Serum-resistant EEV viruses and uses thereof

Abstract

Provided are serum-resistant vaccinia extracellular enveloped viruses (EEV), methods of producing serum-resistant EEVs, and modified EEVs for systemic oncolytic viral therapy. Provided herein are methods to modify vaccinia viruses to produce serum resistant EEVs for systemic oncolytic viral therapy. Compositions containing serum resistant EEVs and methods of treating cancers and other proliferative diseases are provided.

Description

[0001] SERUM-RESISTANT EEV VIRUSES AND USES THEREOF RELATED APPLICATIONS

[0002] Benefit of priority is claimed to U.S. provisional application Serial No. 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., the contents of which is incorporated by reference in its entirety.

[0003] Benefit of priority is claimed to U.S. provisional application Serial No. 63 / 640,822, entitled SERUM-RESISTANT EEV VIRUSES AND USES THEREOF,” filed April 30, 2024, to inventors Antonio Fernandez Santidrian, Duong Hoang Nguyen, and Yunyi Kang, and Applicant Calidi Biotherapeutics (Nevada), Inc., the contents of which is incorporated by reference in its entirety.

[0004] Benefit of priority is claimed to U.S. provisional application Serial No. 63 / 558,577, entitled SERUM-RESISTANT EEV VIRUSES AND USES THEREOF,” filed February 27, 2024, to inventors Antonio Fernandez Santidrian, Duong Hoang Nguyen, and Yunyi Kang, and Applicant Calidi Biotherapeutics (Nevada), Inc., the contents of which is incorporated by reference in its entirety.

[0005] INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

[0006] 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 February 26, 2025, is 43,120,899 bytes in size, and is titled 2613SEQPCl.xml.

[0007] FIELD OF THE INVENTION

[0008] The field is oncolytic viruses and uses thereof for therapy.

[0009] BACKGROUND

[0010] 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. SUMMARY

[0011] Provided are viruses and virus preparations and compositions that have a high percentage of extra enveloped vaccinia (EEV) forms of vaccinia virus and other poxviruses. Also provided are methods of manufacturing such viruses, whereby compositions with high levels of the EEV form are produced.

[0012] 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.

[0013] 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. 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 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 provides 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.

[0014] 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 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.

[0015] The viruses also include knockouts of genes, such as virally-encoded TK, A46, and VEGF, 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. 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:

[0016] (TK -, A46-, VGF-), a high EEV - producing strain RT-01 with the knockouts;

[0017] (TK -, A46-, VGF-) - A33+CD55 - the strain encoding a fusion protein;

[0018] (TK -, A46-, VGF-) + Payload IL-15 (cytokine form) - the strain encoding a cytokine inserted into the VGF locus;

[0019] (TK -, A46-, VGF-) - A33+CD55+ Payload IL-15 (cytokine form) - the strain encoding the fusion protein and the cytokine ;

[0020] (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 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.

[0021] 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 IUD strain NR-52. For example, provide is an EEV particle 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.

[0022] 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 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 between the A56 on the membrane and secreted VCP on EEV particles. US publication US20050208074 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 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 EGFR on the EEV surface. No preferential infection towards ErbB-2 expressing cells of the EEV having the antibody- HA fusion was observed. US publication US20050208074 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 2021 / 0348158, 2019 / 0112388, 20130288927A, and 20130288927). None have exploited EEV transmembrane proteins for modifying EEV outer membranes to increase serum resistance, nor effecting such in virus preparations with a high level of EEV (greater than 1%, generally 5% or more), and certainly not in virus preparations produced as described herein that contain at least 30% and more EEV, as much as al least 80% and more, as shown and provided herein.

[0023] 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 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. 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 SNP in A45R relative to the IHD-W strain.

[0024] 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.

[0025] 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 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. 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.

[0026] 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.

[0027] 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.

[0028] 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 knock-outs. 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.

[0029] 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, 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 (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.

[0030] 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, and 782-793, 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: 1 and 782-790, or a variant of any of SEQ ID NOs: 1, and 782-790, 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 portion of the ITRs or comprise heterologous ITRs, or degenerate sequences of any of the preceding sequences.

[0031] 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, and 485 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.

[0032] 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.

[0033] 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 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. 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 35. Complement regulatory proteins include, but are not limited to, CD35, CD55, CD59, CD46, CR1, Factor H, VCP, MOPICE, SPICE, CCPH, C4- binding protein, CD35, Kaposi-sarcoma associated herpesvirus Kaposica I KCP, Herpesvirus saimiri (HVS) and 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.

[0034] 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 and variants thereof having at least 95% sequence identity thereto, whereby the protein displays the protein that reduces or inhibits humoral immunity or portion thereof. 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, 224, and 225 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, IHD-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, to be high producers, such as by mutation or selection. Other unmodified viruses, include but are not limited to, 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; 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 Al; WO 2022 / 182206 Al; 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.

[0035] 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 791-793 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 increases anti -turn or 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.

[0036] 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 anti-ANGPT2; anti-VEGF, anti-ANGPT-2 and anti-CTL4; anti-VEGF and OX40L; Anti-VEGF, Anti-ANGPT2 and anti-PD-1 products.

[0037] 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.

[0038] 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 1x103and about 1x1015pfu per ml; (ii) between about 1x104and about 1x1014pfu per ml; or (iii) between about 1x106and about 1x1012pfu per ml.

[0039] 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, 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, 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.

[0040] 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 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.

[0041] 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 sufficient portion thereof for display of the CRP on the surface of an EEV. Envelope proteins include for example, vaccinia virus, 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, CD55, CD59, CD46, CR1, 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 envelope protein, whereby the protein or portion thereof that reduces or inhibits humoral immunity is displayed on the EEV outer membrane.

[0042] 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, Volepox virus, Akhmeta virus, Ectromelia virus, Orthopoxvirus Abatino virus, Skunkpox virus, 87 Raccoonpox virus, Yokapox virus, Murmansk poxvirus, NY 014 poxvirus, and Yaba monkey tumor virus, 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.

[0043] Vaccinia virus 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 viruses can include further modifications as described herein and known in the art. For example, the virus can comprise a deletion in or of or insertion in the thymidine kinase (TK) gene. Viruses for used herein include, but are not limited to, 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, 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 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 virus is at greater than 1%, 5%, 10%, 15%, 20%, 25%, 30% or more of the virus population.

[0044] 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 of (i) between about 1x103and about 1x1015pfu per ml; (ii) between about 1x104and about 1x1014pfu per ml; or (iii) between about 1x106and about 1x1012pfu per ml.

[0045] 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 of any of claims 1-63; 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.

[0046] 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. 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. 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.

[0047] 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.

[0048] 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 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, Veto, Vero STAT1 KO, HEK293.STAT1 BAX KO AGEl.CR.pIX, CV1, HELA, HELA S3, CHO, VPCs, VPCs 2.0, FS293, MDCK, and MDCKSTAT1 KO cells. Exemplary of cells for production are iPSCs and HeLa cells. All steps of the methods are performed under low shear force. 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 0.1pm, and collecting the resulting virus composition; e) re-buffering tiie 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 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.

[0049] BRIEF DESCRIPTION OF DRAWINGS

[0050] FIGURE 1 depicts a strategy for selection of a vaccinia virus with high EEV production and resistances to humoral immunity.

[0051] FIGURE 2 shows spreading patterns of the CALI virus (low producer of EEV) and the EEV clone designated RT-01 (high producer of EEV).

[0052] FIGURE 3 shows that EEV, but not IMV are resistant to inhibition by humoral immunity.

[0053] FIGURE 4 show spread of the RT-01 and CAL2 viruses to neighboring cells at 24 and 48 hours. FIGURE 5 shows that the EEV virus RT-01 survives systemic administration and reaches all tumors; the corresponding IMV virus does not.

[0054] 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 et al. Viruses 2016, 8(5), 134.

[0055] 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 IMV virus.

[0056] 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.

[0057] 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.

[0058] FIGURE 10 shows that the N2 virus can amplify and kill mouse cells. 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).

[0059] 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).

[0060] 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. FIGURE 14 depicts the display of hCD55 on the EEV second membrane; the resulting virus is referred to herein as an IV-EEV.

[0061] FIGURE 15 show the structure of A46R-hCD55-B5R as constructed and inserted into the parental virus to produce RT-02.

[0062] 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; infected

[0063] ■ Cells + RT-05.

[0064] 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.

[0065] 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. 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.

[0066] 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; 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.

[0067] 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.

[0068] 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 vitally encoded HER2 is expressed. Such tumor cells express the targeted antigens for treatment with therapeutic products that target the antigens. FIGURE 24 summarizes EEV viruses provided herein, the transfer vectors for generating the viruses, and inserts and loci of the inserts, and nomenclature.

[0069] 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.

[0070] FIGURE 26 presents a graphic representation of major differences in sequence between the virus designated RT-00 (N2 or IHD-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.

[0071] 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).

[0072] 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, Fl 3. Modified from Riccardo et al. Viruses. 2023, Dec (8); 15(12).

[0073] FIGURE 29 shows that incorporation of all or a portion of CD55 into the second membrane of an EEV virus confers increased resistance to human serum of enveloped CD55 virus.

[0074] 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).

[0075] FIGURE 31 sets forth the sequences and components of exemplary transfer vectors for introducing protein encoding genes, such as detectable markers, and transmembrane fusions proteins.

[0076] FIGURES 32A, 32B, and 32C show heat maps of fluorescence signal intensity for single, double, and tripe knockout viruses. FIGURES 32A and 32B 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 32C 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.

[0077] FIGURES 33A, 33B and 33C 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. FIGURE 34 shows that treatment with RT-52 (see Figure 24) and cyclophosphamide as detailed in Example 24 has remarkable therapeutic efficacy after systemic administration. FIGURE 35 depicts complement pathways and components.

[0078] FIGURES 36A, 36B, 36C, and 36D summarize an exemplary method of manufacturing EEV. Figure 36A depicts an upstream culture method / process in a spinner flask; Figure 36B depicts the process in a perfusion reactor; Figure 36C shows the downstream process for isolation of virus from the culture medium harvested from spinner culture and suspension; and Figure 36D shows the process in a wave bioreactor.

[0079] FIGURES 37A, 37B, and 37C examine the effects of CPA treatment. Figure 37A shows that CPA treatment prolonged virus presence at the tumor sites. Figure 37B 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 37C 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.

[0080] FIGURES 38A and 38B show that EEVs released from RT-77-infected Hela or hiPSCs were highly resistant to human serum complement-mediated inactivation compared to EEVs from RT-65. Figure 38A shows results from CBD27 human serum; Figure 38B shows results from AB2391 human serum.

[0081] FIGURES 39A and 39B show that canine cell lines are permissive to RT viruses. Figure 39A 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 39B shows that the CMT-U27 cell line showed resistance at lower MOI, with cytolysis only observed at the highest MOI (MOI 10).

[0082] FIGURES 40A, 40B, and 40C 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 40A shows the quantification by ELISA of IL15 superagonist in the tumors from the RT-96-treated mice at 6 days post treatment; FIGURE 40B shows changes in exemplary cellular composition of the tumor micro environment upon RT-65 and RT-96 administration, FIGURE 40C shows reductions in the growth of LL2 lung cancer tumors upon RT-65 and RT-96 administration.

[0083] DETAILED DESCRIPTION

[0084] Outline

[0085] A. Definitions

[0086] B. Overview

[0087] Vaccinia viruses for Systemic Administration

[0088] C. Vaccinia Viruses and EEV Vaccinia Viruses

[0089] 1. Vaccinia viruses

[0090] 2. Vaccinia Virus EEV and Their Production

[0091] 3. IMV

[0092] 4. Insufficient EEV for System Administration

[0093] 5. Selection of high producing EEV

[0094] 6. Exemplary high EEV-producing viruses

[0095] 7. Modifications of EEV to increase resistance to humoral immunity

[0096] D. Methods of Production of High Amounts of EEVs

[0097] 1. Existing methods result in low production of EEVs

[0098] 2. Methods of manufacturing / producing virus resulting in higher EEV Viruses

[0099] 3. iPSCs and their use for manufacturing EEVs from poxviruses and other viruses

[0100] E. Improved EEV Viruses

[0101] 1. Serum-resistance of EEV viruses is limited by short-term protection from host immune system, such as protection from complement

[0102] 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

[0103] 3. Advantages of the S-R EEVs and IV EEVs

[0104] 4. Complement Regulating Proteins

[0105] 5. Properties of complement regulatory proteins that inhibit complement activation (complement inhibitory proteins)

[0106] 6. EEV Viruses That are Modified to Display Immune Modulating Proteins on the Outer Membrane of the EEV

[0107] 7. Viruses modified to encode Complement Regulating Proteins - Functional or active or sufficient portions of the complement regulating proteins or other such proteins

[0108] 8. Assays for measuring the complement regulating activity of proteins

[0109] 9. Exemplary of immune system regulating proteins are the complement regulatory proteins

[0110] 10. EEV outer membrane proteins

[0111] F. Any Poxviruses Can Be Modified as Described Herein

[0112] 1. Methods for Modifying vaccinia viruses

[0113] 2. Therapeutic, transgenic, and attenuated vaccinia viruses 3. Exemplary Therapeutic Vaccinia Virus

[0114] 4. Virally Encoded Immunomodulators and Effects thereof

[0115] G. EEV Vaccinia Viruses Can Be Modified to Express Heterologous Genes and / or Payloads

[0116] H. Chimeras and Fusions Virally Encoded Membrane Proteins and Proteins That Inhibit or Modulate the Humoral Immunity of the Host

[0117] 1. Fusion proteins and Chimeric proteins

[0118] 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

[0119] 3. Domains of the complement inhibitory / regulatory / control proteins

[0120] I. Pharmaceutical Compositions, Combinations, and Kits

[0121] J. Methods of Treating Cancer and Other Proliferative Diseases, Disorders, and Conditions

[0122] K. Examples

[0123] A. DEFINITIONS

[0124] 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 found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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, IHD-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.

[0129] 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.

[0130] 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 et al. (1995) Vaccine 13:487-493); the Microorganism Collection of FSRI SRC VB Vector (Kozlova et al. (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 et al. (1995) Vaccine 73:487-493; Shchelkunov et al. (1993) Virus Research 28:273-283; 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.

[0131] 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 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.

[0132] 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.

[0133] 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 (i.e., 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 proteins. The proteins can subsequently be processed, such as for activation.

[0134] 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 1x103and 1x1015PFUs; for example, about 1x104, about 1x106, about 1x108, about 1x1010, about 1x1012or about 1x1014or higher, per mL, per kg.

[0135] 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.

[0136] As used herein, an extracellular enveloped virus (EEV) 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.

[0137] 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.

[0138] 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 (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).

[0139] As used herein, complement inhibitor protein refers to any protein, polypeptide, and or portion thereof that can inhibit complement.

[0140] 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. 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.

[0141] 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.

[0142] As used herein, immunomodulatory with respect to combination treatments reduce or modulate the immune response so that the anti-viral response is reduced.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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. 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 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.

[0148] As used herein, “multiplicity of infection (MOI)” refers to the number of virions that are added per cell during infection (z.e., one million virions added to one million cells is an MOI of one).

[0149] 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.

[0150] 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.

[0151] As used herein, a “host cell” or “target cell” are used interchangeably to mean a cell that can be infected by a virus.

[0152] 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.

[0153] 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 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.

[0154] 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.

[0155] 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, y5 (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. 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).

[0156] 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 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 el al., 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.

[0157] 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 (z.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.

[0158] 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.

[0159] 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 (z.e., the preparation is homogenous in sequence) or can have different genomic sequences (z.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)).

[0160] 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.

[0161] 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 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).

[0162] 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.

[0163] 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 (z.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.

[0164] 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. Activities can be observed in in vitro and / or in vivo systems designed to test or use such activities.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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 subject elicits an immune response that clears the virus from the subject. 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.

[0174] 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).

[0175] 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. As used herein, metastasis refers to a growth of abnormal or neoplastic cells distant from the site primarily involved by the morbid process.

[0176] As used herein, malignant tumors can be classified into three major types. 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.

[0177] 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.

[0178] As used herein, neoplastic disease refers to any disorder involving cancer, including tumor development, growth, metastasis and progression.

[0179] 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, 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, 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.

[0180] 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, 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.

[0181] 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. Inhibiting or killing immune effector cells can treat diseases that have an inflammatory component. 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] As used herein, a “normal cell” is a cell that is not derived from a tumor, but is derived from healthy non-diseased tissue.

[0186] 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.”

[0187] 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.

[0188] As used herein, reference to an anticancer or chemotherapeutic agent includes combinations or a plurality of anticancer or chemotherapeutic agents unless otherwise indicated.

[0189] 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.

[0190] As used herein, a patient refers to a human subject exhibiting symptoms of a disease or disorder.

[0191] 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 pharmaceutical use of the cell-assisted viral expression systems described and provided herein.

[0192] As used herein, treatment refers to amelioration of the symptoms of a disease or disorder.

[0193] As used herein, prevention refers to prophylactic treatment to reduce the risk of getting a disease or condition or reducing the severity thereof.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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. 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.

[0201] As used herein, prevent a disease or condition means reduce the probability or rise of getting the disease or condition.

[0202] 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.

[0203] As used herein, a formulation refers to a composition containing at least one active pharmaceutical or therapeutic agent and one or more excipients.

[0204] 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.

[0205] 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 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.

[0206] 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.

[0207] As used herein, direct administration refers to administration of a composition without dilution.

[0208] 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. 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.

[0209] 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.

[0210] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0211] 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.

[0212] 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% is considered about the same. Such amounts can vary depending upon the tolerance for variation in the composition by subjects.

[0213] 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.

[0214] 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).

[0215] 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 (ie., the patient). For example, autologous stem cells are stem cells that are derived from the patient. 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.

[0216] 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.

[0217] 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.

[0218] As used herein, '"immune suppression" or "immunosuppression" refers to the suppression or reduction of the immune response.

[0219] 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. 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.

[0220] 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.

[0221] As used herein, "permissive" with respect to viral infection refers to a cell that is readily infected upon exposure to the virus.

[0222] 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. As used herein, "co-culture" refers to a cell culture in which two or more different populations of cells are grown.

[0223] 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.

[0224] 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.

[0225] 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 CALI virus has the sequence set forth in SEQ ID NO:251. CAL2 is CALI encoding TurboFP635 at the TK locus.

[0226] 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.

[0227] 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. 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.

[0228] As used herein, non-naturally occurring amino acids refer to amino acids that are not genetically encoded.

[0229] 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 nucleotide analogs, single (sense or antisense) and double-stranded nucleic acids. 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.

[0230] 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. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding a MTSP-1 protease provided.

