receptors - host cells can be engineered to express of one or more of: antagonists of MIC-A and MIC-B (NKG2D ligands) (e.g., kK5 (KHSV)); antagonists of the NKG2D receptor (e.g., Cowpox OMCP); antagonists of natural cytotoxicity receptors (NCRs) - targeting NKp30, NKp44, NKp46 receptors (e.g., HA (hemagglutinin - in vaccinia and other viruses)); ligands for the NK inhibitory receptors (KIR) (e.g., HLA-Bw4; HLA-C2); ligands for the NK inhibitory receptors (NKG2a / CD94) (e.g., HLA-E and derivatives alone or combined with 21M HLA-B ligands to generate HLA-E binding peptides and stabilize HLA-E surface expression);128.e) eliminating stress induced NKG2D ligands Membrane-Bound MICA / B (NKG2D Ligands); Membrane-Bound PVR (DNAM-1 Ligand); Membrane-Bound Attorney Docket No. 120276-2615PC129.-526-130.Nectin-2 (DNAM-1 Ligand), MICA, MICB, and ULBP family members, suppression or knockout of NECTIN 2 and PVR;131.f) silencing immunogenic and immune-activating determinants, such as by suppression or deletion of B2M; CIITA; MHC Class I molecules (HLA-A, B, C); MHC Class II molecules (HLA-DP, DQ, DR); MHC -like molecules (CDla / b / c / d); or regulators of transcription or expression of MHC Class I, MHC Class II, MHC-like molecules (e.g., TAP1 / 2, Tapasin, Beta-2 microglobulin, CIITA, RFXANK, RFX5 and RFXAP);132.g) antagonizing immunogenic and immune-activating determinants - express of one or more of: B2M Antagonists of Viral Origin (e.g., ULI 8 (HCMV); and / or MHC Antagonists of Viral Origin (e.g., one or more of A40R MHCI (Vaccinia); Nef, TAT (HIV); E3-19K (Adenovirus); ICP47 (HSV-1 / 2); CPXV012, CPXV203 (Cowpox); EBNA1, BNLF2a, BGLF5, BILF1 (EBV); ORF66 (VZV); US2 / gp24, US3 / gp23, US6 / gp21, US10, USll / gp33 (hCMV); rhl78 / VIHCE (RhCMV); U21 (HHV-6 / 7); LANA1, ORF37 / SOX, kK3 / MIR1, kK5 / MIR2 (KHSV); mK3 (MHV-68); UL41 / vhs (a-herpesvirus, HSV, BHV-1, PRV); UL49.5 (Varicellovirus, BHV-1, EHV-1 / 4, PRV); and m4 / gp34, m6 / gp48, m27, ml52 / gp40 (mCMV));133.h) reducing immune co-stimulation and danger signaling in host cell by silencing or knockout of CD80, CD86, TLR3, TLR7, TLR9, IFH6, AIM2; and134.i) expressing immunosuppressive factors of human or viral origin.

65. The virus or cell or cell line of any of claims 1-64, wherein the host cell-derived virus membrane is modified to enhance anti-tumor immunity by expressing one or more of:136.a co- stimulatory molecules, such as, for example, CD40, CD40L, 4- IBB, 4-1BBL, 4-1BBL with a deletion of the cytoplasmic domain (4-lBBLAcyt), 4-1BBL with a truncated cytoplasmic domain, 0X40 (CD 134), OX40L (CD252), other members of the TNFR superfamily (e.g., CD27, CD27 ligand, GITR, CD30, Fas receptor, TRAIL-R, TNF-R, HVEM, and RANK), B7, CD80, CD86, ICOS, ICOS ligand (B7RP1), and CD28;137.a truncated co-stimulatory molecules (e.g., 4-1BBL, CD80, CD86, CD27L, B7RP1, OX40L), with a full or partial (complete, or truncated, or modified to ensure proper orientation when expressed in a cell) cytoplasmic domain deletion; Attorney Docket No. 120276-2615PC138.-527-139.a TNF superfamily of ligands - CD30, Fas-L, TRAIL-R, and TNF-R, which induce apoptosis, and CD27, OX40L, CD40L, GITR-L, and 4-1 BBL;140.a TGF-beta polypeptide antagonist;141.an immune checkpoint scFv (tethered to a transmembrane protein - an scFv targeting CTLA-4, PD-L1 (B7-H1), PD-L2, PD-1, PD-2, IDO1, IDO2, SIRP alpha (CD47), VISTA (B7-H5), LIGHT, HVEM, CD28, LAG3, TIM3, TIGIT, Galectin-9, CEACAM1, CD155, CD112, CD226, CD244 (2B4), B7-H2, B7-H3, CD137, ICOS, GITR, B7-H4, B7-H6, CD137, CD27, CD40, CD40L, CD48, CD70, CD80, CD86, CD137 (4-1BB), 4-1BBL, CD200, CD272 (BTLA), CD160, A2a receptor, A2b receptor, HHLA2, ILT-2, ILT-4, gp49B, PIR-B, 0X40, OX-40L, HLA-G, ILT-2 / 4, KIR, TIM1, TIM4, and CLEVER- 1 / Stabilin-l;142.a cytokine as a fusion / chimeric protein with a transmembrane protein - IL2, IL4, IL6, IL7, IL7-IL21 fusion proteins, IL9, IL10, IL11, IL12, IL15, IL18, or IL21 can be fused at the cytokine C-terminus, through a linker, to N- or C- terminus of a transmembrane protein or any other membrane bound protein.

66. The virus, cell, or cell line of any of claims 1-65, wherein the cells are selected from among: adult stem cells; embryonic stem cells; fetal stem cells; neural stem cells; mesenchymal stem cells (for example, isolated / derived from: adult bone marrow, adipose tissue, blood, dental pulp, neonatal umbilical cord, umbilical cord blood, placenta, placenta-derived adherent stromal cells, placenta-derived decidual stromal cells, endometrial regenerative cells, placental bipotent endothelial / mesenchymal progenitor cells, amniotic membrane or fluid mesenchymal stem cells, amniotic fluid derived progenitors, Wharton’s Jelly mesenchymal stem cells, pelvic girdle stem cells, Chorionic Villus Mesenchymal Stromal cells, subcutaneous white adipose mesenchymal stem cells, pericytes, adventitial reticular stem cells, hair follicle-derived stem cells, hematopoietic stem cells, periosteum-derived mesenchymal stem cells, lateral plate mesenchymal stem cells, exfoliated deciduous teeth stem cells, periodontal ligament stem cells, dental follicle progenitor cells, stem cells from apical papilla, muscle satellite cells, etc.); totipotent stem cells; pluripotent stem cells; induced pluripotent stem cells (iPSCs); multipotent stem cells; oligopotent stem cells; unipotent stem cells; adipose stromal stem cells; endothelial stem cells (for example, endothelial progenitor cells, placental endothelial progenitor cells, angiogenic endothelial Cells, pericytes); adultAttorney Docket No. 120276-2615PC144.-528-145.peripheral blood stem cells; myoblasts; small juvenile stem cells; skin fibroblast stem cells; tissue / tumor-associated fibroblasts; epithelial stem cells; and embryonic epithelial stem cells.

