SERUM-RESISTANT EEV VIRUSES AND USES THEREOF RELATED APPLICATIONS
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.
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.
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.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY
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.
FIELD OF THE INVENTION
The field is oncolytic viruses and uses thereof for therapy.
BACKGROUND
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
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.
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.
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.
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.
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:
(TK -, A46-, VGF-), a high EEV - producing strain RT-01 with the knockouts;
(TK -, A46-, VGF-) - A33+CD55 - the strain encoding a fusion protein;
(TK -, A46-, VGF-) + Payload IL-15 (cytokine form) - the strain encoding a cytokine inserted into the VGF locus;
(TK -, A46-, VGF-) - A33+CD55+ Payload IL-15 (cytokine form) - the strain encoding the fusion protein and the cytokine ;
(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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 1x103 and about 1x1015 pfu per ml; (ii) between about 1x104 and about 1x1014 pfu per ml; or (iii) between about 1x106 and about 1x1012 pfu per ml.
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.
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.
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.
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.
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.
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 1x103 and about 1x1015 pfu per ml; (ii) between about 1x104 and about 1x1014 pfu per ml; or (iii) between about 1x106 and about 1x1012 pfu per ml.
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.
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.
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.
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.
BRIEF DESCRIPTION OF DRAWINGS
FIGURE 1 depicts a strategy for selection of a vaccinia virus with high EEV production and resistances to humoral immunity.
FIGURE 2 shows spreading patterns of the CALI virus (low producer of EEV) and the EEV clone designated RT-01 (high producer of EEV).
FIGURE 3 shows that EEV, but not IMV are resistant to inhibition by humoral immunity.
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.
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.
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.
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.
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.
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).
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).
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.
FIGURE 15 show the structure of A46R-hCD55-B5R as constructed and inserted into the parental virus to produce RT-02.
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
■ Cells + RT-05.
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.
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.
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.
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.
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.
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.
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.
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).
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).
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.
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).
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.
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.
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.
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.
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.
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.
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).
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.
DETAILED DESCRIPTION
Outline
A. Definitions
B. Overview
Vaccinia viruses for Systemic Administration
C. Vaccinia Viruses and EEV Vaccinia Viruses
1. Vaccinia viruses
2. Vaccinia Virus EEV and Their Production
3. IMV
4. Insufficient EEV for System Administration
5. Selection of high producing EEV
6. Exemplary high EEV-producing viruses
7. Modifications of EEV to increase resistance to humoral immunity
D. Methods of Production of High Amounts of EEVs
1. Existing methods result in low production of EEVs
2. Methods of manufacturing/producing virus resulting in higher EEV Viruses
3. iPSCs and their use for manufacturing EEVs from poxviruses and other viruses
E. Improved EEV Viruses
1. Serum-resistance of EEV viruses is limited by short-term protection from host immune system, such as protection from complement
2. EEV that display or express a CRP (or CRA) or other protein that inhibits or reduces the humoral immune response or sufficient of such proteins on the EEV outer membrane
3. Advantages of the S-R EEVs and IV EEVs
4. Complement Regulating Proteins
5. Properties of complement regulatory proteins that inhibit complement activation (complement inhibitory proteins)
6. EEV Viruses That are Modified to Display Immune Modulating Proteins on the Outer Membrane of the EEV
7. Viruses modified to encode Complement Regulating Proteins - Functional or active or sufficient portions of the complement regulating proteins or other such proteins
8. Assays for measuring the complement regulating activity of proteins
9. Exemplary of immune system regulating proteins are the complement regulatory proteins
10. EEV outer membrane proteins
F. Any Poxviruses Can Be Modified as Described Herein
1. Methods for Modifying vaccinia viruses
2. Therapeutic, transgenic, and attenuated vaccinia viruses 3. Exemplary Therapeutic Vaccinia Virus
4. Virally Encoded Immunomodulators and Effects thereof
G. EEV Vaccinia Viruses Can Be Modified to Express Heterologous Genes and/or Payloads
H. Chimeras and Fusions Virally Encoded Membrane Proteins and Proteins That Inhibit or Modulate the Humoral Immunity of the Host
1. Fusion proteins and Chimeric proteins
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
3. Domains of the complement inhibitory/regulatory/control proteins
I. Pharmaceutical Compositions, Combinations, and Kits
J. Methods of Treating Cancer and Other Proliferative Diseases, Disorders, and Conditions
K. Examples
A. DEFINITIONS
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 1x103 and 1x1015 PFUs; for example, about 1x104, about 1x106, about 1x108, about 1x1010, about 1x1012 or about 1x1014 or higher, per mL, per kg.
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.
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.
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.
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).
As used herein, complement inhibitor protein refers to any protein, polypeptide, and or portion thereof that can inhibit complement.
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.
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.
As used herein, immunomodulatory with respect to combination treatments reduce or modulate the immune response so that the anti-viral response is reduced.
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.
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.
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.
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.
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.
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).
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.
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.
As used herein, a “host cell” or “target cell” are used interchangeably to mean a cell that can be infected by a virus.
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.
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.
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.
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).
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.
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.
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.
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, 3rd edition (1999)).
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
As used herein, neoplastic disease refers to any disorder involving cancer, including tumor development, growth, metastasis and progression.
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.
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.
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.
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.
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.
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.
As used herein, a “normal cell” is a cell that is not derived from a tumor, but is derived from healthy non-diseased tissue.
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.”
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.
As used herein, reference to an anticancer or chemotherapeutic agent includes combinations or a plurality of anticancer or chemotherapeutic agents unless otherwise indicated.
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.
As used herein, a patient refers to a human subject exhibiting symptoms of a disease or disorder.
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.
As used herein, treatment refers to amelioration of the symptoms of a disease or disorder.
As used herein, prevention refers to prophylactic treatment to reduce the risk of getting a disease or condition or reducing the severity thereof.
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.
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.
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.
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.
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.
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.
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.
As used herein, prevent a disease or condition means reduce the probability or rise of getting the disease or condition.
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.
As used herein, a formulation refers to a composition containing at least one active pharmaceutical or therapeutic agent and one or more excipients.
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.
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.
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.
As used herein, direct administration refers to administration of a composition without dilution.
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.
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.
As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
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.
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.
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.
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).
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.
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.
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.
As used herein, '"immune suppression" or "immunosuppression" refers to the suppression or reduction of the immune response.
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.
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.
As used herein, "permissive" with respect to viral infection refers to a cell that is readily infected upon exposure to the virus.
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.
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.
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.
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.
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.
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.
As used herein, non-naturally occurring amino acids refer to amino acids that are not genetically encoded.
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.
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.
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.
As used herein, “polypeptide” refers to two or more amino acids covalently joined. The terms “polypeptide” and “protein” are used interchangeably herein.
As used herein, a “peptide” refers to a polypeptide that is from 2 to about or 40 amino acids in length.
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.
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).
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.
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).
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:
Table of Correspondence
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.
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).
Such substitutions can be made in accordance with the exemplary substitutions set forth in the following Table:
Exemplary Conservative Amino Acid Substitutions
Other substitutions also are permissible and can be determined empirically or in accord with other known conservative or non-conservative substitutions.
As used herein, “naturally occurring amino acids” refer to the 20 L-amino acids that occur in polypeptides.
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).
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).
For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.
B. OVERVIEW
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.
Vaccinia Viruses for Systemic Administration
Oncolytic viral therapy uses naturally occurring or genetically engineered viruses to selectively lyse tumor cells. Oncolytic viruses preferentially accumulate in and then replicate in tumor tissue and in cells and tissues other immunoprivileged and immunosuppressed environments. Oncolytic viruses, including vaccinia viruses, exert anti-tumor effects via oncolysis and activation of anti-tumor immune responses.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
Provided herein are EEV viruses, methods for producing EEV viruses, and methods and modifications for increasing resistance of EEV viruses to humoral immunity.
1. Vaccinia viruses
Vaccinia Virus
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.
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).
Various vaccinia viruses have been demonstrated to exhibit antitumor activities.
In one study, for example, nude mice bearing non-metastatic colon adenocarcinoma cells were systemically injected with a WR strain of vaccinia virus modified by having a vaccinia growth factor deletion and an enhanced green fluorescence protein inserted into the thymidine kinase locus. The virus was observed to have antitumor effects, including one complete response, despite a lack of exogenous therapeutic genes in the modified virus (McCart 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).
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.
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.
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.
(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).
2. Vaccinia Virus EEV and Their Production
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.
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).
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.
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).
Much of the EEV remains attached to the host cell surface and is retained as
CEV. EEV that is released into the extracellular milieu (generally less than about 1% of virions) is responsible for viral spread within the infected host. EEVs are unstable outside of the host and are difficult to manufacture. The co-existence of IMV and EEV forms has been described for vaccinia strains and other poxviruses, including the fowl poxvirus, entomopox, monkeypox, swinepox and pinguin pox. EEV virions exhibit long- range spread in cell culture and systemic dissemination in vivo. The EEV form of poxviruses such as vaccinia virus have been developed for possible systemic delivery of viruses to tumors and tumor metastasis.
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.
EEV has several mechanisms to inhibit its neutralization within the bloodstream.
First, EEV is has resistance to complement due to the incorporation of host cell regulators/inhibitors of complement into its outer membrane coat plus secretion of vaccinia virus complement control protein (VCP) into local extracellular environment. Second, EEV exhibits resistance to neutralizing antibody effects compared to IMV, although smallpox vaccines can generate antibody responses to the EEV form in immunized humans. EEV also is released at earlier time points following infection (e.g., 4-6 hours) than IMV (which is only released during/after infected cell death). The host spread of the EEV virion is faster than IMV (Blasco et al. J. Virology, 61(6)3319-3325, 1993).
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.
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.
3. IMV
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.
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.
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.
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.
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.
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).
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.
5. Selection of high producing EEV
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.
Methods for Selection of high EEV viruses
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.
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.
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%.
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.
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.
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.
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.
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.
6. Exemplary high EEV-producing viruses
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.
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.
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.
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.
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.
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.
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.
Other high EEV viruses
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.
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
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.
7. Modifications of EEV to increase resistance to humoral immunity
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.
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.
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.
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.
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.
The table below, summarizes structures of the above viruses, which are derived from
RT-00/N2, their nomenclature, genotype with reference to the vaccinia virus locus and insert, if present, and heterologous nucleic acid encoded by the virus, (see also Figure 24 for insertion sites for RT-31 through RT-43).
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.
Selection markers include, but are not limited to, EGFP, EmGFP, mNeonGreen, EBFP,
TagBFP, EYFP, TPet, GFP, BFP or TuiboFP635. The RT-00 virus and viruses derived
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,
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-
1BBL, CD40L, B7.1/CD80, GITRL, LIGHT, CD70), bi-specific t-cell engagers (BITEs),
10 therapeutic antibodies, immune checkpoint inhibitors, single chain antibodies such as single chain antibodies against e.g., VEGF, e.g., 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,
15 Aquaporin 1-AQP1, and any other payload of interest, particularly any that have anticancer activity, or promote an anti-cancer phenotype or activity.
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
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.
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.
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.
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.
Generation of the viruses
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.
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.
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.
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:
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
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:
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.
Exemplary protocol for generating viruses provided herein from RT-00.
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.
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.
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.
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.
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.
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.
Mutations that Increase EEV
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.
EEV Biology
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.
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.
Preparing virus with increased EEV production
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.
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.
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.
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
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.
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.
Introduction of mutations in virally encoded EEV membrane proteins (A33R, A34R, A56R, B5R), and combinations of mutations thereof to increase EEV percentages
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:
A33R (A88D, E129M and/or M63R); A34R (K151E, DI ION, K151D, KI 19E, F94H, R91S, T127E, R84G, R91A and/or M66T);
A56 (I269F); and
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.
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.
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
D. METHODS OF PRODUCTION OF HIGH AMOUNTS OF EEVs
1. Existing methods result in low production of EEVs
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)
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.
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.
2. Methods of manufacturing/producing virus resulting in higher EEV
Viruses
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.
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).
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.
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.
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.
3. iPSCs and their use for manufacturing EEVs from poxviruses and other viruses
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.
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.
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.
E. Improved EEV Viruses
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.
1. Serum-resistance of EEV viruses is limited by short-term protection from host immune system, such as protection from complement
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.
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.
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).
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
3. Advantages of the S-R EEVs and IV EEVs
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.
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.
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.
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.
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.
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.
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.
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.
4. Complement Regulating Proteins
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).
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.
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
C5a anaphylatoxins; C3b that can opsonize viruses and promote their immune adherence, direct neutralization, and phagocytic destruction; C3d (the cleaved fragment of C3b) and;
C5b-9 complex or MAC (Agrawal P etal. FEBST-ett. 2020 Aug;594(16):2518-2542).
Activated complement components can result in tissue destruction; they are regulated by host soluble or membrane-bound (s/m) complement regulatory proteins
(complement inhibitory proteins). These include the following proteins:
5. Properties of complement regulatory proteins that inhibit complement activation (complement inhibitory proteins)
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
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
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.
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.
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).
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).
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.
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.
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.
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.
6. EEV Viruses That Are Modified To Display Immune Modulating
Proteins On The Outer Membrane Of The EEV
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.
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.
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.
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)).
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).
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.
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)).
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)).
8. Assays for measuring or assessing the complement regulating activity of portions
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).
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).
9. Exemplary of immune system regulating proteins are the complement regulatory proteins
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.
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).
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%.
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).
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.
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).
10. EEV outer membrane proteins
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.
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.
B5R
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):
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.
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.
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)).
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.
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.
A33R
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
Western Reserve (NCBI: YP_233038.1; SEQ ID NO: 482):
MMTPENDEEQTSVFSATVYGDKIQGKNKRKRVIGLCIRISMVISLLSMIT
MSAFLIVRLNQCMSANEAAITDAAVAVAAASSTHRKVASSTTQYDHKES CNGLYYQGSCYILHSDYQLFSDAKANCTAESSTLPNKSDVLITWLIDYVE DTWGSDGNPITKTTSDYQDSDVSQEVRKYFCVKTMN
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
RL. et al. Journal of Virology, 1998, 72(5):4192-4204).
A56R
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):
MTRLPILLLLTSLVYATPFPQTSKKIGDDATLSCNRNNTNDYWMSAWY KEPNSIILLAAKSDVLYFDNYTKDKISYDSPYDDLVTTITIKSLTARDAGT YVCAFFMTSTTNDTDKVDYEEYSTELIVNTDSESTIDIILSGSTHSPETSSK KPDYIDNSNCSSVFEIATPEPITDNVEDHTDTVTYTSDSINTVSASSGESTT DETPEPITDKEDHTVTDTVSYTTVSTSSGIVTTKSTTDDADLYDTYNDND TVPPTTVGGSTTSISNYKTKDFVEIFGITALIILSAVAIFCITYYIYNKRSRK YKTENKV
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.
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)).
F13L
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):
MWPFASVPAGAKCRLVETLPENMDFRSDHLTTFECFNEIITLAKKYIYIA SFCCNPLSTTRGALIFDKLKEASEKGIKIIVLLDERGKRNLGELQSHCPDIN FITVNIDKKNNVGLLLGCFWVSDDERCYVGNASFTGGSIHTIKTLGVYSD YPPLATDLRRRFDTFKAFNSAKNSWLNLCSAACCLPVSTAYHIKNPIGGV FFTDSPEHLLGYSRDLDTDWIDKLKSAKTSIDIEHLAIVPTTRVDGNSYY WPDIYNSIIEAAINRGVKIRLLVGNWDKNDVYSMATARSLDALCVQNDL SVKVFTIQNNTKLLIVDDEYVHITSANFDGTHYQNHGFVSFNSIDKQLVS EAKKIFERDWVSSHSKSLKI
Host Protein
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.
F. ANY POXVIRUSES CAN BE MODIFIED AS DESCRIBED HEREIN
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.
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.
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.
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).
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.
Various vaccinia viruses have been demonstrated to exhibit antitumor activities.
In one study, for example, nude mice bearing non-metastatic colon adenocarcinoma cells were systemically injected with a WR strain of vaccinia virus modified by having a vaccinia growth factor deletion and an enhanced green fluorescence protein inserted into the thymidine kinase locus. The virus was observed to have antitumor effects, including one complete response, despite a lack of exogenous therapeutic genes in the modified virus (McCart et al. (2001) Cancer Res. 1 :8751-8757). In another study, vaccinia melanoma oncolysate (VMO) was injected into sites near melanoma positive lymph nodes in a Phase 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.
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.
1. Methods for modifying vaccinia viruses
Methods of modifying the vaccinia virus genome are well-known (see e.g., U.S.
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:
(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).
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).
(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)).
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.
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).
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.
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.
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).
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.
3. Exemplary Therapeutic Vaccinia Virus
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;
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.
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).
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.
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.
Virally Encoded Immunomodulators and Effects thereof
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.
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 engineered to express intracellular and/or extracellular immunomodulators, increasing their virulence, or alternatively, they can be attenuated by deletion of the genes encoding immunomodulatory proteins.
Extracellular poxvirus immunomodulators that can be expressed by the viruses herein include the chemokine inhibitor/binding protein A41; the TNF inhibitors/binding proteins CrmB, CrmC, CrmD and CrmE; the MHC-like TNF-alpha inhibitor TPXV 2; the IL- 18 binding protein C12; VACV CC chemokine inhibitor (vCCI), which prevents leukocyte recruitment; IFN-gamma binding protein (IFN-y BP), which blocks binding of IFN-gamma to its receptor; the IL-10-binding protein Bl 5; the type I IFN-binding protein Bl 8; the type II IFN-binding protein B8; complement control protein VCP (C21/B27); and the semaphorin 7 A mimic A39 (Bahar et al. (2011) J. Struct. Biol. 175(2-2): 127-134; Sumner et al. (2016) Foccz/ze 34:4827-4834; Nichols e/ a/. (2017) Viruses 9, 215; Albamaz etal. (2018) Viruses 10, 101).