[0231] 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.

[0232] As used herein, “polypeptide” refers to two or more amino acids covalently joined. The terms “polypeptide” and “protein” are used interchangeably herein.

[0233] As used herein, a “peptide” refers to a polypeptide that is from 2 to about or 40 amino acids in length.

[0234] 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 (see Table of Correspondence). The nucleotides which occur in the various nucleic acid fragments are designated with the standard single-letter designations used routinely in the art.

[0235] 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 (see Table of Correspondence), non-natural amino acids and amino acid analogs (i.e., amino acids wherein the a-carbon has a side chain).

[0236] 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 of Correspondence). The nucleotides, which occur in the various nucleic acid molecules and fragments, are designated with the standard single-letter designations used routinely in the art.

[0237] 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 etal. (1994) Biopolymers 34:1681).

[0238] 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. As used herein, “amino acid residue” refers to an amino add formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are generally in the “L” isomeric form. Residues in the “D” isomeric form can be substituted for any L-amino add 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., 243:3557-59 (1968) and adopted at 37 C.F.R. §§ 1.821- 1.822, abbreviations for amino acid residues are shown in the following Table:

[0239] Table of Correspondence

[0240] 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. The phrase “amino acid residue” is defined to include the amino acids listed in the above Table of Correspondence, as well as modified, non-natural, and unusual amino acids. 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.

[0241] 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).

[0242] Such substitutions can be made in accordance with the exemplary substitutions set forth in the following Table:

[0243] Exemplary Conservative Amino Acid Substitutions

[0244] Other substitutions also are permissible and can be determined empirically or in accord with other known conservative or non-conservative substitutions.

[0245] As used herein, “naturally occurring amino acids” refer to the 20 L-amino acids that occur in polypeptides.

[0246] As used herein, the term “non-natural amino add” refers to an organic compound that has a structure similar to a natural amino acid, but that 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, 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-Methylisoleucine (Melle), 6-N- Methyllysine (MeLys), N-Methylvaline (MeVal), Norvaline (Nva), Norleucine (Nle), and Ornithine (Om).

[0247] 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).

[0248] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.

[0249] B. OVERVIEW

[0250] Provided are vaccinia viruses for systemic administration and methods for manufacturing viruses. Provided are compositions of viruses that contain high percentages of EEV forms of poxviruses, such as vaccinia virus. As described herein, provided is a high EEV and derivatives thereof. These viruses were selected to produce a high level of EEVs and to have 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. Also provided are derivatives of these viruses and any vaccinia virus 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 between a viral 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. All of these aspects and others are detailed in the sections and Examples that follow.

[0251] Vaccinia Viruses for Systemic Administration

[0252] 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.

[0253] Upon infection of tumors with vims, the tumor microenvironment is remodeled to stimulate an anti-tumor immune response, the viruses amplify, and lyse tumor cells. Thereby releasing vims and tumor antigens; the vimses, 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-tumor response. For successful therapy, the viruses must be able to disseminate and must survive attack by the immune system of the host.

[0254] Oncolytic therapy has had limited success. The successful oncolytic virotherapies require administration by intratumoral or local inj ection. Approved oncolytic vimses for cancer therapy require local or intratumorally administration. These viruses include Rigvir® (enteric cytopathic human orphan vims 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.

[0255] 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.

[0256] 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 of oncolytic viruses can target cancer cells that are inaccessible by localized therapies and provides the ability to target multiple tumor sites upon administration.

[0257] 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.

[0258] 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. (2Q\4)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.

[0259] 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 administrate virus products for use as therapeutics and vaccines.

[0260] 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 vims 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 vims 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 vims. Seven virus-encoded proteins have been identified that are components of IEV, and five of them are present in CEV or EEV.

[0261] Intracellular mature vims (IMV) particles are formed in the from non-infectious precursors called crescents and immature vims (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. 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 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.

[0262] 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.

[0263] 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 expressed on the EEV membrane. Exemplary of this, the RT-00 virus has been modified by introducing a CD55 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.

[0264] 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.

[0265] 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 adds, 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.

[0266] 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 viruses that exhibit 80% and greater serum resistance. These and other properties are described and exemplified in the sections and Examples below. c. VACCINIA VIRUSES AND EEV VACCINIA VIRUSES

[0267] Provided herein are EEV viruses, methods for producing EEV viruses, and methods and modifications for increasing resistance of EEV viruses to humoral immunity.

[0268] 1. Vaccinia viruses

[0269] Vaccinia Virus

[0270] 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 (MVA), 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 vimses 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.

[0271] Vaccinia vimses also can be easily modified by insertion of heterologous genes. This can result in the attenuation of the vims 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 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 eZa / . (2001) Biotechniques 31:534- 540).

[0272] Various vaccinia viruses have been demonstrated to exhibit antitumor activities.

[0273] 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 etal. (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 in clinical trial of melanoma patients. As a control, a New York City Board of Health strain vaccinia virus (W) 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 W control (Kim et al. (2001) Surgical Oncol. 10:53-59).

[0274] 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 et al. (2007) Surgery 142:976-983; Lin etal. (2008) J. Clin. Endocrinol. Metab. 93:4403-7; Kelly era / . (2008) Hum. Gene Ther. 19:774-782; Yu etal. (2009) Mol. Cancer Ther. 8:141-151; Yu et al. (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.

[0275] 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.

[0276] Vaccinia vims 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 vims has a unique history in its widespread application as a smallpox vaccine that has resulted in an established track record of safety in humans. Toxicides related to vaccinia administration occur in less than 0.1% of cases, and can be effectively addressed with immunoglobulin administration. In addition, vaccinia vims 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 etal. (2001) Riotechniques 3V. 534-540). Vaccinia virus strains have been shown to specifically colonize solid tumors, while not infecting other organs (see, e.g., Zhang etal. (2007) Cancer Res. 67:10038-10046; Yu et al.

[0277] (2004) Nat. Biotech. 22:313-320; Heo et al. Qti\V)Mol. Ther. 19:1170-1179; Liu et al. (200S)Mol. Ther. 16:1637-1642; Park er a / . (2008) Lancet Oncol. 9:533-542).

[0278] 2. Vaccinia Virus EEV and Their Production

[0279] Vaccinia virus (W) 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.

[0280] 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).

[0281] 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.

[0282] 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 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).

[0283] Much of the EEV remains attached to the host cell surface and is retained as

[0284] 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.

[0285] 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.

[0286] EEV has several mechanisms to inhibit its neutralization within the bloodstream.

[0287] 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, 61(6)3319-3325, 1993).

[0288] 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 (CRTs) or 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., (A.998) Proc Natl Acad Sci USA. (13):7544-9; Smith er al. (1998) Adv Exp Med Biol. 440: 395-414). This plays a role for repeat administration once neutralizing antibody levels have increased and when anti-cancer therapies require repeat administration.

[0289] 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.

[0290] 3. IMV

[0291] 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 DSL 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 Song K. etal.

[0292] Biomedicines. 2020 8(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, MOPICE, SPICE and CCPII, as a chimeric proteins with an IMV membrane protein (see, International 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.

[0293] 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.

[0294] 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.

[0295] 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.

[0296] Insufficient EEV for Systemic Administration The systemic capabilities of oncolytic vaccinia viruses are limited by the relatively small amount of EEV generated by an infected cell, fiirther 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 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 et al., 1998 Adv Exp Med Biol. 1998:440:395-414).

[0297] 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.

[0298] 5. Selection of high producing EEV

[0299] 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.

[0300] Methods for Selection of high EEV viruses

[0301] 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.

[0302] 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. W02013038066A1; Published International PCT application No. WO2023128672A1; U.S. Publication Nos. US 2022 / 0049228A1 US 2023 / 0002740 Al). 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. W02013038066A1; Published International PCT application No. WO2023128672A1; and U.S. Publication Nos. US 2022 / 0049228A1 and US 2023 / 0002740A1. For example, US 22023002740 describes viruses that produce high levels of EEV, such as a vaccinia virus (OW), 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.

[0303] 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 EMV. 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- derived secondary plaques (Blasco et al. 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%.

[0304] 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.

[0305] 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.

[0306] 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 -LI 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 -LI NR-45114 antibody or an anti-Vaccinia Immune Globulin (VIG) antibody. Higher plaque formation indicates greater EEV production and resistance to neutralizing antibodies.

[0307] Virus samples containing enrichment of IMV or enrichment of EEV can be separated and purified using a CsCl density gradient ultracentrifugation method. Vims 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.

[0308] 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 vims sized particles (VSP) and VSP containing the B5 antigen can then be quantified by flow cytometry.

[0309] 6. Exemplary high EEV-producing viruses

[0310] Provided herein are RT-00 vaccinia vims (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 vimses are summarized in Figure 24.

[0311] RT-00 was selected for high EEV production and high tumor selectivity. It can be used as a systemically administered extracellular enveloped vims (EEV) for oncolytic vims therapy. As detailed herein, the RT vimses 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 vims provided herein, including modified RT vimses, and can be introduced into any therapeutic vaccinia vims. 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.

[0312] 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 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.

[0313] 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 (IHD)-J and W strains (IHD-J and IHD-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.

[0314] The genome of RT-00 is related IHD-W but possesses key distinguishing features. IHD-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.

[0315] 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 IHD-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.

[0316] When compared with the available IHD-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 SNP in A45R.

[0317] Other high EEV viruses

[0318] 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.

[0319] 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. SillaJen / Jennerex viruses

[0320] Another virus derived from an IHD-J virus preparation (parent IHD-J obtained from ATCC® Catalog No. VR-156) is detailed in International PCT publication No. W02024 / 011250 (see, also US publication No. US20240033347), 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 (zn 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.

[0321] 7. Modifications of EEV to increase resistance to humoral immunity

[0322] 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.

[0323] 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 Bl 9R. 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 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.

[0324] 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.

[0325] 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.

[0326] 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 add encoding the noted polypeptide and promoter, and includes flanking vaccinia vims DNA to insert into the noted loci (i.e., TK, A46R, etc.) SEQ ID NOs: 2-21, 518-524, refer to the sequences of the transfer vectors or inserts, which 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.

[0327] The table below, summarizes structures of the above viruses, which are derived from

[0328] 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).

[0329]

[0330] RT-00 or a vims derived therefrom can be and has been modified or engineered for recombinant expression of selection markers or detectable and encoded payloads.

[0331] Selection markers include, but are not limited to, EGFP, EmGFP, mNeonGreen, EBFP,

[0332] TagBFP, EYFP, TPet, GFP, BFP or TuiboFP635. The RT-00 virus and viruses derived

[0333] 5 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,

[0334] IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, TNF, MIPla, FLt3L, IFN-b, IFN-g), chemokines (CC15, CC12, CC119, CXC111, RANTES), co-stimulators (OX40L, 4-

[0335] 1BBL, CD40L, B7.1 / CD80, GITRL, LIGHT, CD70), bi-specific t-cell engagers (BITEs),

[0336] 10 therapeutic antibodies, immune checkpoint inhibitors, single chain antibodies such as single chain antibodies against e.g., VEGF, e.g., VEGFA, VEGFB, PGF, VEGFR2,

[0337] 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,

[0338] 15 Aquaporin 1-AQP1, and any other payload of interest, particularly any that have anticancer activity, or promote an anti-cancer phenotype or activity.

[0339] RT-00 or a vims 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

[0340] 20 viral promoters (e.g., Pel, pL, CMV). The vimses 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-VEGF A 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.

[0341] 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.

[0342] 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 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.

[0343] 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 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.

[0344] Generation of the viruses

[0345] 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.

[0346] 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 Set.; 89: 5.13.1-5.13.18; Broder and Earl, (1999)MoZ. 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.

[0347] 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.

[0348] 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:

[0349] Transgene insertion sites generally are selected from the non-essential (for viral propagation)sites, which are well-known to those of skill in the art, within the vaccinia virus genome. The vaccinia virus genome has several such well-known insertion sites where transgenes can be inserted. Exemplary insertion sites in the vaccinia virus genome include A46R, B8R, J2R (thymidine kinase, TK), A52R, F1L, VACV growth factor gene (VGF), B19R, Ig, BamHL, hemagglutinin gene (A56), ribonucleotide reductase (RR), and

[0350] A27L. The insertion site choice is user selected, depends on a variety of parameters, such as the intended application of the transfer vectors for use in the production of recombinant vaccinia viruses here include, but are not limited to:

[0351] Homologous recombination can be effected by infection of tissue culture cells, typically a monolayer of cells, parental strain of vaccinia virus followed by transfection with the transfer vector containing the transgene of interest. Homologous recombination between the vaccina virus DNA and the transfer vector results in incorporation of the transgene into the viral genome, and, in general, is expected to yield about 1 recombinant virion in 1000 progeny. The desired recombinant vaccinia virus is plaque purified by several rounds of selection and / or screening, such as by fluorescent microscopy to detect a fluorescent transgene followed by propagation of the recombinant virus. High titer recombinant virus stocks can be prepared from infected cell lysates and stored at -80°C. For purposes herein, the fusion proteins that comprise the humoral immune modulators, such as a complement resistance protein (CRP), are inserted into virally encoded proteins that, when expressed, display the humoral immune modulator protein on the surface of the virus. Display of such proteins is detailed elsewhere herein. For payloads, such as therapeutic proteins, such as anti-cancer proteins, including cytokines, such as the IL-15 / IL-15R alpha chain complex (also referred to as an IL-15 superagonist), they are inserted the viral genome, generally into a non-essential locus. . In some examples, the heterologous nucleic acid, such as that encoding a heterologous gene product, is inserted into or in place of a non-essential gene or region in the genome of the virus. These regions / genes are well-known. For example, the nucleic acid encoding the heterologous gene product can be inserted into or in place of all or a portion of the hemagglutinin (HA), thymidine kinase (TK), F14.5L, vaccinia growth factor (VGF), A35R, NIL, E2L / E3L, K1L / K2L, superoxide dismutase locus, 7.5K, C7-K1L, B13R+B14R, A26L, andI4L gene loci in the genome of the virus.

[0352] Exemplary protocol for generating viruses provided herein from RT-00.

[0353] In general, the skilled person can modify any virus known in the art as described herein to increase serum resistance of the virus. In general, the starting virus is on that is selected to have high EEV production (greater than 1%, generally greater than 5% or 10% EEV) or so-modified by methods known in the art and / or methods detailed herein. The viruses can be propagated by the methods provided herein that preferentially isolate EEV viruses and preserve the EEV viruses during propagation, isolation, and storage. The methods (1) culturing the virus under gentle conditions that retain the second membrane as the viruses are produced; (2) isolating virus from culture medium by selecting parameters, such as m.o.i. and time for culturing such that the virus does not lyse the cells, and the EEV virus is released into the medium, and isolated with minimal shear forces so that the EEV membrane is preserved; and (3) stored under conditions that retain the EEV membrane.

[0354] Exemplary of the protocols that can be employed to generate a virus, including any in the above tables, is preparation of the virus designated RT-52. RT-52 is a triple modified vaccinia virus produced from RT-00 with the genotype [eGFP(A46R) / (TK-) / Turbo(VGF)]. RT-52 is produced from the virus RT-21 with the genotype [(TK) / Turbo(VGF)] and homologous recombination with the transfer vector pUC-eGFP (A46R) (SEQ ID NO: 3) to add pSEL-eGFP into the A46R loci of RT-21.

[0355] To produce RT-52, a monolayer of CV1 cells is seeded in 6-well tissue culture plates and infected with RT-21 (2e5 PFU / mL) and standard methods. The infected monolayer of CV1 cells is then transfected with the transfer vector pUC-eGFP (A46R) using TurboFect™ Transfection Reagent in accord with the manufacturer’s protocol. Infected / transfected cells are incubated at 37°C for up to 24 hours. Following incubation, the cells are dislodged and infected / transfected, the cells are lysed followed by several rounds of plaque purification and selection by fluorescent microscopy for eGFP and TurboFP635 dual positive populations. Alternatively, infected / transfected cells are lysed by one or more freeze-thaw cycles and the cell lysate can be stored at -80°C for later use. Isolated recombinant virus is amplified by further rounds of CV1 infection with the purified virus.

[0356] RT-21 [(TK-) / Turbo(VGF)], which was derived from RT-12 [eGFP(TK) / Turbo(VGF] and CRE-LOX to knockout pSEL-eGFP from the thymidine kinase loci (IK or J2R). pSEL-eGFP in TK loci of RT-12 is flanked by loxP sites, whereby knockout of pSEL-eGFP in the TK loci is achieved by infection of a cell line, such as HEK cells, expressing CRE recombinase. Selection of RT-21 is achieved by cell lysis, plaque purification, and selection by fluorescent microscopy for TurboFP635 positive plaques.

[0357] RT-12 [eGFP(TK) / Turbo(VGF] was derived from RT-14 [eGFP (TK)] and homologous recombination with the transfer vector pUC-Turbo(VGF) (SEQ ID NO: 12) to add pSEL-TurboFP635 to the VGF loci of RT-14. CV1 cells infected with RT-14 are transfected with the transfer vector pUC-Turbo(VGF), followed by plaque purification and selection by fluorescent microscopy for eGFP and TurboFP635 dual positive populations.

[0358] RT-14 [eGFP (TK)] was derived from RT-01 [Turbo (TK)] and homologous recombination with the transfer vector pUC-eGFP (TK) (SEQ ID NO: 14) to replace pSEL-TurboFP635 with pSEL-eGFP in the TK loci of RT-01. CV1 cells infected with RT-01 are transfected with the transfer vector pUC-eGFP (TK), followed by plaque purification and selection by fluorescent microscopy for eGFP positive populations. RT-01 [Turbo (TK)] was derived from RT-00 [Wild-Type] and homologous recombination with the transfer vector pUC-Turbo (TK) (SEQ ID NO: 2) to insert pSEL- TurboFP635 into the TK loci of RT-00. CV1 cells infected with RT-00 are transfected with the transfer vector pUC-Turbo (TK) followed by plaque purification and selection by fluorescent microscopy for eGFP positive populations.

[0359] Mutations that Increase EEV

[0360] Provided herein are methods to modify any poxvirus, such as vaccinia virus, for high EEV production and resistance to host immunity, such as humoral immunity. Poxviruses, such as vaccinia virus, can be modified by replacement of the A34R gene with the A34R gene from a different strain to increase the production of EEV. Exemplary mutations that can be introduce into any poxvirus, vaccinia virus and / or smallpox vaccine. Such mutations include for example A34R with a mutation at codon 151 of A34R from a lysine to glutamic acid substitution (A34R / K151E). Such A34R mutation renders the A34R protein less able to tether EEV to the infected cell membrane, resulting in the release of EEV particles from the infected host cell. In this way, higher EEV production can be achieved. Additional genome modifications can be introduced into such viruses to increase serum resistance or modify other viral properties resulting, for example, in increased tumor selectivity, increased tumor colonization, and / or reduced toxicity. EEV viruses with this mutation are known, but have not been effective anticancer therapeutics when systemically administered.