67. The virus, cell, or cell line of any of claims 1-66 selected from among iPSCs, stem cells, and cell lines, such as HEK293, HEK293T, A549, PerC6, Vero, Vero STAT1 KO, HEK293. STAT1 BAX KO AGE1.CR.pIX, CV1, HELA, HELA S3, CHO, VPCs, VPCs 2.0, FS293, MDCK, and MDCK. STAT1 KO cells.

68. The vims, cell, or cell line of any of claims 1-67, wherein the host cell-derived membrane or the host cell-derived membrane and ae virally encoded membrane protein are modified to display a fusion or chimeric polypeptides that:148.a) attenuate or block anti-viral immunity, such as by providing:149.enhanced resistance to complement and / or attenuated complement deposition and / or attenuating or eliminating neutralizing antibody binding and / or antibody dependent opsonization;150.b) attenuate or modulate phagocytic uptake;151.c) enhance, increase specificity, or increase delivery to tumors, such as by including tissue and tumor homing proteins;152.d) comprise targeting and / or binding domains, such as those that target solid tumor markers, hematologic malignancy markers, or autoimmune or inflammatory disease markers selected from among: solid tumor markers, hematologic malignancy markers, and autoimmune or inflammatory disease markers, where the solid tumor markers, hematologic malignancy markers, and autoimmune or inflammatory disease markers are targeted by approved or investigational monoclonal antibodies, antibodydrug conjugates (ADCs), cell therapies, CAR T-cells, CAR-NK cells, and CAR-macrophages, and transgenic T-cell receptors;153.e) increase or enhance anti-tumor immunity and / or the anti-tumor response of the immune system of the host, such expressing a co-stimulatory molecule or truncated costimulatory molecule (e.g., 4-1BBL, CD80, CD86, CD27L, B7RP1, and OX40L) and / or to display or contain a single-chain antibody or antigen binding portion thereof, such as an immune check point inhibitor; and / or display or contain a cytokine or chemokine.

69. The vims, cell, or cell line of any of claims 1-68, wherein:Attorney Docket No. 120276-2615PC155.-529-156.the host cell membrane is modified to express a transmembrane protein that comprises a peptide or polypeptide or portion thereof that is displayed on the membrane; and157.the displayed peptide or polypeptide or portion thereof alters a property and / or activity of a virus that comprises the membrane.

70. The virus, cell, or cell line of any of claims 1-69, wherein the genome of the virus is modified to encode the membrane protein to be expressed on the virus membrane.

71. The virus, cell, or cell line of any of claims 1-70, wherein the cell membrane comprises an immunomodulatory protein, a receptor, a ligand, an antigenbinding portion of an antibody, a chimeric receptor, or an immune modulator.

72. The virus, cell, or cell line of any of claims 1-71, wherein the cell membrane and / or the virally encoded polypeptide comprises a chimeric of fusion protein that comprises one or more of all or an active portion of CD55, CD47, and / or a CAR.

73. The virus, cell, or cell line of any of claims 1-72, wherein the cell membrane and / or the virally encoded polypeptide comprises a target polypeptide or peptide selected from ALIX, KCNJ2, MICA, NECTIN2, or PVR.

74. The virus, cell, or cell line of any of claims 1-73, wherein the polypeptide expressed on the membrane is a chimeric antigen receptor (CAR).

75. The virus, cell, or cell line of claim 73, wherein the CAR is selected from one or more of an epidermal growth factor receptor (EGFR) CAR, TROP2, CD38, a HER2 CAR, a CD 19 CAR, a mesothelin CAR, a GD2 CAR, and a BCMA CAR.

76. The virus, cell, or cell line of any of claims 1-75, wherein the EEV is vaccinia virus (EEV) particle that has higher anti-tumor activity and higher EEV production than the virus IHD-W, wherein the EEV is a clone of the polyclonal vaccinia IHD strain NR-52.

77. The virus, cell, or cell line of any of claims 1-76, wherein the EEV virus genome has an intact A56 locus, whereby the virus has increased serum stability compared to the polyclonal IHD strain.

78. The virus, cell, or cell line of any of claims 1-77, wherein:167.the extra enveloped vaccinia virus (EEV) that has higher anti-tumor activity and higher EEV production than the virus H4D-W; Attorney Docket No. 120276-2615PC168.-530-169.the EEV is a clone of the polyclonal vaccinia IHD strain NR-52; and contains a deletion of at least one amino acid in the K7R gene, a TLR modulator receptor; and / or170.contains a gene identical to the gene encoding RPXV102, a cell surface-binding protein and carbonic anhydrase homolog, which does not occur in IHD-W, and which is in the IMV and binds to chondroitin sulfate on the cell surface, providing virion attachment to a target cell; and / or has 2 SNPs in the A30L gene compared with IHD-W; and / or171.when its sequence is compared with the sequences of each of A33R, A34R, A36R, A56R, B5R, F13L, A45R, A29L A31R A30L, A32L, and / or A13L in the strain IHD- J, the genome of the clonal EEV has 2 SNPs in A30L and 1 in A45R.

79. The virus, cell, or cell line of any of claims 1-78, wherein the EEV differs from IHD-W as shown in Figure 26.

80. The virus, cell, or cell line of any of claims 1-79, wherein the virus is designated RT-00 or a virus having at least 95% sequence identity thereto or comprising degenerate codons, and having at least one of the properties recited in any of claims 15-18.

81. The virus, cell, or cell line of claim 80 that is RT-00 or a derivative virus thereof.

82. The virus, cell, or cell line of any of claims 1-80, wherein the virus is an EEV vaccinia virus that comprises or further comprises knockouts of one or more genes selected from among A46R, B8R, J2R / TK, A52R, F1L, VGF, and B19R; wherein the knockouts are effected by gene deletions, insertions, or disruptions to render encoded products inactive or not produced.

83. The virus, cell, or cell line of any of claims 1-82, wherein the EEV has higher anti-tumor activity and EEV production than the virus IHD-W, wherein the EEV is a clone of the polyclonal vaccinia IHD strain NR-52, wherein the EEV further comprises modifications in the genome, whereby an EEV outer membrane transmembrane protein comprises a protein or portion thereof that, when administered to a host, reduces or inhibits humoral immunity, wherein the portion is sufficient to inhibit or reduce humoral immunity; and the protein or portion thereof is display on the outer membrane of the EEV.Attorney Docket No. 120276-2615PC177.-531-84. The virus, cell, or cell line of any of claims 1-83, wherein the virus is an EEV vaccinia virus that comprises or further comprises modifications in the genome, whereby an EEV outer membrane transmembrane protein comprises a protein or portion thereof that, when administered to a host, reduces or inhibits humoral immunity, wherein the portion is sufficient to inhibit or reduce humoral immunity; and the protein or portion thereof is displayed on the outer membrane of the EEV.

85. The virus, cell, or cell line of any of claims 1-84, wherein the EEV genome comprises an intact A56 gene.

86. The virus, cell, or cell line, of any of claims 1-85, wherein the virus is a vaccinia virus preparation that, when propagated, comprises, EEV particles that comprise more than 1%, 5%, 10%, 15%, 20%, 25%, 30% or more of the virus population.