Intracellular poxvirus immunomodulators that can be expressed by the viruses herein include hemagglutinin (HA, A56); thymidine kinase (TK); B5 (promotes viral dissemination); N1 (Bcl-2-like inhibitor of apoptosis and inhibitor of NF-KB/IRF3 activation); B14 and A52 (Bcl-2-like inhibitors of NF-KB); K7 (Bcl-2-like inhibitor of NF-KB and IFN-beta); Fl and Ml 1 (Bcl-2-like anti-apoptotics); E3 (inhibitor of PKR activation, dsRNA binding protein); K3 (inhibits PKR mediated phosphorylation of eIF2a); C4 (inhibitor of NF-KB activation); C6 (IRF3/7 and JAK/STAT inhibitor); VH1 (dephosphorylates STAT1 and blocks expression of IFN-induced genes); A35 (inhibitor of MHC class II antigen presentation); B13 (SPI-2/CrmA) and B22 (SPI-1), which inhibit caspase activity; N2 (IRF3 inhibitor); D9 and D10 (de-capping enzymes); C16 (inhibitor of DNA sensing and promoter of hypoxic response); A49, KI and M2 (inhibitors of NF-KB activation); protein 169 (inhibitor of translation); vGAAP (inhibitor of apoptosis); A44 (3P-hydroxysteroid dehydrogenase); and A46 (TLR signaling, NF-KB, IRF3 and MAPK inhibitor) (Bahar etal. (2011) J. Struct. Biol. 175(2-2):127-134; Sumner et al. (2016) Vaccine 34:4827-4834; Nichols et al. (2017) Viruses 9, 215; Albamaz e/ al. (2018) Viruses 10, 101).
VCP (C3L)
The vaccinia virus complement control protein (VCP; encoded by C3L, C21L) is the major protein secreted from cells infected with vaccinia virus, and interacts with heparan sulfate proteoglycans (HSPGs) on the surfaces of uninfected cells. VCP also can be expressed on the surfaces of VACV infected cells, independently of HSPGs. The surface expression of VCP is dependent on its interaction with another viral protein, A56 (also known as hemagglutinin), present on the surface of vaccinia virus-infected cells and extracellular enveloped virus (EEV) particles. VCP inhibits the activation of classical and alternative complement pathways by accelerating the decay of C3 and C5 convertases, which is irreversible, and by acting as a cofactor for the factor I-mediated cleavage and inactivation of C3b and C4b (Girgis et al. (2008) J. Virol. 82(8):4205-4214; Smith et al. (2013) Journal of General Virology 94:2367-2392). A deletion mutant, lacking the C21L gene that encodes VCP, was attenuated in rabbits, and was associated with increased infiltration of CD4+ and CD8+ T-cells, reduced viral titers and increased antibodies against VACV (Albamaz et al. (2018) Viruses 10, 101).
Studies have shown that when VCP is engineered to contain a transmembrane domain that allows it to be expressed on the cell surface, it is capable of protecting cells from complement-mediated lysis, demonstrating a threefold decrease in lysis in the presence of VCP. (Rosengard etal. (1999) Afo/. Immunol. 36(10):685-697). By protecting vaccinia-infected cells from lysis, surface-bound VCP prolongs viral production and results in increased viral titers. The reduction in complement activation on the cell surfaces also reduces the production of proinflammatory peptides, such as C3a and C5a, which reduces local inflammation and immune system activation (Girgis et al. (2008) J. Virol. 82(8):4205-4214).
B5
B5 (encoded by B5R), a member of the complement protein family, is a type I integral membrane glycoprotein present in the extracellular enveloped virus (EEV) outer envelope that is needed for the formation of EEV and promotes viral dissemination (Smith etal (2013) Journal of General Virology 94:2367-2392).
Thymidine Kinase (TK)
VACV thymidine kinase (TK), encoded by the early VACV J2R gene, is a virulence factor that, when deleted from the viral genome, results in attenuated vaccinia virus strains (Yakubitskiy etal. (2015) Acta Naturae 7(4): 113-121).
HA (A56)
Natural killer (NK) cells, which play an important role in the immune defense against orthopox family members such as vaccinia virus (VACV or W), are regulated through inhibitory and activating signaling receptors. The activating signaling receptors include NKG2D and natural cytotoxicity receptors (NCRs) such as NKp46, NKp44 and NKp30. NCRs are important activating receptors for the anti-viral and anti-tumor activity of NK cells (Jarahian et al. (2011) PLoS Pathogens 7(8):e 1002195). Hemagglutinin (HA) (encoded by A56R), also known as A56, is a protein that mediates viral attachment to host cells, inhibits fusion of infected cells, and promotes proteolytic activation of infectivity (Y akubitskiy et al. (2015) Acta Naturae 7(4): 113- 121). HA, which is expressed as a late-phase product on the surface of VACV-infected cells, is a viral ligand for the activating receptors NKp30 and NKp46. HA/A56 has been shown to block NKp30-triggered activation, resulting in a decreased susceptibility of infected cells to NK lysis at late time points of VACV expression, when HA expression is pronounced. Thus, HA is a conserved ligand of NCR and results in immune escape through its blocking effect on NKp30-mediated activation at a late stage of infection (Jarahian etal. (2011) PLoS Pathogens 7(8):el002195).
Deletion of A56R from the VACV genome resulted in a 40-fold decrease in the LDso in mice, compared to the parent strain. Thus, inactivation of the HA gene in VACV leads to significant attenuation (Y akubitskiy et al. (2015) Acta Naturae 7(4): 113-121).
B18
VACV protein B18 (encoded by B18R) is a soluble extracellular immunomodulatory protein that binds type-I interferon and exhibits activity as a “decoy IFN receptor” in solution, and when associated with the cell surface via glycosaminoglycans (GAGs), sequestering type-I IFNs produced by uninfected cells, particularly IFN-a. When Bl 8 binds to cell surfaces, preventing the induction of the IFN- mediated antiviral state in uninfected cells, the cells remain susceptible to viral infection and replication (Smith et al. (2013) Journal of General Virology 94:2367-2392; Albamaz etal. (2018) Viruses 10, 101).
B8
B8 (encoded by B8R) is a soluble VACV decoy type-II IFN receptor that binds IFN-y extracellularly. Deletion of B8, which is a homologue to the extracellular domain of the IFN-y receptor, resulted in attenuation of VACV, in comparison to wild-type VACV, in mouse infection studies (Yakubitskiy etal. (2015) Acta Naturae 7(4):113- 121). Unlike cellular IFN-yR, B8 can dimerize in the absence of IFN-y (Smith et al. (2013) Journal of General Virology 94:2367-2392).
B15
VACV protein B15 (encoded by B15R) is a soluble IL-1R that is secreted by infected cells and binds IL- 10 with high affinity, preventing it from binding to its natural receptor. Studies have shown that viruses lacking the B15R gene exhibit reduced virulence (Smith etal. (2013) Journal of General Virology 94:2367-2392).
A39/A39R
A39/A39R is a secreted immunomodulatory glycoprotein, similar in amino acid sequence to glycophosphatidylinositol-linked cell surface semaphorin. A39R is expressed late during infection and has been shown to have pro-inflammatory properties, and to affect the outcome of infection, in a murine intradermal model (Gardner et al. (2001) J. Gen. Virol. 82:2083-2093).
CrmA/B13/SPI-2
Cytokine response modifier A (CrmA) (also known as B13/B13R or serine proteinase inhibitor 2 (SPI-2)) is an orthopoxvirus protein that is expressed early in the viral infection process and remains inside the host cell. CrmA binds caspase- 1 and blocks pro-IL-ip cleavage to IL- 10, a proinflammatory cytokine that is important in controlling poxvirus infections. By inhibiting the activation of multiple caspases (eg., caspase 1, caspase 8), CrmAZB13 also inhibits apoptosis. B13 additionally inhibits the formation of mature IL-18 (Smith etal. (2013) Journal of General Virology 94:2367-2392; Nichols et al. (2017) Viruses 9, 215).
SPI-1/B22R
Vaccinia vims SPI-1 (also known as B22 or B22R) is an intracellular immunomodulatory protein similar to SPI-2/CrmA, that inhibits caspase activity. Studies have shown that a mutated VACV, lacking the SPI-1/B22R gene displayed lower viral replication in A549 cells. Infected cells are sensitive to TNF-induced apoptosis, indicating the significant role that SPI-1 plays in virulence (Nichols et al. (2017) Viruses 9, 215).
Viral TNF Receptors (vTNFRs)
Viral TNF receptors (vTNFRs) are soluble, secreted decoy receptors that bind TNFo, preventing it from binding to its natural receptor, and mitigating its antiviral effects. The vTNFRs, which include cytokine response modifier B (CrmB), CrmC, CrmD and CrmE (A53), mimic the extracellular domain of the cellular TNF receptors TNFR1 and TNFR2, and differ in their ligand affinity and expression in orthopoxviruses. Studies have shown that vTNFRs enhance the virulence of recombinant VACV. For example, VACV strain USSR mutants lacking CrmE were attenuated, while a recombinant strain of VACV WR expressing CrmE displayed increased virulence (Nichols etal. (2017) Viruses 9, 215; Smith etal. (2013) Journal of General Virology 94:2367-2392; Bahar et al. (2011) J. Struct. Biol. 175(2-2): 127-134).
C12
C12 (encoded by C12L) is a soluble orthopoxvirus protein that binds IL-18 in solution, preventing it from interacting with its natural receptor, IL-18R. C12 increases the virulence of VACV by inhibiting the IL- 12 induced production of JFN-y, which inhibits NK cell and VACV-specific CD8+ T-cell responses (Smith etal. (2013) Journal of General Virology 94:2367-2392). Deletion of the C12L gene has been shown to result in viral attenuation, with increased levels of IL- 18 and IFN-y, and enhanced NK-cell cytotoxicity and CTL response after intranasal infection of mice (Albamaz et al. (2018) Viruses 10, 101).
VACV CC chemokine inhibitor (vCCI)
Chemokines are small chemo-attractant cytokines that recruit leukocytes to sites of infection and inflammation. Chemokines binds to GAGs on the surfaces of adjacent endothelial cells, creating a concentration gradient, and recruiting circulating leukocytes by binding to their chemokine receptors. VACV CC chemokine inhibitor (vCCI), also known as VACV chemokine-binding protein (vCKBP), which is secreted by virus- infected cells during the early stages of infection, binds CC chemokines, preventing them from binding to their receptors. This prevents the recruitment of leukocytes to the site of infection, reducing inflammation (Smith et al. (2013) Journal of General Virology 94:2367-2392).
A41
Whereas most viral CC chemokine inhibitors bind chemokines at their receptorbinding sites, preventing their interaction with, and recruitment of leukocytes to sites of inflammation, A41 (encoded by A41L) is an extracellular VACV immunomodulatory protein that binds chemokines at their GAG-binding site, rather than the receptor-binding site (Bahar et al. (2011) J. Struct. Biol. 175(2-2):127-134). A41 also is secreted by infected cells during the early stages of infection, but binds chemokines with a lower affinity than vCCI, and does not prevent chemokines from binding to their respective chemokine receptors. Instead, A41 disrupts the chemokine concentration gradients on the surfaces of endothelial cells, which are important for the recruitment of leukocytes (Smith et al. (2013) Journal of General Virology 94:2367-2392; Albamaz et al. (2018) Viruses 10, 101).
VH1
VACV VH1 is an intracellular immunomodulatory protein (phosphatase) that inhibits the transcription factors STAT1 (signal transducer and activator of transcription
1) and STAT2 by dephosphorylation, inhibiting signaling from all IFN receptors and preventing the expression of antiviral genes (Bahar et al. (2011) J. Struct. Biol. 175(2-
2): 127-134; Smith etal. (20Y3) Journal of General Virology 94;2361 -2392).
K3
K3 is a VACV intracellular immunomodulatory protein that inhibits PKR- mediated phosphorylation of eIF2a. In virus-infected cells, STAT1 induces expression of dsRNA-dependent protein kinase (PKR), which detects dsRNA produced during VACV transcription, and phosphorylates and inhibits the host protein translation factor eIF2a (eukaryotic translation initiation factor 2 alpha), arresting the synthesis of host and viral proteins in infected cells, and leading to apoptosis. K3 is a viral mimic of the N-terminal 88 amino acids of eIF2a, that binds PKR by acting as a non-phosphorylatable pseudosubstrate, and prevents PKR-induced apoptosis by inhibiting the phosphorylation of eIF2a by PKR (Bahar eta/. (2011) J. Struct. Biol. 175(2-2): 127-134; Smith etal. (2013) Journal of General Virology 94:2367-2392).
N1
B-cell lymphoma 2 (Bcl-2) proteins can be pro- or anti-apoptotic, and regulate the release of pro-apoptotic molecules from the mitochondria. Several viruses, including herpesvirus, adenovirus and VACV, express anti-apoptotic Bcl-2 and Bcl-2-like proteins to evade host cell death. For example, N1 (encoded by NIL) is a VACV virulence factor that functions as an intracellular immunomodulator and is similar in structure to anti- apoptotic Bcl-2 proteins. N1 binds BIB motifs of pro-apoptotic proteins, inhibiting apoptosis in VACV-infected cells. N1 has been shown to interact with host pro-apoptotic Bcl-2 proteins such as Bid, Bad, Bak and Bax, and has been shown to inhibit innate immune signaling pathways by binding to the IxB kinase (IKK) complex and TANK binding kinase 1 (TBK1), inhibiting activation of nuclear factor (NF)-xB and IRF3 (Bahar etal. (2011) J. Struct. Biol. 175(2-2): 127-134).
Fl Fl (encoded by ML) is a VACV intracellular immunomodulator that is a poxviral Bcl-2-like family protein and inhibits the apoptosis of virus-infected cells. Fl binds to the BH3 motifs of pro-apoptotic Bcl-2 proteins and, unlike Nl, which is found in the cytosol, is localized to the mitochondrial membrane, where it interacts with host pro-apoptotic Bcl-2 proteins such as Bak and Bax, which initiate apoptosis at the mitochondrial membrane (Bahar et al. (2011) J. Struct. Biol. 175(2-2): 127-134). Fl also reduces the inflammatory response by binding to NLRP-1, which is an upstream activator of caspase- 1 (Smith et al. (2013) Journal of General Virology 94:2367-2392), and binds to, and inhibits, caspase 9 (Albamaz et al. (2018) Viruses 10, 101).
Bcl-2-like proteins that inhibit the NF-KB pathway
Nuclear factor (NF)-KB is a transcription factor complex that stimulates the innate and adaptive immune responses to infection. Receptors for proinflammatory cytokines such as TNFa and IL-1, and Toll-like receptors (TLRs), which recognize pathogen associated molecular patterns (PAMPs), activate signaling pathways that lead to NF-KB activation. VACV encodes several Bcl-2-like proteins, including Nl, A52, B14 and K7, that inhibit the NF-KB signaling pathway. While Nl inhibits apoptosis, A52, B14 and K7, which lack the BH3-binding grooves, do not. B14 inhibits NF-KB activation and acts at the IKK complex by binding IKK0 and preventing its phosphorylation and the phosphorylation of IxBa. A52 and K7 inhibit signaling upstream of B14, by inhibiting TLR-induced signaling and TLR- and IL-ip-mediated NF-KB activation (via binding to TRAF6 and IRAK2). K7 (encoded by K7L) also forms a complex with the human DEAD-box RNA helicase 3 (DDX3), antagonizing IFN-beta promoter induction and inhibiting the production of pro-inflammatory cytokines (Bahar et at (2011) J. Struct. Biol. 175(2-2): 127-134; Smith et al. (2013) Journal of General Virology 94:2367-2392; Albamaz et al. (2018) Viruses 10, 101).
A46
A46 (encoded by A46R) is an intracellular VACV immunomodulatory protein that binds to Toll/IL-IR (TIR) domain-containing adaptor molecules (such as, for example, MyD88, MAL, TRIP and TRAM) that associate with the cytoplasmic tails of TLRs. This, in turn, inhibits activation of MAP kinases, NF-KB, and IRF3, which inhibits the induction of IFN-beta (Smith et al. (2013) Journal of General Virology 94:2367- 2392). A VACV WRA46R deletion mutant was found to be attenuated in comparison to control viruses (Albamaz et al. (2018) Viruses 10, 101).
Other proteins that inhibit NF-KB activation
A49 is an intracellular VACV protein that stabilizes phosphorylated IKBO (inhibitor of KB), by preventing its recognition and degradation, such that IKBO remains bound to NF-KB in the cytoplasm. Intracellular VACV protein C4 inhibits NF-KB activation at, or downstream of, the IKK complex, but the mechanism remains unknown. VACV protein E3 (encoded by E3L) inhibits NF-KB activation by PKR-dependent and independent mechanisms and by antagonizing the RNA polymerase m-dsDNA sensing pathway. E3 binds and sequesters dsRNA from cellular pattern recognition receptors (PRRs), preventing the cell from identifying viral dsRNA. In addition to E3, the VACV de-capping enzymes D9 and D10 prevent the accumulation of dsRNA by de-capping viral mRNAs, preventing the activation of PKR and dsRNA induced anti-viral pathways. VACV protein KI inhibits NF-KB activation by preventing the degradation of IKBO. Protein M2 reduces phosphorylation of extracellular signal-regulated kinase 2 (ERK2) induced by phorbol myristate acetate, and prevents p65 nuclear translocation (Smith et al. (2013) Journal of General Virology 94:2367-2392; Nichols et al. (2017) Viruses 9, 215; Albamaz etal. (2018) Viruses 10, 101).