[0361] EEV Biology

[0362] It is shown herein that EEVs are difficult to isolate or purify with intact outer membranes. Methods for manufacturing viruses that produce EEVs to the extent that known strains with the K151E mutation do, and to increase yield of EEVs (as a percentage of total virus) are provided. The methods include steps in which the viruses are treated gently, such as by employing low shear pumps, and appropriate buffers. Additional mutations are introduced into the virus including one or more knockouts described herein, and those that resulted from selection of RT-00 for increased tumor selectivity.

[0363] As detailed herein, and known in the art, poxviruses such as vaccinia virus, produce four different types of virions from each infected cell: intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), and extracellular enveloped virus (EEV). EEV provide rapid and efficient spread through solid tumors locally and to regional or distant tumor sites. Vaccinia viral polypeptides act to inhibit the release of infectious virus from cells. For example, viral polypeptides prevent EEV detachment from the host cell surface. Polypeptides involved in the modulation of EEV release include, but are not limited to, the EEV membrane proteins A33R, A34R, A36R, B5R and F13L. Loss of A33R can effect a 3-fold increase in EEV. The total deletion of A34R can lead to increased EEV release, but markedly reduced infectivity of the viruses, while the KI 5 IE mutation increases EEV release while maintaining infectivity of the released viruses. Deletion of A36R and B5R can reduce EEV particle formation by 5-fold and 5- to 10-fold, respectively. Replacement of the B5R short consensus repeat (SCR) domains with the extracellular domain of A56R can reduce EEV formation by 25-fold. Overexpression of F13L reduces EEV production.

[0364] Preparing virus with increased EEV production

[0365] Different strains of vaccinia vims produce different ratios of IMV and EEV particles following infection. For example, rabbitpox and IHD-J release relatively high levels of EEV and produce the characteristic comet-shaped plaques. The difference in EEV formation is due to the retention of EEV virions on the host cell surface whereby the association and retention of the EEV virion to the host cell membrane is regulated by the A34R gene (Balasco et al. J. Virol. 66:4170-4179, 1992). An exemplary A34R protein sequence (NCBI Protein ID: BAD97823) is set forth below (SEQ ID NO: 191): The release of EEV virions can be effected by incubation with a protease or peptidase such as trypsin or by modification of the A34R lectin homology / carbohydrate binding domain. A substitution mutation that is present in the International Health Department (IHD)-J strain of vaccinia vims (IHD-J), but not in WR, in the A34R protein (K151E). Enhancing the release of EEV-particles produced by vaccinia vims infected cells can be achieved by silencing the A34R gene. Repressed expression of the A34R gene in WR leads to small plaque formation. Western Reserve A34R deletion mutant vims can release up to 25-fold more EEVs from infected cells than A34R wild-type Western Reserve virus. Deletion of A34R leads to increased EEV release but reduces infectivity of the virus (Mcintosh A. A. J Virol 1996, 70, 272-81). Vaccinia viruses can be modified or engineered to include substitution mutation (A34R / K151E) to render the A34R protein less able to tether the EEV to the infected host cell membrane to provide for the release of EEV particles. The A34R / K151E substitution mutation can be introduced into the genome of any known poxvirus or vaccinia virus by known methods of genetic engineering or modification to produce EEV.

[0366] The A34R / K151E substitution mutation can be introduced alone or in combination with other A34R mutations, such as the A34RZD110N substitution mutation, and / or other A34R mutations known in the art to enhance EEV production. A strain derived from the Western Reserve (WR) virus containing the A34R (K15 IE) alone or in combination with a second A34R substitution mutation at codon 110 from aspartate to asparagine (DI 1 ON mutation) from IHD-J recombined into the WR A34R gene locus was shown to increase EEV production by comet formation (Blasco etal. J. Virology, 67(6):3319-3325, 1993). The A34R / K15 IE isoform, alone or in combination with the A34R / D1 ION isoform, increases EEV production while maintaining infectivity and resistance to humoral immunity of the released viruses. Any poxvirus, vaccinia virus and / or smallpox vaccine can be modified or engineered to comprise a nucleic acid encoding A34R / K151E, A34R / D1 ION or both, to obtain enhanced EEV production.

[0367] Virus with increased EEV production can be prepared by selecting clones from existing viral strains and selecting for those with higher EEV production, and / or by introducing a mutation or mutations that confer such phenotype. As shown in the Examples, the RT-00 virus was selected from a polyclonal strain that produced high EEV. Sequencing revealed that it comprises the KI 5 IE mutation, and some additional differences.

[0368] Additional mutations can be introduced as demonstrated herein to increase EEV production of any poxvirus, such as a vaccinia virus. Such mutations can be combined with the A34R isoforms A34R / K15 IE and A34R / D 11 ON of IHD-J. Such mutations include substitution mutations of like amino acids such as, for example, a mutation at codon 151 of A34R from a lysine to aspartic acid (A34R / K151D). Other exemplary mutations include A34R substitution mutation (KI 19E), and a double mutation in A34R (K119E and K151E) termed “W034.” W034 mutants displayed enhanced therapeutic effect and significant increase in EEV particle production. Modified vaccinia virus strains with the A34R substitution mutations KI 5 IE and KI 19E can release 10-fold to

[0369] 200-fold higher levels of EEV particles, compared to an identical vaccinia virus strain that do not comprise the A34R double mutant (see, e.g., U.S. Patent App. No. 2022 / 0049228).0ther identified mutations in A34R that increase EEV production include F94H, R91S, T127E, R84G, R91A, M66T and others disclosed in U.S. Patent App. No. 2023 / 0002740.

[0370] Modified vaccinia with EEV production and resistance to host or humoral immunity effected for vaccinia virus. For example, any vaccinia virus can be modified to enhance EEV production by introducing the mutations (A34R / K151E), (A34R / D110N), (A34R / K151D), (A34R / K119E), (A34R / F94H), (A34RZR91S), (A34R / T127E), (A34R / R84G), (A34R / R91A), (A34R / M66T) or combinations thereof into the wild-type A34R gene. Any vaccinia virus can comprise a nucleic acid encoding (A34R / K151E), (A34R / D110N), (A34R / K151D), (A34R / K119E), (A34R / F94H) or combinations thereof. The modified vaccinia virus strain comprising one or more mutation(s) in the A34R protein can release higher amounts of EEV particles and possess greater resistance to complement and neutralizing antibodies relative to the unmodified vaccinia virus. The modified vaccinia virus strain comprising one or more mutation(s) in the A34R protein can release higher amounts of EEV virions and possess greater resistance to complement and neutralizing antibodies relative to IMV virions.

[0371] Introduction of mutations in virally encoded EEV membrane proteins (A33R, A34R, A56R, B5R), and combinations of mutations thereof to increase EEV percentages

[0372] Mutations in vaccinia virus encoded EEV proteins can be introduced into the viral genome. These mutations, including deletions, disruptions, and insertions to alter properties of the encoded product, can be introduced separately and in combination into the viral genome For example, mutations in A34R can be combined with mutations in A34R, A56R and B5R to enhance EEV production. Combinations of mutations in others of the EEV membrane proteins can be introduced. Thus, viruses with higher EEV production can be obtained by introducing one or more mutations or combinations thereof into the viral genome, such as, by introducing mutations in:

[0373] A33R (A88D, E129M and / or M63R); A34R (K151E, DI ION, K151D, KI 19E, F94H, R91S, T127E, R84G, R91A and / or M66T);

[0374] A56 (I269F); and

[0375] B5R (S197F, S197V, N39G, S273I, S199M, L90R, S273V, K229C, S273L, S273I, I236P, V238R, T240R, E243G, V233D, I236L, V238W, T240Y, E243R, D263V, E268T, E270G, E272P, E275S, N94T, N241G, E243S, V247W, D248Y, G250A, A276F, D263A, E270S, E272G, E275F, N241T, E243V, V247S, G250R and / or A276F) and those described in US 2023 / 0002740 Al); and conservative substitutions of any of the above.

[0376] These mutations increase the percentage of EEV viruses. Conservative amino acid substitutions are well known in the art (see, Table in the definitions section), and are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity charge, size. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Mutations in A34R or combinations thereof can be combined with one or more amino acid substitutions of relative similarity into the EEV proteins A33R, A34R, A56R and B5R to enhance EEV production.

[0377] Effects of other mutations in A33R, A34R, A56 and B5R mutation on EEV production and virus spreading can be assessed using known methods described in the Examples and provided in the specification, as well as any other suitable methods known in the art. Many such mutations are known in the art

[0378] D. METHODS OF PRODUCTION OF HIGH AMOUNTS OF EEVs

[0379] 1. Existing methods result in low production of EEVs

[0380] EEV with intact outer envelope are needed for effective systemic oncolytic viral therapy. Unmodified or “wild-type” vaccinia virus strains generate relatively low amounts of EEV. For example, often only about 1% of the virus particles are EEV (or precursors thereof)

[0381] Standard vaccinia virus manufacturing and purification methods result in EEV inactivation and outer membrane disruption. For example, EEV can be separated from IMV by cesium chloride density gradient centrifugation because of its lower buoyant density (1.23 g / ml for EEV compared with 1.27 g / ml for IMV) or by sucrose gradients (Dorns et al., J Virol. 1990 Oct;64(10):4884-92). The EEV envelope is easily ruptured by either freeze-thawing, sonication, or ultracentrifugation, so that although the particles purify as EEV; ruptured EEV biologically functions as IMV due to exposure of IMV surface proteins.

[0382] Non-human cell lines frequently are used to manufacture vaccinia virus although the EEV generated from the non-human cell generally are not protected from complement-mediated clearance since complement inhibitory proteins acquired by EEV from an infected host cell have species restricted effects (Vanderplasschen et al, (1998) Proc Natl Acad Sci USA. (13):7544-9). Resistance to complement neutralization is dependent upon the cell line used for manufacturing.

[0383] 2. Methods of manufacturing / producing virus resulting in higher EEV

[0384] Viruses

[0385] Provided herein are methods to manufacture or produce and obtain EEV with an intact EEV outer membrane from any vaccinia virus, and particularly from high EEV producing vaccinia virus. The methods provided herein are designed to culture EEVs and isolate them with intact outer membranes.

[0386] In general, vaccinia virus about 1% (or less) of the viral particles produced are EEVs, but once the virus is amplified or cultured, the percentage of EEVs recovered is much lower because the fragile second envelope does not survive culturing and isolation. The methods provided herein result in higher level of EEV relative to the amounts of EEV produced by any vaccinia virus. For example, high EEV-producing virus generally produce more than 1%, typically 4% or 5% or more, generally 5% to 10%, EEV. The methods herein preserve as much as 60% or more (more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%) of the EEV produced by any virus. By virtue of the methods, the EEV can be obtained in a ready-to-use injection solution ready for IV or other injection or for storage for later use, while retaining at least about 70%, 75%, 80% or more lytic potency after thawing. These methods combined with selection of an appropriate cell line for manufacture (or modification or selection of a cell line to express high levels of CD55 or other such complement inhibitory or resistance protein) or modification of the viruses to display / express a complement inhibitory or resistance protein in an EEV transmembrane, or combinations thereof, results in compositions containing virus with relatively high levels of EEV (60% or more). Modifications of the virus as detailed herein, including particular knockouts, and display of humoral immunity resistance proteins on the outer membrane, particularly virally-encoded fusion proteins for displaying the humoral immunity resistance proteins, increases the serum resistance / stability of resulting EEVs so that upon administration a high percentage of virus (as much as more than 80%, 85%, 90%, or 95% of the EEV in any preparation).

[0387] Any EEV producing vaccinia virus can be propagated in cell line that expresses a humoral resistance proteins, such as a CRP, such as CD55, in relatively high amounts so that the resulting EEVs are serum-resistant for example, Hela S3, HEK 293 T / 17SF, MDA-MB-231, iPSCs, and other such cell lines. A cell line that produces at least EEVs that are about 40%, or at least about 50% serum resistance can be employed.

[0388] For viruses that are modified as described herein to be IV-EEVs so that they express a complement inhibitory or resistance protein, such as CD55, or other fusion protein as detailed herein, displayed in the second membrane of EEV, any host cell line can be used, such as, for example A549, CV-1 Vero, Hek293, or a stem cell line, including an iPSC, particularly a cell line modified to increase membrane-expression of a humoral resistance protein, such as CD55.

[0389] Also provided are methods for rendering and manufacturing viruses resistant to complement. These methods allow not only the expression of a complement inhibitor, such as CDS 5, during the manufacturing process, but permit use of other host cell lines, including non-tumor cell lines, such as, for example, stem cells, and CD55 high- expressing cells, in addition to cells such Hela cells and other cells that produce higher levels of CD55, thereby generating serum-resistant EEVs in vitro. Other exemplary cell lines include HEK293, HEK293T, A549, PerC6, Vero, Vero STAT1 KO, HEK293.STAT1 BAX KO AGEl.CR.pDC, CV1, HELA, HELA S3, CHO, VPCs, VPCs 2.0, FS293, MDCK, and MDCK.STAT1 KO. Cells also include iPSC for manufacturing EEVs; the resulting resistance to serum complement, while lower than in Hela cells (70%-80% ) is high (40% to 50%) iPSCs, thus, can serve as non-tumor cells for manufacturing EEV. Protection is significantly increased when the EEVs express fusion proteins with complement resistance proteins, such as A33-CD55 expressing virus on iPSC. Provided are EEVs that comprise iPSC proteins from the iPSCs in which they are produced. iPSCs can be modified to express fusion proteins that comprise complement resistance proteins. As shown in the examples, human iPSCs can be used as a manufacturing host cell line to produce EEVs that are protective against complement neutralization. The data show that human iPSCs (hiPSCs) can be used to manufacture enveloped viruses; they incorporate the iPSC functionality into the virus envelope, including protections against serum-induced inhibition. Oher functionalities such as stealth, immunomodulation, targeting, and tumor homing present in various iPSCs can be added to the extracellular membrane of enveloped viruses. iPSCs can be modified to express fusion proteins that comprise complement resistance proteins. iPSCs can be modified to express human receptors that enhance the functions of enveloped viruses, such as targeting receptors, chimeric antigen receptors, integrins, chemokine receptors, and anti-immunomodulatory proteins.

[0390] 3. iPSCs and their use for manufacturing EEVs from poxviruses and other viruses

[0391] Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell that can be generated directly from adult somatic cells. Introducing four specific genes, known as Yamanaka factors (Oct3 / 4, Sox2, Klf4, and c-Myc), can reprogram somatic cells into pluripotent stem cells. iPSCs have the ability to propagate indefinitely and differentiate into any cell type in the body. iPSCs can be generated from various somatic cell types, Sources include fibroblasts, keratinocytes, blood cells, urine-derived cells, adipose- derived stem cells, and hepatocytes. The tissue of origin impacts reprogramming efficiency, epigenetic memory, and potential applications. For instance, fibroblasts are easily accessible and have a high proliferation rate, while blood cells and keratinocytes reprogram more efficiently. Non-invasive sources like urine-derived and blood cells are particularly advantageous for generating patient-specific iPSCs. iPSCs can be used to manufacture enveloped viruses, such as vaccinia virus, for therapeutic purposes. This approach involves using iPSCs as host cells to produce viruses that incorporate iPSC-derived proteins into their envelopes, enhancing their therapeutic properties and immune evasion capabilities. This method can be applied to other enveloped viruses. iPSCs as host cells for virus production offers several advantages compared to tumor cell. iPSCs are derived from non-tumor cells, thereby reducing the risk of introducing tumorigenic properties. iPSCs can be genetically modified to express immune-modulatory proteins, enhancing the therapeutic efficacy of the produced viruses. iPSCs provide high production of enveloped viruses. They can be engineered to produce high levels of extracellular enveloped viruses (EEVs). The incorporation of iPSC-derived immune-modulatory proteins into the viral envelope enhances the virus's ability to evade the host immune system, increasing its therapeutic potential. iPSCs provide a consistent and safe source of cells for virus production, reducing the risk of introducing tumorigenic properties.

[0392] In general in accord with methods herein, EEVs with an intact EEV outer membranes can be obtained by methods that include 1) high density cell culture in a perfusion suspension reactor, such as for example, at a cell density, such as, of about or more than 20xl0e6 cells / mL or other such cell format that does not disrupt the outer membrane as the EEV is produced and budded out and released into the medium; 2) direct infection in the reactor for a sufficient time, such that, secreted or released EEV particles are in the supernatant, but prior to lysis of the cells by the virus; 3) pre-filtration of the cell culture medium to separate cells / cell material from the EEV viruses; 4) benzonase or other such nuclease digestion to eliminate or reduce host cell DNA i; 5) nearly shear force-free concentration of viruses by Tangential Flow Filtration ( I FF) or other such separation method that purifies EEVs but does not disrupt the outer membrane; 6) nearly sheer force-free re-buffering of the viruses in a formulation buffer, such as, but not limited to, for example, buffer containing lOmM Tris / HCl, 1% sucrose, 2% trehalose, 5% mannitol and 300 mM glycine, 0.1% recombinant human albumin; and, if needed, 7) filling containers for storage at -80°C (or, depending on storage time and expected or planned use, -20°C). The methods employ low- or sheer force-free steps to avoid disruption of the second membrane to minimize or eliminate or substantially eliminate loss of the second membrane.

[0393] The combination of the high EEV producing virus manufactured by the methods provided herein that do not disrupt the second membrane results in preparations of virus that contain very high percentages of EEV. Such EEV viruses can be modified as described herein by genome modifications that increase anti-tumor activity, delivery of therapeutic payloads, delivery of payloads that enhance various therapies, and modifications in which the EEV second membrane displays proteins or portions thereof that inhibit host humoral anti-viral immunity so that the virus has increased serum resistance, and also that preserve such resistance when amplified in tumors.

[0394] E. Improved EEV Viruses

[0395] Provided are modified EEVs that not only are produced at high levels relative to strains such as WR, but also are modified so that they have increased serum resistance.

[0396] 1. Serum-resistance of EEV viruses is limited by short-term protection from host immune system, such as protection from complement

[0397] The extracellular enveloped virus (EEV) form of poxvirus is responsible for viral spread within the infected host while being relatively easy to degrade outside of the host making it an effective viral form for systemic therapy. In general vaccinia viruses have a low percentage (less than 1%) EEV virus. As discussed above, there are examples in the art of vaccinia viruses that produce higher percentages of EEV viruses or that are engineered to do so.