87. The virus, cell, or cell line of claim 86, wherein the EEV particles comprise more than 30% of the population88. The virus, cell, or cell line of any of claims 1-87, wherein that EEV is a vaccinia virus and its genome comprises the sequence set forth in any of SEQ ID NOs: 1-21, 518-523, 628-634, 782-790, and 887-898 or a variant of any of SEQ ID NOs: 1, 782-790, and 887-898 or a virus set forth in Figure 24 and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, or 99% sequence identity therewith, or variants lack all or a portion of the ITRs or comprise heterologous ITRs, or degenerate sequences of any of the preceding sequences.

89. The virus, cell, or cell line of any of claim s 1-88, wherein EEV is a vaccinia virus designated RT-00, and the genome comprises the sequence set forth in SEQ ID NO: 1, or variants thereof whose genome comprises at least one degenerate codon, or has at least 90% or at least 95% or at least 98.5% or at least 99% sequence identity thereto.

90. The vims, cell, or cell line of any of claims 1-89, wherein the vims comprises nucleic acid encoding a chimeric protein, whereby a humoral immunity regulatory protein or portion thereof, such as a complement regulatory protein (CRP) or portion thereof is displayed on the surface of the EEV, wherein a portion is sufficient to reduce or inhibit a response of the immune system.Attorney Docket No. 120276-2615PC185.-532-91. The virus, cell, or cell line of claim 90, wherein the chimeric protein comprises a transmembrane protein or a fusion protein that displays the fused protein on the second membrane.

92. The virus, cell, or cell line of claim 91, wherein the transmembrane protein or fusion protein is encoded by the virus.

93. The virus, cell, or cell line of claim 92, wherein the transmembrane protein is selected from among A33R, A34R, A56R, B5R, and F13L, such as A33 or B5R.

94. The virus, cell, or cell line of any of claims 1-93, wherein the virus is selected from among viruses designated190.i) RT-01, RT-02, RT-03, RT-04, RT-06, RT-07, RT-08, RT-09, RT-10, RT-11, RT-12, RT 13, RT-14, RT-15, RT-16, RT-17, RT-18, RT-19, RT-20, RT-21, RT-22, RT-23, RT-24, RT-25, RT-26, RT-27, RT-28, RT-29, RT-30, RT-31, RT-33, RT-34, RT-35, RT-36, RT-37, RT-38, RT-39, RT-40, RT-41, RT-42, RT-43, RT-44, RT-45, RT-46, RT-47, RT-48, RT-49, RT-50, RT-51, RT-52, RT-53, RT-54, RT-55, RT-56, RT-57, RT-58, RT-59, RT-60, RT-61, RT-62, RT-63, RT-64, RT-65, RT-66, RT-67, RT-68, RT-69, RT-70, RT-71, RT-72, RT-73, RT-74, RT-75, RT-76, RT-77, RT-78, RT-79, RT-80, RT-81, RT-82, RT-83, RT-84, RT-85, RT-86, RT-87, RT-88, RT-89, RT-90, RT-91, RT-92, RT-93, RT-94, RT-95, RT-96, RT-97, RT-98, RT-99, RT-100, RT-101, RT-102, RT-103, RT-104, RT-105, RT-106, RT-107, RT-108, RT-109, RT-110, RT-111, RT-112, RT-113, RT-114, RT-114b, RT-115, RT-116, RT-117, RT-118, RT-118b, RT-118c, RT-119, RT-120, RT-121, RT-122, RT-123, RT-124, RT-125, RT-126, RT-127, RT-128, RT-129, and RT-134; or191.ii) variants of i) produced upon propagation of the virus or modification of the virus to encode additional proteins or replacement of all or part of non-essential genes, whereby the virus is substantially as resistant or more resistant to human serum than RT-00 and / or produces at least or at least about the same level of EEVs as RT-00; or192.iii) variants of i) or ii) that encode the same proteins, but by virtue of the genetic code, comprise on or more degenerate codons thereof.

95. The virus, cell, or cell line of any of claims 1-94, wherein: each virus comprises the inserts as set forth in Figure 24 or a variant genome thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto andAttorney Docket No. 120276-2615PC194.-533-195.retaining high EEV level and displaying a complement resistance protein or portion thereof, whereby the virus is substantially as resistant or more resistant to human serum than RT-00 and / or produces at least or at least about the same level of EEVs as RT-00.

96. The virus, cell, or cell line thereof of any of claims 1-95, wherein the virus comprises the inserts encoded in the transfer vectors set forth in any of SEQ ID NOs:2-21 and 518-524 or a variant thereof having at least 70%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, or 99% sequence identity thereto and retaining high EEV level and displaying a complement resistance protein or portion thereof, whereby the virus is more resistant to human serum than is RT-00 and / or produces at least or at least about the same level of EEVs as RT-00.

97. The virus, cell, or cell line of any of claims 1-86, wherein the virus comprises ab EEV transmembrane protein and a complement regulatory protein (CRP, also referred to as a complement resistance protein or complement inhibitory protein complement protein or regulator of complement activation (RCA)) or portion thereof sufficient to inhibit complement, wherein the transmembrane protein is a vaccinia virus or poxvirus encoded outer membrane protein.

98. The virus, cell, or cell line of claim 97, wherein the CRP or regulator of complement activity (RCA) or complement resistance protein is selected from among the proteins that reduce or inhibit humoral immunity or portion thereof is selected among one or more of CD35 / CR1, CD55, CD59, CD46, Factor H, VCP, MOPICE, SPICE, ORF4, CCPH, C4- binding protein, Kaposi -sarcoma associated herpesvirus Kaposica I KCP, Herpesvirus saimiri (HVS)-CD59, Rhesus rhadinovirus RCP-H and RCP-1, murine gamma herpesvirus 68 (yHV-68) RCA, Influenzavirus Ml, EMICE and IMP, as well as modified sequences thereof, or functional portions thereof, such as, for example, selected from among one or more of: CD35, CD55, VCP, mutated VCP, SPICE, CCPH and ORF4, and functional portions thereof.

99. The virus, cell, or cell line claim 97 or claim 98, wherein the EEV transmembrane protein is selected from among from among A33R, A34R, B5R, and F13L.

100. The virus, cell, or cell line of any of claims 1-99, wherein polypeptide expressed on the EEV or host-derived membrane comprises a complement regulatory protein that comprises the sequence of amino acids (excluding the signal sequence) setAttorney Docket No. 120276-2615PC201.-534-202.forth in any of SEQ ID NOs.:238, 241, 244, 247, 492, 493, 498-502, or 638-650 or a sequence having at least 95% sequence identity thereto or a portion thereof, which retains complement resistance activity of at least 10% of the full-length protein.

101. The virus, cell, or cell line of any of claims 1-100, wherein the virus comprises or encodes a fusion protein between a complement regulatory protein (CRP) that comprises the sequence of amino acids (excluding the signal sequence) set forth in any of SEQ ID NOs.:238, 241, 244, 247, 492, 493, 498-502, or 638-650 or a sequence having at least 95% sequence identity thereto or a portion thereof, which retains complement resistance activity of at least 10% of the full-length protein and an EEV transmembrane protein selected from A33R, A34R, A56R, B5R, and F13L or a sufficient portion thereof to effect display of the CRP on the outer membrane of an EEV.