C6
C6 (encoded by C6L) is an intracellular immunomodulatory protein that enhances virulence and inhibits activation of IRK3 and IRF7 by binding to the adaptor proteins needed to activate the upstream kinases TANK-binding kinase 1 (TBK1) and IKKE. This results in the inhibition of type-I IFN production. C6 also inhibits the activation of the JAK/STAT signaling pathway after type I IFNs bind to their receptors, preventing the transcription of interferon-stimulated genes (ISGs). Deletion of C6L has been shown to enhance CDS"1" and CD4+ T-cell responses (Albamaz etal. (2018) Viruses 10, 101).
C16
Cl 6, an intracellular immunomodulatory protein, inhibits DNA sensing that leads to IRF3 -dependent innate immunity, by binding to the proteins Ku70 and KuSO, which are subunits of the DNA-PK complex (a DNA sensor). C16 also binds the oxygen sensor prolylhydroxylase domain-containing protein 2 (PHD2), preventing the hydroxylation of hypoxia-inducible transcription factor (HIF)-la. This prevents the ubiquitylatian and degradation of HIF-la, and the stabilized HIF-la induces transcription of genes that lead to a hypoxic response. Deletion of Cl 6 has been shown to result in faster pulmonary recruitment and activation of CD8+ and CD4+ T-cells (Albamaz et al. (2018) Viruses 10, 101).
N2
Protein N2 is an intracellular immunomodulatory protein with a Bcl-2 fold that inhibits IRF3 activation by an unknown mechanism. Deletion of N2 from VACV strain WR resulted in decreased virulence and increased pulmonary cell infiltration (Albamaz etal. (2018) Viruses 10, 101).
Protein 169
Protein 169 is an intracellular immunomodulatory protein that suppresses the immune response by inhibiting the initiation of cap-dependent and cap-independent translation (Albamaz et al. (2018) Viruses 10, 101).
Protein A35
Protein A35, which is encoded by A35R, is an intracellular immunomodulatory protein that restricts antigen presentation to T-cells via MHC class n molecules. A3SH deletion mutants were attenuated, and resulted in lower VACV-specific antibodies, decreased IFN-y secretion and decreased lysis by splenocytes (Albamaz et al. (2018) Viruses 10, 101).
Protein A44
Protein A44 is an intracellular immunomodulator that is a 30-hydroxysteroid dehydrogenase (30-HSD) and promotes virulence. A VACV strain WR mutant lacking protein A44 resulted in an enhanced inflammatory response, increased IFN-y levels, rapid recruitment of CD8+ and CD4+ T-cells, and a stronger cytolytic T-cell response to VACV-infected cells (Albamaz et al. (2018) Viruses 10, 101). vGAAP
The viral Golgi anti-apoptotic protein (vGAAP) is a hydrophobic protein that localizes predominantly to the Golgi and inhibits apoptosis. VACV vGAAP inhibits both the intrinsic and extrinsic apoptotic pathways, induced by staurosporine, TNFo/cycloheximide (CHX), Fas antibodies, doxorubicin, cisplatin, and C2 ceramide, as well as apoptosis induced by the overexpression of Bax. vGAAP forms ion channels that result in the leakage of Ca2+, reducing its concentration in the Golgi apparatus, and affecting apoptotic pathways that are mediated by the release of Ca2+ (Nichols et al.
(2017) PHUWS 9, 215).
G. EEV VACCINIA VIRUSES CAN BE MODIFIED TO EXPRESS
HETEROLOGOUS GENES AND/OR PAYLOADS
Any of the viruses provided herein, the EEV viruses (the S-R EEVs, such as the RT viruses), and the EEV viruses (designated IV-EEV) that comprise the chimeric transmembrane proteins to display inhibitors or regulators of the host immune response can be modified to encoded and deliver a payload. Payloads include therapeutic products and detectable products for monitoring therapy and/or diagnosis.
An engineered virus or viral genome can comprise a deletion of one or more endogenous genes, one or more heterologous genes, and comprise one or more expression construct or polynucleotide sequence that can be inserted into the viral genome, for example, through homologous recombination or any other suitable mechanism known in the art. The genome of any transgenic or recombinant vaccinia virus can be further modified to comprise any modification to enhance EEV production, systemic spread, and resistance to humoral immunity. For example, any transgenic or recombinant vaccinia virus can be further modified to comprise an A34R mutation, such as the mutation KI 5 IE, or one or more mutations in A33R, A34R, A56R and B5R to enhance EEV production, systemic spread, and resistance to complement.
The genome of any transgenic or recombinant vaccinia virus EEV can be further modified to overexpress a regulator of complement activation (RCA) or complement regulatory protein (CRP) or a functional portion thereof, or other humoral immunity modulator that results in increased serum resistance or stability, on the EEV outer membrane, to evade complement and to enable the replication of complement resistant EEV viruses.
In some embodiments, the EEV outer membrane can comprise one or more modified EEV membrane proteins, such as a modified EEV membrane protein covalently linked to a regulator of complement activation (RCA) or complement regulatory protein (CRP) or a functional portion thereof to evade complement and to enable the replication of complement resistant EEV viruses. In some embodiments, the genome of any transgenic or recombinant vaccinia virus can be modified to comprise an A34R/K151E mutation and can be modified to overexpress CRP such as CD55/DAF or a functional portion thereof. In some embodiments, the genome of any transgenic or recombinant vaccinia virus can be modified to comprise an A34R/K151E mutation and can be modified to overexpress a modified EEV protein such as B5R covalently linked to a complement regulatory protein (CRP) or regulator of complement activation (RCA), such as CDS 5/D AF or a functional portion thereof.
Immune Checkpoint Inhibitors and other immune regulatory proteins
Immune checkpoint inhibitors are immune suppression antagonists that are critical for the maintenance of self-tolerance, but that can be overexpressed by tumors to evade detection by the immune system (Meyers et al. (2017) Front. Oncol. 7: 114). Programmed cell death protein 1 (PD-1; also known as CD279) and its cognate ligand, programmed death-ligand 1 (PD-L1; also known as B7-H1 and CD274), are two examples of numerous inhibitory “immune checkpoints,” which function by downregulating immune responses. For example, upregulation of PD-1 on T cells, and its binding to PD-L1, which is expressed on both antigen presenting cells (APCs) and tumor cells, interferes with CD8+ T cell signaling pathways, impairing the proliferation and effector function of CD8+ T cells, and inducing T cell tolerance. Anti-PD-1 antibodies (for example, pembrolizumab, nivolumab, pidilizumab) and anti-PD-Ll antibodies (for example, atezolizumab, BMS-936559, avelumab (MSB0010718C) and durvalumab) can be expressed by the viruses herein to enhance the antitumor effect.
Another inhibitory immune checkpoint is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4; also known as CD 152), which is expressed on T cells and binds to and inhibits co-stimulatory receptors on APCs, such as CD80 or CD86, out-competing the co-stimulatory cluster differentiation 28 (CD28), which binds the same receptors, but with a lower affinity. This blocks the stimulatory signal from CD28, while the inhibitory signal from CTLA-4 is transmitted, preventing T cell activation (see, Phan et al. (2003) Proc. Natl. Acad. Set. U.S.A. 100:8372-8377). Inhibition of CTLA-4 enhances immune responses mediated by CD4+ T helper cells, and leads to the inhibition of the immunosuppressive effects of Tregs. (Pardoll, D. M. (2012) Nat. Rev. Cancer 12(4):252- 264). Anti-CTLA-4 antibodies, such as ipilimumab and tremelimumab, can be encoded by the viruses herein to enhance the antitumor effect. Lymphocyte-activation gene 3 (LAG3; also known as CD223) is another T-cell associated inhibitory molecule, which is expressed by T cells and NK cells following MHC class II ligation, and has a negative regulatory effect on T cell function. Monoclonal antibodies against LAG-3 can be used to inhibit LAG-3. Additionally, LAG- 3-Ig fusion protein (IMP321, Immutep®), a soluble form of LAG-3 that upregulates costimulatory molecules and increases IL- 12 production, enhancing tumor immune responses, has been shown to increase tumor reactive T cells in clinical trials (Marin- Acevedo et al. (2018) Journal of Hematology & Oncology 11 :39).
T cell immunoglobulin and mucin-domain containing-3 (TIM-3, also known as hepatitis A virus cellular receptor 2 (HAVCR2)), is a direct negative regulator of T cells that is expressed on NK cells and macrophages and promotes immunosuppression by inducing expansion of myeloid-derived suppressor cells (MDSCs). Monoclonal antibodies against TIM-3, such as MBG453, can increase T cell proliferation and cytokine production (Marin- Acevedo et al. (2018) Journal of Hematology & Oncology 11:39).
V-domain Ig suppressor of T cell activation (VISTA), also known as programmed death- 1 homolog (PD-1H), suppresses T cell activation and proliferation and cytokine production. Studies have shown that blocking VISTA increases TIL activation and enhances tumor-specific T cell responses. Monoclonal antibodies against VISTA (for example, JNJ-61610588) and inhibitors (for example, the oral inhibitor CA- 170) are being investigated in clinical trials (Marin- Acevedo et al. (2018) Journal of Hematology & Oncology 11 :39).
B7-H3 (also known as CD276) is expressed on APCs, NKs, B cells and T cells, and inhibits T cell activation and proliferation, as well as cytokine production. B7-H3 is overexpressed in several types of cancer, including melanoma, NSCLC, prostate, pancreatic, ovarian and colorectal cancer. Enoblituzumab (MGA271), a humanized monoclonal antibody against B7-H3, and 8H9, an anti-B7-H3 antibody labeled with radioactive iodine, have shown anti-tumor activity (Marin- Acevedo et al. (2018) Journal of Hematology & Oncology 11:39).
B- and T-lymphocyte attenuator (BTLA, or CD272) is an inhibitory receptor that is expressed by the majority of lymphocytes, that, when bound by its ligand, herpes virus entry mediator (HVEM), blocks B and T cell activation, proliferation and cytokine production (Marin-Acevedo et al. (2018) Journal of Hematology & Oncology 11 :39).
Killer-cell immunoglobulin-like receptors (KIRs, also known as CD158) are expressed by NK and T cells and decrease lymphocyte activation, cytotoxic activity and cytokine release. Antibodies against KIRs include lirilumab and IPH4102 (Marin- Acevedo etal. (2018) Journal of Hematology & Oncology 11:39).
Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme that is involved in immunosuppression and is overexpressed in several tumor types, including melanoma, chronic lymphocytic leukemia, ovarian cancer, CMC and sarcomas. IDO inhibitors can be used in immune checkpoint therapy, and include BMS-986205, indoximod and epacadostat (Marin- Acevedo et al. (2018) Journal of Hematology & Oncology 11:39).
The adenosine receptor A2aR inhibits T cell responses, and its deletion has been shown to enhance inflammatory responses to infection. A2aR can be inhibited by antibodies that block adenosine binding, or by adenosine analogs (Pardoll, D. M. (2012) Nat. Rev. Cancer 12(4):252-264).
Other inhibitory immune checkpoint molecules that can be targeted for cancer immunotherapy by the viruses herein include, but are not limited to, signal regulatory protein a (SIRPa), programmed death-ligand 2 (PD-L2), indoleamine 2,3-dioxygenase (IDO) 1 and 2, galectin-9, T cell immunoreceptor with Ig and HIM domains (TIGIT), herpesvirus entry mediator (HVEM), CTNNB1 (P-catenin), TIM1, TIM4, CD39, CD73, B7-H4 (also called VTCN1), B7-H6, CD47, CD48, CD80 (B7-1), CD86 (B7-2), CD112, CD 155, CD 160, CD200, CD244 (2B4), and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, or CD66a).
While immune checkpoint inhibitors have demonstrated success for anti-cancer therapy in responding patients, many patients do not respond. Reasons for lack of response include the lack of active tumor-specific T cells in the tumor microenvironment. Because oncolytic virotherapy induces antitumor adaptive immunity, oncolytic virotherapy has been combined with immune checkpoint inhibitors. For example, a combination of T-VEC and CTLA-4 inhibition has shown results in the treatment of melanoma (Meyers etal. (2017) Front. Oncol. 7:114). The viruses provided herein can be engineered to express inhibitors of immune checkpoints. Such targets for inhibition include, but are not limited to, PD-1 , PD-L1 , CTLA-4, IDO, TING, LAG3, TIGIT, BTLA, VISTA, ICOS, KIRs, CD39, and PD- 1/VEGF. Immune checkpoint inhibitors can include, for example, antibodies, such as anti -PD-1 antibodies (e.g., pembrolizumab, nivolumab), anti-PD-Ll antibodies (e.g, atezolizumab, avelumab and durvalumab), anti -CTLA-4 antibodies (e.g., ipilimumab), anti-TIM-3 antibodies (e.g., MBG453), and anti-LAG-3 antibodies (e.g., relatlimab/BMS-986016), and functional portions, such as antigen binding portions, thereof.
Co-Stimulatory Molecules
While inhibitory pathways attenuate the immune system, co-stimulatory molecules enhance the immune response against tumor cells. Co-stimulatory pathways thus are inhibited by tumor cells to promote tumorigenesis. Viruses described and provided herein can be modified or engineered to express co-stimulatory molecules, such as, for example, CD27, CD70, CD28, CD30, CD40, CD40L (CD154), CD122, CD137 (4- IBB), 4-1BBL, 0X40 (CD134), OX40L (CD252), OX40L, B7.1/CD80, GITRL, LIGHT, CD226, glucocorticoid-induced TNFR family related gene (GITR), herpes-virus entry mediator (HVEM), LIGHT (also known as TNFSF14), B7-H2, and inducible T-cell costimulatory (ICOS; also known as CD278). It has been shown, for example, that the expression of 4-1BBL in murine tumors enhances immunogenicity, and intratumoral injection of dendritic cells (DCs) with increased expression of OX40L can result in tumor rejection in murine models. Studies have also shown that injection of adenovirus expressing recombinant GITR into B16 melanoma cells promotes T cell infiltration and reduces tumor volumes.
The co-stimulatory molecule B7.1 (CD80) is an integral membrane protein found on activated antigen-presenting cells that binds CD 152 or CD28 on the surface of T cells to produce co-stimulatory signals. Recombinant vaccinia virus expressing B7.1/CD80 (rV-B7.1) was well-tolerated and induced objective partial response was observed in 1 patient and disease stabilization in 2 patients a phase I trial in metastatic melanoma patients. All patients demonstrated an increase in post-vaccination antibody and T cell responses against vaccinia virus (Kaufman, J Clinlnvest. 2005 Jul;115(7):1903-12). Stimulatory antibodies to molecules such as 4- IBB, 0X40 and GITR also can be encoded by the viruses to stimulate the immune system. For example, agonistic anti-4- 1BB monoclonal antibodies have been shown to enhance anti-tumor CTL responses, and agonistic anti-OX40 antibodies have been shown to increase anti-tumor activity in transplantable tumor models. Additionally, agonistic anti-GITR antibodies have been shown to enhance anti-tumor responses and immunity (Peggs et al. (2009) Clinical and Experimental Immunology 157:9-19). As another example vaccinia virus, TRICOM, encodes three co-stimulatory molecules B7.1, ICAM (Intercellular adhesion molecule) and LFA-3 (Lymphocyte function association antigen-3).
0X40 (CD134) is a member of the TNF receptor superfamily, which, together with its ligand (OX40L) results in the activation, potentiation, proliferation and survival of T cells, as well as the modulation of NK cell function. Agonistic monoclonal antibodies can be used to activate 0X40, increasing antitumor activity by the immune system. These include, for example, MOXR 0916, PF-04518600 (PF-8600), MEDI6383, MEDI0562, MEDI6469, INCAGN01949 and CSK3174998 (Marin-Acevedo et al. (2018) Journal of Hematology & Oncology 11:39). Other TNF superfamily member protein include TRAIL, Fas ligand, LIGHT (TNFSF-14), TNF-alpha, and 4-1BB ligand.
Glucocorticoid-induced TNFR family-related protein (GITR), is a co-stimulatory cell surface receptor that is expressed by T and NK cells, and whose expression increases after T cell activation. Its ligand, GITRL, is expressed by APCs and endothelial cells, and plays a role in the upregulation of the immune system, leukocyte adhesion and migration. Agonistic GITR antibodies include TRX-518, BMS-986156, AMG228, MEDI1873, MK-4166, INCAGN01876 and GWN323 (Marin-Acevedo et al. (2018) Journal of Hematology & Oncology 11:39).
Inducible T-cell co-stimulator (ICOS; also known as CD278), which is primarily expressed by CD4+ T cells, is a co-stimulator of proliferation and cytokine production. Agonistic antibodies of ICOS include JTX-2011, GSK3359609 and MEDI-570 (Marin- Acevedo et al. (2018) Journal of Hematology & Oncology 11 :39).
4-1BB (CD 137) is an inducible co-stimulatory receptor that is expressed by T cells, NK cells and APCs, which binds its ligand, 4-1BBL to trigger immune cell proliferation and activation. Anti-4-lBB agonists have been shown to increase immune- mediated antitumor activity, and include utomilumab (PF-05082566) and urelumab (Marin-Acevedo et al. (2018) Journal of Hematology & Oncology 11 :39). As another example, vaccinia vims engineered to express 4-1BB ligand (rV-4-lBBL) improved antitumor responses compared with parent transgenic rV-LacZ vims in a B 16 melanoma model. rV-4-lBBL promoted MHC I expression, reduced antiviral antibody titers, promoted viral persistence, and rescued effector memory CD8+ T cells, leading to significant improvement of the therapeutic effectiveness in the context of host lymphodepletion.