[0398] Examples of previously engineered or modified EEV viruses include, but are not, limited to: IHDJ-B5RcoASCRI and IHDJ-B5RcoASCRl (modified IHDJ viruses where the neutralizing antibody binding site on the surface of the EEV (B5R SCR1) was deleted); WR.TK-A34R KI 5 IE; WR.TK-PH20+ (a TK deficient Western Reserve virus optionally with PH-20 incorporated into the EEV outer envelope); GLV-H69 (a Lister strain vaccinia virus, where the A34R gene is replaced by the A34R gene from vaccinia IHD-J strain); IGV-006; IGV-007; IGV-013; IGV-023; IGV-033; IGV-034; IGV-035; IGV-038; IGV-051; IGV-052; IGV-059; IGV-060; IGV-061; IGV-062; IGV-063; IGV- 064; IGV-065; IGV-066; IGV-067; IGV-068; IGV-069; IGV-070; IGV-071; IGV-072; IGV-073; IGV-077; IGV-084; IGV-085; IGV-086; IGV-087; IGV-101; IGV-102; IGV- 103; IGV-104; IGV-105; IGV-106; IGV-107; IGV-108; IGV-110; IGV-111; IGV-112; IGV-114; IGV-116; IGV-117; IGV-118; IGV-119; IGV-120; Cop.Luc-GFP.A34- K151E; Cop.mGM-CSF.A34-K151E; Cop.mIL-2v.A34-K151E; WR.Luc-GFP.A34R- K151E; WR.mIL-2v.A34R-K151E; MD-RW-A34R; MD-RW-ARR-A34R; MD- RVV-EEV6; MD-RVV-ARR-EEV6; MD-RVV-EEV7; MD-RVV-ARR-EEV7; an EEV virus expressing a chimeric B5R-HTV protein; an EEV virus expressing a single-chain antibody fused to hemagglutinin (HA); an EEV virus expressing an integral membrane protein; and, for example, those described in: U.S. Patent Nos. 8,329,164, 9,708,601, 10,550,199, 10,577,427, 11,529,402, and 11,685,904; US Patent Publication Nos: 2009 / 0053244; US2022 / 0049228; and US 2023 / 0002740; Published International PCT application Nos; WO 2013 / 038066, and WO 2019 / 089755.

[0399] Vaccinia EEV particles resistance to complement can depend on the presence of CDS 5 and other anti-complement human factors encoded by the host cell line, and such presence is not specific to a tumor type. Production of EEVs depends on the cell in which virus is propagated. EEV, such as SR-EEV, can be resistant to complement neutralization due to host Complement regulatory proteins (CRPs or RCAs) that are incorporated from the host cell membrane into the EEV outer envelope or outer membrane. Because resistance depends upon expression of host cell proteins, EEV resistance to complement can be species or homologous-restricted. EEV can be resistant to neutralization by complement only when its cellular origin and challenging complement are form the same species because the incorporation of host cell CRPs into the EEV outer membrane. EEVs can be relatively resistant to complement neutralization when it is grown in a cell type from the same species. In addition to host CRPs (e.g, CD55, CD46 and CD59), host cell membrane proteins CD71, CD81, and major histocompatibility complex class I (MHC class I) were detected in purified EEV, butnot in IMV (Smith et al. (1998) Adv Exp Med Biol. 440: 395-414; Vanderplasschen e / a / ., (1998) Proc Natl Acad Sci USA. (13):7544-7549).

[0400] Although CRPs are expressed by every cell in the body, as shown herein expression varies across tissue and cell type. The expression of human CRP on the EEV outer membrane is depends upon the expression of CRP by the infected host cell. It is shown herein that the pattern of expression of human CRP across cell types (e.g., tumor cells) and within tumor types (e.g, among tumor cells from the same cancer type) is highly variable. For example, as shown herein, CD55 expression is highly variable in every individual tumor cell line of breast cancer. Because EEV resistance to complement neutralization derives from the incorporation of host CRPs into the EEV outer membrane, the levels of CRP incorporated into the EEV outer membrane and, therefore, resistance to complement neutralization is dependent upon the expression of CRP by the infected host cell.

[0401] Complement resistance in vivo of standard EEV preparations or EEVs prepared from CRP-high cells is transient. Once administered to a tumor, the EEV expression of CRP of virus progeny EEV is dependent upon the expression levels of subsequent host tumors / cells. For example, subsequent infection of an CRP-low cell results in the loss of CRP on the progeny EEV outer membrane and loss of complement resistance even if the EEV was produced from CRP-high cells, e.g., SR-EEV.

[0402] Upon propagation in a tumor or cell that does not express high levels of CRP (e.g., CDS 5) the resulting EEV or SR-EEV viruses are not serum or complement resistant, which can limit the effectiveness of single doses. The therapeutic potential of systemically administered EEV or SR-EEV virions is dependent upon the expression of CRPs, e.g.,CD55, on the tumor or cell line or tumor metastasis. In addition, pre-existing immunity to a specific oncolytic virus can be established following repeated doses which can further limit the effectiveness of repeated doses (Bell, E. et al Virology (2004) 325:425-431; Putz, M. M. etalNat Med. (2006) 12:1310-1315); Lawrence, S. J. etal. J. Infect. Dis. (2007) 196:220-229). Improved systemically administered EEV viruses must resist neutralization by host immunity, such as complement, for a sufficient time so that effective amounts of virus can reach tumors and metastasis.

[0403] 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

[0404] To produce EEVs that can resist the immune response of the treated host or subject, nucleic acid encoding an CRP or other such protein that modulates humoral immunity to increase serum stability or a portion thereof is encoded in the viral genome, whereby the protein or portion thereof is displayed or expressed on the EEV membrane. To effect such display, the CRP or other such protein can be introduced into an EEV transmembrane protein to produce a chimeric protein that is expressed or displayed on the EEV membrane. Any vaccinia virus can be so-modified. If the virus is not one that produces a higher percentage of EEVs, the virus can be modified by mutation of proteins, such as A34R, to increase EEV production, or can be subject to selection to select clones with such property. The viral genome EEV-producing viruses then can be modified to express a chimeric transmembrane protein so that the EEV has increased serum resistance or resistance to humoral immunity. The resulting viruses can be grown or propagated in any cell, in vivo or in vitro to produce serum resistant EEVs. An advantage and property of these viruses is that they will propagate and continue to exhibit the serum resistance or resistance to humoral immunity because the resistance protein (or portion thereof) is encoded by the virus, and is independent of the cell in which the virus propagates. These viruses are referred to herein as IV-EEVs because they are suitable for systemic administration. Any vaccinia virus, including any known therapeutic virus developed for oncolytic therapy or other purpose, can be modified so that the serum resistance or humoral immunity resistance protein or portion thereof is displayed on the EEV membrane and will be so-displayed in any progeny. Hence provided are methods for producing vaccinia viruses that can be systemically administered, and provided are the resulting viruses.

[0405] Provided herein are complement resistant EEV viruses and methods of producing complement resistant EEV viruses, the viruses are designated S-REEV and IV-EEV. The complement resistant EEV viruses are well-suited for systemic cancer therapy and have the ability for wide-spread host distribution which is desirable for the treatment of metastatic or I ate- stage cancers.

[0406] The viruses, such as the SR-EEV and IV-EEV, EEV viruses provided herein are resistant to complement independent of CRP expression level or target tumor owing to the sable expression of CRP s on the EEV particle. It is also shown herein that complement resistance is transient even if the production of EEV virions from CRP -high cells. By expressing CRP s on the EEV outer membrane, the EEV viruses provided herein can now produce complement resistant vial progeny in vivo; protection from inactivation is maintained as the virus self-amplifies and spreads in tumors cells and tumor metastasis.

[0407] EEV production can be increased in a poxvirus, such as a vaccinia virus, can be effected by selecting for increased EEV production and / or by introducing a mutation or mutations into an EEV membrane protein, in particular by introducing one or more mutations into one or more of A33R, A34R, A56R and / or B5R to increase the EEV form of the virus. For example, the A34R polypeptide can include a mutation corresponding to KI 5 IE, which results in increased EEV production. The resulting virus can be further modified or engineered to encode and express one or more host CRP s or complement inhibiting portions thereof on the EEV outer membrane to mediate resistance to complement. This results in virus that have high EEV production independent of the level of CRP expression level of the infected cell or tumor. Stable expression of CRP s can be achieved by engineering or modifying any EEV poxvirus or vaccinia vims to overexpress CRP s or a portion thereof on the EEV outer membrane or as a fusion protein that is covalently linked to a protein expressed or present on the EEV outer membrane.

[0408] Complement regulatory proteins (CRPs or RCAs) or functional portion thereof can be selected from among CD55, CD46, CD59, CD35 and / or other such complement regulatory proteins such as, for example, Cl inhibitor (Cl -INH), C4 binding protein (C4BP), Complement Factor H, Complement factor H related 1 (CFHR1), Complement Factor I, Vitronectin, Clusterin, and / or Carboxypeptidase N.

[0409] The CRP s can be overexpressed on the EEV outer membrane or expressed as a fusion protein, such that the CRP or functional portion thereof is covalently linked to an EEV outer membrane protein. EEV outer membrane proteins include, but are not limited to, among viral proteins such as, for example, A33R, A34R, A56R, B5R, F13L and / or host cell membrane proteins, such as, for example, CD71, CD81, major histocompatibility complex class I (MHC class I) and / or other such proteins that are incorporated into or become part of the EEV outer membrane.

[0410] Any combination of CRP s or functional portions thereof can be overexpressed on the EEV outer membrane or overexpressed as a fusion protein that is covalently linked to an EEV outer membrane by any EEV producing virus using known methods of genetic engineering. For example, CRP s or a functional portion thereof, e.g., CD55, can be covalently linked to an EEV-specific membrane protein, e.g., B5R. A nucleic acid molecule encoding an EEV fusion protein, e.g.,CD55-B5R, can be inserted into a region of the vaccinia virus genome such as A46R, B8R, J2R, A52R, F1L, VGF and / or B19R.

[0411] 3. Advantages of the S-R EEVs and IV EEVs

[0412] For use in the treatment of cancer, the EEV viruses (e.g., S-R EEV and IV -EEV) provided herein offer several advantages over standard vaccinia viruses or standard EEV preparations. It is demonstrated herein that the expression of human CRP (RCA; e.g., CDS 5) on the EEV outer membrane is highly dependent upon the expression of CRP in the infected host cell. Every tumor type and cells of the same tumor type have highly variable CRP expression. Consequently, EEV spread and protection from complement depends on the tumor type, infected cell, and subject to be treated. Overexpressing one or more CRP s, alone or as a fusion protein, on the EEV outer membrane overcomes inconsistencies in cell, tumor, and subject RCA expression by providing viruses that stably expresses one or more CRP s on the EEV outer membrane. One or more CRP s can be expressed at higher density on the EEV surface than with standard EEV preparations.

[0413] The viruses provided herein produce EEV with stable CRP expression such that any tumor type can be targeted irrespective of CRP expression of the tumor or metastasis or cell line from which the EEV was produced. Any vaccinia virus can be modified to (1) enhance EEV production and (2) overexpress one or more CRP s on the EEV outer membrane (e.g., as an CRP alone or as a fusion protein with an outer membrane protein) to achieve the stable expression.

[0414] Stable CRP expression by the EEV overcomes dilutive effects of subsequent rounds of infection and provides the benefit that EEV progeny retain protection from complement and neutralization by the host immune system so that enough virus can reach and amplify in tumors and tumor metastasis following systemic administration.

[0415] It is also shown herein that cells infected with CRP armed viruses express high levels of CRP. For example, cells infected with B5R-CD55 armed virus express high levels of CD55 regardless of prior CD55 cell expression. Virus expressing CD55 increase expression of CD55 in host cells and EEV particles produced from the host cells. EEV producing host cells also are protected against complement mediated killing allowing for further amplification and spread of the virus which can support the continuous production of complement resistant virus following a single systemic dose.

[0416] It is demonstrated herein that poxviruses, such as vaccinia virus, can be modified or engineered to increase the EEV form of the virus and modified or engineered to overexpress, on the EEV extemal / outer membrane, one or more CRP s or functional portion(s) thereof on the EEV outer membrane. In some embodiments, the CRP or functional portion thereof can be selected from among CD55, CD46, CD59, CD35 and / or other such complement regulatory proteins such as, for example, Cl inhibitor (Cl -INH), C4 binding protein (C4BP), Complement Factor H, Complement factor H related 1 (CFHR1), Complement Factor I, Vitronectin, Clusterin, and / or Carboxypeptidase N.

[0417] In some embodiments one or more CRP s or functional portion(s) thereof can be covalently linked to a protein expressed or present on EEV outer membrane. Exemplary EEV outer membrane proteins can be selected from among viral proteins such as, for example, A33R, A34R, A56R, B5R, F13L and / or host cell membrane proteins, such as, for example, CD71, CD81, major histocompatibility complex class I (MHC class I) and / or other such proteins.

[0418] In some embodiments, an CRP or functional portion thereof can lack a signal peptide. In some embodiments an CRP or functional portion thereof can be covalently linked to the transmembrane domain of an EEV outer membrane. In some embodiments an CRP or functional portion thereof can be covalently linked to an EEV outer membrane selected from among A33R, A34R, A56R, B5R, and F13L. In some embodiments an CRP or functional portion thereof can be covalently linked to the extracellular domain (ED) of an EEV outer membrane selected from among A33R, A34R, A56R, B5R, and F13L. In some embodiments an CRP or functional portion thereof can be covalently linked to the C-terminal domain of A33R or A34R; or covalently linked to the N-terminal domain of A56R or B5R.

[0419] In some embodiments, the CRP or functional portion thereof is covalently linked to the transmembrane domain of the EEV outer membrane protein B5R. In some embodiments the CRP or functional portion thereof is covalently linked to the N-terminal region of B5R. In other embodiments, CD55 or a functional portion thereof is covalently linked to the N-terminal region of B5R.

[0420] 4. Complement Regulating Proteins

[0421] Complement is a component of the innate immune system, targeting the virus for neutralization and clearance from the circulatory system. The complement system destroys virus-infected cells and increases opsonization and phagocytosis of virus in circulation. Complement can enhance neutralization and antibody-mediated immunity induced by smallpox vaccination. The complement system can be activated by three pathways including the classical (CP), lectin pathway (LP), and alternative pathway (AP).

[0422] Vaccinia virus activates the CP arm of the complement pathways by forming antigen-antibody complexes that are recognized by the Cl complex. Cl recognizes the Fc region of antibodies that are bound to viral epitopes and activates an enzyme cascade of proteins which result in the formation of the C3 convertase C4b2b and cleavage of C3 and deposition of opsonic C3b fragments on cell surfaces (Classical Pathway). Alternatively, C3 can be activated though spontaneous hydrolysis of its internal thioester bond and reacting with a hydroxyl or amino group on the surface of a pathogen. The bound C3b serves as an opsonin for phagocytic cells and as a component of C3 convertase enzyme C3bBb (Alternative Pathway). Further cleavage and binding of C3b leads to formation of C5 convertase. Subsequent cleavage of C5 leads to assembly of the membrane attack complex (MAC) (C5b, 6, 7, 8, 9), which disrupts lipid bilayers.

[0423] Complement activation by viruses can result in their neutralization by various mechanisms, such as Clq-mediated aggregation and opsonization, which leads to immune adherence and phagocytosis of a virus, and / or lysis an infected cell owing to the formation of the membrane attack complex at the infected cell surface. Other effector molecules that are generated against viruses by complement activation include: C3a and

[0424] C5a anaphylatoxins; C3b that can opsonize viruses and promote their immune adherence, direct neutralization, and phagocytic destruction; C3d (the cleaved fragment of C3b) and;

[0425] C5b-9 complex or MAC (Agrawal P etal. FEBST-ett. 2020 Aug;594(16):2518-2542).

[0426] Activated complement components can result in tissue destruction; they are regulated by host soluble or membrane-bound (s / m) complement regulatory proteins

[0427] (complement inhibitory proteins). These include the following proteins:

[0428] 5. Properties of complement regulatory proteins that inhibit complement activation (complement inhibitory proteins)

[0429] Complement activation is negatively regulated by several Complement regulatory proteins (CRTs) or, including but not limited to: CD55, CD46, CD59, CD35 and / or other such complement regulatory proteins. CRP s downregulate complement activation through several mechanisms, which include, for example: 1) by CD35 (complement receptor 1) and CD55 (decay-accelerating factor) inhibiting the formation and accelerating the decay of C3 convertases (decay accelerating activity); 2) by catabolizing

[0430] C3b and C4b through the action of CD35 and CD46 (membrane cofactor protein) acting as cofactors for the serine proteases factor I and factor H to inhibit the formation of the

[0431] C3 convertases C4b2a and C3bBb (co-factor activity) by proteolytic cleavage; and 3) by preventing the formation of the membrane attack complex through CD59 activity.

[0432] Of particular interest herein, are complement regulator proteins that can be displayed on the surface of the EEV by encoding them in the virus in a EEV gene, such as a gene encoding a transmembrane protein so that the chimeric protein is displayed on the surface of the EEV to inhibit complement activation. Similarly other proteins that inhibit complement or other components of the immune system of the host involved in inhibiting, inactivating, or eliminating viruses can be displayed on the EEV surface by encoding it in the virus, such as part as part of the transmembrane protein. Hence proteins such as CD55, CD46, CD59, and CD35, which act as surface proteins, are exemplary of such proteins.

[0433] One or more complement regulatory proteins (CRPs) or regulators of complement activation (RCAs), such as, but not limited to, CD55, CD35 and CD46, which block or inhibit complement activation are encoded in the virus genome. These proteins regulate C3 convertases (C4b2a and C3bBb). Human CRP proteins target and regulate C3 convertases (C4b2a and C3bBb) by two mechanisms: decay-accelerating activity (DA A) and cofactor activity (CEA).

[0434] During the decay-accelerating activity (DAA), an CRP protein binds to the 03 convertases and irreversibly dissociates the catalytic subunit (C2a / Bb) from its noncatalytic subunit (C3b / C4b). Current models propose DAA is effected by first docking onto 03 convertases followed, whereby the complement control protein (COP) domains or sushi domains of the CRP act as an allosteric modulator (by binding the von Willebrand factor type A domain of the 03 convertase catalytic subunit (C2a / Bb)) to induce a conformational change in the C3 convertase catalytic subunit (C2a / Bb). This causes the dissociation of the C3 convertase catalytic subunit (C2a / Bb) from its noncatalytic subunit (C3b / C4b).