102. The virus, cell, or cell line of claim 101, wherein the fusion protein comprises the sequence of amino acids (excluding the signal sequence) set forth in any of SEQ ID NOs.:238, 241, 244, 247, 492, 493, 498-502, or 638-650 or a sequence having at least 95% sequence identity thereto or a portion thereof, which retains complement resistance activity of at least 10% of the full-length protein and an EEV transmembrane protein selected from A33R, A34R, A56R, B5R, and F13L or a sufficient portion thereof to effect display of the CRP on the outer membrane of an EEV.

103. The virus, cell, or cell line of claim 101 or claim 102, wherein the transmembrane protein comprises all or sufficient portion, sufficient for display of the CRP, of the polypeptide of any of SEQ ID NOs:651-775 or polypeptide having at least 95% sequence identity sufficient to display the CRP.

104. The virus, cell, or cell line of any of claims 1-103, wherein the EEV is a poxvirus or a vaccinia virus nucleic acid encoding a chimeric or fusion protein, whereby the genome is modified, wherein:207.the chimeric protein comprises all or a functional portion of an EEV outer membrane transmembrane protein and all or a functional portion of a protein that reduces or inhibits humoral immunity in a host upon expression of the protein in the host;208.the functional portion of the transmembrane protein is a sufficient portion to display the protein or portion thereof that reduces or inhibits humoral immunity on the surface of an EEV particle comprising the genome; and Attorney Docket No. 120276-2615PC209.-535-210.the functional portion of the protein that reduces or inhibits humoral immunity is a sufficient portion to reduce or inhibit humoral immunity in the host.

105. The virus, cell, or cell line of any of claim s 1-104, wherein:212.the virus genome comprises a mutation(s) that renders the virus a high EEV producer; and213.a high level is higher than that produced by the WR strain, or is higher than 10%, 15%, 20%, 25%, or 30%.

106. The vaccinia virus genome, vaccinia virus, derivative virus, fusion protein, or nucleic acid molecule, of any of claims 1-105, wherein the virus encodes a fusion protein that comprises the sequence of amino acids (excluding the signal sequence) set forth in any of SEQ ID NOs.:238, 241, 244, 247, 492, 493, 498-502, or 638-650 or a sequence having at least 95% sequence identity thereto or a portion thereof, which retains complement resistance activity of at least 10% of the full-length protein and an EEV transmembrane protein selected from A33R, A34R, A56R, B5R, and F13L or a sufficient portion thereof to effect display of the CRP on the outer membrane of an EEV.

107. The virus, cell, or cell line of any of claims 1-106, wherein the virus or its genome comprises or encodes a fusion protein, wherein the transmembrane protein comprises all or sufficient portion, sufficient for display of the complement resistance protein (CRP), of the polypeptide of any of SEQ ID NOs:651-775 or polypeptide having at least 95% sequence identity sufficient to display the CRP.

108. The virus, cell, or cell line of any of claims 1-107, wherein:217.the virus genome comprises a knockout or insertion in one or more of the A46, TK, and VGF locus or loci whereby the virus is one or more of A46-, TK-, and / or VGF-;218.knockouts are effected by gene deletions, insertions, or disruptions to render encoded products inactive or not produced; and219.EEV production is increased compared to the virus that does not comprise the knockout or knockouts.

109. The virus, cell, or cell line of any of claims 1-108, wherein the vims is: an IV-EEV is an EEV that comprises nucleic encoding a chimeric transmembrane protein;Attorney Docket No. 120276-2615PC221.-536-222.the transmembrane protein when transcribed and translated is expressed in the second membrane; and223.the chimeric transmembrane protein comprises a polypeptide that confers humoral immunity or comprises sufficient portion thereof to confer humoral immunity when expressed.

110. The virus, cell, or cell line of any of claim 109, wherein the transmembrane protein is selected from among A33R, A34R, A56R, B5R, and F13L.

111. The virus, cell, or cell line of any of claims 1-110, wherein the virus comprises encodes a fusion protein of a transmembrane protein that is A33R, A34R, A56R, B5R, and F13L with a complement regulatory protein selected from among CD35 / CR1, CD55, CD59, CD46, Factor H, VCP, MOPICE, SPICE, CCPH, C4- binding protein, Kaposi-sarcoma associated herpesvirus Kaposica I KCP, Herpesvirus saimiri (HVS)-CD59, Rhesus rhadinovirus RCP-H and RCP-1, murine gamma herpesvirus 68 (yHV-68) RCA, Influenzavirus Ml, EMICE, IMP, and functional portions thereof, and variants thereof that have at least 95% amino acid sequence identity with any of the preceding and have complement regulatory activity, whereby complement is inhibited.

112. The virus, cell, or cell line of any of claims 1-111, wherein the unmodified or modified genome has the sequence set forth in any of SEQ ID NOs: 1, 782-790, and 887-898 or a variant thereof that retains the at least the level of EEV production of RT-00 and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% nucleotide sequence identity thereto, excluding the ITRs, or degenerates thereof that comprise one or more degenerate codons in protein-encoding sequences.

113. The virus, cell, or cell line of any of claims 1-112, wherein the virus that comprises one or more knockouts of a gene, wherein:228.the knockout increases the resistance of a virus comprising the genome to the humoral immunity of a host or increases tumor selectivity accumulation of the virus or increases anti-tumor activity of the virus; and229.a knockout comprises an insertion or deletion or rearrangement of the knocked-out gene, whereby a native encoded product is not produced.

114. The virus, cell, or cell line of any of claims 1-113, wherein the virus comprises one or more knockouts that inactivate one or more genes selected from among: A46R, B8R, J2R / TK, A52R, F1L, VGF, and B19R.Attorney Docket No. 120276-2615PC231.-537-115. The virus, cell, or cell line of any of claims 1-114, wherein:233.the virus genome comprises two or three knockouts of genes; and234.the knockouts are effected by gene deletions, insertions, or disruptions.

116. The virus, cell, or cell line of any of claims 1-115, wherein:236.double knockouts comprise a) TK, A46R; b) TK, A52R; c) TK, B8R; d) TK, VGF; e) TK, F1L; or f) TK, B19R; and237.three / triple knockouts comprise g) TK, A46R, VGF; h) TK, A52R, VGF; i)TK, B8R, VGF; j)TK, F1L, VGF; k)TK, B8R, B19R; 1)TK, A46R, B19R; m)TK, A52R, B19R; orn) TK, F1L, B19R.