CD27 is a member of the TNF receptor family, which, after binding its ligand, CD70, results in the activation and differentiation of T cells into effector and memory cells and the boosting of B cells. Agonistic CD-70 antibodies include ARGX-110 and BMS-936561 (MDX-1203), and agonistic CD27 antibodies include varlilumab (Marin- Acevedo et al. (2018) Journal of Hematology & Oncology 11 :39).
CD40 is another member of the TNF receptor family. CD40 is expressed by APCs and B cells, while its ligand, CD154 (CD40L), is expressed by activated T cells. Interaction between CD40 and CD154 stimulates B cells to produce cytokines, resulting in T cell activation and tumor cell death. Monoclonal antibodies against CD40 include CP-870893 (agonistic), APX005M (agonistic), ADC-1013 (agonistic), lucatumumab (antagonistic), Chi Lob 7/4 (agonistic), dacetuzumab (partial agonist), SEA-CD40 (agonistic) and R07009789 (agonistic) (Marin- Acevedo et al. (2018) Journal of Hematology & Oncology 11 :39).
Interleukins and chemokines
Interieukin-2 (IL-2), which was the first cytokine approved for the treatment of cancer, is implicated in the activation of the immune system by several mechanisms, including the activation and promotion of CTL growth, the generation of lymphokine- activated killer (LAK) cells, the promotion of Treg cell growth and proliferation, the stimulation of TILs, and the promotion of T cell, B cell and NK cell proliferation and differentiation. Recombinant IL-2 (rIL-2) is FDA-approved for the treatment of metastatic renal cell carcinoma (RCC) and metastatic melanoma (Sheikhi etal. (2016) Iran J. Immunol. 13(3): 148-166). Viral delivery of IL-2 acts to increase the amount of tumor specific T-cells while limiting the severe side effects associated with systemic IL- 2 administration. Several recombinant vaccinia viruses that express IL-2 or variants thereof include for example wDD-EL-2 RG, which expresses at the tumor cell surface a cell-membrane bound IL-2 in addition to TK and VGF deletions. Treatment of mice baring colon cancer tumors resulted in tumor regression and activation of tumor-specific T cells (Liu, Z. et al. Nat. Comm. (2018), 9, 4682). As another example, the vaccinia virus TG-1031, which expresses human IL-2 and epithelial membrane antigen Mucin 1 (MUC 1). Vaccination in patients with recurrent breast cancer was shown to stimulate the immune system resulting in tumor regression in a subset of patients (Scholl et al. J. Biomed. Biotechnol. (2003) 194-201).
IL-10 is a cytokine that results in the inhibition of secretion of proinflammatory cytokines such as IFNy, TNFo, IL-113 and IL-6, and the inhibition of expression of MHC molecules and co-stimulatory molecules, which results in the inhibition of T cell function. Studies have shown that IL- 10 induces the activation and proliferation of CDS, resulting in an antitumor effect. Studies using AM0010, a PEGylated recombinant human IL-10, in combination with pembrolizumab (anti-PD-1 antibody) in melanoma patients (Marin-Acevedo et al. (2018) Journal of Hematology & Oncology 11 :39). Exemplary IL- 10 -armed vaccinia viruses include WLATK-IL-10 which is a TK-deleted replicating lister virus expressing IL-10. WLATK-IL-10 demonstrated superior anti-tumor activity compared to the unarmed WLATK in mice bearing pancreatic ductal adenocarcinoma tumors (Chard, L. S. etal. Clin. Cancer Res. (2015), 21, 405-416).
IL- 12, which is secreted by antigen-presenting cells, promotes the secretion of IFN-y by NK and T cells, inhibits tumor angiogenesis, results in the activation and proliferation of NK, CD8+ T cells and CD4+ T cells, enhances the differentiation of CD4+ ThO cells into Thl cells, and promotes antibody-dependent cell-mediated cytotoxicity (ADCC) against tumor cells (Sheikhi et al. (2016) Iran J. Immunol. 13(3): 148-166). IL-12 has been shown to exhibit antitumor effects in murine models of melanoma, colon carcinoma, mammary carcinoma and sarcoma (Lee, S. and Margolin, K. (2011) Cancers 3:3856-3893). IL-12 expressing vaccinia viruses include, for example, ivKT0327mIL-12.
IL-15 enhances antitumor immunity by activating NK and CD8+ T cells and induces long-term antitumor immunity by activating memory T cells. (Sheikhi et al. (2016) Iran J. Immunol. 13(3): 148-166). Although IL-15 is related to IL-2, it functions through alternative pathways in adaptive immune responses in which its bioactivity in vivo in which IL-15 is presented in a complex with the o-subunit of soluble IL-15 receptor to target cells such as NK, NKT and T cells, rather than binding membranebound IL-15 receptor. Soluble IL-15-IL-15Ra (IL-15 superagonist) complexes enhance the bioavailability and IL- 15 half-life in vivo and can revive tumor-resident CD8+ T cells (Epardaud M. et al. Cancer Res. (2008), 68, 2972-2983). The vacdnia virus wDD-IL- 15Ra is a TK and VGF defident vaccinia virus that encodes the IL-15 super agonist, IL- 15-IL-15Ra. wDD-IL-15Ra demonstrated anti-tumor activity in mice with ovarian cancer and colon adenocarcinoma, increased survival and activation of T- andNK-cells (Kowalsky, S. J. etal. Mol. Ther. (2018) Oct 3; 26(10): 2476-2486).
IL-21, which is produced by activated CD4+ T cells, promotes the proliferation of CD4+ and CD8+ T cells and enhances CD8+ T cell and NK cell cytotoxicity. IL-21 has demonstrated antitumor effects in murine models of melanoma (Lee, S. and Margolin, K. (2011) Cancers 3:3856-3893). Exemplary vaccinia virus encoding IL-21 include, but are not limited to, rll VATK-IL21 and WATK-STCAN1L-IL21.
Inhibitors against Immunosuppressive or pro-tumorigenic cytokine/growth factors
Included are inhibitors against Immunosuppressive or pro-tumorigenic cytokine/growth factors, such as IL-4, IL-6, IL-10, IL-11, IL-13, IL-17, IL-32, IGF, TGF-P, VEGF, PGF, and chemokines, including CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11.
Cytokines and Chemokines
Vaccinia viruses, such as RT vaccinia viruses or EEV produced therefrom, can be modified, or engineered to encoded or express a cytokine by the insertion of one or more heterologous nucleic acids inserted into the viral genome. In some embodiments, the heterologous nucleic add comprises a transgene encoding a cytokine protein to improve anti-tumor immune responses. In some embodiments, the inserted nucleic add comprises a transgene encoding a cytokine. In some embodiments, the cytokine can be selected from among: GM-CSF, IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-y, IFN-o, IFN-P, TNF-a, TGF-p.
Oncolytic viruses encoding and expressing these cytokines, such as GM-CSF, EL- 2, IL-6, IL-12, IL-15, IL-23, and IL-24, have demonstrated anti-cancer effects and safety in clinical and preclinical studies. These viruses include, for example, rV-GM-CSF; W- GMCSF-Lact; JX-594 (Park, B-H, et al. Lancet Oncol. 2008; 9:533^42); JX-954 (Parato etal. (2012) Mol Ther. 20:749-58); ASP9801 (Nakao, S„ etal. Sci TranslMed 2020;! 2: eaax7992); VG9-IL24 (Deng, L., etal. Virol J. 2022;! 9:44) and those described in: U.S. Patent Nos. 8,980,246; 9,180,149; 9,226,977; and 9,919,062; US Patent publications Nos.: 2010/0303714 ; US 2018/0256751; US 2020/0009269; and US 2021/0322578. For example, clinical trials have been performed with JX-594 in various cancers in adult and the virus is generally well-tolerated.
Chemokines are chemotactic cytokines that regulate the trafficking and positioning of cells by activating the seven-transmembrane spanning chemokine receptors. Chemokines can be divided into four subfamilies based on the position of the first two N-terminal cysteine residues, including the CC, CXC, CX3C and XC subfamilies. Differential expression of chemokine receptors on immune cells results in the selective recruitment or homing of immune cells to an appropriate location to effect an immune response. Chemokines are essential coordinators of cellular migration and cell-cell interactions and therefore have great impact on tumor development and on the composition of the tumor microenvironment. In addition to their primary role as chemoattractants, chemokines, in many cases, are also involved in other tumor-related processes, including tumor cell growth, angiogenesis and metastasis. Tumor cells have been shown to acquire the ability to produce growth-promoting chemokines. For instance, melanoma has been found to express chemokines, including CXCL1, CXCL2, CXCL3, CXCL8, CCL2 and CCL5, which have been implicated in tumor growth and progression. In some embodiments, the viruses described herein can be engineered to express chemokines, such as, for example, RANTES, CCL2, CCL5, CCL19 and CXCL11.
Inhibitors against Immunosuppressive and/or pro-tumorigenic cytokine/growth factors
These include, but are not limited to, IL-4, IL-6, IL-10, IL-11, IL-13, IL-17, IL- 32, IGF, TGF-P, VEGF, PGF, CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11.
Immunocytokines
These include, but are not limited to, CCL21, IL-1, IL-2, IL-3, IL-7, IL-12, IL-15, IL-15/IL15a receptor complex (referred to as IL 15 superagonist) and variants thereof, IL-18, IL-21, IFN-o, IFN-P, IFN-y, TNF-o, EPO, GM-CSF, G-CSF, Flt3L, FGF, EGF, IL-4, IL-6, IL-10, IL-11, IL-13, IL-17, IL-32. The IL-15 superagonist is Other IL-15 receptor complex polypeptides comprise the IL-15 sushi domain linked, via a linker, to a modified IL-15, such as, for example, IL-15Ra sushi-domain-linker-IL-15N72D, which is an IL-15 superagonist, where the Sushi domain is
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH
WTTPS LKCIRDPALVHQRPAPPSTVTTAGV (SEQ ID NO:635) the linker is SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO:636), and IL-15 N72D is
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIHDTVENLnLANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS (SEQ ID NO:637). See also U.S. Patent No. 10858452, which describes H-15 sushi domain- linker- IL-15 where Q108 is modified. See also, US publications 20180258174, 20180155439 US 20160318986, 20180312560, 20210147503, 20240083963, and 20090238791, and U.S. Patent Nos. 10626155, 1089981, 10358477. Variations in such sequences and linkers are known in the art.
Fms-like tyrosine kinase-3 ligand (FTL3L)
Integrin alphavbeta3 has a role in enhancing beta-catenin signaling in acute myeloid leukemia harboring Fms-like tyrosine kinase-3 internal tandem duplication mutations. Flt3 ligand (FLT3L) is a cytokine that promotes the growth and development of hematopoietic stem cells, progenitor cells, and the immune system. FLT3L binds to the Flt3 tyrosine kinase receptor on the outer membrane of certain cell types, which activates the FLT3 protein and a series of signaling pathways. FLT3L is expressed in various human and mouse tissues, including bone marrow, thymic fibroblasts, and endothelial cells. FLT3 ligand is s a transmembrane protein with an extracellular domain, a transmembrane segment, and a cytoplasmic tail; it promotes the proliferation, survival, and differentiation of hematopoietic stem cells. Interactions include working synergistically with other hematopoietic growth factors, such as IL-3, IL-6, IL-11, IL-12, KIT Ligand, and GM-CSF.
Interferons
Type I interferons (IFNs), including IFN-a and IFN-P, are secreted by almost all cell types and are potent immunomodulators with anti-proliferative and pro-apoptotic effects on tumors. Type I IFNs induce the expression of MHC class I molecules on tumor cell surfaces, mediate DC maturation, activate cytotoxic T lymphocytes (CTLs), NKs and macrophages, can have anti-angiogenic effects on tumor neovasculature, and can exert cytostatic and apoptotic effects on tumor cells (Lee, S. and Margolin, K. (2011) Cancers 3:3856-3893).
IFN-a, such as the interferon Intron®ZRoferon®-A, is approved for the treatment of hairy cell leukemia, malignant melanoma, AIDS-related Kaposi's sarcoma, and follicular Non-Hodgkin’s lymphoma, and is also used in the treatment of chronic myelogenous leukemia (CML), renal cell carcinoma, neuroendocrine tumors, multiple myeloma, non-follicular Non-Hodgkin’s lymphoma, desmoid tumors and cutaneous T- cell lymphoma.
TFN-y, a type II IFN, is secreted by NK cells, NKT tells, CD4+ T cells, CD8+ T cells, APCs and B cells. IFN-y activates macrophages, induces the expression of MHC class I and II molecules on APCs, promotes Thl differentiation of CD4+ T cells and activates the JAK/STAT signaling pathway. Additionally, IFN-y has anti- angiogenic properties, has been shown to be cytotoxic to some malignant cells, and can regulate the anti-tumor activities mediated by other cytokines, such as IL-2 and IL- 12 (Lee, S. and Margolin, K. (2011) Cancers 3:3856-3893).
TNF-alpha
Tumor necrosis factor alpha (TNF-a) is produced by activated macrophages, T cells and NK cells and exhibits antitumor activity via the induction of apoptosis by binding to tumor cell surface receptors, the blocking of T-Reg cells and the activation of macrophages and NK cells, the disruption of tumor vasculature and prevention of angiogenesis, the attraction and stimulation of neutrophils and monocytes, the promotion of tumor associated macrophages to the Ml antitumor stage, and the downregulation of IL- 13 expression by eosinophilic-like cells and inhibition of tumor induced monocyte differentiation to immunosuppressive phenotypes. Clinical trials using systemically administered TNF-a were limited by dose-limiting toxicides, but studies in which TNF-a was administered intratumorally have been successful in the treatment of Kaposi's sarcoma and liver metastases, for example (Josephs et al. (2018) J. Transl. Med. 16:242).
TGF-beta
TGF-P is a cytokine that can promote the differentiation of inflammatory T cells such as T-helper 17 (Thl7), Th9 and resident memory T cells (Trm), and promotes the survival of CD4+ and CD8+ T cells (Dahmani and Delisle (2018) Cancers 10(6): 194). Bispecific T-Cell Engagers
Bi-specific T cell engager (BITE®) constructs are a class of artificial bispecific monoclonal antibodies that are used in cancer immunotherapy, and are formed by linking two single chain variable fragments (scFv), such that one scFv binds CD3 on the surface of cytotoxic T cells and the other binds a specific tumor-associated antigen. BiTEs® thus target T cells to tumor cells, stimulating T cell activation, cytokine production and tumor cell cytotoxicity independently of MHC class I or co-stimulatory molecules. The bispecific T-cell engagers (available under the trademark BiTEs®) in clinical trials include blinatumomab (MT- 103), which is has been developed for the treatment of NonHodgkin’s lymphoma and acute lymphoblastic leukemia, and is directed towards CD 19; solitomab (MT110), which is for the treatment of gastrointestinal and lung cancers and is directed towards the EpCAM antigen; MT-111, which targets carcinoembryonic antigen (CEA) and has been developed the treatment of gastrointestinal adenocarcinoma; and BAY2010112 (AMG112), which targets prostate-specific membrane antigen (PSMA) and has been developed for the treatment of prostate cancer. Catumaxomab (sold under the trademark Removab®) is a bi-specific rat-mouse hybrid monoclonal antibody which targets CD3 and EpCAM and is used in the treatment of malignant ascites. Other bi- specific T-cell engagers include those targeting EGFR, EphA2, Her2, ADAM 17/T ACE, prostate stem cell antigen (PSCA) and melanoma-associated chondroitin sulfate proteoglycan (MCSP) (Huehls etal. (2015) Immunol. Cell Biol. 93(3):290-296).
Vaccinia viruses provided herein can be modified or engineered to express bispecific T-cell engagers targeting, for example, an immune cell and a tumor antigen or marker to enhance intratumor T-cell infiltration, modulate the immune suppressive microenvironment and enhance antitumor immunity tumors. For example, recombinant vaccinia viruses have been engineered to encode single-chain variable fragments (scFv) specific, CD3 and tumor antigens to for example EphA2 (Yu F, et al. Mol Ther. 2014 22(1): 102-11), EpCAM (Wei M, et al. Front Immunol. 2022 13: 1017574) and CD19 (Lei W, et al. Blood Cancer J. 2022 12(2): 35).
Angiogenesis Inhibitors / Tumor Blood Vessels Reprogramming / Vascular Normalization
In some embodiments, the viruses provided herein can encode angiogenesis inhibitors (for tumor blood vessels reprogramming, e.g.). Angiogenesis is a well-known contributor to the progression of cancer, and angiogenesis inhibitors, particularly when used in combination with other anti-cancer therapies, can be used to prevent the formation of new blood vessels during cancer therapy, thereby blocking the supply of nutrients and/or oxygen to the tumor. Proteins that act as angiogenesis activators, and can be targeted by angiogenesis inhibitors, include vascular endothelial growth factor (VEGF; e.g., (VEGF A, VEGFB), vascular endothelial growth factor receptor (VEGFR2), basic fibroblast growth factor (bFGF, FGF2), angiogenin, transforming growth factor (TGF)-a, TGF-P, tumor necrosis factor (TNF)-a, platelet derived endothelial growth factor, granulocyte colony-stimulating factor (GM-CSF), interleukin-8 (IL-8), hepatocyte growth factor (HGF), angiopoietin (e.g., ANGPT-1, ANGPT-2), placental-derived growth factor (PDGF) and PDGF receptor (PDGFRa), and epidermal growth factor (EGF) (Rajabi, M. and Mousa, S.A. (2017) Biomedicines 5, 34; Kong etal. (2017) Int. J. Mol. Sci. 18(8): 1786).