[0435] During cofactor activity (OF A), an CRP protein binds to the noncatalytic subunit of the 03 convertases and recruits a serine protease factor I (FI) or factor H to inactivate the noncatalytic subunit (C3b / C4b) by cleavage of the complement Clr / Cls, Uegf, Bmpl (CUB) domain of C3b / C4b.

[0436] Other than DAA and OF A, the glycosylphosphatidylinositol (GPI)-anchored molecule, CD59 (Protectin), blocks or inhibits the formation of the membrane attack complex on the host cell. Like the CRP proteins, human CD59 exhibits species restriction in that it shows less inhibitory activity towards complement from other species.

[0437] Complement regulating proteins that can be encoded in the EEV include, but are not limited to, CD35, CD55, CD59, CD46, CR1, Factor H, VCP, MOPICE, SPICE and CCPH. Vaccinia virus IMV have been modified to encode in the IMV membrane one or more of these proteins as chimeric proteins with an IMV protein (see, International PCT application No. WO2023 / 118603). The IMV have increased resistance to complement. A problem, however, is that IMV cannot be systemically administered and do not disseminate systemically. Hence the problems of host immunity to virus and problems with systemic administration have not been solved.

[0438] The EEV genome is modified to encode chimeric proteins between an EEV envelope protein and a complement regulating protein linked to or in the EEV envelope protein, whereby the complement regulating protein is displayed on the surface of the outer EEV membrane. Examples of such are chimeric proteins are set forth in SEQ ID NOs: 252 and 503-507. Any viral genome is modified to encode these proteins and display them in the outer EEV membrane whereby viral resistance to humoral immunity is increased and is passed on to progeny viruses.

[0439] 6. EEV Viruses That Are Modified To Display Immune Modulating

[0440] Proteins On The Outer Membrane Of The EEV

[0441] Provided are modified EEV viruses that display an immunomodulatory protein on the membrane to inhibit or reduce that humoral immune response of the host. Any protein that can effect a reduction in such response can be displayed. This is effected by introducing an immune response modulating portion of such protein into a virally- encoded transmembrane protein, whereby the immune response modulating protein portion thereof is presented or displayed on the EEV membrane so that it interacts with the host immune system. The immune response modulating proteins are so-displayed by encoding them in an EEV transmembrane protein as a chimera, whereby the transmembrane protein is expressed in the EEV membrane and the immune response modulating protein is expressed / displayed on the membrane. An advantage of this modification is that the virally encoded chimeric protein is amplified with the virus, and resulting EEVs express the proteins on their surfaces. As a result, the EEV can be propagated in any cell in vitro to produce viruses that are resistant have increased resistance to the host immune system, such as serum-resistance, and amplified in vivo such that the progeny virus express these proteins. It is not necessary to manufacture EEVs in cells that express the high levels of such proteins. Upon systemic administration, not only will the EEVs have resistance to the human immune response of the host, when the EEVs propagate in tumors, the progeny viruses will retain the resistance of the administered viruses.

[0442] Chimeric proteins are encoded by EEV viruses. The chimeric proteins comprise an EEV membrane protein and all or a portion of a protein that increases the resistance to the immune system of the host. Exemplary of such proteins are the complement regulating proteins, such as CD55, CD46, and CD35. Provided are nucleic acid molecules encoding the chimeric proteins and the encoded proteins. Nucleic acid encoding the inhibitory or resistance proteins are introduced into nucleic acid encoding an EEV membrane protein in the virus genome so that upon expression the protein that inhibits an immune response or increases resistance of the immune system of the host is displayed on the surface of the EEV. EEV transmembrane proteins include F13L, A56R, B5R, A33R, A34R and A36R. The skilled person can select a site in the transmembrane protein for insertion of the heterologous nucleic acid encoding the immune system modulating protein so that it is displayed on the surface of the EEV.

[0443] Viruses modified to encode Complement Regulating Proteins — Functional or active or sufficient portions of the complement regulating proteins or other such proteins Three mechanisms by which CRP s block or inhibit complement are: decayaccelerating activity (DAA), cofactor activity (CFA), and MAC inhibition. DAA refers to irreversible dissociation of C3 convertases by the CRP protein and CFA refers to inactivation of the non-catalytic subunit (C3b / C4b) of C3 convertases by the serine protease factor I due to its recruitment onto the C3b / C4b- CRP protein complex. Complement regulatory proteins (CRPs) or regulators of complement activation (RCAs) block or inhibit complement activation by targeting and regulating C3 convertases (C4b2a and C3bBb) or by inhibiting MAC formation.

[0444] The structure of human CRP s are known. For example, the structures of DAF / CD55, MCP / CD46, CR1 / CD35 bound to complement C3b are known and the location of the functionally important residues and of conserved functional sites on of CCP domains central to complement inhibition have been mapped (Agrawal P et at. FEBSTjett. 2020 Aug;594(16): 2518-2542; Forneris F, etal. EMBO J 2016, 35, 1133- 1149; and Qjha et al. Communications Biology 2, 290 (2019)).

[0445] CRP proteins are formed by tandemly repeating units called complement control protein (CCP) domains (also known as short consensus repeats or sushi domains) that can bind C3 convertases to mediate the inhibition of C3 convertases by decayaccelerating activity (DAA) and cofactor activity (CFA). CCP domains contain ~60 amino acids, with four invariant cysteines that pair (Cysl to Cys3 and Cys2 to Cys4) and 10-18 other highly conserved residues. For example, in human CRP (huRCA) proteins, the number of CCP domains range from 4 to 59. For example, the number of CCP modules in human CRP (or RCA) proteins vary from 4 to 59 (CD55ZDAF and CD46 / MCP, 4 CCPs; FH, 20 CCPs; C4BP, 59 CCPs; CD35 / CR1, 30 CCPs).

[0446] Deletion mutagenesis showed that a minimum of 3-4 successive CCP domains in CRP proteins contribute to complement regulation and cell protection from complement- mediated damage (Qjha et al. Communications Biology, 2: 290 (2019)). The location of the functionally important residues and of conserved functional sites and motifs on of CCP domains central to complement inhibition by DAA and CFA have been mapped for human CRP s and viral homologs (Agrawal P etal. FEES Lett. 2020 Aug; 594(16):2518- 2542; Qjha etal. Communications Biology, 2: 290 (2019)). The CCP domains that impart regulatory activities are known.

[0447] The structures of DAF / CD55, MCP / CD46, CR1 / CD35 bound to C3b and the structures of FH in complex with C3b and factor I are known. The structures show that all the CRP proteins bind in an extended orientation to C3b and share the same binding platform (Wu, J. et al. Nat. Immunol. 10, 728-733 (2009); Forneris, F. et al. EMBO J. 35, 1133-1149 (2016); Xue, X. et al. Nat. Struct. Mol. Biol. 24, 643-651 (2017)).

[0448] Other than DAA and CFA, CD59 (Protectin), blocks or inhibits the formation of the membrane attack complex (MAC or C5b-9) on host cell membranes. The MAC is formed by the self-assembly of complement proteins C5b, C6, C7, C8, and from 1 to 18 molecules of C9. Host cells are protected from MAC-mediated lysis by CD59, a 18-21- kDa glycophosphatidylinositol-linked membrane glycoprotein. The structural characteristics of CD59 and complement inhibiting mechanism is well-known. For example, it previously had been determined that CD59 functions by binding to the o- subunit of C8 in the C5b-8 complex and preventing the binding of C9, and / or by binding to C9 in the C5b-9 complex and preventing recruitment of additional C9 molecules. CD59 can only bind to C8a and C9 in the nascent complex after conformation rearrangements of the two proteins that occur during MAC assembly; CD59 binding sites map to residues 359-384 in C9, and to residues 364-415 in C8a. It previously had been determined that a 6-residue consensus sequence of VSLAFS in human C9, spanning residues 365-371 of C9, is the primary CD59 recognition domain involved in CD59- mediated regulation of MAC formation (Huang Y. et al. J Biol Chem. 2006 Sep 15;281(37):27398-404). Structures of two inhibited MAC precursors known as C5b8 and C5b9 previously have been determined (Couves etal. Nat. Comm. 14, 890 (2023)).

[0449] 8. Assays for measuring or assessing the complement regulating activity of portions

[0450] Assays to define functioning of CRP s and of anti-complement proteins are known and have been used to measure or access the contribution of CCP domains to the decay accelerating activity or cofactor activity of the complement pathways. Exemplary assays include Alternative pathway DAA assay (AP-DAA); classical pathway DAA assay (CP-DAA); cofactor activity assay (CFA) are known (see. e.g., Qjha et al. Communications Biology volume 2, Article number: 290 (2019) and Liszewski et al. JBC 215, 48, 1 2000, 37692-37701).

[0451] The anti-complement activity of CD59 can be measured by complement lysis assays. CHO cells (wild type or expressing CD59 or a fragment thereof) can be sensitized to complement lysis with rabbit anti-CHO membrane serum and incubated, e.g., at 37 °C for 60 min, and cell viability determined by Trypan Blue staining (live and dead cells were counted). Wild-type CHO cells served as negative control for CD59 complement inhibitory function. Complement inhibitory fragments retain cell viability, while cell death indicates the lack of complement protection (see, e.g., Huang et al. JBC 2006, 281, 37, 27398-27404).

[0452] 9. Exemplary of immune system regulating proteins are the complement regulatory proteins

[0453] Exemplary of such regulatory proteins are CD35, CD55, CD59, CD46, CR1, Factor H, VCP, MOPICE, SPICE and CCPH. Complement regulatory proteins (CRPs, also referred to as regulators of complement activation RCAs) of viral origin include: Astrovirus capsid protein (CaPt); Influenza virus matrix protein 1 (Ml); Flavivirus non- structural protein (NS1); Hepatitis C virus non-structural 3 / 4a protease (NS3 / 4A); Hepatitis C virus core protein (HCV-CP); Hepatitis C virus non-structural 5 A protein (NS5 A); Factor I mimetics of Nipah and Chikungunya viruses; Vaccinia virus complement control protein (VCP); Smallpox inhibitor of complement enzymes (SPICE); KSVH inhibitor of complement activation (Kaposcia / KCP); Murine y- herpesvirus 68 regulator of complement activation (y-HV68 RCA); Herpesvirus saimiri complement control protein homolog (HVS CCPH); Ectromelia virus inhibitor of complement enzymes (EMICE); Monkeypox inhibitor of complement enzymes (MOPICE); Rhesus rhadinovirus complement control protein H (RCP-H); Rhesus rhadinovirus complement control protein- 1 (RCP-1); Herpes simplex virus glycoprotein C-l (gCl); E protein; Zika virus E protein; Herpes virus saimiri CD59 homolog (HVS CD59); Hepatitis B virus X protein (HBx); and ORF4.

[0454] Poxviruses and herpesviruses encode homologs of the human regulators against C3 convertases. Viral CRP s possess four or fewer complement control protein (CCP) domains. Although resembling human DAF / CD55 and MCP / CD46 molecules, which have decay-accelerating activity (DAA) and cofactor activity (CFA), respectively, viral CRP s, demonstrate dual-activity and demonstrate DAA (primarily against C4b2a; Classical Pathway-DAA) and CFA (against C3b as well as C4b) mechanisms of complement inhibition. MOPICE is the exception which exhibits only CFA. Mechanistic studies of viral CRP proteins and C3b / C4b showed that: (a) largely all the four CCP domains participate in binding, (b) fast on- and off-rates and transient binding to target proteins, (c) low micromolar binding affinities, and (d) like human CRP s, ionic and long-range electrostatic interactions (Agrawal P et al. FEBS Lett. 2020 Aug;594(16):2518-2542). Structures of NUMEROUS proteins (CD46 / MCP, CD55 / DAF and CD35 / CR1) and a v CRP protein (SPICE) bound to complement C3b showed that hu CRP and v CRP proteins share a common complement inhibitory binding mechanism (Forneris F etal. EMBO J (2016) 35, 1133-1149).

[0455] CRP homologs, which inhibit C3 convertases, identified from poxviruses include, for example, the secreted protein vaccinia virus complement control protein (VCP); inflammatory modulatory protein (IMP) isolated form cowpox; smallpox inhibitor of complement enzymes (SPICE); monkeypox inhibitor of complement enzymes (MOPICE); and ectromelia virus inhibitor of complement enzymes (EMICE). Poxviral complement regulators possess high sequence similarity to each other; their sequence similarity exceeds 90%.

[0456] Poxvirus CRP proteins are soluble, but can anchor and protect the virus-infected cells. For example, SPICE, VCP and MOPICE bind to heparin sulfate proteoglycans, and VCP binds to VACV protein A56 / haemagglutinin that is exposed on the surface of infected cells thereby aiding in the EEV-mediated inhibition of complement. SPICE was shown to be 100-fold and 6-fold more potent than VCP in inactivating human C3b and C4b, respectively (Agrawal P et al. FEES Lett. 2020 Aug; 594(16):2518-2542).

[0457] Herpes viruses encode soluble and membrane-bound Complement regulatory proteins (CRPs). Functional homologs identified in herpes viruses include, for example, herpesvirus saimiri complement control protein homolog (HVS-CCPH); Kaposi’s sarcoma-associated herpesvirus (KSHV / HHV-8) RCP homolog named (Kaposcia / KCP); murine gamma herpesvirus 68 RCA (MHV68 RCA); and rhesus rhadinovirus RCP (RRV-RCP). Apart from the CRP homologs, the homolog of the cellular membrane glycoprotein CD59 (encoded by ORF 15) that targets the MAC was identified and characterized in herpesvirus saimiri. HVS-CD59 displays 64% and 48% nucleotide and amino acid sequence identity, respectively, to human CD59.

[0458] The structures of viral CRP homologs ARE n (Agrawal P et al. FEES Lett. 2020 Aug;594(16):2518-2542). In viral CRP (v CRP) homologs the number of CCP domains vary from 2 to 8. In poxviruses, the 4 CCP-containing CRP homologs reside in vaccinia virus, variola virus and cowpox virus (CPXV), whereas 3 CCP-containing homologs are observed in monkeypox (MPXV), yatapox, leporipox and deerpox viruses. Poxviral complement regulators are highly similar, having 90% sequence similarity. Most poxviral inhibitors of complement enzymes possess four CCP domains as do the human regulators MCP and DAF. Capripox and suipox have been shown to encode the homologs having 2 CCPs. 4 CCP-containing CRP homologs are observed in HVS, KSHV, MHV-68, Herpesvirus ateles, RRV isolate H26-95 (RCP-H) and wood mouse herpes viruses (WoHV), whereas 8 CCP-containing CRP is seen in RRV isolate 17577 (RCP-1).

[0459] 10. EEV outer membrane proteins

[0460] Provided are methods of producing complement resistant (or other humoral immune responses) EEV viruses that have increased resistance to complement independent from host CRP expression level of the infected cell or tumor. Stable expression of CRP s on the EEV can be achieved by engineering or modifying an EEV outer membrane protein encoded by the virus to express as a fusion protein a regulatory protein (CRP or other humoral modulatory protein) or functional portion thereof covalently linked to an EEV outer membrane protein. Complement proteins are target for increasing serum resistance. Figure 35 summarizes complement pathways and exemplary complement proteins. Complement proteins can be targets for inhibiting complement. Complement inhibiting proteins, such as CDS 5, are known in the art. As detailed herein, fusion proteins (chimeric proteins) between viral EEV membrane proteins and proteins that inhibit complement can be displayed in the EEV membrane to inhibit complement and thereby reduce or eliminate serum inactivation of EEVs. As detailed elsewhere herein, the cells in which the virus is produced also can be modified to express complement inhibitors or other humoral immunity modulating (inhibiting) proteins in the cell membranes for incorporation into the viruses.

[0461] Six vaccinia virus genes encode EEV outer membrane proteins, e.g., F13L, A56R, B5R, A33R, A34R and A36R A33R, A34R, A56R, and B5R are exposed to the outside of the CEVZEEV outer membrane while F 13L is located between the EEV outer envelope and the IMV surface. A33R, A34R, A56R and B5R are glycoproteins, A36R is a non-glycosylated transmembrane protein, and F13L is a palmitoylated peripheral membrane protein. The proteins encoded by F13L, A33R, A34R, A56R and B5R are present in IEV, CEV and EEV particles and approximately one-third of EEV particles lack A56R (Krauss O. etal. J. Gen. Virol. (2002) 83, 2347-2359). The EEV outer membrane proteins B5R, A33R, A56R and F13L are targeted by anti-viral neutralizing antibodies.

[0462] B5R

[0463] The B5R gene of vaccinia virus contains an open reading frame (ORF) which encodes an EEV-membrane bound a 42-kDa glycoprotein that is essential for EEV formation. For example, the vaccinia virus strain IHD-W B5R protein sequence is set forth (Gen Bank protein_id= AAN78219.1 (SEQ ID NO: 481):

[0464] The extracellular portion of B5R comprises four short consensus repeat (SCRs 1- 4; SCR 1, amino acids 20-72; SCR2, amino acids 76-125; SCR3, amino acids 130-182; SCR4, amino acids 186-237) domains that are present in complement regulatory proteins.

[0465] The B5R protein further comprises a signal peptide (SP, amino acids 1-20; SEQ ID NO: 232), a stalk region (STALK, amino acids 237-275; SEQ ID NO: 233), a transmembrane region (TM, amino acids 275-303; SEQ ID NO: 234), and a cytoplasmic tail (CT, amino acids 303-317; SEQ ID NO: 235). B5R is N-terminus is extracellular exposed outside of CEV / EEV, while the C-terminus is located between the EEV outer envelope and the IMV surface.

[0466] The B5R protein is related to the RCA protein family and contains four copies of the complement control protein module (CCP) and shows sequence homology to Vaccinia virus complement control protein (VCP). Deletion of B5R leads to small plaque size, normal IMV formation, few IEV and CEV formation, results in 5-10-fold less EEV formation and attenuated virulence in vivo (Blasco, R. and Moss, B., J. Virol. 65, 5910- 5920 (1991); Engelstad, M. and Smith G.L., Virology, 194, 627-627 (1993)).

[0467] Sequences within the transmembrane and cytoplasmic tail of B5R are important for targeting the protein and wrapping membrane (Sanderson et al., J. Gen. Virol. 79 (6), 1415- 1425 (1998); Mathew et al., J. Virol. 72, 2429-2438 (1998)). Mutant vaccinia virus with deletion in SCR4; SCIO, 4; or SCR-2,3,4 generate small comet-shaped plaques, but produce ~50-fold more EEV virions than wild-type virus (Sanderson el al., J. Gen. Virol. 79 (6), 1415- 1425 (1998); Mathew et al., J. Virol. 72, 2429-2438 (1998)). Deletions in the SCR3 and SCR4 domains can partially disrupt binding of neutralizing antibodies to B5R. SCR3 can contain a P189S mutation that can result in increased EEV release. As stated above, B5R mutations can be combined with mutations in A33R, A34R, A56R and B5R to enhance EEV production, see e.g., US 2023 / 0002740; WO 2019 / 089755 Al. B5R mutations that remove binding sites for neutralizing antibodies, such as removal of antibody binding site B5R SCR1, can be combined with other EEV enhancing mutations, such as mutations in A34R. B5R can be expressed as a truncated polypeptide. B5R can be expressed without short consensus repeat (SCR) domains SCR2, SCR3, and SCR4 (B5R S-STC) or expressed without four short consensus repeat (SCR) domains or modified as described in WO 2020 / 074902.