117. The virus, cell, or cell line of any of claims 1-116, wherein:239.the viral genome comprises heterologous nucleic acid encoding a therapeutic protein and / or a diagnostic or detectable or product or a reporter; and240.the nucleic acid encoding the heterologous nucleic acid is inserted into or in place of nucleic acid in a non-essential gene locus, or is inserted to effect a knockout of one or more of: A46R, B8R, J2R, A52R, F1L, VGF, and B19R; or241.the heterologous nucleic acid encodes one or more of EGFP, EmGFP, mNeonGreen, EBFP, TagBFP, EYFP, TPet, GFP, BFP or TurboFP635, and the RT-00 virus and viruses derived therefrom also can encode therapeutic or diagnostic payloads; for example, therapeutic proteins include, but are not limited to, cytokines (GM-CSF, IL-2, IL-7, IL-10, IL-12, IL-15, IL-15 / IL-15R alpha chain complex, IL-17, IL-18, IL-21, an IL-7-IL21 fusion protein, TNF, MIPla, FLt3L, IFN-b, IFN-g), chemokines (CC15, CC12, CC119, CXC111, RANTES), co-stimulators (OX40L, 4-1BBL, CD40L, B7.1 / CD80, GITRL, LIGHT, CD70), bi-specific t-cell engagers (BITEs, such as anti-CD3 -DLL bispecific constructs), therapeutic antibodies, immune checkpoint inhibitors, single chain antibodies such as single chain antibodies against VEGF, VEGFA, VEGFB, PGF, VEGFR2, PDGFR, Ang-1, Ang-2, ANGPT1, ANGPT2, HGF, TGF-P and immune checkpoint inhibitors, such as inhibitors of PD-1, PD-L1, CTLA4, or TIM-3, prodrug activators, such as lacZ, cytosine deaminase enzymes, human sodium iodide symporter, hNIS, and Aquaporin 1-AQP1.

118. The virus, cell, or cell line of claim 117, wherein the heterologous nucleic acid encodes one or more of modulators of angiogenesis, immune system co-stimulators, or checkpoints inhibitors, such as, Anti -VEGF A and VEGFB and PGF; anti -VEGF andAttorney Docket No. 120276-2615PC243.-538-244.anti-ANGPT2; anti-VEGF, anti-ANGPT-2 and anti-CTL4; anti-VEGF and OX40L; Anti-VEGF, Anti-ANGPT2 and anti-PD-1 products.

119. A method of treatment of cancer, comprising systemically administering a virus, an EEV particle, or vaccinia virus, or virus produced from the cells or cell line, or that is a programmed EEV of any of claims 1-118 to a subject in need thereof.

120. The virus, EEV particle, vaccinia virus, virus produced from the cells or cell line, or virus that is a programmed EEV any of claims 1-119 for use for treating cancer, wherein the vaccinia virus genome comprises a vaccinia virus particle.

121. The method, use, virus, EEV particle, vaccinia virus, or virus produced from the cells or cell line, or virus that is a programmed EEV of claim 119 or claim 120, wherein the cancer comprises a solid tumor, or metastases, or is a hematological malignancy, including metastatic cancers, lymphatic tumors, and blood cancers, such as a cancer is selected from among type of malignant tumor or hematological malignancy, including metastatic cancers, lymphatic tumors, and blood cancers. Exemplary cancers include, but are not limited to, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoma, adrenal cancer, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brainstem glioma, brain cancer, carcinoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway or hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, Burkitt’s lymphoma, carcinoid tumor, carcinoma, central nervous system lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, epidermoid carcinoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer / intraocular melanoma, eye cancer / retinoblastoma, gallbladder cancer, gallstone tumor, gastric / stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, giant cell tumor, glioblastoma multiforme, glioma, hairy-cell tumor, head and neck cancer, heart cancer, hepatocellular / liver cancer, Hodgkin’s lymphoma, hyperplasia,Attorney Docket No. 120276-2615PC248.-539-249.hyperplastic corneal nerve tumor, in situ carcinoma, hypopharyngeal cancer, intestinal ganglioneuroma, islet cell tumor, Kaposi's sarcoma, kidney / renal cell cancer, laryngeal cancer, leiomyoma tumor, lip and oral cavity cancer, liposarcoma, liver cancer, nonsmall cell lung cancer, small cell lung cancer, lymphomas, macroglobulinemia, malignant carcinoid, malignant fibrous histiocytoma of bone, malignant hypercalcemia, malignant melanomas, marfanoid habitus tumor, medullary carcinoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic skin carcinoma, metastatic squamous neck cancer, mouth cancer, mucosal neuromas, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disorder, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neck cancer, neural tissue cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial tumor, ovarian germ cell tumor, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma, pituitary adenoma, pleuropulmonary blastoma, polycythemia vera, primary brain tumor, prostate cancer, rectal cancer, renal cell tumor, reticulum cell sarcoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, seminoma, Sezary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck carcinoma, stomach cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, throat cancer, thymoma, thyroid cancer, topical skin lesion, trophoblastic tumor, urethral cancer, uterine / endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia or Wilms tumor; or cancers commonly diagnosed in humans include, but are not limited to, cancers of the bladder, brain, breast, bone marrow, cervix, colon / rectum, kidney, liver, lung / bronchus, ovary, pancreas, prostate, skin, stomach, thyroid, or uterus; or cancers commonly diagnosed in dogs, cats, and other pets include, such as, lymphosarcoma, osteosarcoma, mammary tumors, mastocytoma, brain tumor, melanoma, adenosquamous carcinoma, carcinoid lung tumor, bronchial gland tumor, bronchiolar adenocarcinoma, fibroma, myxochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma, Wilms tumor, Burkitt's lymphoma, microglioma, neuroblastoma, osteoclastoma, oral neoplasia, fibrosarcoma, osteosarcoma and rhabdomyosarcoma, genital squamous cell carcinoma, transmissible venereal tumor, testicular tumor, seminoma, Sertoli cell tumor, hemangiopericytoma, histiocytoma, Attorney Docket No. 120276-2615PC250.-540-251.chloroma (e.g., granulocytic sarcoma), corneal papilloma, corneal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, stomach tumor, adrenal gland carcinoma, oral papillomatosis, hemangioendothelioma and cystadenoma, follicular lymphoma, intestinal lymphosarcoma, fibrosarcoma and pulmonary squamous cell carcinoma. Exemplary cancers diagnosed in rodents, such as a ferret, include, but are not limited to, insulinoma, lymphoma, sarcoma, neuroma, pancreatic islet cell tumor, gastric MALT lymphoma and gastric adenocarcinoma.

122. The method or use of claims 119-121, wherein the subject is human.

123. The method or use, of any of claims 119-120, wherein the subject is a non-human animal and the cancers are selected from among leukemia, hemangiopericytoma and bovine ocular neoplasia (in cattle); preputial fibrosarcoma, ulcerative squamous cell carcinoma, preputial carcinoma, connective tissue neoplasia and mastocytoma (in horses); hepatocellular carcinoma (in swine); lymphoma and pulmonary adenomatosis (in sheep); pulmonary sarcoma, lymphoma, Rous sarcoma, reticulo-endotheliosis, fibrosarcoma, nephroblastoma, B-cell lymphoma and lymphoid leukosis (in avian species); retinoblastoma, hepatic neoplasia, lymphosarcoma (lymphoblastic lymphoma), plasmacytoid leukemia and swimbladder sarcoma (in fish), caseous lymphadenitis (CLA): chronic, infectious, contagious disease of sheep and goats caused by the bacterium Corynebacterium pseudotuberculosis, and contagious lung tumor of sheep caused by jaagsiekte.