Studies indicate that in addition to blocking blood vessel formation, cancer immunotherapy using angiogenesis inhibitors can be enhanced by their effects on stabilizing and/or normalizing tumor vasculature (such doses can sometimes be lower than doses that block blood vessel formation) (see, e.g., Huang et al., Cancer Res., 73(10):2943-2948 (2013); Matuszewska eta/., Clin. Cancer Res., 25(5): 1624-1638 (2019); Lanitis et al, Curr. Opin. Immunol., 33:55-63 (2015); and Bykov et al., Clin. Cancer Res., 25(2): 1446-1448 (2019), the contents of which are incorporated in their entirety by reference herein). To meet oxygen and nutrient demands, tumors initiate a version of angiogenesis by secreting factors such as VEGF in response to hypoxia, oncogenes and the loss of tumor suppressor genes. The resulting blood vessels are marked by structural abnormalities such as irregular branching, loss of basement membrane integrity, and inadequate or absent perivascular cells, leading to inefficiencies in the delivery of cancer therapies to the tumor core. In addition, the limited vascular perfusion in the tumors selects for hypoxia and acidity in the tumor microenvironment, which can limit the effectiveness of a therapeutic agent and exacerbate metastasis, (see, e.g., Matuszewska etal., Clin. Cancer Res., 25(5): 1624-1638 (2019), and references cited therein). Furthermore, endothelial cells lining the vessels can suppress T cell activity, target them for destruction and block them from gaining entry into the tumor through the deregulation of adhesion molecules (Lanitis etai, Curr. Opin. Immunol., 33:55-63 (2015).
The efficacy of OV (oncolytic virus) therapy can be increased by downregulating I inhibiting angiogenesis and/or upregulating anti-angiogenesis. While the administration of OV therapy as a single agent can be effective at reducing tumor growth, administration of the virus initiates a vascular shutdown. Previously, the shutdown was viewed as a potential benefit that maximized direct oncolysis by facilitating sequestering of the virus. OV therapy often is administered in multiple doses for optimum efficacy; when vascular disruption is induced, it can impair the uptake of subsequent doses of the OV. In addition, the delivery of immune cells to the tumor site can become impaired. (see, e.g., Matuszewska et al., Clin. Cancer Res., 25(5): 1624-1638 (2019), and references cited therein). Matuszewska etal. (Clin. Cancer Res., 25(5): 1624-1638 (2019) found that in an in vivo mouse model of ovarian cancer, co-administering an oncolytic virus (NDV; Newcastle Disease Virus) with 3TSR protein, an anti-angiogenic protein, led to enhanced tumor perfusion, with normalization of vascular structure and reduction of hypoxia within the tumor, which in turn resulted in improved reduction in primary tumor growth, ascites and metastases when compared to either treatment alone.
Included are viruses that encode molecules that inhibit angiogenesis, including those that downregulate pro-angiogenic factors and/or upregulate anti-angiogenic factors. Alternately, or, in addition, the viruses provided herein can be administered in combination with angiogenesis inhibitors. The angiogenesis inhibitors can induce vascular normalization, repairing tumor vasculature (tumor blood vessel reprogramming) by restoring balance in the cascade of signals initiated by the interplay of tumor cells with their local cellular environment.
Anti-VEGF agents (also called VEGF inhibitors) slow the abnormal angiogenesis/growth of blood vessels associated with certain cancers and degenerative diseases. Anti-VEGF agents block or inhibit the vascular endothelial growth factor (VEGF) or by blocking VEGF receptors so that VEGF cannot bind to the VEGF receptor to effect signaling. Exemplary Anti-VEGF agents include antibodies such as Avastin® (bevacizumab) and Lucentis® (ranibizumab).
Direct inhibitors of angiogenesis, which target the endothelial cells in the growing vasculature, include angiostatin, endostatin, arrestin, canstatin and tumstatin. Indirect angiogenesis inhibitors, which target tumor cells or tumor-associated stromal cells, act by blocking the expression or activity of pro-angiogenic proteins. For example, gefitinib is a small molecule EGFR tyrosine kinase inhibitor (TKI) used in the treatment of colon, breast, ovarian and gastric cancers. Bevacizumab (Avastin®) is a recombinant humanized monoclonal antibody against VEGF, which blocks tumor-derived VEGF-A, preventing the development of new blood vessels and resulting in tumor growth inhibition. Other angiogenesis inhibitors include thalidomide (Tmmunoprin), imatinib, lenalidomide, sorafenib (Nexavar®), sunitinib, axitinib (Inlyta®), temsirolimus (Torisel®), pazopanib, cabozantinib, everolimus, ramucirumab (Cyramza®), regorafenib, vandetanib, tanibirumab, olaratumab (Lartruvo®), nesvacumab, AMG780, MEDI3617, vanucizumab, rilotumumab (AMG102), ficlatuzumab, TAK-701, onartuzumab (MetMab), emibetuzumab and aflibercept (Eylea, Zaltrap®) (Rajabi, M. andMousa, S.A. (2017) Biomedicines 5, 34; Kong et al. (2017) Int. J. Mol. Sci. 18(8):1786).
VEGF is a potent angiogenic activator in neoplastic tissues and plays an important role in tumor angiogenesis. For example, studies have shown that: VEGF receptors (VEGFRs) are expressed in leukemia, non-small cell lung cancer (NSCLC), gastric cancer and breast cancer; higher levels of VEGF mRNA are correlated with decreased 5-year survival rates in NSCLC; VEGF-A expression in breast cancer promotes proliferation, survival and metastasis of breast cancer cells; and VEGF-A and VEGF-C overexpression in gastric cancer is associated with poor prognosis, while silencing of VEGF-A and VEGF-C significantly inhibits proliferation and tumor growth. Bevacizumab (Avastin®), which is a recombinant humanized immunoglobulin G (IgG) antibody that inhibits the formation of the VEGF-A and VEGFR-2 complex, was approved by the FDA in 2004 for the treatment of metastatic colorectal cancer in combination with chemotherapy, and is used to treat various other cancers, including metastatic non-squamous NSCLC, metastatic renal cell carcinoma, breast cancer, epithelial ovarian cancer and glioblastoma. Aflibercept (Zaltrap®) is an Fc fusion protein that inhibits the activity of VEGF-A, VEGF-B and Pl GF, and was FDA-approved in 2012 for the treatment of metastatic colorectal cancer that is resistant to, or has progressed following treatment with oxaliplatin. Ramucirumab (Cyramza®) is a fully human monoclonal antibody that inhibits the interaction of VEGFR-2 with VEGF ligands, and was FDA-approved in 2014 for the treatment of advanced gastric or gastroesophageal junction adenocarcinoma and metastatic NSCLC. Tanibirumab is a fully human monoclonal antibody that binds VEGFR-2, blocking its interaction with ligands such as VEGF-A, VEGF-C and VEGF-D (Kong etal. (2017) Int. J. Mol. Sci. 18(8):1786).
In addition to promoting tumor angiogenesis, VEGF is immunosuppressive and can inhibit the function of T cells, increase the recruitment of Tregs and MDSCs, and prevent the differentiation, maturation and activation of DCs. VEGF A was found to enhance the expression of inhibitory checkpoints such as PD-1, CTLA-4, TIM-3 and LAG-3, which was reversed by antibodies against VEGFR2. Thus, antitumor immunity can be enhanced by targeting VEGF/VEGFR. For example, targeting VEGF/VEGFR has been shown to promote T-cell infiltration in the TME. Therapy with bevacizumab was found to increase B and T cell compartments in patients with metastatic colorectal cancer, and improve cytotoxic T-lymphocyte responses in patients with metastatic NSCLC. Bevacizumab was also found to increase the number of DCs and promote their activation. Axitinib, a small molecule inhibitor of VEGFR1, VEGFR2 and VEGFR3, was found to reduce the number and suppressive capacity of MDSCs, and induce differentiation of MDSCs toward an antigen-presenting phenotype. Sorafenib, a multikinase inhibitor that targets VEGFR2, VEGFR3 and PDGFRP, among others, was found to restore the differentiation of DCs. Sunitinib, a tyrosine kinase inhibitor that blocks VEGFR1, VEGFR2, VEGFR3, platelet-derived growth factor receptors a and P, stem cell factor receptor and Flt3, was found to reduce expression of IL-10, Foxp3, PD- 1, CTLA-4 and BRAF, increase the proportion of CD4+ and CD8+ TILs, reduce the number of Tregs, and increase cytotoxic T cell activity against tumor cells in mice. Sunitinib also was found to decrease the number of MDSCs in various tumor models.
Thus, sunitinib can be used to modify the TME, altering cytokine and costimulatory molecule expression profiles and resulting in favorable T-cell activation and Thl responses. Sunitinib in combination with an oncolytic reovirus was shown to significantly decrease tumor burden and increase the lifespan in a pre-clinical murine model of renal cell carcinoma, while the combination of sunitinib with an oncolytic VSV was found to eliminate prostate, breast and kidney malignant tumors in mice (Meyers et al. (2017) Front. Oncol 7:114; Yang et al. (2018) Front. Immunol. 9:978). These results indicate that combination therapy that includes angiogenesis inhibitors with oncolytic viruses can improve anti-cancer therapeutic efficacy.
PDGF/PDGFR signaling is associated with angiogenesis, tumor growth and decreased patient survival, with PDGFR and/or PDGF overexpression being observed in colorectal cancer, prostate cancer and glioblastomas, for example. Small molecules that target PDGFRs include imatinib, sunitinib, regorafenib and pazopanib, which inhibit the activation of PDGFRs and other kinases, such as VEGFR and FGFR. These molecules have been approved for the treatment of metastatic colorectal cancer, metastatic renal cell carcinoma and gastrointestinal stromal tumors. Antibodies targeting PDGF and PDGFR include olaratumab (Lartruvo™), which targets PDGFRa and has been approved by the FDA for the treatment of soft tissue sarcoma (Kong et al. (2017) Int. J. Mol. Sci. 18(8):1786).
Hepatocyte growth factor (HGF), a motility and morphogenic factor, interacts with c-MET and results in various biological responses, such as embryonic development, epithelial branching morphogenesis, wound healing and tumor development. HGF/c- MET signaling is thus a target for cancer therapy. Rilotumumab, a fully human monoclonal antibody, binds HGF, blocking its interaction with c-MET, and resulting in anti-tumor effects such as tumor growth inhibition, tumor regression, apoptosis and abrogation of cell proliferation. Other humanized monoclonal antibodies against HGF include ficlatuzumab and TAK-701 (L2G7), while humanized monoclonal antibodies against c-MET include onartuzumab (MetMab) and emibetuzumab (LY-2875358) (Kong etal. (2017) /nr. J. Mol. Sci. 18(8):1786).
Other Therapeutic Antibodies
Monoclonal antibodies can be used to target antigens expressed by cancer cells for cancer therapy. In certain embodiments, the viruses herein can be engineered to express other therapeutic anti-cancer antibodies, in addition to the angiogenesis inhibitors, BiTEs, and immune checkpoint inhibitors/stimulators discussed above, including, for example, humanized or chimeric monoclonal antibodies, such as, but not limited to alemtuzumab (Campath®; anti-CD52), trastuzumab (Herceptin®; anti-HER2), cetuximab (Erbitux®; anti-EGFR), panitumumab (Vectibix®; anti-EGFR), ofatumumab (Arzerra®; anti-CD20), rituximab (Rituxan®/Mab Thera®; anti-CD20), gemtuzumab ozogamicin (Mylotarg®; anti-CD33), brentuximab vedotin (Adcetris®; anti-CD30), tositumomab (anti-CD20), daratumumab (Darzalex®; anti-CD38); dinutuximab (Unituxin®; anti-GD2); elotuzumab (Empliciti®; anti-SLAMF7); necitumumab (Portrazza™; anti-EGFR); obinutuzumab (Gazyva®; anti-CD20); and pertuzumab (Peg eta®; anti-HER2).
Monoclonal antibodies have been successful in the treatment of several types of cancers, alone and in combination therapies. For example, rituximab, also known as IDEC-C2BB, was the first monoclonal antibody to be approved by the FDA, and is used for the treatment of Non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. Trastuzumab is an FDA-approved monoclonal antibody used to treat HER2+ breast cancer. Additionally, cetuximab is used in the treatment of colorectal cancer, metastatic NSCLC and head and neck cancer; panitumumab is used to treat metastatic colorectal cancer; alemtuzumab is used to treat chronic lymphocytic leukemia (CLL), cutaneous T- cell lymphoma and T-cell lymphoma; ofatumumab is used in the treatment of CLL; gemtuzumab ozogamicin is used in the treatment of acute myeloid leukemia; brentuximab vedotin is used in the treatment of relapsed or refractory Hodgkin’s lymphoma, systemic anaplastic large cell lymphoma and cutaneous T-cell lymphoma; tositumomab, in combination with iodine-labeled tositumomab (Bexxar), is used in the treatment of chemotherapy and rituximab-refractory Non-Hodgkin’s lymphoma; daratumumab is used in the treatment of multiple myeloma, diffuse large B cell lymphoma, follicular lymphoma and mantle cell lymphoma; dinutuximab is used in the treatment of pediatric neuroblastoma; elotuzumab is used in the treatment of multiple myeloma; necitumumab is used in the treatment of metastatic squamous NSCLC; obinutuzumab is used in the treatment of chronic lymphocytic leukemia and follicular lymphoma; and pertuzumab is used in the treatment of HER2+ breast cancer.
Other antibodies include DI (Al 2), which targets the TACE ectodomain, and was shown to inhibit the proliferation and motility of cancer cells in head and neck squamous cell carcinoma; FsnO5O3h, an antibody against Cathepsin S, which has been shown to suppress angiogenesis and metastases in vivo; and ATN-658, an antibody against urokinase plasminogen activator receptor (uPAR), which has been shown to inhibit invasion, metastasis and tumor proliferation and induce apoptosis (Neves and Kwok (2015) BBA CZzzzzcaZ 3:280-288). Reporter Genes
In certain embodiments, the viruses can be engineered to express reporter genes, including imaging molecules/agents, such as, for example, fluorescent proteins (for example, GFP, YFP, RFP, TurboFP635); luminescent proteins (for example, luciferase); and magnetic resonance, ultrasound or tomographic imaging agents, including radionuclides. The viruses herein also can be engineered to express human sodium iodide symporter (hNIS) or aquaporin 1 (AQP1), which facilitate the detection of viruses via deep tissue non-invasive imaging techniques, such as PET, SPECT/CT, y-camera or MRI.
NIS
The Na+/T symporter (NIS) is a transmembrane glycoprotein that mediates the transport of iodide anions into cells, for example, in the thyroid and other tissues, such as salivary glands, the stomach, kidneys, placenta, lactating mammary glands and small intestine. Radioisotopes such as123I,124I,125I,1311 and99mTc are transported via NIS, which, when encoded by the virus, is expressed on the surface of infected cells, allowing for non-invasive imaging, using, for example, PET, SPECT/CT, and y-camera (Msaouel etal. (2013) Expert Opin. Biol. Ther. 13(4):483-502).
NIS is useful as a reporter gene because it accumulates radiolabeled substrates, concentrating and amplifying the signal, can be used to monitor the delivery of other genes and, upon expression in the tumor, can be used to monitor tumor size using diagnostic scintigraphic imaging. For example, adenovirus expressing human NIS (hNIS) has been delivered intranasally into the lungs of rats, and an124I" PET signal was detectable for up to 17 days following administration. A lentiviral vector expressing NIS was used to detect transplanted rat cardiac-derived stem cells with single-photon emission computed tomography (PET) imaging using99mTcC>4" or124I"(Portulano etal. (2014) Endocr. Rev. 35(1): 106-149).
NIS-expressing viruses can be used to combine oncolytic and radiation therapies, which has been shown to enhance oncolytic efficacy pre-clinically. For example, an adenoviral vector expressing NIS under a CMV promoter was injected into the portal vein of hepatocarcinoma-bearing rats, and following131I" therapy, potent inhibition of tumor growth and prolonged survival were observed. Administration of an adenoviral vector expressing NIS under the MUC1 promoter to mice with pancreatic carcinoma resulted in significant tumor regression following131I" treatment (Portulano et al (2014) Endocr. Rev. 35(1):106-149). Vaccinia virus encoding the human NTS gene (W-NIS) has been studied for the treatment and monitoring of endometrial cancer, pancreatic cancer, malignant pleural mesothelioma, colorectal cancer, anaplastic thyroid cancer, prostate cancer and gastric cancer. W-NIS also has been used as a reporter gene to identify positive surgical margins of breast cancer in a murine model with124I microPET imaging (Ravera etal. (2017) Anriu Rev Physiol. 79:261-289).
Oncolytic measles virus (MV) expressing NTS (MV-NIS) for radio- virotherapy with 1-131 also has demonstrated results pre-clinically in multiple myeloma, glioblastoma multiforme, head and neck cancer, anaplastic thyroid cancer, ovarian cancer, pancreatic cancer, mesothelioma, hepatocellular carcinoma, osteosarcoma, endometrial cancer and prostate cancer models. Several Phase I/n clinical trials have investigated the use of MV-NIS in multiple myeloma (NCT00450814, NCT02192775), mesothelioma (NCTO 1503177), head and neck cancer (NCT01846091) and in ovarian cancer using virus-infected MSCs (NCT02068794).
Aquaporin 1 (AQP1)
Aquaporins are integral membrane proteins that mediate the transport of water across the plasma membrane in cells. Human aquaporin 1 (AQP1) can be used as a genetically encoded reporter for diffusion-weighted MRI, and is advantageous due to its non-toxicity, metal-free nature, and sensitivity. Because it is an autologous reporter gene, there is no risk of immunogenicity. Studies have shown that AQP1 enables gene expression imaging in tumor xenografts (Mukherjee et al. (2016) Nature Communications 7: 13891).