[0468] A nucleic acid molecule encoding B5R or a modified B5R can encode an CRP or functional portion thereof as a fusion protein. B5R or modifications thereof can be linked to an CRP, e.g., CD55, without the signal peptide, by a linker to express the CRP on the surface membrane on EEV vaccinia enveloped virus.

[0469] A33R

[0470] A33R is a 20.5kDa glycosylated EEV outer membrane protein that is exposed on the outside of the EEV outer membrane. Exemplary of A33R protein sequence is that of

[0471] Western Reserve (NCBI: YP_233038.1; SEQ ID NO: 482):

[0472] MMTPENDEEQTSVFSATVYGDKIQGKNKRKRVIGLCIRISMVISLLSMIT

[0473] MSAFLIVRLNQCMSANEAAITDAAVAVAAASSTHRKVASSTTQYDHKES CNGLYYQGSCYILHSDYQLFSDAKANCTAESSTLPNKSDVLITWLIDYVE DTWGSDGNPITKTTSDYQDSDVSQEVRKYFCVKTMN

[0474] A33R is involved in actin tail formation during the budding of EEV from the host cell membrane. A36R is a key protein for induction of actin tails; A33R protein is required for normal plaque formation, actin tails, and specialized virus-tipped microvilli (Roper

[0475] RL. et al. Journal of Virology, 1998, 72(5):4192-4204).

[0476] A56R

[0477] The A56R protein is the vaccinia virus hemagglutinin and is a standard type I integral membrane protein comprising an amino-terminal extracellular (“extramembrane”) domain, a single transmembrane domain, and a cytoplasmic (“intramembrane”) domain. A56R contains an N-terminal signal peptide of about 33 amino acids, an Ig-like domain extending from about amino acid 34 to about amino acid 103, a stalk region extending from about amino acid 121 to about amino acid 275, a transmembrane domain extending from about amino acid 276 to about amino acid 303, and an cytoplasmic (“inter-membrane”) domain extending from about amino acid 304 to amino acid 314 (DeHaven et al., J. Gen Virol. 92: 1971-1980 (2011). Exemplary of A56R protein sequence is that of Western Reserve (NCBI: YP 233063.1; SEQ ID NO:483):

[0478] MTRLPILLLLTSLVYATPFPQTSKKIGDDATLSCNRNNTNDYWMSAWY KEPNSIILLAAKSDVLYFDNYTKDKISYDSPYDDLVTTITIKSLTARDAGT YVCAFFMTSTTNDTDKVDYEEYSTELIVNTDSESTIDIILSGSTHSPETSSK KPDYIDNSNCSSVFEIATPEPITDNVEDHTDTVTYTSDSINTVSASSGESTT DETPEPITDKEDHTVTDTVSYTTVSTSSGIVTTKSTTDDADLYDTYNDND TVPPTTVGGSTTSISNYKTKDFVEIFGITALIILSAVAIFCITYYIYNKRSRK YKTENKV

[0479] In summary, the A56R domains are as follows: amino acids 1-33 N-terminal domain; 34-103 Ig-like domain; 121-275 stalk domain; 276-303) transmembrane domain; and 304-314) cytoplasmic domain.

[0480] A56R can be modified to express single chain antibodies or display membrane proteins as target antigens for screening libraries of binding molecules, e.g., antibody display libraries see e.g., US 2005 / 0208074 Al; Galmiche etal. J. Gen. Virol., 1997, 78, 3019-3027; US 2021 / 0348158 Al; US 2019 / 0112388 Al; US20130288927A; US 2013 / 0288927 Al. A56R can bind or localize secreted VCP to protect EEV from complement (DeHaven et al., J. Gen Virol. 92:1971-1980 (2011)).

[0481] F13L

[0482] The F13L protein is associated with the interior surface of the outermost EEV membrane through palmitoylation of cysteines 185 and 186 (Smith, Trends in Microbiol. 16:472-479 (2008)). Vaccinia viruses in which the gene encoding F13L is deleted produce tiny plaques and the number of EEV produced is reduced significantly. Since F13L does not cross the membrane, it does not have a transmembrane domain or signal peptide. Because of the orientation F13L the interior surface of the outermost EEV membrane F13L is suited for the expression of membrane proteins such as integral membrane proteins in a conformationally intact state / native conformation such as multipass integral membrane proteins as F13L fusion proteins see e.g., US 2019 / 0112388 Al. Exemplary of F13L protein sequences is that of Western Reserve (NCBI YP 232934.1; SEQ ID NO:484):

[0483] MWPFASVPAGAKCRLVETLPENMDFRSDHLTTFECFNEIITLAKKYIYIA SFCCNPLSTTRGALIFDKLKEASEKGIKIIVLLDERGKRNLGELQSHCPDIN FITVNIDKKNNVGLLLGCFWVSDDERCYVGNASFTGGSIHTIKTLGVYSD YPPLATDLRRRFDTFKAFNSAKNSWLNLCSAACCLPVSTAYHIKNPIGGV FFTDSPEHLLGYSRDLDTDWIDKLKSAKTSIDIEHLAIVPTTRVDGNSYY WPDIYNSIIEAAINRGVKIRLLVGNWDKNDVYSMATARSLDALCVQNDL SVKVFTIQNNTKLLIVDDEYVHITSANFDGTHYQNHGFVSFNSIDKQLVS EAKKIFERDWVSSHSKSLKI

[0484] Host Protein

[0485] Host proteins can be incorporated into the EEV outer membrane. Such proteins are not restricted to complement regulators, and can include CD71, CD81 and major histocompatibility class I (MHC-I) SEQ ID NOs: 226-231 and host cell membrane proteins. Other such proteins are apparent to those of skill in the art. Any protein that will result in a decrease in the host response to systemically administered virus can be displayed on the EEV membrane to modulate or reduce or inhibit the response of the immune system of the host so that systemically administered and disseminated virus is not inhibited or eliminated.

[0486] F. ANY POXVIRUSES CAN BE MODIFIED AS DESCRIBED HEREIN

[0487] The skilled person understands that any poxviruses can be modified as described herein so that it can be administered systemically. For purposes herein the poxviruses are exemplified by vaccinia viruses. To effect such modification, an EEV virus is chosen for modification, or is produced as described herein by selection or mutation, or any other method known to those of skill in the art. All therapeutic poxviruses, particularly vaccinia viruses that are known or that have been developed or that will be developed can be modified to express host immune system resisting proteins. Any virus is modified to be EEV, or is a known EEV virus, such that it produces more than 1%, generally at least 10% EEVs upon propagation. The genome is modified to encode and express on the EEV membrane a chimeric transmembrane protein that includes a protein or portion thereof that inhibits or increases resistance of the virus to the immune system of the host. A skilled person with the knowledge of the disclosure herein regarding the transmembrane chimeric proteins can so-modify any poxvirus, and particularly, any therapeutic vaccinia virus.

[0488] 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-NR52, IHD-J, IHD-W, Brighton, Ankara, Modified Vaccinia Ankara (MV A), Dairen I, LIPV, LC16M8, LC16M0, LIVP, WR 65-16, EM63, Bern, Paris, CVA382, NYVAC, ACAM2000, ACAM1000, CVA382, JX-594, GL-ONC1, wDD TK mutant, GL-ONC1, wDD TK mutant, VET2-L2 and Connaught strains. Discussed below are other known therapeutic viruses that can be so-modified.

[0489] Vaccinia is a cytoplasmic virus; 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. 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.

[0490] 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. Toxicides 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 etal. (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 etal. (2007) Cancer Res. 67:10038-10046; Yu et al. (2004) Nat. Biotech. 22:313-320; Heo etal. (2011)AfoZ. Ther. 19:1170-1179; Liu etal. (2008) Mol. Ther. 16:1637-1642; Park et al. (2008) Lancet Oncol. 9:533-542). Vaccinia virus and engineered strains thereof have been extensively described (see, e.g., U.S. Patent Nos. 4,603,112; 4,722,848; 4,769,330; 5,110,587; 5,338,683; 5,378,457; 5,482,713; 5,505,941; 5,583,028; 5,972,597; 5,997,878; 6,267,965; 6,340,462; 6,723,325; 6,998,252; 7,015,024; 7,045,136; 7,045,313; 7,115,270; 7,645,456; 7,767,449; and 10,238,700).

[0491] 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.

[0492] Various vaccinia viruses have been demonstrated to exhibit antitumor activities.

[0493] 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 in clinical trial of melanoma patients. As a control, a New York City Board of Health strain vaccinia virus (W) 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 W control (Kim et al. (2001) Surgical Oncol. 10:53-59). LIVP strains of vaccinia vims 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 et al. (2007) Surgery 142:976-983; Lin etal. (2008) J. Clin. Endocrinol. Metab. 93:4403-7; Kelly et al. (2008) Hum. Gene Ther. 19:774-782; Yu etal. (2009) A / oZ. Cancer Ther. 8:141-151; Yu et al. (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 and its production are described, for example, in U.S. Pat. Nos. 7,588,767, 7,588,771, 7,662,398 and 7,754,22. LIVP strains of vaccinia, including attenuated forms thereof, exhibit less toxicity than WR strains of vaccinia vims, and result in increased and longer survival of treated tumorbearing animal models (see, e.g., U.S. Publication No. 2011 / 0293527). Wyeth strains of vaccinia vims, such as JX-594, also exhibit lower toxicity, and have been used for the treatment of cancers.

[0494] Companies and institutions that have developed viruses include, but are not limited to, Kalivir, IGNITE, Sillajen, Transgene, VacVBTX, KM Biologies, Kolon BioScience, NIH, StratosVir, Viromissile, Imugen, Turnstone, Bionoxx, Anovac / Icellkealex, AstraZeneca, Genelux, and Vac Biotherapeutics. Such viruses are described publications, including patents and applications, discussed below. Any of these vaccinia viruses can be improved by the modifications as described herein to encode and express a chimeric EEV transmembrane protein-immune resistance or immune inhibiting protein as described herein. Sequences thereof include the viruses of SEQ ID NOs: 616- 627.

[0495] 1. Methods for modifying vaccinia viruses

[0496] Methods of modifying the vaccinia virus genome are well-known (see e.g., U.S.

[0497] Patent No. 8,329,164; 10,584,317; 11,655,455; U.S. Patent App. No. 2022 / 0049228). Methods for production of modified vaccinia strains by genetic engineering are well- known (see, e.g., Moss, Curr. Opin. Genet. Dev. 3 (1993), 86-90; Broder etal., Mol. Biotechnol. 13 (1999), 223-245, and many others). Methods for engineering oncolytic viruses include, but are not limited to:

[0498] (1) Homologous recombination requires the use of a donor vector, containing transgene flanked by two DNA areas homologous to the recipient viral DNA in the location where the transgene is to be inserted (Kaufman, H.L., F.J. Kohlhapp, and A. Zloza, Oncolytic viruses: a new class of immunotherapy drugs. Nat Rev DrugDiscov, (2015) 14(9):642-62). Recombinant viruses can be then selected by several approaches, including TK-positive / negative, beta-galactosidase, dominant selective markers such as green fluorescent protein (GFP or eGFP), blue fluorescent protein (BFP) or TuiboFP635, or with transient dominant selection (IDS) with phosphoribosyltransferase (gpt).

[0499] The CRE / lox system, derived from Pl bacteriophage, is a site-specific recombinase technology used to cany out deletions, insertions, translocations, and inversions at specific sites in the DNA of cells (Kleinstiver, B.P., etal., High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529(7587): 490-495 (2016)). This tool works both in eukaryotic and prokaryotic organisms and has been well established, creating a variety of transgenic animal models (Ran et al., Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell, 2013. 154(6): p. 1380-9). This system is based on a site-specific Cre recombinase (~1 kb) that requires a 34 bp specific loxP sequences which are easy to incorporate into any target DNA. One of the advantages of the Cre / lox recombination system is that there is no need for additional cofactors or sequence elements for efficient recombination regardless of the cellular environment (Schaefer, K.A., etal., Unexpected mutations after CRISPR-Cas9 editing in vivo. Nat Methods, 2017. 14(6): p. 547-548). Analysis showed that the mutations of loxP sequence such as m2, m3, m7, mil, lox5171 recombine readily with themselves but have a markedly low recombination with the wild-type site. Therefore, those sequences can be used for gene insertion via recombinase-mediated cassette exchange (RMCE) with high efficiency fidelity and in a site-specific manner (Oberstein, A., etal., Site-specific transgenesis by Cre-mediated recombination in Drosophila. Nat Methods, (2005) 2(8):583-5). Nakano and colleagues demonstrated the RMCE can be used to produce adenovirus vectors by the replacement of a specific gene in the replicating adenovirus genome with a gene of interest using a nuclear Cre recombinase and incompatible loxP and loxP 2272 system (Kuhn, R. and R.M. Torres, Cre / loxP recombination system and gene targeting. Methods Mol Biol, 2002. 180: pp. 175-204).

[0500] (2) CRISPR / Cas9 has been used to generate recombinant vaccinia viruses. CRISPR / Cas9 has recently emerged as a method to edit genomes from various organisms due to the ability of Cas9 protease to cut at a defined site in DNA genomes marked by a signal guide RNA (Wyatt, L.S., P.L. Earl, and B. Moss, Generation of Recombinant 1982 Viruses. CurrProtoc Mol Biol, 2017. 117: p. 16 17 1-16 17 18). Introduction of a double-strand break by Cas9 in the target DNA facilitates insertion of the desired gene. Yuan and colleagues showed improved efficiency in the generation of recombinant virus (more than 50 times) by using CRISPR / cas9 system as compared to with homologous recombination approach (Falkner, F.G. and B. Moss, Transient dominant selection of recombinant vaccinia viruses. J Virol, 1990. 64(6): p. 3108-11). To overcome off-target- induced mutations in mammalian cells, several mutations in Cas9 nuclease have been introduced: N497A, R661A, Q695A, and Q926A (Cas9 high fidelity) resulting in a more precise cut (Mali, P., K.M. Esvelt, and G.M. Church, Cas9 as a versatile tool for engineering biology. Nat Methods, 2013. 10(10): p. 957-63) or D10A mutated Cas9, which provides a single-stranded break (Yuan, M., et al., A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors. J Vis Exp, 2016(116)).

[0501] A nucleic acid molecule can be inserted into vaccinia virus, under control of a transcriptional control region. Vaccinia virus transcriptional control regions can comprise a promoter and a transcriptional terminational signal. Gene expression in vaccinia virus can be temporally regulated, and promoters for early, intermediate, and late genes poses varying structures. Certain viral genes are expressed constitutively, and promoters for these “early-late” genes bear hybrid structures. Synthetic early-late promoters are also known (Hammond J. M., et al., J. Virol. Methods 66: 135-8 (1997); Chakrabarti S., et al., Biotechniques 23:1094-7 (1997). The viruses provided herein can contain one or more nucleic acid molecules inserted into the genome of the virus. A nucleic acid molecule can contain an open reading frame or can be a non-coding sequence. The nucleic acid can replace all or a portion of an endogenous viral gene. The viral gene can be replaced with homologous gene or a heterologous gene.

[0502] 2. Therapeutic, transgenic, and attenuated vaccinia viruses 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 strain can be used for designing and generating modified viruses in other strains. Techniques for production of modified vaccinia strains by genetic engineering are well- established (Moss (1993) CMTT. Opin. Genet. Dev. 3:86-90; Broder and Earl, (1999) Mol. Biotechnol. 13:223-245; Timiryasova et al. (2001) Biotechniques 31:534-540).

[0503] Therapeutic or transgenic virus can be modified or engineered to express a myriad of payloads, therapeutic proteins and / or combinations / multiple transgenes. Recombinant vaccinia virus is known for its safety, potential for systemic delivery and capacity for transgene insertion and has been used as a platform to express heterologous genes, such as therapeutic genes. Exemplary recombinant therapeutic vaccinia viruses that have been administered to humans and that are known in the art include, for example: JX-594, GLV-lh68, TroVax, TG4010, PROSTVAC, PANVAC, MVA-5T4, rV-B7.1, IN rW, and W-IL-2.

[0504] The therapeutic genes generally can be inserted in the virus genome in a manner that disrupts the expression of one or more viral genes, thereby attenuating the virus and improving tumor selectivity. Vaccinia viruses can be modified by insertion of heterologous genes into the genome to improve tumor selectivity, reduce toxicity, immunogenicity, and to express heterologous genes. This can result in the attenuation of the virus and / or permit delivery of therapeutic proteins to tumors. Viruses for use in the methods provided herein include those well-known to those skilled in the art and include, for example, those described in: 7,354,591; 8,329,164; 8,986,674; 10,584,317; 11,452,770; 11,529,402; 11,655,455; 11,685,904; US 2019 / 0218522 Al; US 2019 / 0209629 Al; US 2020 / 0392535 Al; US 2021 / 0388388 Al; US 2022 / 0049228 Al; US 2023 / 0002740 Al; US 2023 / 0201283 Al; WO 2013 / 038066 Al; WO 2019 / 089755 Al; WO 2020 / 074902 Al; WO 2020 / 086423 Al; WO 2022 / 182206 Al. Vaccinia viruses can be engineered or modified to contain one or more genomic deletions to increase tumor selectivity and / or to reduce toxicity to non-tumor cells (attenuation) and / or reduce immunogenicity of a vaccina vims compared to an unmodified vims. Attenuated vimses can be safer for systemic administration in addition to being vehicles for carrying exogenous genes, such as therapeutic genes. For example, the viruses can be modified or engineered for conditional replication such that the virus selectively replicates in a proliferative cell, such as a cancer cell, relative to a normal non-cancer cell.