124. The virus, cell, or cell line, method, or use of any of claims 1-123, wherein the nucleic acid encoding the chimeric or fusion protein in the virus replaces the respective envelope protein-encoding nucleic acid or is inserted into a gene locus to knockout the activity of the protein encoded at the locus; optionally254.the virus comprises a deletion of or inactive thymidine kinase (TK) gene; or the genome comprises one or more knockouts whereby active gene products are not expressed, and the knockouts are in the TK, A46, and VGF loci.

125. The virus, cell, or cell line, or method, or use of any of claims 1-124, wherein the virus comprises a fusion protein between an EEV transmembrane protein and a CRP or other humoral immunity modulator to increase serum resistance, and / or the virus or genome comprises nucleic acid encoding a cytokine or other anti-tumor therapeutic.Attorney Docket No. 120276-2615PC256.-541-126. The virus, cell, cell line, composition, or method, or use of any of claims 1-125, wherein the EEV transmembrane protein is A33, and the genome comprises one or more knockouts whereby active gene products are not expressed, and the knockouts are in the TK, A46, and VGF loci.

127. The virus, cell, cell line, composition, method, or use of any of claims 1-126, wherein the vims encodes a cytokine; optionally,259.the cytokine is an IL-15 or an IL-15 / IL-15R alpha chain complex; optionally an IL-15 superagonist (IL-15 / IL-15R alpha chain complex) or a modified form thereof that has increased activity, optionally comprising the replacement atN72, G78, and / or N79, including N72D, G78A, G78V, G78L, G78I, N79Q, N79H, N79M, G78A, and / or N79Q.

128. The vims, cell, cell line, composition, or method, or use of any of claims 1-127, wherein the vims selected from a high EEV vaccinia vims comprising knock outs (KOs) and / or payloads of a)-f):261.a) 3KO (TK -, A46-, VGF-);262.b) 3KO (TK -, A46-, VGF-) - A33+CD55;263.c) 3KO (TK -, A46-, VGF-) + Payload IL-15 (cytokine form);264.d) 3KO (TK -, A46-, VGF-) - A33+CD55+ Payload IL-15 (cytokine form); e) 3KO (TK -, A46-, VGF-)+ Payload IL- 15 superagonist; and265.f) 3KO (TK -, A46-, VGF-) - A33+CD55+ Payload IL-15 superagonist.

129. The EEV vims, cells, cell line, composition, or method, or use of any of claims 1-128, comprising a transmembrane fusion protein as described above and herein, wherein the unmodified virus genome comprises the sequence set forth in any of SEQ ID NOs: 1, 22-165, 251, 485, 616-627 or a sequence having at least 95% sequence identity thereto, whereby the modified vims is a high EEV vims and has increased semm resistance compared to the unmodified vims.

130. The vims, cell, cell line, composition, method, or use of any of claims 1-129, wherein the vims is a high EEV vaccina vims encoding a fusion protein of the EEV transmembrane protein with a complement regulatory protein or other humoral immunity modulating protein, optionally, wherein the payload encoding nucleic acid is inserted into in place of all or a portion of the vims VGF encoding nucleic acid locus or to render the vims VGF':Attorney Docket No. 120276-2615PC268.-542-269.the genome of the unmodified virus comprises a genome is selected from among SEQ ID NOs: 1, 22-165, 251, 485, and 616-627, or a genome having at least 95% sequence identity thereto excluding the ITRs; and270.the modified virus retains the high EEV phenotype and encoded fusion protein whereby the virus has increased serum resistance compared to a virus comprising the unmodified genome.

131. A composition, comprising the cell, cell line, or virus of any of claims 1-130.

132. A pharmaceutical composition comprising the virus or virus produced from the cells or cell lines of any of claims 1-130.

133. A method of treating cancer and / or proliferative diseases, disorders, and / or conditions, comprising administering the pharmaceutical composition of claim 132.

134. The pharmaceutical composition of claim 132 for use for treating cancer and / or proliferative disorders.

135. The method or use of claim 133 or claim 134, wherein the pharmaceutical composition or virus is systemically administered.

136. The method, or use, or programmed virus, or virus produced from the cell or cell line, or pharmaceutical composition of any of claims 1-135 for use for treating cancer in combination with a second anti-cancer agent or treatment.

137. A method of treating cancer, comprising:278.a) systemically administering an EEV programmed virus, or virus produced from the cell or cell line, or pharmaceutical composition virus, of any of claims 1-73; and b) administering a second agent or treatment, wherein:279.a) and b) are effected serially, simultaneously, or intermittently, or a) is effected before b), or b) is effected before a).

138. The method of claim 136 or use of claim 137, wherein the second anti¬ cancer agent or treatment is chemotherapy, or immunotherapy, or cell therapy, or an antibiotic, or radiation therapy, or surgery, or combinations of two or more thereof.

139. The method or use of claim 136 or claim 137, wherein the second agent effects lymphodepletion to thereby inhibit anti-viral activity of a treated subject for a time at least sufficient to effect delivery of virus to a tumor in the subject.Attorney Docket No. 120276-2615PC282.-543-140. The method or use of any of claims 136-139, wherein the second agent or treatment is selected from among, ceftazidime, cefepime, imipenem, aminoglycoside, vancomycin and antipseudomonal P-lactam; with antifungal agents which can be included in a combination with a virus provided herein include, but are not limited to, amphotericin B, dapsone, fluconazole, flucytosine, griseofulvin, itraconazole, ketoconazole, miconazole, clotrimazole, nystatin, and combinations thereof, such as, for example, antiviral agents that can be included in a combination with a virus provided herein, such as but are not limited to, cidofovir, alkoxyalkyl esters of cidofovir (CDV), cyclic CDV, and (S)-9-(3 -hydroxy -2 phosphonylmethoxypropyl)adenine, 5-(dimethoxymethyl)-2'-deoxyuridine, isatin-beta-thiosemicarbazone, N-methanocarbathymidine, brivudine, 7-deazaneplanocin A, ST-246, Gleevec, 2'-beta-fluoro-2', 3 '-dideoxyadenosine, indinavir, nelfinavir, ritonavir, nevirapine, AZT, ddl, ddC, and combinations thereof; combinations with an antiviral agent contain an antiviral agent known to be effective against the virus of the combination, such as combinations can contain a vaccinia virus with an antiviral compound, such as cidofovir, alkoxyalkyl esters of cidofovir, ganciclovir, acyclovir, ST-246, Gleevec, and derivatives thereof.

141. The method or use of any of claims 133-140, comprising lymphodepletion; optionally lymphodepletion is effected by administration of a chemotherapeutic agent before or with treatment with the virus, wherein lymphodepletion optionally is effected by administration of cyclophosphamide; or an immunomodulator is administered or used, optionally, the immunomodulator is a chemotherapeutic agent and administered in a dose sufficient to achieve an immunomodulatory effect but not lymphodepletion, or the immunomodulator is an immunotherapeutic agent.

142. The method or use of any of claims 133-140, further comprising administering an anti-viral agent or an anti-viral antibody to modulate the level of virus or to eliminate the virus.