Tumor homing proteins.
In other examples, Virus can encode alone or in combination with other payloads, a tumor homing protein, nanobodies or receptors or chimeric receptors. Viral-encoded tumor homing proteins will be expressed byAn infected host cell line and be integrated in the envelop of the virus to redirect or enrich homing to define tissue, TME or tumors.
Tumor homing proteins include, but are not limited to, CXCR1, CXCR2, CXCR3, CXCR4, CCR4, CCR5, CD44, ITGB1, other integrins.
Additionally, virus can encode targeting antibodies against Tumor-associated antigens. Antibodies would be express and attached to the enveloped of the virus. Antibodies against :ADAM9, AFP, ALB, AMHR2, AMIGO2, B7-H3, B7-H4, BCMA, CAIX, CD105, CD142, CD155, CD2, CD22, CD228, CD30, CD33, CD43, CDS, CD7, CD70, CD71, CDCP1, CDH11, CDH17, CDH3, CDH6, CEACAM5, CEACAM6, Claudin 19.2, CLDN6, DLK1, DLL3, EGFR, EPCAM, EPHA2, EPHB2, FAP, FLT3, FOLR1, GP 100, GPC-1, GPRC5D, GUCY2C, HER2, HER3, HLA-G, IL3RA, ITGB6, KIT, KREMEN2, LGR5, LILRB4, LIV-1, LRRC15, LUNX, LYPD3, MET, MSLN, MUC1, MUC16, MUC18, Nectin-4, PALP, PRLR, PSMA, PTK7, RAGE, ROR1, SEZ6, SLC34A2, SSTR2, TIM1, 5T4 (TPBG), TREM2, TROP2, WT1
In other examples, Virus can encode Chimeric proteins consisting of TM of a EEV protein F12L, F13L, A33R, A34R, A36R, A56R, B5R fused to antibody, nanobody, single chain antibody against Tumor-associated antigens
ADAM9, AFP, ALB, AMHR2, AMIGO2, B7-H3, B7-H4, BCMA, CAIX, CD105, CD142, CD155, CD2, CD22, CD228, CD30, CD33, CD43, CDS, CD7, CD70, CD71, CDCP1, CDH11, CDH17, CDH3, CDH6, CEACAM5, CEACAM6, Claudin 19.2, CLDN6, DLK1, DLL3, EGFR, EPCAM, EPHA2, EPHB2, FAP, FLT3, FOLR1, GP100, GPC-1, GPRC5D, GUCY2C, HER2, HER3, HLA-G, IL3RA, ITGB6, KIT, KREMEN2, LGR5, LILRB4, LIV-1, LRRC15, LUNX, LYPD3, MET, MSLN, MUC1, MUC16, MUC18, Nectin-4, PALP, PRLR, PSMA, PTK7, RAGE, ROR1, SEZ6, SLC34A2, SSTR2, TIM1, 5T4 (TPBG), TREM2, TROP2, WT1
Or Chemokines receptors and tumor matrix or TME molecules, CXCR1, CXCR2, CXCR3, CXCR4, CCR4, CCR5, CD44, ITGB1, other integrins.
Tumor antigens
In other examples, the virus can express tumor-associated antigens to express in tumors define set or 1 or multiple tumor-associated antigens to redirect into the tumor ADC, CAR-T or other immunotherapy design against those tumor antigens : ADAM9, AFP, ALB, AMHR2, AMIGO2, B7-H3, B7-H4, BCMA, CAIX, CD105, CD142, CD155, CD2, CD22, CD228, CD30, CD33, CD43, CDS, CD7, CD70, CD71, CDCP1, CDH11, CDH17, CDH3, CDH6, CEACAM5, CEACAM6, Claudin 19.2, CLDN6, DLK1, DLL3, EGFR, EPCAM, EPHA2, EPHB2, FAP, FLT3, FOLR1, GP100, GPC-1, GPRC5D, GUCY2C, HER2, HER3, HLA-G, IL3RA, ITGB6, KIT, KREMEN2, LGR5, LILRB4, LIV-1, LRRC15, LUNX, LYPD3, MET, MSLN, MUC1, MUC16, MUC18, Nectin-4, PALP, PRLR, PSMA, PTK7, RAGE, ROR1, SEZ6, SLC34A2,
SSTR2, TTM1, 5T4 (TPBG), TREM2, TROP2, WT1
H. CHIMERAS AND FUSIONS VIRALLY ENCODED MEMBRANE
PROTEINS AND PROTEINS THAT INHIBIT OR MODULATE THE
HUMORAL IMMUNITY OF THE HOST
As discussed, all therapeutic vacdnia viruses can be modified to produce EEVs and to also encode an immune regulating/inhibiting protein, such as a complement regulatory protein, or an immune regulating or inhibiting portion thereof on the EEV outer membrane.
The extracellular enveloped virus (EEV) form of vaccinia virus is responsible for viral spread within the infected host while being relatively easy to degrade outside of the host. EEV is relatively resistant to complement compared to IMV owing to the acquisition of a second outer membrane which contain Complement regulatory proteins (CRPs, also referred to as regulators of complement activation (RCAs)) acquired from an infected host cell making it an ideal viral form for systemic oncolytic viral therapy.
Provided herein are modified or engineered vaccinia viruses that overexpress one or more CRPs (RCAs) on the EEV membrane. The modified or engineered viruses can be vaccinia viruses in which the A34R polypeptide is modified, such as with the mutation K151E (see, U.S. Pat. No. 8,329,164, which describes vaccinia viruses with increased infectivity by virtue of modification of the virus) and other modifications to increase EEV production. For example, the virus can include one or more mutations in A33R, A34R, A56R or B5R to improve EEV production. These viruses can be further modified or engineered to contain one or more nucleic acid molecules, such as a heterologous nucleic add, to express recombinant, therapeutic, and/or immunomodulatory protdns.
The viruses provided herein can be engineered by methods known in the art or as provided herdn. The genome of any vaccinia virus can be modified or engineered to comprise any modification to enhance EEV production, systemic spread, and resistance to humoral immunity. For example, any transgenic or recombinant vaccinia virus can be further modified to comprise an A34R mutation, such as the mutation K151E, or one or more mutations in A33R, A34R, A56R and B5Rto enhance EEV production, systemic spread, and resistance to complement. The genome of any EEV producing vaccinia virus can be further modified overexpress a complement regulatory protein (CRP) on the EEV outer membrane. These regulatory proteins include 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 and IMP, as well as modified sequences thereof, and functional portions thereof, as well as variants thereof that have at least 95% amino acid sequence identity therewith and complement regulatory activity. The chimeric proteins generally, although not necessarily, include the CRAs that are membrane bound, rather than soluble.
The genome of any EEV producing vaccinia virus can be further modified or engineered to comprise one or more modified EEV membrane proteins, such as a modified EEV membrane protein covalently linked to a complement regulatory protein (CRP) or a functional portion thereof to evade complement and to enable the replication of complement resistant EEV viruses. For example, the genome of any transgenic or recombinant vaccinia virus can be modified to comprise an A34R KI 5 IE mutation and can be modified to express a modified EEV protein such as B5R covalently linked to a complement regulatory protein (CRP) such as CD55ZDAF or a functional portion thereof. As noted herein CRPs also are referred to as regulators of complement activation (RCAs)
1. Fusion proteins and Chimeric proteins
Provided are nucleic acid molecules encoding fusion proteins and chimeric proteins containing a virally-encoded EEV transmembrane protein and a protein that inhibits or reduces anti-viral responses or other humoral immune response of the immune system of the host, such as by inhibiting complement. The fusion proteins and chimeric proteins can contain the full length of each and can be provided as fusion proteins, such that, upon expression, the immune response inhibiting protein is displayed on the surface of the EEV membrane, such A33R, A34R, A56R, B5R, A36R,F13L. The fusion/chimeric proteins can contain a sufficient portion or portions of the transmembrane protein to display the immune response inhibiting protein, and contain a sufficient portion of the immune response inhibiting portion to inhibit the immune response, such as to inhibit complement, such as assessed by serum resistance. Details of such fusion and chimeric proteins are provided herein. There are a least six virus-encoded proteins that occur as components of the EEV envelope membrane of vaccinia virus. A33R, A34R, A56R, and B5R are glycoprotein, A36R is a non-glycosylated transmembrane protein, and F13L is a palmitoylated peripheral membrane protein. During infection, these proteins localize to the Golgi complex, where they are incorporated into infectious virus that is then transported and released into the extracellular medium. Other transmembrane proteins expressed in the cells in which the EEVs are manufactured or produced that are incorporated into the EEV membrane also can be employed to display immune response inhibiting proteins or portions thereof.
The fusion/chimeric proteins can contain sufficient portions of one or both of the proteins, whereby the immune response inhibiting protein is displayed on the surface of the EEV. Exemplary of the fusion/chimeric proteins are fusions/chimeras between the EEV transmembrane proteins and a complement regulatory protein, such as, but not limited to, CD55, DAF-2, CD46, CD59, and CD35. The following is a summary of sequences of EEV outer membrane transmembrane proteins and portions thereof, and complement resistance proteins. Exemplary active portions are described. For example, the complement resistance protein or portion thereof can be linked, via peptide bonds, to the N-terminal of B5R, A56R or the C-terminal of A33R or A34R of the protein or the transmembrane domain, or can be inserted into the stalk of such protein.
EEV outer membrane proteins and active portions and encoding nucleic acid from, for example, Wyeth vaccinia virus proteins:
A33R: SEQ ID NOs: 168- 174 set forth sequences of vaccinia virus Copenhagen, Ankara, IHD-J, lister, tian tan, WR and Wyeth DNA and AA sequences;
A34R: SEQ ID NOs: 182-188 set forth sequences of vaccinia virus Copenhagen, Ankara, IHD-J, lister, tian tan, WR and Wyeth DNA and AA sequences;
A56R: 196-202 set forth sequences of vaccinia virus Copenhagen, Ankara, IHD-J, lister, tian tan, WR and Wyeth DNA and AA sequences;
B5R: SEQ ID NOs:210-216 set forth sequences of vaccinia virus Copenhagen, Ankara, IHD-J, lister, tian tan, WR and Wyeth DNA and AA sequences; and F13L - SEQ ID NOs: 224 and 225 set forth WR nucleic acid and encoded amino acid sequences.
Exemplary portions of the transmembrane proteins include the stalk, transmembrane, and intra-membrane domains. For example: SEQ ID NO:232 B5R signal peptide domain (protein) SEQ ID NO:233 B5R stalk domain (protein) SEQ ID NO:234 B5R transmembrane domain (protein) SEQ ID NO:235 B5R C-terminal domain (protein) SEQ ID NO:236 B5R STC (protein) SEQ ID NO:486 B5R signal peptide domain (nucleotide) SEQ ID NO:487 B5R stalk domain (nucleotide) SEQ ID NO:488 B5R transmembrane domain (nucleotide) SEQ ID NO:489 B5R C-terminal domain (nucleotide) SEQ ID NO:490 B5R STC (protein)
The fusion, for example, can be in the stalk, in place of the stalk or a portion thereof such that the fused the immune response inhibiting or portion thereof sufficient to inhibit the immune response, such as a complement protein.
Immune response inhibitor proteins, which include any that inhibit complement, such as complement regulatory proteins, can be fused to an EEV second envelope transmembrane protein so that the immune response protein is displayed. These include, but are not limited to the following proteins and variants thereof, such as those having at least 95% sequence identity thereto and retaining the inhibitory activity: SEQ ID NO:237 human CD55 (DNA) SEQ ID NO:238 Human complement decay-accelerating factor (CD55) isoform 1 preproprotein
CCP (complement control protein) and active potions -CCP domains a: AAs 36..94; 98..280
SEQ ID NO:239 Human complement decay-accelerating factor isoform 2 precursor Active portions/ CCP domains: 36..94; 98..158; 225..284
SEQ ID NO:240 CD46 (DNA) SEQ ID NO:241 Membrane cofactor protein (CD46) isoform 1 precursor
Active portions/ CCP domains: 35..88; 99..158
SEQ ID NO:242 Membrane cofactor protein (CD46) isoform 4 precursor Active portions/ CCP domains: 35..88; 99..158
SEQ ID NO:243 CD59 (DNA)
SEQ ID NO:244 CD59 glycoprotein preproprotein
SEQ ID NO:245 CD35 (DNA) SEQ ID NO:246 CD35/CR1 isoform F precursor
Active portions/ CCP domains: 1650..1706 SEQ ID NO:247 CD35/CR1 isoform S precursor
Active portions/ CCP domains: 2100..2156 SEQ ID NO:248 CD35/CR1 isoform 3
Active portions/ CCP domains: 1650..1706.
An exemplary nucleic acid encoding a chimeric/fusion protein is set forth in SEQ ID NO:252 and 503-507, which encodes B5R (STC)-hCD55 DNA. Functional domains are as follows: Signal peptide- human CD55- B5R stalk- transmembrane domain- C- terminal tail. Complement resistance proteins can be inserted into the stalk or transmembrane domains of A33R, A34R, A56R, B5R or F13L (F13L is associated with the inner side of the EEV outer membrane via palmitoylation).
A skilled person can combine these proteins to produce chimeric and/or fusion proteins that display the complement regulatory portion on the surface of the outer membrane of the EEV.
Nucleic acid encoding these proteins, and other such proteins that confer resistance to host humor immunity, is inserted into the genome of a vaccinia virus, such as one that produces a high level (greater than 1%, 5%, 10%, or 20%, or 30% or more EEVs) than unmodified WK, or than a parental strain that is not selected from higher production of EEVs. The resulting viruses are IV-EEV viruses because they result from a higher producing virus, and they display a host immune system inhibiting protein or portion thereof whereby the virus is therapeutically effective upon systemic administration for treating a cancer. Effectiveness for treatment of cancer can be assessed by standard parameters include symptom-free survival, reduction of tumor size, and/or elimination of tumors. For example, the oncolytic virus, T-VEC was the first oncolytic immunotherapy to demonstrate therapeutic benefit against melanoma in a phase m clinical trial in melanoma, leading to its FDA approval. T-VEC was considered well- tolerated and is therapeutically effective 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.
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. The complement inhibitory protein or regulatory protein is introduced into the EEV transmembrane protein in any locus so that it the protein is produced and the complement inhibitory/regulatory protein is displayed on the surface of the virus when the envelope is produced. It can be linked to the terminus of the transmembrane protein; the selected terminus depends on the orientation of the transmembrane protein in the membrane. The orientation should be such that the fusion protein is expressed to the outside of the CEVZEEV outer membrane.
For example, A33R, A34R, A56R, and B5R are exposed, and F13L is located between the EEV outer envelope and the IMV surface. The N-terminus of B5R, A56R and the C-term of A33R and A34R are exposed to the outside of the EEV. The complement inhibitory protein can be fused to the exposed terminus. Alternatively, it can be inserted into the transmembrane protein so that it is displayed on the EEV surface or in the transmembrane domain. As exemplified herein, the complement inhibitory/regulatory protein can be inserted into the EEV membrane protein at the STALK domain. Exemplary is CD55 inserted into the B5R STALK-TM-C term (STC) or CD55-B5R(STC), and CD55 inserted into the STALK of A33R
3. Domains of the complement inhibitory/regulatory/control proteins
Domains include short consensus repeats (SCR domains; also called complement control protein (CCP) or Sushi domains) inhibit complement. B5R has 4 SCR domains that can be replaced by the 4 SCR domains of CD55, which can be inserted into or fused to the transmembrane domain. It is possible to truncate the and just inserted SCR domain 1 or SCR 1,2 or SCR 1,2 and 3. The configuration and structure is depicted Figure 28A. Figure 28B provides a schematic representation of the expression of a CD55- transmembrane protein (B5R) on the EEV second membrane, and depicts the structure of CD55. This is exemplary of fusion proteins and their expression on the EEV membrane. The N-terminal segment of CD55, which comprises four SCR domains, is fused with the transmembrane region of B5R. Subsequently, this hybrid or chimeric protein is inserted into specific loci — TK, A46R, VGF, or B19R — within the genomes of recombinant viruses. The resulting viruses can be propagated in any cell line in vitro to produce viruses that have high serum resistance. In vivo, these viruses amplify and retain the high serum resistance of the administered virus. As depicted in this figure, the complement resistance protein contains several domains, and a subset thereof can be included in the transmembrane chimeric protein; it is not necessary to insert the full-length protein; a sufficient portion to provide increased serum resistance is required. Sequences of EEV transmembrane proteins and domains thereof are set forth in the following table:
To prepare fusion proteins (or chimeras) the SCR domains of the complement regulatory protein and the signal peptide of the EEV protein, which gets cleaved, is attached the stalk or transmembrane portion of the EEV protein in the appropriate orientation for display on the surface of the EEV.
Figure 28B provides a schematic representation of CDS 5 with another enveloped membrane vaccinia virus protein, A33R. This has a structure similar to the CD55-B5R construct. The extracellular portion of CD55 (SCR1-4) is fused with intracellular and transmembrane portions of A33R protein under control of 3 different promoters, pSE, pSEL, and pSL. The CD55 portion is inserted in other EEV envelope proteins, including
A34, A56, F13.