[0505] Examples of such modifications include: modification that renders the virus deficient in the function of vaccinia growth factor (VGF) (McCart et al. (2001) Cancer Research 61 :8751); modification to the vaccinia virus TK gene to render the virus TK deficient, or modifications to the hemagglutinin (HA) gene, or F3 gene or an interrupted F3 locus (WO 2005 / 047458 A2); modification that renders the vaccinia virus deficient in the function of VGF and OIL (WO 2015 / 076422 Al); insertion of a micro RNA whose expression is decreased in cancer cells into the 3' noncoding region of the B5R gene (WO 2011 / 125469 Al); modifications that render the vaccinia virus deficient in the function of B 18R (Kim et al. (2007) PLoS Medicine 4:e353), ribonucleotide reductase (Gammon etal. (2010) PLoS Pathogens 6:el000984), serine protease inhibitor (e.g., SPI- 1, SPI-2) (Guo etal. (2005) Cancer Research 65:9991), SPI-1 and SPI-2 (Yang etal. (2007) Gene Therapy 14:638), ribonucleotide reductase genes F4L or I4L (Child et al. (1990) Virology 174:625; Potts et al. (2017) EMBOMol. Med. 9:638), B18R (B19R in Copenhagen strain) (Symons etal. (1995) Cell 81:551), A48R (Hughes etal. (1991) J. Biol. Chem. 266:20103); B8R (Verardi etal. (2001) J. Virol. 75:11), B15R (B16Rin Copenhagen strain) (Spriggs et al. (1992) Cell 71 : 145), A41R (Ng et al. (2001) Journal of General Virology 82:2095), A52R (Bowie et al. (2000) Proc. Natl. Acad Sci. USA 97: 10162), F1L (Gertie et al. (2013) Proc. Natl. Acad Sci. USA 110:7808), E3L (Chang etal. (\991)Proc. Natl. Acad Set. USA 89:4825), A44R-A46R (Bowie etal. (2000) Proc. Natl. Acad Sci. USA 97: 10162), K1L (Bravo Cruz et al. (2017) Journal of Virology 91:e00524), A48R, B18R, C11R, and TK (Mejias-Perez etal. (20VT) Molecular Therapy: Oncolytics 8:27), E3L and K3L regions (WO 2005 / 007824 A2), or OIL (Schweneker et al. (2012) J. Virol. 86:2323). Moreover, a recombinant vaccinia vims can comprise a modification that renders the vaccinia vims deficient in the extracellular region of B5R (Bell et al. (2004) Virology 325:425), deficient in the A34R region (Thirunavukarasu et al. (2013) Molecular Therapy 21 :1024), or deficient in interleukin- 1P (IL-1 P) receptor (WO 2005 / 030971 Al).

[0506] Engineered or attenuated vaccinia viruses have been identified that exhibit one or more of improved oncolytic viral activity, replication in tumors, infectivity, immune evasion, tumor persistence, capacity for incorporation of exogenous DNA sequences, and amenability for large scale manufacturing when the viruses are engineered to comprise deletions in one or more of the following genes: C2L, C1L, NIL, N2L, MIL, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, B14R, B15R, B16R, B17L, B18R, B19R, B20R, K ORF A, K ORF B, B ORF E, B ORF F, B ORF G, B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R, and B29R, see, e.g., WO 2019 / 134049 Al.

[0507] 3. Exemplary Therapeutic Vaccinia Virus

[0508] Factors that promote virus-specific replication in cancers can include for example, EGFR / RAS pathway activity and tumor thymidine kinase (TK) levels. Thymidine kinase (TK) encoded by the early vaccinia virus (J2R) gene is a key virulence factor and enzyme for the synthesis of vaccinia virus DNA. Deletion in the J2R gene enhances tumor-selective viral replication and decreased virulence (attenuation) of recombinant vaccina viruses (Yakubitskiy e / al. (2015) Acta Naturae 7(4): 113-121). Replication of vaccinia viruses lacking TK are restricted to cells with elevated levels of thymidine kinase, such as in proliferating cells such as tumor cells. Exemplary sequences of the TK gene include but are not limited to GenBank: AAR17937.1 or AY313847.1. The J2R region can also be modified by inserting a heterologous nucleic acid into the deleted loci thereby resulting in reduced vaccinia virus TK expression or activity and transgene expression. Such recombinant or transgenic viruses include, but are not limited to, JX-594 (Pexastimogene Devacirepvec, Pexa-Vec); LIVP GLV-lh68 (GLV-ONC1 or GL-ONC1); wDD; TG6002; VG9-GM-CSF; CW; deW5; CF33; Guang9; INrW; T601; vA34R; aCEA TCE; a modified WR.TK-GMCSF vaccinia virus;

[0509] WR.B5Rmut.1K-; mCCR5 / TK- virus; mCXCR4 / TK- virus; TK- PH20 DCK virus and KLS-3010 and those described in: 8,980,246; US 2019 / 0218522 Al; WO 2022 / 182206 Al; WO 2023 / 118603.

[0510] Vaccinia vims uses the epidermal growth factor (EGF) receptor signaling pathway to promote the spread of the vims through rapid and direct motility of infected cells. Cl 1R protein, a vaccinia virus growth factor (VGF) that has high homology to EGF is secreted at an early stage of vaccinia virus infection. VGF binds to an EGF receptor on infected and surrounding cells to initiate signal transduction through the MAP kinase cascade (Ras / RafZMEK / ERK metabolic pathway). VGF deletions have been shown to restrict viral replication to cells with mutations in the Ras / MAPK / ERK pathway, to enhance tumor selective infection. For example, vaccinia viruses with deletions in the genes J2R, VGF and Cl 1R (KLS-3010) show selective replication in cancer cells and reduced pathogenicity compared with the wild-type virus (see, e.g., Shin etal. Hum Gene Ther. 202; 32(9-10): 517-527; and 11,452,770).

[0511] VGF deletion can be further combined with additional deletions in the vaccinia virus genome and / or combined with the expression with one or more heterologous genes. In for example, the OIL protein of vaccinia virus constitutively activates extracellular signal-regulated kinase (ERK) in infected cells and promotes the pathogenicity of the virus. A Mitogen-activated protein kinase-Dependent Recombinant Vaccinia Virus (MD- RW) is an oncolytic vaccinia virus in which both genes encoding VGF and OIL protein are deleted to restrict growth in normal cells and to specifically proliferate in cancer cells with abnormally activated ERK pathways or combined with attenuation of ribonucleotide reductase to enhance tumor selectivity see, e.g., WO2015 / 076422A1; US2022 / 0275347A1; US2023 / 0086531A1; W02013 / 038066A1.

[0512] LIVP GLV-lh68 (also designated GLV-0NC1) is an LIVP virus that contains ruc-gfp (a luciferase and green fluorescent protein fusion gene (see e.g., U.S. Pat. No. 5,976,796), beta-galactosidase (Lac Z) and beta-glucuronidase (gusA) reporter genes inserted into the F14.5L, J2R (thymidine kinase) and A56R (hemagglutinin) loci, respectively. The genome of GLV-lh68 has a sequence of nucleotides set forth in SEQ ID NO: 485 (GenBank Acc. No. EU410304) or a sequence of nucleotides that has at least 97%, 98% or 99% sequence identity to the sequence of nucleotides set forth in SEQ ID NO: 485. Recombinant derivatives of GLV-lh.68 and GLV-lh64 (set forth in SEQID NOs: 42-53 and 122 exhibit tumor targeting properties and an improved safety profile compared to its parental LIVP strain (set forth in SEQ ID NO: 113) and the WR strain (Zhang etal. (2009) Mol. Genet. Genomics, 282:417-435). References that describe engineered therapeutic vaccinia virus include those described in 7,588,767; 8,221,769; 8,021,662; 7,754,221; 7,662,398; 8,221,769; 7,588,771; 8,784,836; 8,323,959; 9,492,534; 10,463,730; 10,584,317. The patents show that tumors treated with vaccinia viruses (GLV-lh68 virus and derivatives thereof), results in tumor-specific vims replication, which can lead to tumor antigen release and viral protein production in the tumors.

[0513] Virally Encoded Immunomodulators and Effects thereof

[0514] Viruses, including vaccinia virus (VACV), encode several host range immunomodulators that block the initial anti-viral response in the tumor microenvironment, and protect infected cells against neutralization by complement and natural killer (NK) cells. Genes encoding these immunomodulators can be modified to modulate the response of the immune system of the host to the virus and the effects of the virus on the host.

[0515] These immunomodulators can be intracellular (non-secreted) or extracellular (secreted). For example, infected cells secrete proteins that bind to and dismpt the function of complement, interferons (IFNs), cytokines and chemokines, and interfere with semaphorin signaling. Secreted (extracellular) immunomodulators can prevent the interaction between chemokines and their host receptors on leukocytes, interfering with the migration of leukocytes into areas of infection and inflammation. Extracellular immunomodulators also can counteract the proinflammatory cytokine-induced antiviral state, for example, by disrupting TNF-alpha induced apoptosis in virus-infected cells. Intracellular immunomodulators inhibit apoptosis, modulate the antiviral effects of IFNs, and interfere with innate immune signaling and host gene transcription. For example, intracellular immunomodulators inhibit signaling pathways that lead to the production of interferons and proinflammatory chemokines and cytokines (Bahar et al. (2011) J. Struct. Biol. 175(2-2): 127-134; Smith et al. (2013) Journal of General Virology 94'2361 -2392). Different vaccinia strains encode different immunomodulators; thus, each viral strain interacts differently with host cells. The viruses herein can be genetically...

Claims

WHAT IS CLAIMED:

1. An extra enveloped vaccinia virus (EEV) particle that has higher antitumor activity and higher EEV production than the virus IHD-W, wherein the EEV is a clone of the polyclonal vaccinia IHD strain NR-52.

2. The EEV of claim 1, wherein the EEV virus genome has an intact A56 locus, whereby the virus has increased serum stability compared to the polyclonal IHD strain.

3. An extra enveloped vaccinia virus (EEV) 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; 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 SNP in A45R.

4. The EEV particle of claim 1 that differs from IHD-W as shown in Figure26.

5. The EEV virus of any of claims 1-4 that is the virus 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 1-4.

6. The EEV of any of claims 1-5 that is RT-00 or a derivative virus thereof.

7. The EEV particle of claim 1 or claim 2 that further comprises knockouts of one or more genes selected from among A46R, B8R, J2R, A52R, F1L, VGF, TK, and B19R; wherein the knockouts are effected by gene deletions, insertions, or disruptions to render encoded products inactive or not produced.

8. An extra enveloped vaccinia virus (EEV) particle that has higher antitumor activity and EEV production than the virus IHD-W, wherein the EEV is a clone ofthe 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.

9. An EEV particle any of claims 1-8, further comprising 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.

10. An EEV particle of any of claims 1-9 whose genome comprises an intactA56 gene.

11. A vaccinia virus preparation, comprising, upon propagation, EEV particles of any of claims 1-10, wherein the EEV particles comprise more than 1%, 5%, 10%, 15%, 20%, 25%, 30% or more of the virus population.

12. A vaccinia virus genome or a vaccinia virus comprising the genome, wherein the virus genome comprises the sequence set forth in any of SEQ ID NOs: 1-21, or a variant of any of SEQ ID NOs: 1, and 782-790 or a virus 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 portion of the ITRs or comprise heterologous ITRs, or degenerate sequences of any of the preceding sequences.

13. The vaccinia virus genome or vaccinia virus that comprises the genome, wherein the virus is 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% sequence identity thereto.

14. A derivative virus of the vaccinia virus genome or vaccinia virus of claim 13 that 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.

15. The derivative virus or virus genome of claim 14, wherein the chimeric protein comprises a transmembrane protein or a fusion protein that displays the fused protein on the second membrane.

16. The derivative virus or virus genome of claim 15, wherein the transmembrane protein or fusion protein is encoded by the virus.

17. The derivative virus or virus genome of claim 15, wherein the transmembrane protein is selected from among A33R, A34R, A56R, B5R, and F13L.

18. The vaccinia virus genome or vaccinia virus or derivative virus thereof of any of claims 1-17 selected from among viruses designated 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; or ii) variants thereof 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.

19. The vaccinia virus genome or vaccinia virus or derivative virus thereof of any of claims 1-18, 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 and retaining high EEV level and displaying a complement resistance protein or portion thereof, whereby the virus is substantially asresistant 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.

20. The vaccinia virus genome or vaccinia virus or derivative virus thereof of any of claims 1-19, 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%, 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.

21. A fusion protein or nucleic acid molecule encoding the fusion protein, wherein the fusion protein is encoded by a virus or variant thereof set forth in claim 20 or is a fusion protein comprising an 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.

22. The fusion protein or nucleic acid molecule of claim 21, 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, CD55, CD59, CD46, CR1, Factor H, VCP, MOPICE, SPICE, COPY, ORF4, CCPH, C4- binding protein, CD35, Kaposi-sarcoma associated herpesvirus Kaposica I KCP, Herpesvirus saimiri (HVS) and 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, among one or more of: CD35 CD55, VCP, mutated VCP, SPICE, CCPH and ORF4, and functional portions thereof.

23. The fusion protein or nucleic acid molecule of claim 21 or claim 22, wherein the EEV transmembrane protein is selected from among from among A33R, A34R, A56R, B5R, and F13L.

24. The derivative virus, or virus genome, fusion protein or nucleic acid molecule of any of claims 14-23, wherein the complement regulatory protein comprises the sequence of amino acids (excluding the signal sequence) set forth in any of SEQ IDNOs.: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.

25. The derivative virus, or virus genome, fusion protein or nucleic acid molecule of any of claims 1-24, comprising or encoding 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.

26. The derivative virus, or virus genome, fusion protein, or nucleic acid molecule of claim 25, 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.

27. The derivative virus, or virus genome, fusion protein, or nucleic acid molecule of claim 25 or claim 26, 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.

28. A vaccinia virus genome or vaccinia virus comprising the genome or a poxvirus or genome thereof, comprising nucleic acid encoding a chimeric or fusion 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.

29. The vaccinia virus genome or vaccinia virus or poxvirus comprising the genome, comprising nucleic acid of any of claims 21-28.

30. The vaccinia virus genome or vaccinia virus or poxvirus comprising the genome of claim 28 or claim 29, wherein: the vaccinia virus or poxvirus 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.

31. The vaccinia virus genome, vaccinia virus, derivative virus, fusion protein, or nucleic acid molecule of any of claims 21-30, wherein: the virus genome comprises a mutation(s) that renders the virus a high EEV producer; and a high level is higher than that produced by the WR strain.

32. The vaccinia virus genome, vaccinia virus, derivative virus, fusion protein, or nucleic acid molecule, of any of claims 1-31, 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.

33. The vaccinia virus genome, vaccinia virus, derivative virus, fusion protein, or nucleic acid molecule of any of claims 1-32, wherein the virus or 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.

34. The derivative virus, or virus genome, fusion protein, or nucleic acid molecule of any of claims 1-33, wherein: 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-; knockouts are effected by gene deletions, insertions, or disruptions to render encoded products inactive or not produced; andEEV production is increased compared to the virus that does not comprise the knockout or knockouts.

35. An IV-EEV, wherein: 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 transmembrane protein comprises a polypeptide that confers humoral immunity or comprises sufficient portion thereof to confer humoral immunity when expressed.

36. The IV-EEV of claim 35, wherein the transmembrane protein is selected from among A33R, A34R, A56R, B5R, and F13L.

37. The EEV of claim 35 or claim 36, 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.

38. The EEV of any of claims 34-37, wherein the protein or portion thereof is a complement regulatory protein.

39. The EEV particle, or vaccinia virus genome, or vaccinia virus or derivative virus thereof, or fusion protein of any of claims 1-38, wherein: the virus produces a high level of EEV virus; and a high level is more than 4%, 5%, 10%, or 20%, or is 30% or more of the total virus particles produced.

40. The IV-EEV, EEV, or vaccinia virus genome, or vaccinia virus or derivative virus thereof, or fusion protein of any of claims 1-32, wherein the protein that reduces or inhibits humoral immunity is a complement regulating protein that inhibits complement.

41. The IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus or derivative virus thereof, or fusion protein of any of claims 1-40, wherein the EEV transmembrane protein is selected from among A33R, A34R, A56R, B5R, and F13L and variants thereof having at least 95% sequence identity thereto, whereby the protein displays the protein that reduces or inhibits humoral immunity or portion thereof.

42. The IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus or derivative virus thereof, or fusion protein of claim 41, wherein 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, 224, and 225 and variants thereof having at least 90% or at least 95% sequence identity.

43. The IV-EEV or EEV, or vaccinia virus genome, or vaccinia virus or derivative virus thereof, or fusion protein of any of claims 1-42, wherein the complement regulatory protein is selected from among CD35, CD55, CD59, CD46, CR, Factor H, VCP, MOPICE, SPICE, CCPH, C4- binding protein, CD35, Kaposi-sarcoma associated herpesvirus Kaposica I KCP, Herpesvirus saimiri (HVS) and 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.

44. The IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus or derivative virus thereof, or fusion protein of any of claims 1-43, 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, IHD-J, IHD-W, Brighton, Ankara, modified vaccinia Ankara (MVA), CVA382, Dairen I, LIPV, LC16M8, LC16M0, ACAM, WR 65-16, Connaught, JX-594 (pexastimogene devacirepvec), GL-ONC1, wDD TK mutant, New York City Board of Health (NYCBH), EM-63, and NYVAC vaccinia virus strains, and variants thereof that producevirus particles that produce EEV particles that display the protein or portion thereof that reduces or inhibits humoral immunity.

45. The IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus or derivative virus thereof, or fusion protein of any of claims 1-44, wherein the unmodified genome is the genome of a virus selected from among JX-594 (Pexastimogene Devacirepvec, Pexa-Vec); LIVP GLV-lh68 (GLV-ONC1 or GL-ONC1); wDD; TG6002; VG9-GM-CSF; CW; deW5; CF33; Guang9; IN rW; T601; vA34R; aCEA TCE; a modified WR.TK-GMCSF vaccinia virus; 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 Al; WO 2022 / 182206 Al; WO 2023 / 118603.

46. The IV-EEV, or EEV, or vaccinia virus genome or vaccinia virus or derivative virus thereof of any of claims 1-44, wherein the unmodified genome or modified genome is from a vaccinia virus selected from among an RT virus and derivatives thereof that retain substantially the level of EEV production as RT-00.

47. The IV- EEV, or EEV, or vaccinia virus genome, or vaccinia virus or derivative virus thereof, or fusion protein of claim 46, wherein the unmodified or modified genome has the sequence set forth in any of SEQ ID NOs: 1 and 782-790 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.

48. The IV- EEV, or EEV, or vaccinia virus genome, or vaccinia virus or derivative virus thereof, or fusion protein of any of claims 1-47 that comprises 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 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.

49. The IV- EEV, or EEV, or vaccinia virus genome or vaccinia virus or derivative virus thereof of any of claims 1-48 that comprises one or more knockouts thatinactivate one or more genes selected from among: A46R, B8R, J2R, A52R, F1L, VGF, TK, and B19R.