143. The method or use of claim 142, wherein the anti-viral agent, or antibody is selected from among cidofovir, alkoxyalkyl esters of cidofovir, ganciclovir, acyclovir, ST-246, Gleevec, and derivatives thereof.Attorney Docket No. 120276-2615PC287.-544-144. The method or use of any of claims 133-143, wherein treatment or use comprises a regimen of systemic viral administration and an immunomodulatory agent, wherein the regiment comprises:289.a) treatment with an immunomodulatory agent; then treatment with virus; and then clear vi s with ST-246 or other anti-viral agent; or290.b) regimen a) further comprising administration of an immunomodulatory agent after treatment with the vims, or after viral treatment, not before.

145. The vims, cell, or cell line, method, or use of any of claims 1-144, wherein:292.the vims comprises the fusion protein, or composition, or vims or genome thereof of any of claims 144;293.the genome of the vims is modified to encode a target antigen that, upon expression, is expressed on the surface of a cell infected with the vims; and294.the target antigen is a target for a therapy selected from among an immunotherapy or cell therapy, or antibody, or antibody-dmg conjugate for treating cancer.

146. The virus, cell, or cell line, method, or use of claim 145, wherein the therapy is a checkpoint inhibitor, CAR-T cell therapy, NK cell therapy, gene-editing therapy, or TIL cell therapy.

147. The vims, cell, or cell line, method, or use of any of claims 133-146, wherein the target antigen is a tumor-specific antigen or neoantigen.

148. The virus, cell, or cell line, method, or use of any of claims 133-147, wherein the target antigen is CD20 and HER2.

149. The vims, cell, or cell line, or method, or use of any of claims 1-148, wherein the vims encodes a heterologous product.

150. The virus, cell, or cell line, or composition, or method, or use of any of claims 1-149, wherein the product targets the vims, interacts with a host receptor, is a therapeutic or bioactive product, targets a therapeutic protein to cells comprising the virus, or a modified receptor.

151. The vims, cell, or cell line, or composition, or method, or use of any of claims 1-150 wherein the product comprises a co-stimulatory molecule, an immune checkpoint inhibitors or other immune regulatory protein, or an interleukins, cytokine, chemokine, growth factor, inhibitors against immunosuppressive or pro-tumorigenicAttorney Docket No. 120276-2615PC301.-545-302.cytokine / growth factors, angiogenesis inhibitors, tumor blood vessels reprogramming / vascular normalization products, Fms-like tyrosine kinase-3 ligand (FTL3L), TNF-alpha, TNF-beta, Bispecific T-Cell Engagers, therapeutic antibodies, reporter genes, Tumor homing proteins, and tumor antigens.

152. The virus, cell, or cell line, or composition, or method, or use of any of claims 1-151, wherein virus encodes a product that comprises one or more of IL-4, IL-6, IL-10, IL-11, IL-13, IL-17, IL-32, IGF, TGF-P, VEGF, PGF, CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11, CCL21, IL-1, IL-2, IL-3, IL-7, IL-12, IL-15, IL-15 / IL15a receptor complex, IL15 super agonist, IL-18, IL-21, IFN-a, IFN-P, IFN-y, TNF-a, EPO, GM-CSF, G-CSF, Flt3L, FGF, EGF, IL-4, IL-6, IL-10, IL-11, IL-13, IL-17, IL-32, an IL-7-IL-21 fusion protein, and IL-15 sushi domain linked, via a linker, or to a modified IL-15.

153. A method for manufacturing EEV virus, wherein the resulting product comprises at least 60% EEV virus, comprising:305.culturing cells infected with vaccinia virus for a time sufficient for virus to replicate and to be released into the medium without lysing the cells;306.collecting the culture medium and filtering, under low shear force, through a filter that captures particulates; and307.purifying the virus from the culture medium with low shear force filtration.

154. The method of claim 153, wherein the cells are modified so that the membrane displays a product that alters a property and / or activity of the virus propagated in the cells.

155. The method of claim 153 or claim 154, wherein the cells are cells of any of claims 1-89.

156. The method of any of claims 153-155, wherein the purified virus is formulated in formulation buffer for administration and / or storage at low temperature.

157. The method of any of claims 153-156, wherein the cells are selected from among ipSCs, stem cells, and cell lines, such as HEK293, HEK293T, A549, PerC6, Vero, Vero STAT1 KO, HEK293. STAT1 BAX KO AGE1.CR.pIX, CV1, HELA, HELA S3, CHO, VPCs, VPCs 2.0, FS293, MDCK, and MDCK. STAT1 KO cells are modified, as described herein, to express a polypeptide or peptide in the cell membrane that, when on the EEV virus, alters a property and / or activity of the virus.Attorney Docket No. 120276-2615PC312.-546-158. The method of any of claims 153-156, wherein all steps of the method are performed under low shear force; optionally, wherein low shear force is less than 100 shear / seconds or low shear force is from 10 to less than 100 shear / seconds.

159. The method of any of claims 153-158, wherein the cells are HeLa cells or human ipSCs.

160. The method of any of claims 153-159, comprising:316.a) infecting cultured cells with an IMV crude lysate and culturing the cells for a time sufficient for production of EEV particles and release thereof into the cell culture medium without lysing the cells, wherein the conditions are low shear force conditions;317.b) harvesting the culture medium; adding 5-10% sucrose; and filtering the resulting mixture under low shear force to remove particulates;318.c) treating the mixture with a DNAase to digest any host cell DNA in the mixture; d) low or shear force free concentration of viruses by a tangential flow filtration (TFF), wherein the pore size is about 0.05pm to about 0.1pm, and collecting the resulting virus composition;319.e) re-buffering the virus into a storage and injectable formulation buffer;320.f) optionally filling a vial or vials or other container for low temperature storage.

161. The method of claim 160, comprising:322.a) culturing cells in suspension spinner flasks to achieve S cell densities of 2xl0e6 cells per mL, wherein the culture conditions are 37°C and 5% CO2;323.b) directly infecting the cells with IMV crude lysates with at a multiplicity of infection (MOI) of about 0.1 to 1 virus particles per cell, such as at about 0.5 virus particles per cell, and culturing for about 35-50 hours, wherein the culturing is sufficient for release of EEV into the medium without lysing the cells to avoid release of IMV into the cell culture medium;324.c) harvesting the culture medium, adding 5-10% sucrose, and pre-filtering with a filter to remove cells and cell material from viruses to produce filtered medium;325.d) adding a DNAase, such as benzonase enzyme, to digest any host cell DNA in the filtered medium;326.e) concentrating the viruses by TFF under low shear force or force free conditions; Attorney Docket No. 120276-2615PC327.-547-328.f) shear force free or low shear force re-buffering of the viruses into a storage and IV injectable formulation buffer.

162. The method of claim 160 or claim 161, wherein the formulation buffer comprises lOmM Tris / HCl, 1% sucrose, 2% trehalose, 5% mannitol, 300 mM glycine, and 0.1% recombinant human albumin.

163. The method of any of claims 153-162, further comprising filling a vial or vials for storage at low temperature and subsequent injection.

164. The method of any of claims 153-163, wherein the infecting virus is a vaccinia virus or genome or derivative or IV-EEV or composition of any of claims 1-105.