Donor vector sequence (of the vector in Figure 28B) of CD55-A33R to insert in TK locus:
The Tables below provide sequences of exemplary complement regulatory proteins, EEV transmembrane proteins, exemplary chimeric/fusion proteins of the CRTs with EEV transmembrane proteins, and SEQ ID NOs. Exemplary proteins include
CD35/CR1, CD55, CD59, CD46, Factor H, Vaccinia Virus VCP, Modified vaccinia virus VCP, Variola Virus VCP (SPICE), monkeypox inhibitor of complement enzymes
(MOPICE), Herpesvirus saimiri (HVS) CCPH, C4BPo, C4BPp, Kaposi-sarcoma associated Herpesvirus Kaposica I (KCP), Herpesvirus saimiri (HVS) -CD59 (HVS-
CD59), Rhesus rhadinovirus RCP-H (RCPH), Rhesus rhadinovirus RCP-1 (RCP1),
Murine gamma herpesvirus (yHV-68) RCA, Influenza virus Ml (Ml), ORF 4
[Retroperitoneal fibromatosis-associated herpesvirus], EMICE Full-length, EMICE- truncated, CPXV034 -full, CPXV034-truncated, and CRASP-2 [Borrelia burgdorferi].
Examples of exemplary chimeric (fusion) proteins are set forth in the table below
(see, also SEQ ID NOs: 252, 503-507, and 651-775). In this table, the amino acid single letter codes for CRP residues are depicted with lowercase letters. The amino acid single letter codes for EEV proteins are depicted with uppercase letters where the portions of the transmembrane proteins are represented with bold, underlined, and italicized uppercase letters to depict the stalk region, transmembrane domain, and N- or C- terminal residues, respectively.
L PHARMACEUTICAL COMPOSITIONS, COMBINATIONS, AND KITS
Provided herein are pharmaceutical compositions, combinations and kits containing the EEV viruses provided herein. Pharmaceutical compositions can include EEV and any of the oncolytic viruses provided herein, and a pharmaceutical carrier. The pharmaceutical compositions can be at room temperature to about 37° C, at refrigeration temperatures e.g., from 0-5° C, e.g., 4° C, or under freezing or cryopreservation conditions, e.g., -20 to -80° C. In embodiments, the pharmaceutical compositions are at -80° C.
Combinations can include, for example, an EEV virus; and at least one protein that blocks or inhibits complement that is encoded by the oncolytic virus. Combinations can include any of the viruses provided herein, a virus-encoded protein and a detectable compound; a virus and an additional therapeutic compound; a virus and a viral expression modulating compound, or any combination thereof. Kits can include one or more pharmaceutical compositions or combinations provided herein, and one or more components, such as instructions for use, a device for administering the pharmaceutical composition or combination to a subject, a device for administering a therapeutic or diagnostic compound to a subject or a device for detecting a virus in a subject. A virus contained in a pharmaceutical composition, combination or kit can include any oncolytic virus provided herein. An oncolytic virus contained in a pharmaceutical composition, combination or kit can include any EEV virus provided herein.
Pharmaceutical Compositions
Provided herein are pharmaceutical compositions containing an EEV virus, including any of the viruses provided herein, and a suitable pharmaceutical carrier. A pharmaceutically acceptable carrier includes a solid, semi-solid or liquid material that acts as a vehicle carrier or medium for the virus. Pharmaceutical compositions provided herein can be formulated in various forms, for example in solid, semi-solid, aqueous, liquid, powder or lyophilized form. Exemplary pharmaceutical compositions containing any virus provided herein include, but are not limited to, sterile injectable solutions, sterile packaged powders, eye drops, tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, and suppositories.
Examples of suitable pharmaceutical carriers are known in the art and include, but are not limited to, water, buffers, saline solutions, phosphate buffered saline solutions, various types of wetting agents, sterile solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, gelatin, glycerin, carbohydrates, such as lactose, sucrose, dextrose, amylose or starch, sorbitol, mannitol, magnesium stearate, talc, silicic add, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, and powders, among others. Pharmaceutical compositions provided herein can contain other additives including, for example, antioxidants, preserving agents, analgesic agents, binders, disintegrants, coloring, diluents, excipients, extenders, glidants, solubilizers, stabilizers, tonicity agents, vehicles, viscosity agents, flavoring agents, sweetening agents, emulsions, such as oil/water emulsions, emulsifying and suspending agents, such as acacia, agar, alginic add, sodium alginate, bentonite, carbomer, carrageenan, carboxymethylcellulose, cellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, octoxynol 9, oleyl alcohol, povidone, propylene glycol monostearate, sodium lauryl sulfate, sorbitan esters, stearyl alcohol, tragacanth, xanthan gum, and derivatives thereof, solvents, and miscellaneous ingredients, such as, but not limited to, crystalline cellulose, microcrystalline cellulose, citric acid, dextrin, liquid glucose, lactic acid, lactose, magnesium chloride, potassium metaphosphate, starch, among others. Such carriers and/or additives can be formulated by conventional methods and can be administered to the subject at a suitable dose. Stabilizing agents such as lipids, nuclease inhibitors, polymers, and chelating agents can preserve the compositions from degradation within the body. Other suitable formulations for use in a pharmaceutical composition can be found, for example, in Remington: The Science and Practice of Pharmacy (2005, Twenty-first edition, Gennaro & Gennaro, eds., Lippincott Williams & Wilkins).
Pharmaceutical formulations that include an EEV provided herein for injection or mucosal delivery typically include aqueous solutions of the virus provided in a suitable buffer for injection or mucosal administration or lyophilized forms of the virus for reconstitution in a suitable buffer for injection or mucosal administration. Such formulations optionally can contain one or more pharmaceutically acceptable carriers and/or additives as described herein or known in the art. Liquid compositions for oral administration generally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as com oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
Pharmaceutical compositions provided herein can be formulated to provide quick, sustained or delayed released of an EEV virus as described herein by employing procedures known in the art. For preparing solid compositions such as tablets, an EEV virus provided herein is mixed with a pharmaceutical carrier to form a solid composition. Optionally, tablets or pills are coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action in the subject. For example, a tablet or pill contains an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer, for example, which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials are used for such enteric layers or coatings, including, for example, a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. These liquid or solid compositions optionally can contain suitable pharmaceutically acceptable excipients and/or additives as described herein or known in the art. Such compositions are administered, for example, by the oral or nasal respiratory route for local or systemic effect. Compositions in pharmaceutically acceptable solvents are nebulized by use of inert gases. Nebulized solutions are inhaled, for example, directly from the nebulizing device, from an attached face mask tent, or from an intermittent positive pressure breathing machine. Solution, suspension, or powder compositions are administered, orally or nasally, for example, from devices which deliver the formulation in an appropriate manner such as, for example, use of an inhaler. Pharmaceutical compositions provided herein can be formulated for transdermal delivery via a transdermal delivery devices (“patches”). Such transdermal patches are used to provide continuous or discontinuous infusion of a virus provided herein. The construction and use of transdermal patches for the delivery of pharmaceutical agents are performed according to methods known in the art (see, for example, U.S. Pat. No. 5,023,252). Such patches are constructed for continuous, pulsatile, or on-demand delivery of an EEV virus provided herein.
Colloidal dispersion systems that can be used for delivery of viruses include macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions (mixed), micelles, liposomes and lipoplexes. An exemplary colloidal system is a liposome. Organ-specific or cell-specific liposomes can be used to achieve delivery only to the desired tissue. The targeting of liposomes can be carried out by the person skilled in the art by applying commonly known methods. This targeting includes passive targeting (using the natural tendency of the liposomes to distribute to cells of the reticuloendothelial system (RES) in organs which contain sinusoidal capillaries) or active targeting (for example, by coupling the liposome to a specific ligand, for example, an antibody, a receptor, sugar, glycolipid and protein, by methods known to those of skill in the art). Monoclonal antibodies can be used to target liposomes to specific tissues, for example, tumor tissues, via specific cell-surface ligands.
Generally, the virus is administered in an amount that is at least or about or 1 x 10s pfu at least one time over a cycle of administration. Exemplary minimum levels for administering a virus to a 65 kg human can include at least about 1 x 105 plaque forming units (pfu), at least about 5x 105 pfu, at least about 1 x 106 pfu, at least about 5xl06pfu, at least about lxl07pfu, at least about lxl08pfu, at least about lxl09pfu, or at least about 1 x IO10 pfu. For example, the virus is administered in an amount that is at least or about or is lxl05pfu, lxK^pfu, lxl07pfu, lx108pfu, lx109pfu, lxlOlopfu, 1 x 1011 pfu, 1 x 1012 pfu, 1 x 1013 pfu, or 1 x 1014 pfu at least one time over a cycle of administration.
Combinations
Provided herein are complement resistant EEV viruses and methods of producing complement resistant EEV viruses, e.g., RT, S-R EEV and IV-EEV. A combination with an EEV virus can include a second, third or fourth agent, such as a second virus or other therapeutic or diagnostic agent. A combination can contain pharmaceutical compositions containing an EEV virus provided herein. A combination also can include any reagent for effecting treatment or diagnosis in accord with the methods provided herein such as, for example, an antiviral or chemotherapeutic agent. Combinations also can contain a compound used for the modulation of gene expression from endogenous or heterologous genes encoded by the virus.
Combinations provided herein can contain an EEV virus and a therapeutic compound. Therapeutic compounds for the compositions provided herein can be, for example, an anti-cancer or chemotherapeutic compound. Exemplary therapeutic compounds include, for example, cytokines, growth factors, photosensitizing agents, radionuclides, toxins, siRNA molecules, enzyme/pro E drug pairs, anti-metabolites, signaling modulators, anti-cancer antibiotics, anti-cancer antibodies, angiogenesis inhibitors, chemotherapeutic compounds, anti-metastatic compounds, immune checkpoint inhibitors, co-stimulatory pathway agonists, innate immune agonists, or a combination of any thereof.
EEV viruses provided herein can be combined with an anti-cancer compound, such as a platinum coordination complex. Exemplary platinum coordination complexes include, for example, cisplatin, carboplatin, oxaliplatin, DWA2114R, NK121, IS3 295, and 254-S. Exemplary chemotherapeutic agents also include, but are not limited to, methotrexate, vincristine, Adriamycin, non-sugar containing chloroethylnitrosoureas, 5- fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin, carmustine, polifeprosan, MM1270, BAY 12-9566, RAS famesyl transferase inhibitor, famesyl transferase inhibitor, MMP, MTA/LY231514, lometrexol/LY264618, Glamolec, CI-994, TNP-470, Hycamtin/topotecan, PKC412, Valspodar/PSC833, Novantrone/mitoxantrone, MetaretO/suramin, BB-94Aatimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel/VX-710, VX-853, ZD0101, IS1641, ODN 698, TA 2516/marimastat, BB2516/marimastat, CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317, picibanil/OK-432, valrubicin/AD 32, strontium-89/Metastron, Temodal/temozolomide, Yewtaxan/paclitaxel, Taxol/paclitaxel, Paxex/paclitaxel, Cyclopax/oral paclitaxel, Xeloda/capecitabine, Furtulon/doxifluridine, oral taxoids, SPU-077/cisplatin, HMR 1275/flavopiridol, CP-358 (774)ZEGFR, CP-609 (754)/RAS oncogene inhibitor, BMS-182751/oral platinum, UFT (Tegafur/Uracil), Ergamisol/levamisole, Campto/levamisole, Eniluracil/776C85/5FU enhancer, Camptosar/irinotecan, Tomudex/raltitrexed, Leustatin/cladribine, Caelyx/liposomal doxorubicin, Myocet/liposomal doxorubicin, Doxil/liposomal doxorubicin, Evacet/liposomal doxorubicin, Fludara/fludarabine, Pharmorubicin/epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphthalimide, LU 103793/Dolastatin, Gemzar/gemcitabine, ZD0473/AnorMED, YM 116, Iodine seeds, CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/dexifosfamide, Ifex/Mesnex/ifosfamide, Vumon/teniposide, Paraplatin/carboplatin, Platinol/cisplatin, VePesid/Eposin/Etopophos/etoposide, ZD 9331, Taxotere/docetaxel, prodrugs of guanine arabinoside, taxane analogs, nitrosoureas, alkylating agents such as melphalan and cyclophosphamide, aminoglutethimide, asparaginase, busulfan, carboplatin, chlorambucil, cytarabine HC1, dactinomycin, daunorubicin HC1, estramustine phosphate sodium, etoposide (VP 16-213), floxuridine, fluorouracil (5-FU), flutamide, hydroxyurea (hydroxycarbamide), ifosfamide, interferon alfa-2a, interferon alfa-2b, leuprolide acetate (LHRH-releasing factor analogue), lomustine (CCNU), mechlorethamine HC1 (nitrogen mustard), mercaptopurine, mesna, mitotane (o,p'-DDD), mitoxantrone HC1, octreotide, plicamycin, procarbazine HC1, streptozocin, tamoxifen citrate, thioguanine, thiotepa, vinblastine sulfate, amsacrine (m- AMSA), azacitidine, erythropoietin, hexamethyl melamine (HMM), interleukin 2, mitoguazone (methyl-GAG; methyl glyoxal bis-guanyl hydrazone; MGBG), pentostatin (2'deoxycoformycin), semustine (methyl-CCNU), teniposide (VM-26), vindesine sulfate, and RAS inhibitors. Additional exemplary therapeutic compounds for use in pharmaceutical compositions and combinations provided herein can be found elsewhere herein.
In some examples, the combination can include additional therapeutic compounds such as, for example, compounds that are substrates for enzymes encoded and expressed by the virus, or other therapeutic compounds provided herein or known in the art to act in concert with a virus. For example, the virus can express an enzyme that converts a prodrug into an active chemotherapy drug for killing the cancer cell. Hence, combinations provided herein can contain a therapeutic compound, such as a prodrug. An exemplary virus/therapeutic compound combination can include a virus encoding Herpes simplex virus thymidine kinase with the prodrug ganciclovir. Additional exemplary enzymeZpro-drug pairs, for the use in combinations provided include, but are not limited to, varicella zoster thymidine kinase/ganciclovir, cytosine deaminase/5- fluorouracil, purine nucleoside phosphorylase/6-methylpurine deoxyriboside, beta lactamase/cephalosporin-doxorubicin, carboxypeptidase G2/4-[(2-chloroethyl)(2- mesyloxyethyl)amino]benzoyl-L-glutamic acid, cytochrome P450/acetaminophen, horseradish peroxidase/indole-3 -acetic acid, nitroreductase/CB1954, rabbit carboxylesterase/7-ethyl-l 0-[4-( 1 -piperidino)- 1 -piperidinojcarbonyloxycamptothecin (CPT-11), mushroom tyrosinase/bis-(2-chloroethyl)amino-4- hydroxyphenylaminomethanone 28, beta galactosidase/l-chloromethyl-5-hydroxy-l,2- dihydro-3H-benz[e]indole, beta glucuronidase/epirubicin-glucuronide, thymidine phosphorylase/S'-deoxy-S-fluorouridine, deoxycytidine kinase/cytosine arabinoside, beta-lactamase and linamerase/linamarin. Additional exemplary prodrugs, for the use in combinations also can be found elsewhere herein (see e.g., Section E). Any of a variety of known combinations provided herein or otherwise known in the art can be included in the combinations provided herein.
In some examples, the combination can include compounds that can kill or inhibit viral growth or toxicity. Such compounds can be used to alleviate one or more adverse side effects that can result from viral infection (see, e.g., U.S. Patent Pub. No. US 2009- 016228-Al). Combinations provided herein can contain antibiotic, antifungal, anti- parasitic or antiviral compounds for treatment of infections. In some examples, the antiviral compound is a chemotherapeutic agent that inhibits viral growth or toxicity.
Exemplary antibiotics which can be included in a combination with a virus provided herein include, but are not limited to, 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, griseoftdvin, 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, 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 ddofovir, alkoxyalkyl esters of cidofovir, ganciclovir, acyclovir, ST-246, Gleevec, and derivatives thereof.
In some examples, the combination can include a detectable compound. A detectable compound can include, for example, a ligand, substrate or other compound that can interact with and/or bind specifically to a protein or RNA encoded and expressed by the virus, and can provide a detectable signal, such as a signal detectable by tomographic, spectroscopic, magnetic resonance, or other known techniques. In some examples, the protein or RNA is an exogenous protein or RNA. In some examples, the protein or RNA expressed by the virus modifies the detectable compound where the modified compound emits a detectable signal. Exemplary detectable compounds can be, or can contain, an imaging agent such as a magnetic resonance, ultrasound or tomographic imaging agent, including a radionuclide. The detectable compound can include any of a variety of compounds as provided elsewhere herein or are otherwise known in the art. Exemplary proteins that can be expressed by the virus and a detectable compound combinations employed for detection include, but are not limited to luciferase and luciferin, ^-galactosidase and (4,7,10-tri(acetic acid)-l-(2-P- galactopyranosylethoxy)-l,4,7,10-tetraazacyclododecane) gadolinium (Egad), and other combinations known in the art.
In some examples, the combination can include a gene expression-modulating compound that regulates expression of one or more genes encoded by the virus. Compounds that modulate gene expression are known in the art, and include, but are not limited to, transcriptional activators, inducers, transcriptional suppressors, RNA polymerase inhibitors and RNA binding compounds such as siRNA or ribozymes. Any of a variety of gene expression modulating compounds known in the art can be included in the combinations provided herein. Typically, the gene expression-modulating compound included with a virus in the combinations provided herein will be a compound that can bind, inhibit or react with one or more compounds, active in gene expression such as a transcription factor or RNA of the virus of the combination. An exemplary virus expression modulator combination can be a virus encoding a chimeric transcription factor complex having a mutant human progesterone receptor fused to a yeast GAL4 DNA-binding domain an activation domain of the herpes simplex virus protein VP16 and also containing a synthetic promoter containing a series of GAL4 recognition sequences upstream of the adenovirus major late E1B TATA box, where the compound can be RU486 (see, e.g., Yu etal. (2002) Mol Genet Genomics 268:169-178). A variety of other virus cell/expression modulator combinations known in the art also can be included in the combinations provided herein.