50. The IV- EEV, or EEV, or vaccinia virus genome, or vaccinia virus or derivative virus thereof, or fusion protein of any of claims 1-49, wherein: the virus genome comprises two or three knockouts of genes; and the knockouts are effected by gene deletions, insertions, or disruptions.

51. The IV- EEV, or EEV, or vaccinia virus genome, or vaccinia virus or derivative virus thereof, or fusion protein of claim 50, wherein: double knockouts comprise a) TK, A46R; b) IK, 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; orn) TK, F1L, B19R.51a. The IV- EEV, or EEV, or vaccinia virus genome, or vaccinia virus or derivative virus thereof, or fusion protein of any of claims 1-50, wherein: the viral genome comprises heterologous nucleic acid encoding a therapeutic protein and / or a diagnostic or detectable or product or a reporter; and 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.

52. The IV- EEV, or EEV, or vaccinia virus genome, or vaccinia virus or derivative virus thereof, or fusion protein of claim 51a, wherein 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-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, such as anti-CD3 -DLL bi-specific 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-0 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.

53. The IV- EEV, or EEV, or vaccinia virus genome, or vaccinia virus or derivative virus thereof, or fusion protein claim 51a, 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 and anti-ANGPT2; anti-VEGF, anti-ANGPT-2 and anti-CTL4; anti-VEGF and OX40L; Anti-VEGF, Anti-ANGPT2 and anti-PD-1 products.

54. An isolated EEV virus particle that comprises the virus genome of a virus or genome of any of claims 1-53.

55. A high EEV producing vaccinia virus whose genome comprises knockouts of at least two of A33R, A34R, A36R, A56R, B5R, F13L, A45R, A29L.

56. A composition, comprising virus particles that comprise the virus, virus genome or derivative virus thereof or IV-EEV or EEV virus particle of any of claims 1- 55.

57. The composition of claim 56, wherein EEVs comprise at least 50%, 60%, 70%, 85%, 90%, 95%, or more of the virions in the composition.

58. The composition of claim 57, comprising purified EEV virus particles.

59. The composition of claim 57 or claim 58, consisting essentially of EEV virus particles.

60. The composition of any of claims 56-59 that is formulated for systemic administration.

61. The composition of any of claims 56-60, comprising the EEV virion particles in an amount that is:(i) between about lxlO3and about 1x1015pfu per ml;(ii) between about lxlO4and about lxlO14pfu per ml; or(iii) between about lxlO6and about lxlO12pfu per ml.

62. A method of treatment of cancer, comprising systemically administering aEEV particle or vaccinia virus or virus comprising the fusion protein, vaccinia virus composition of any of claims 1-61 to a subject in need thereof.

63. The IV-EEV, or EEV, or vaccinia virus genome or vaccinia virus or virus comprising the fusion protein, or composition of any of claims 1 -62 for use for treating cancer, wherein the vaccinia virus genome comprises a vaccinia virus particle.

64. The method, or use, or IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus, vaccinia virus comprising the fusion protein, or composition of claim 62 or claim 63, wherein the cancer comprises a solid tumor, or metastases, or is a homological malignancy.

65. The method, or use, or IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus, vaccinia virus comprising the fusion protein, or composition of any of claims 63-64, wherein the cancer comprises a malignant tumor or hematological malignancy, including metastatic cancers, lymphatic tumors, and blood cancers.

66. The method, or use, or IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus, vaccinia virus comprising the fusion protein, or composition of any of claims 62-65, wherein the 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 / cardnoid, Burkitt’s lymphoma, carcinoid tumor, cardnoma, 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 comeal 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, 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.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.

67. The method, or use, or IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus, vaccinia virus comprising the fusion protein, or composition of any of claims 62-66 wherein the subject is human.

68. The method, or use, or IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus, or vaccinia virus comprising the fusion protein, or composition of any of claims 62-67, 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 fin 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.

69. A nucleic add molecule encoding a fusion polypeptide, comprising an EEV or poxvirus- or 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 that increases or effects serum resistance of a poxvirus or vaccinia virus expressing the fusion polypeptide70. A nucleic add molecule encoding a fusion polypeptide, comprising a complement regulatory protein (CRP) or sufficient portion thereof for activity or other humor immunity modulating protein, and an extracellular enveloped vaccinia virus (EEV) transmembrane protein or suffident portion thereof for display of the CRP or humoral modulating protein or portion thereof on the surface of an EEV.

71. The isolated nucleic acid of claim 69 or claim 70, wherein the poxvirus is vaccinia virus, and the envelope protein is B5R, A33R, A34R, A56R or F13L.

72. The nucleic acid molecule of any of claims 69-71, wherein the protein that reduces or inhibits humoral immunity or portion thereof is selected among one or more of : CD35, CD55, CD59, CD46, CR1, Factor H, VCP, MOPICE, SPICE, COPY, ORF4, CCPH, C4- binding protein, CD35, Kaposi-sarcoma associated herpesvirus Kaposica I KCP, Herpesvirus saimiri (HVS) and 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 a functional portion thereof.

73. The nucleic acid molecule of claim 72, wherein 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.

74. The nucleic acid molecule of any of claims 69-73, wherein the protein that reduces or inhibits humoral immunity or portion thereof is fused to a transmembrane region of the EEV envelope protein, whereby the protein or portion thereof that reduces or inhibits humoral immunity is displayed on the EEV outer membrane.

75. The nucleic acid molecule of any of claims 69-74, wherein the envelope protein is from Vaccinia Copenhagen virus, Camelpox virus, Variola virus, Cowpox virus, Taterapox virus, Monkeypox virus Zaire-96-1-16, Volepox virus, Akhmeta virus, Ectromelia virus, Orthopoxvirus Abatino virus, Skunkpox virus, 87 Raccoonpox virus, Yokapox virus, Murmansk poxvirus, NY 014 poxvirus, and Yaba monkey tumor virus or part thereof.

76. The nucleic acid molecule of any of claims 69-75, wherein 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 transmembrane region of the envelope protein or is covalently linked to the C-terminus of the envelope protein.

77. A vaccinia virus or genome thereof, or a poxvirus or genome thereof, wherein the virus genome comprises the nucleic acid of any of claims 69-76.

78. A vaccinia virus or genome thereof, or a poxvirus or genome thereof, of claim 77, wherein the nucleic acid encoding the chimeric or fusion protein 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.

79. The virus or virus genome of any of claims 69-78 wherein the vaccinia virus vector comprises a deletion of or inactive thymidine kinase (TK) gene.

80. The virus or virus genome of any of claims 69-79, wherein: 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 (MVA), CVA382, Dairen I, LIPV, LC16M8, LC16M0, ACAM, WR 65-16, Connaught, JX-594 (pexastimogene devacirepvec), GL-ONC1, wDD 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; and a high level of EEV virus is at greater than 1%, 5%, 10%, 15%, 20%, 25%, 30% or more of the virus population.

81. The vaccinia virus or virus genome of any of claims 69-75, wherein a high level of EEV virus is greater than 25% or 30% of the virus population.

82. The method, or use, or IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus, or vaccinia virus comprising the fusion protein, or composition, or virus or genome thereof of any of claims 1-81, wherein: 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.

83. The method, or use, or IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus, or vaccinia virus comprising the fusion protein, or composition, or virus or genome thereof claim 82, wherein the virus comprises a fusion protein between an EEV transmembrane protein and a CRP or other humoral immunity modulator toincrease semm resistance, and / or the virus or genome comprises nucleic acid encoding a cytokine or other anti-tumor therapeutic.

84. The method, or use, or IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus, or vaccinia virus comprising the fusion protein, or composition, or virus or genome thereof of claim 83, wherein the EEV transmembrane protein is A33.

85. The method, or use, or IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus, or vaccinia virus comprising the fusion protein, or composition, or virus or genome thereof of claim 83 or 84, wherein the cytokine is an IL-15 or an IL-15ZIL- 15R alpha chain complex.

86. The method, or use, or IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus, or vaccinia virus comprising the fusion protein, or composition, or virus or genome thereof of any of claims 82-85 selected from a vaccinia virus of a)-f): a) 3KO (TK -, A46-, VGF-); b) 3KO (TK -, A46-, VGF-) - A33+CD55; c). 3KO (TK -, A46-, VGF-) + Payload IL-15 (cytokine form); d). 3KO (TK -, A46-, VGF-) - A33+CD55+ Payload IL-15 (cytokine form); e). 3KO (TK -, A46-, VGF-)+ Payload IL- 15 superagonist; and f).3KO (TK -, A46-, VGF-) - A33+CD55+ Payload IL-15 superagonist.

87. The vaccinia virus or genome of claim 86, wherein the payload is an IL- 15 superagonist or a modified form thereof that has increased activity.

88. The method, or use, or IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus, or vaccinia virus comprising the fusion protein, or composition, or virus or genome thereof of any of claims 83-87, 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 virus is a high EEV virus and has increased serum resistance compared to the unmodified virus.

89. The method, or use, or IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus, or vaccinia virus comprising the fusion protein, or composition, or virus or genome thereof of any of claims 83-88 that is a high EEV virus encoding a fusion protein of the EEV transmembrane protein with a complement regulatory protein or other humoral immunity modulating protein, wherein the payload encoding nucleic acid isinserted into in place of all or a portion of the virus VGF encoding nucleic acid locus or to render the virus VGF": 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; 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.

90. A pharmaceutical composition comprising the virus of any of claims 1-89.

91. The pharmaceutical composition of claim 90 formulated to have a unit dose of:(i) between about 1x103and about 1x1015pfu per ml;(ii) between about 1x104and about 1x1014pfu per ml; or(iii) between about 1x106and about 1x1012pfu per ml.

92. The virus or pharmaceutical composition of any of claims 1-91, for use for treating cancer and / or proliferative diseases, disorders, and / or conditions in a subject.

93. A method of treating cancer and / or proliferative diseases, disorders, and / or conditions, comprising administering the pharmaceutical composition or virus of any of claims 1-92.

94. The use of claim 92, or method of claim 93, wherein the pharmaceutical composition or virus is systemically administered or is for systemic administration.

95. The method, or use, or IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus, or vaccinia virus comprising the fusion protein, or composition, or virus or genome thereof of any of claims 1 -94 for use for treating cancer in combination with a second anti-cancer agent or treatment.

96. A method of treating cancer, comprising: a) systemically administering an EEV, virus, or composition of any of claims 1- 92; 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).

97. The method of claim 96 or use of claim 94, wherein the second anticancer 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.

98. The method or use of claim 96 or claim 97, 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.

99. The method or use of any of claims 96-98, wherein the second agent or treatment is selected from among, ceftazidime, cefepime, imipenem, aminoglycoside, vancomycin and antipseudomonal 0-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. 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, i satin-b eta-thiosemi carb azone, N- methanocarbathymidine, brivudine, 7-deazaneplanocin A, ST-246, Gleevec, 2 -beta- fluoro-2',3'-dideoxyadenosine, indinavir, nelfmavir, 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. 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.

100. The method of any of claims 92-99, comprising administering an agent to effect lymphodepletion to reduce or eliminate an anti-viral immune response.

101. The method of claim 100, wherein 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.

102. The method or use of any of claims 62-67 and 82-101, further comprising administering an anti-viral agent or an anti-viral antibody to modulate the level of virus or to eliminate the virus.

103. The method or use of claim 102, wherein the anti-viral agent or antibody is selected from among cidofovir, alkoxyalkyl esters of cidofovir, ganciclovir, acyclovir, ST-246, Gleevec, and derivatives thereof.103a. The method or use of any of claims 62-67, and 82-103, comprising administering or use of an immunomodulator.

104. The method or use of claim 103a, wherein the immunomodulator is a chemotherapeutic agent and administered in a dose sufficient to achieve an immunomodulatory effect but not lymphodepletion.

105. The method or use of claim 104, wherein the immunomodulator comprises immunotherapy.

106. The method or use of any of claims 62-67 and 82-105, wherein treatment or use comprises 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 agent; or b) regimen a) further comprising administration of an immunomodulatory agent after treatment with the virus, or after viral treatment, not before.

107. The method, or use, or IV-EEV, or EEV, or vaccinia virus genome, or vaccinia virus, or vaccinia virus, or composition, wherein: the virus comprises the fusion protein, or composition, or virus or genome thereof of any of claims 1-106; 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; and the target antigen is a target for a therapy selected from among an immunotherapy or cell therapy, or antibody, or antibody-drug conjugate for treating cancer.

108. The virus, virus genome, EEV, composition, method, or use of claim 107, wherein the therapy is a checkpoint inhibitor, CAR-T cell therapy, NK cell therapy, gene-editing therapy, or TIL cell therapy.

109. The virus, virus genome, EEV, composition, method, or use of claim 107 or claim 108, wherein the target antigen is a tumor-specific antigen or neoantigen.

110. The virus, virus genome, EEV, composition, method, or use of any of claims 107-109, wherein the target antigen is CD20 and HER2.

111. The virus, virus genome, or derivative thereof, EEV, composition, method, or use of any of claims 1-110, wherein the virus encodes a heterologous product.

112. The virus, virus genome, or derivative thereof, EEV, composition, method, or use of any of claims 1-111, wherein the product targets the virus, interacts with a host receptor, is a therapeutic or bioactive product, targets a therapeutic protein to cells comprising the virus, or a modified receptor.

113. The virus, virus genome, or derivative thereof, EEV, composition, method, or use of any of claims 1-112, wherein the product comprises a co-stimulatory molecule, an immune checkpoint inhibitors or other immune regulatory protein, or an interieukins, cytokine, chemokine, growth factor, inhibitors against immunosuppressive or pro-tumorigenic 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.

114. The virus, virus genome, or derivative thereof, EEV, composition, method, or use of any of claims 1-113, wherein the product comprises one or more of IL- 4, IL-6, IL-10, IL-11, IL-13, IL-17, IL-32, IGF, TGF-β, 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-o, IFN-P, IFN-y, TNF-o, EPO, GM-CSF, G-CSF, Flt3L, FGF, EGF, and IL-15 sushi domain linked, via a linker, or to a modified IL-15.

115. A transfer vector, comprising the sequence of nucleotides set forth in any of SEQ ID NOs: 2-21 and 518-524.

116. A chimeric protein or encoding nucleic acid, wherein: the chimeric protein comprises an EEV transmembrane protein and a protein that reduces or inhibits humoral immunity or humoral immunity inhibiting portion thereof; and the EEV transmembrane protein is expressed on the outer membrane of an EEV.

117. The chimeric protein or nucleic acid of claim 116 wherein: the transmembrane protein is selected from among A33R, A34R, A56R, B5R, andF13L.

118. The chimeric protein or nucleic acid of claim 116 or claim 100, wherein the protein that reduces or inhibits humoral immunity or a portion thereof is selected from among wherein the protein that inhibits humoral immunity is complement regulatory protein that is selected from among CD35, CD55, CD59, CD46, CR, Factor H, VCP, MOPICE, SPICE, CCPH, C4- binding protein, CD35, Kaposi-sarcoma associated herpesvirus Kaposica I KCP, Herpesvirus saimiri (HVS) and 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, and portions thereof sufficient to reduce or inhibit humoral immunity.

119. The chimeric protein or nucleic acid of any of claims 116-118, wherein the protein that reduces or inhibits humoral immunity or a portion thereof is linked to the N- or C-terminus of the transmembrane protein or inserted into the stalk domain of the transmembrane protein, whereby a sufficient portion of the protein that reduces or inhibits humoral immunity or a portion thereof is displayed on the second membrane on the surface of the EEV.

120. A method for manufacturing EEV virus, wherein the resulting product comprises at least 60% EEV virus, comprising: 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.

121. The method of claim 120, wherein the purified virus is formulated in formulation buffer for administration and / or storage at low temperature.

122. The method of claim 120 or claim 121, wherein the cells are cultured in a perfusion suspension reactor, or in a spinner flask, or in a wave bioreactor.

123. The method of any of claims 120-122, 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 AGEl.CR.pIX, CV1, HELA, HELA S3, CHO, VPCs, VPCs 2.0, FS293, MDCK, and MDCK.STAT1 KO cells.

124. The method of any of claims 120-123, wherein all steps of the method are performed under low shear force.

125. The method of claim 124, wherein low shear force is less than 100 shear / seconds.

126. The method of claim 124 or 125, wherein low shear force is from 10 to less than 100 shear / seconds.

127. The method of any of claims 120-126, wherein the cells are HeLa cells or human iPSCs.

128. The method of any of claims 120-127, comprising: a) infecting cultured cells with an IMV cmde 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 0.05 pm to about 0.1pm, and collecting the resulting virus composition; e) re-buffering the virus into a storage and injectable formulation buffer; f) optionally filling a vial or vials or other container for low temperature storage.

129. The method of claim 128, comprising: 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 IFF 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.

130. The method of claim 128 or claim 129, wherein the formulation buffer comprises lOmM Tris / HCl, 1% sucrose, 2% trehalose, 5% mannitol, 300 mM glycine, and 0.1% recombinant human albumin.

131. The method of any of claims 128-130, further comprising filling a vial or vials for storage at low temperature and subsequent injection.

132. The method of any of claims 120-131, wherein the infecting virus is a vaccinia virus or genome or derivative or IV-EEV or composition of any of claims 1- 114.

133. The method of any of claims 120-132, wherein cells are infected with a red tail (RT) virus.

134. The method of claim 133, wherein the RT virus is RT-00- RT-114.

135. The method of any of claims 120-134, wherein the EEV virus is comprises three knockouts of genes TK, A46, and VGF.

136. The virus, genome, EEV, composition, or method, or use of any of claims 1-135, wherein the virus encodes a therapeutic product.

137. The virus, genome, EEV, composition, or method or use of claim 136, 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.

138. The virus, genome, EEV particle, composition, or method or use of any of claims 1-137, 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".

139. The virus, genome, EEV particle, composition, or method, or use of any of claims 1-138, wherein the virus encodes a therapeutic protein that that is an immunostimulatory protein and / or has anti-cancer activity.

140. The virus, genome, EEV particle, composition, or method, or use of claim 139, wherein the therapeutic protein is an IL-15 or IL-15ZIL-15R alpha chain complex or modified form thereof that comprises a mutation that increased activity.

141. The virus, genome, EEV particle, composition, or method, or use of any of claims 1 -140, wherein the EEV virus genome comprises the sequence set forth in SEQ ID NOs: 782-790 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.

142. The virus, genome, EEV particle, composition, or method, or use of claim 141 that comprises a fusion protein encoding IL-15 or IL-15 / IL-15R alpha chain complex and / or a fusion protein with a transmembrane protein and a humoral immunity modulator, such as a CRP, such as CDS 5.