165. The method of any of claims 153-164 wherein cells are infected with a red tail (RT) virus.

166. The method of claim 163, wherein the RT virus is:334.i) one of RT-00 - RT-129 and RT-134;335.ii) variants of i) produced upon propagation of the virus or modification of the virus to encode additional proteins or replacement of all or part of non-essential genes, whereby the virus is substantially as resistant or more resistant to human serum than RT-00 and / or produces at least or at least about the same level of EEVs as RT-00; or iii) variants of i) or ii) that encode the same proteins, but by virtue of the genetic code, comprise on or more degenerate codons thereof.

167. The method of any of claims 153-166, wherein the EEV virus is comprises three knockouts of genes TK, A46, and VGF.

168. The method of any of claims 153-167, wherein the cells are HeLa cells or are iPSCs or HeLa cells or iPSCs modified whereby that the membrane displays a product that alters a property and / or activity of the virus propagated in the cells.

169. The method of claim 168, wherein:339.a) culturing HeLa S3 cell in suspension in a 100 mL spinner flask with animal-derived serum to reach a density of approximately 1-4 x 10A6, such as 3.0 x 10A6, cells / mL; following addition of fresh medium, infecting the cells with a programmed EEV virus at a multiplicity of infection (MOI) of about 0.1 to 1, such as 0.5; and the harvesting the filtrated suspension containing the EEV virus at about 48 ± 1 hours postinfection; or Attorney Docket No. 120276-2615PC340.-548-341.seeding iPSCs adherently in a coated in a xeno- and serum-free medium; at 80-90% confluency, infecting the cells with a high EEV producing virus at an MOI of about 0.1 to 1, such as 0.5, following a complete medium exchange; and collecting the medium containing the EEV virus at least 24 hours post-infection.

170. The method of claim 168 or claim 169, wherein the cells are iPSC; and the method comprises:343.growing iPSCs in 3D aggregates;344.infecting the cells with a high EEV producing virus at an MOI of about 0.1 to 1, such as 0.5, following a complete medium exchange; and345.collecting the medium comprising the EEVs at least 24 hours post-infection.

171. The virus, cell, cell line, composition, method, or use of any of claims 1-170, wherein the virus encodes a therapeutic product.

172. The virus, cell, cell line, composition, method, or use of claim 171, wherein the nucleic acid encoding the therapeutic protein is inserted into or in place of all or a portion of a non-essential viral locus.

173. The virus, cell, cell line, or composition, method, or use of any of claims 1-172, wherein the nucleic acid encoding therapeutic protein is inserted into or in place of all or a portion of the VGF encoding nucleic acid rendering the virus VGF'.

174. The virus, cell, cell line, composition, method, or use of any of claims 1-173, wherein the virus encodes a therapeutic protein that that is an immunostimulatory protein and / or has anti-cancer activity.

175. The virus, cell, cell line, composition, method, or use of claim 174 wherein the therapeutic protein is an IL-15 or IL-15 / IL-15R alpha chain complex or modified form thereof that comprises a mutation that increased activity.

176. The virus, cell, or cell line, or composition, method, or use of any of claims 1-175, wherein the EEV virus genome comprises the sequence set forth in SEQ ID NOs: 782-790 or 887-898 or a sequence having at least 90%, 95%, 98%, or 99% sequence identity thereto, excluding ITRs, and having at least the same anti-tumor activity or serum resistance as the virus designated RT-00.

177. The virus, cell, cell line, composition, method, or use of claim 176 that comprises a fusion protein encoding IL-15 or IL-15 / IL-15R alpha chain complex and / orAttorney Docket No. 120276-2615PC352.-549-353.a fusion protein with a transmembrane protein and a humoral immunity modulator, such as a CRP, such as CD55.

178. The virus, cell, cell line, composition, method, or use of any of claims 1-177, wherein the virus is poxvirus or herpes virus, or single membrane virus, such as lentivirus, encased in a host cell membrane.

179. The virus, cell, or cell line, or composition, method, or use of claim 178, wherein the virus is vaccinia virus.

180. The virus, cell, cell line, composition, method, or use of any of claims 1- 179, wherein the virus is a vaccinia that, upon propagation, produces a high level of EEV; and357.a high level is higher than that produced by the Western Reserve (WR) strain virus or is greater than 5%, 10%, 15%, 20%, 25%, or 30% of the progeny produced.

181. The virus, cell, cell line, composition, method, or use of any of claims 1- 180, wherein the unmodified genome is from a vaccinia virus selected from among a Western Reserve (WR), Copenhagen (Cop), Bern, Paris, Tashkent, Tian Tan, Lister, Wyeth, H4D-J, H4D-W, Brighton, Ankara, modified vaccinia Ankara (MV A), CVA382, Dairen I, LIPV, LC16M8, LC16M0, AC AM, WR 65-16, Connaught, JX-594 (pexastimogene devacirepvec), GL-ONC1, vvDD TK mutant, New York City Board of Health (NYCBH), EM-63, and NYVAC vaccinia virus strains, and variants thereof that produce virus particles that produce EEV particles that display the protein or portion thereof that reduces or inhibits humoral immunity.

182. The virus, cell, cell line, composition, method, or use of any of claims 1- 181, wherein the unmodified virus genome is the genome of a virus selected from among JX-594 (Pexastimogene Devacirepvec, Pexa-Vec); LIVP GLV-lh68 (GLV-ONC1 or GL-ONC1); vvDD; TG6002; VG9-GM-CSF; CVV; deVV5; CF33; Guang9; IN rVV; T601; vA34R; aCEA TCE; a modified WR. TK-GMCSF vaccinia virus;360.WR. B5Rmut. TK-; mCCR5 / TK- virus; mCXCR4 / TK- virus; TK- PH20 DCK virus and KLS-3010 and those described in: 8,980,246; 12,364,724; US 2019 / 0218522;361.2023 / 0002740; WO 2022 / 182206; WO 2023 / 118603; and WO 2023 / 064793.

183. The virus, cell, cell line, composition, method, or use of any of claims 1- 182, wherein:Attorney Docket No. 120276-2615PC363.-550-364.the virus is a Western Reserve (WR), Copenhagen (Cop), Bern, Paris, Tashkent, Tian Tan, Lister, Wyeth, IHD-J, IHD-W, Brighton, Ankara, modified vaccinia Ankara (MV A), CVA382, Dairen I, LIPV, LC16M8, LC16M0, ACAM, WR 65-16, Connaught, JX-594 (pexastimogene devacirepvec), GL-ONC1, vvDD TK mutant, New York City Board of Health (NYCBH), EM-63, and NYVAC vaccinia virus strains, and variants thereof that produce virus particles that produce a high level of EEV particles, or that produce a high level of EEV particles and that display the protein that reduces or inhibits humoral immunity or portion thereof; and365.a high level of EEV virus is at greater than 1%, 5%, 10%, 15%, 20%, 25%, 30% or more of the virus population.

184. The virus, cell, cell line, composition, method, or use of any of claims 1-182, wherein a high level of EEV virus is greater than 25% or 30% of the virus population.

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