In some examples, the combination can contain nanoparticles. Nanoparticles can be designed such that they carry one or more therapeutic agents provided herein. Additionally, nanoparticles can be designed to cany a molecule that targets the nanoparticle to the tumor cells. In one non-limiting example, nanoparticles can be coated with a radionuclide and, optionally, an antibody immunoreactive with a tumor-associated antigen.
Tumor Antigens
Vaccinia viruses, such as RT vaccinia viruses or EEV produced therefrom, can be modified or engineered to express tumor-associated surface markers, tumor-associated antigens, or cancer neo-antigens molecules by the insertion of one or more heterologous nucleic acids inserted into the viral genome. For example, a phase n trial employing a recombinant Wyeth strain with deleted TK and containing transgenes for prostatespecific antigen (PSA) and three immune co-stimulatory molecules (B7.1, ICAM-1, and LFA-3) (PROSTAVAC-VF) demonstrated enhanced median overall survival in patients with metastatic castrate-resistant prostate cancer. Patients received boosters with recombinant fowl pox containing the same four transgenes. 12 of 32 patients showed declines in serum PSA post-vaccination and 2/12 showed decreases in lesions. Patients with greater PSA-specific T-cell responses showed a trend toward enhanced survival (Kantoff, P.W. et al. J Clin Oncol. 2010; 28 :1099-105).
The viruses described and provided herein can be engineered of modified to encode tumor-associated antigens (TAA) or tumor-specific antigens (neo-antigens), including, but not limited to, for example, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, cancer testis antigens (CTAs), New York esophageal squamous cell carcinoma- 1 (NY-ESO-1), E6/E7, SV40, MART-1, FRAME, CT83, SSX2, BAGE family, CAGE family, epithelial tumor antigen (ETA), prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), melanoma-associated antigens (MAGEs), TACE/ADAM17, tyrosinase, CD19, GP100, telomerase, cyclin Bl, survivin, mesothelin, EPHA2, B-cell maturation antigen (BMCA), and HER2. Other such exemplary tumor-associated antigens (TAA) or tumor-specific antigens (neoantigens) include, but are not limited to, CD19, CD20, CD33, EpCAM, CEA, PSMA, EGFRvIII, CD133, EGFR, CDH19, ENPP3, DLL3, MSLN, ROR1, HER2, HLAA2, EpHA2, EpHA3, MCSP, CSPG4, NG2, RON, FLT3, BCMA, CD20, FAPa, FRa, CA-9, PDGFRa, PDGFR0, FSP1, S100A4, ADAM12m, RET, MET, FGFR, INSR, and NTRK. Exemplary antigens targeted by antibody drug conjugate drugs include by are not limited toHER2, TROP2, Nectin4, CD 19, CD20, CD22, CD33, CD30, tumor neoantigens and cancer stem cell antigens.
Antibody-Drug Conjugates
An antibody-drug conjugate (ADC) generally comprises and antibody, a payload (e.g, and anti-cancer agent such as an anti-cancer small molecule), and a linker connecting the antibody to the payload. Targets of ADCs generally are antigen expressed exclusively on the surface of tumor cells. Antibodies incorporated into ADCs generally are mainly humanized antibodies, significantly less immunogenic than murine and chimeric monoclonal antibodies (mAb). ADC antibodies generally are based on the IgGl isotype. Linkers generally can be biochemical compounds connecting the antibody to the payload. Linkers guarantee ADC stability in the bloodstream and promote efficient cleavage upon internalization into tumor cells. Linkers can be classified into cleavable and non-cleavable according to their chemical properties (Criscitiello, C. et al. JHematol Oncol. 2021; 14: 20). For example, an ADC can act to target a specific antigen expressed by a tumor by the antibody portion and deliver a small molecule payload, such as a chemotherapy agent, to selectively eliminate the tumor. The small molecules of ADCs can penetrate the cell membrane to further kill surrounding tumor cells, which is termed the bystander killing effect, some ADCs have antibody immune effects, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP) (see, e.g., Ruan, D-Y, et al. Cancer Commun (Lond). 2024 Jan; 44(1): 3-22; Chau, CH, et al. Lancet.
2019;394(10200):793-804; Giugliano, F., et al. Curr Oncol Rep. 2022;24(7):809-817; Khera, E., etal. BioDrugs. 2018;32(5):465-480; Liu, R., et al Antibodies (Basel). 2020;9(4):64). ADCs also can include conditional antibody ADCs, bispecific ADCs, nonintemalized ADCs (see, e.g., Drago. J.Z., et al. Nat Rev Clin Oncol.
344; Autio, K.A., et al. Clin Cancer Res. 2020;26(5):984-989; and Kast, F., etal. Nat Commun. 2021;12(l):3790).
Exemplary ADCs suitable for use in combinations with EEV can be selected from among: Trastuzumab emtansine, Trastuzumab deruxtecan, Disitamab vedotin, Trastuzumab duocarmazine, TAA013, SHR-A1811, MRG002, LCB14-0110, ARX788, DX126-262, DP303c, A166, Sacituzumab govitecan, SKB264, Datopotamab deruxtecan, Cetuximab saratolacan, MRG003, AVID 100, Telisotuzumab vedotin, RC108, Moxetumomab pasudotox, Inotuzumab ozogamicin, Vobramitamab duocarmazine, Ifinatamab deruxtecan, Tusamitamab ravtansine, Labetuzumab govitecan, Mirvetuximab soravtansine, Farietuzumab ecteribulin, Mecbotamab vedotin, Belantamab mafodotin, Pivekimab sunirine, Indatuximab ravtansine, Loncastuximab tesirine, Eramkafusp alfa, Camidanlumab tesirine, Brentuximab vedotin, Gemtuzumab ozogamicin, Naratuximab emtansine, Polatuzumab vedotin, L-DOS47, OBI-999, Patritumab deruxtecan, Ladiratuzumab vedotin, Upifitamab rilsodotin, Enfortumab vedotin, Zilovertamab vedotin, Ozuriframab vedotin, Tisotumab vedotin, Cetuximab sarotalocan and those described in (see, e.g., Ruan, D-Y, etal. Cancer Commun (Lond). 2024 Jan; 44(1): 3-22; Chau, C.H., etal. Lancet. 2019;394(10200):793-804; Giugliano, F., etal. Curr Oncol Rep. 2022;24(7):809-817; Khera, E., etal. BioDrugs. 2018;32(5):465-480; Liu, R., etal Antibodies (Basel). 2020;9(4):64).
CAR-T Cell Therapies and TIL cell Therapies and other Cell Therapies
The EEV viruses provided herein can express virally encoded proteins that can be targeted by cell therapies, such as, for example, CAR-T cells engineered to target a viral protein. Virally encoded proteins can include a viral protein. Virally encoded payloads can include tumor antigens, such that the virus or EEV expressing a tumor antigen can be combined with an anti-cancer therapy that targets a tumor antigen. Suitable antigen- targeted therapy can include any cell therapy, such as for example, chimeric antigen receptor (CAR)-T cell therapy. EEV producing viruses can be modified or engineered to deliver tumor-associated antigens (TAA) or tumor-specific antigens (neo-antigens) such as a surface antigen to a target tumor or metastatic tumor that are recognized by CAR-T cells. CAR-T cells are a recognized therapeutic approach for the treatment of patients with blood cancers, such as chemotherapy-refractory or relapsed blood cancers, including acute lymphoblastic leukemia, diffuse large B cell lymphoma, follicular lymphoma, mantie cell lymphoma and multiple myeloma. Despite FDA approvals of anti-CD19 CAR T cell products for both ALL and certain types of B cell lymphoma, brief remission has been observed in patients, owing to poor CAR T cell persistence and/or cancer cell resistance resulting from antigen loss or modulation. Even the most advanced cell therapeutics have difficulty infiltrating solid tumors unless combined with systemic enveloped virotherapies. EEV viruses can be modified or engineered to express surface antigens for CAR-T cells to overcome these problems.
For example, thymidine kinase-deficient (TK-) oncolytic vaccinia virus (mCD19 W) can be engineered to selectively deliver CD 19 to malignant cells. The combination of CD19 CAR-T cells and mCD19 W delayed tumor growth and improved median survival compared with antigen-mismatched combinations in an immunocompetent mouse model (B16 melanoma). Vaccinia viral delivery of CD19 improved CD19-CAR T cell activity against tumor cells expressing low CD19 levels (Aalipour, A. et al, Mol Ther Oncolytics. 2020 Apr 7: 17:232-240). Other recombinant vaccinia viruses that have been engineered to express tumor antigens include, for example: PANVAC, MVA-5T4, TroVax and those described in US 2020/0215132 Al.
Oncolytic viruses can be engineered to produce cytokines or chemokines that enhance CAR T cell function and anti-tumor efficacy. There can be a mismatch in chemokine/chemokine receptor in the tumor microenvironment. Tumors producing minute amounts of chemokines can result in inefficient targeting of effector cells, such as T cells, to tumors. Vaccinia virus can be engineered to produce CXCL11 (W.CXCL11), a CXCR3 ligand which is highly expressed on effector T cells. Intravenously (iv) administered W.CXCL11 to mice bearing established subcutaneous TCI tumors (which expressed meso antigen) significantly increased CXCL11 protein levels within tumors increased number of total T cells. W.CXCL11 increased antigen-specific T cells within tumors after CAR T cell injection or vaccination and significantly enhanced anti-tumor efficacy (Moon, E.K., etal. Oncoimmunology. 2018;7(3):el395997). Exemplary CAR-T cell include, for example, Abecma® (idecabtagene vicleucel), Breyanzi® (lisocabtagene maraleucel), Kymriah® (tisagenlecleucel), Carvykti® (ciltacabtagene autoleucel), Tecartus® (brexucabtagene autoleucel), Yescarta® (axicabtagene ciloleucel), single- and multi-antigen targeted CAR-T cells.
Lymphodepletion and Immunomodulation
For systemic administration, the viruses provided herein are modified or selected so that they have resistance to the immune system of the host. Additionally, upon administration, or prior to, or during, treatment with the virus, the host can be treated so the immune system of the host is less active. This can be effected by lymphodepletion. Advantageously, complete lymphodepletion is not needed; instead, the immune system of the host can be modulated so that the anti-viral response of the host is muted so that the virus is not eliminated, but the immune system is still functional. This can be achieved, for example, by low dose (lower than used for lymphodepletion) chemotherapy. Examples of such treatment are detailed in the Examples. As treatment proceeds and virus amplifies in the tumors and is dissemination, virus levels can be controlled, as needed, such as by administration of anti-virals, including but are not limited to, ST246. In this way the levels of virus can be controlled throughout the treatment, permitting high levels of virus in the tumors, but as needed, systemic levels of virus can be eliminated or reduced if toxic effects occur. Selection of such treatments, for modulating the immune system and/or administering anti-virals is within the judgement and skill of the skilled artisan, such as the physician.
Vaccinia virus promotes an anti-tumor microenvironment by stimulating immune responses to convert the tumor microenvironment into an anti-tumor environment. Upon lysis, following amplification of virus in tumors, tumor-associated antigens (TAAs), and/or virally encoded immunomodulators are released. It is shown herein that treatment of tumors, such as by systemic administration of EEV such as those derived from RT vaccinia viruses, promote anti-tumor immune cell infiltration into tumors. EEV, such as those derived from RT vaccinia viruses, such as, for example, EEV produced from RT- 05 or any other EEV viruses modified to encode complement inhibitory proteins and to display them on the cell surface, increase the infiltration of anti-tumor immune cells such as CD8+ and effector CD4+ T cells, and reduce pro-tumor immune cells such as CD4+ CD25+ Foxp3+ regulatory T cells, and reduce myeloid populations in tumors. Administration of EEV, such as those derived or produced from RT vaccinia virus, promote an anti-tumor microenvironment by reducing immunosuppressive cell types, such as T regs and myeloid derived suppressor cells, by releasing pro-inflammatory cytokines and damage associated molecular products (DAMPs). The triggered anti-tumor cellular immunity, however, can decrease viral replication and persistence even though this can serve as a safety mechanism to eliminate virus-induced toxicity.
Administration of chemotherapeutic agents, such as cyclophosphamide (Cy, CPA, or CTX) at high doses deplete lymphocytes, thereby eliminating anti-virus (and hence anti-tumor) immunity. Alkylating agents, such as cyclophosphamide, at high doses are potent cytotoxic and lympho-ablative drugs, used in immunosuppressive regimens in the oncological and internal medicine. For example, lymphodepletion (LD) or conditioning can be used as an initial step prior to the administration of autologous and allogeneic CAR-T cell therapies. Lymphodepletion or conditioning can promote (1) the reduction of endogenous lymphocytes to prepare a niche for engraftment of CAR-T infusions; (2) the long-term activity of CAR-T; (3) reduce the amount of tumor cells to avoid rapid exhaustion of CAR-T; and (4) prepare/prime and reprogram the microenvironment and soluble factors promote engraftment, homing and long-term survival of CAR-T. Chemotherapeutic agents with both anti-tumor activity and T-cell reducing activity commonly are used in lymphodepletion. Schedules for lymphodepletion can involve fludarabine (Flu) and cyclophosphamide (alone or in combination). Commonly used dosing schedules are based on clinical trials involving trials with tumor-infiltrating lymphocytes (see, e.g., Muranski, P., etal. Nat Clin Pract Oncol (2006) 3(12):668— 81; Dudley, M.E., etal. Science (2002) 298(5594):850-4) as well as for allogeneic hematopoietic stem cell transplantation (see, e.g., Gyurkocza, B., et al. Blood (2014) 124(3):344-53) and those described in (Lickefett, B., etal. Front. Immunol. 14:1303935). Exemplary lymphodepleting doses can include fludarabine at doses of 90mg/kg and CPA at a dose of 900-1500mg/kg and bendamustine at a dose of 180mg/kg in human subjects. Exemplary lymphodepleting treatments include, but are not limited to, fludarabine, cyclophosphamide, bendamustine, alemtuzumab, oxaliplatin/cyclophosphamide, clofarabine, radiation therapy and those described in (Lickefett, B., etal. Front. Immunol. 14:1303935).
It is described and shown herein, that lymphodepletion is not needed. These agents can be used in moderate or low doses in combination with the virus can be used to modulate or mute the immune system response of the host so systemic viral levels are controlled. Immunomodulation can be effected by administration of chemotherapeutic agents, such as cyclophosphamide, at lower or moderate doses can result in sustained persistence of the virus in the tumor microenvironment, in the expression of the viral payload genes and increased populations of IFNy-producing CD8+ cytotoxic T cells, and decrease regulatory T cell and myeloid cell populations in the tumor microenvironment. In this way, lower doses of chemotherapeutic agents, such as cyclophosphamide provide an immunomodulatory effect or promote an anti-tumor immune microenvironment. Combination therapy of the virus and low dose chemotherapy or other such immunomodulating agent can solve the problem of virus clearance by the immune system, by reducing viral clearance but not eliminating the immune populations that are important for an anti-tumor response. For example, a low dose of CPX can be about 1-5 mg/kg/day to about 250 mg/kg/day, such as, for example, 2mg/kg/day in human subjects (see, e.g. , Zhu et al. J Clin Invest. 1987 Apr;79(4): 1082-90) to generally about 250 mg/kg or lower (see, e.g., Sistigu, A. et. al. Semin Immunopathol (2011) 33:369-383). Immunomodulation can be effected by other immunomodulators, such as immunotherapy agents, such as an agonist antibody of a co-stimulatory pathway and/or immune checkpoint inhibitors, and cell therapies, such as CAR-T therapies and TIL cell therapies. Therapy can include combinations of low dose chemotherapeutic agents and the other immunomodulatory agents. Combinations can also include anti-virals, as needed to control systemic viral loads.
Natural Killer Cell Therapy
Natural killer (NK) cells are an innate immune cell that participate in immune surveillance, pathogen clearance, and anti-tumor immunotherapy without prior sensitization or antigen recognition. Viral infection, such as vaccinia viral infection results in the accumulation of NK cells prior to activation T- and B-cells. Vaccinia viruses can modulate the activation of NK cells, and does so through several mechanism. For example, the EEV surface protein hemagglutinin (HA, A56) is a ligand for NK cell receptors NKp30 and NKp46 that trigger NK cytotoxicity. A56 binding to these receptors can modulate NK cell activation (Kirwan, S. et al. Virology (2006), 347, 75-87; Chisolm, S.E. et al. J. Virology (2006) 80, 2225-2233). Vaccinia viruses can be modified to express recombinant immunomodulatory agent to modulate the activation of NK cells and increase NK cell numbers in the tumor microenvironment. For example, vaccinia virus can be modified or engineered to express the chemotactic cytokine CCL5 which directs NK cells to the infected tumor microenvironment or to express interleukins (IL) such as IL-2 and IL- 15 which promote NK cell proliferation, infiltration, distribution.
NK cell therapies can include, for example, CAR-NK therapy. Compared with CAR-T therapy, CAR-NK therapy has advantages due to the replacement of T cells with NK cells, such as for example, the benefit of low risk of allogeneic rejection of NK cells which allows NK cells in CAR-NK to be generated from a variety of off-the-shelf sources. CAR-NK cells are engineered to express chimeric antigen receptors recognizing surface antigens of target cells. Such modification can counteract inhibitory receptors on the NK cell, thereby enhancing NK cell-specific killing effect of targeted cells (Shimasaki, N., et al. Nat Rev Drug Discov. 2020;19:200-18).
The immunosuppressive effect of the microenvironment is the biggest obstacle to the application of CAR-NK in solid tumor (Wang et al. Cell Death Discov. 2024; 10:40). It is shown herein that such limitations can be overcome by combination by EEV viruses. It is shown herein, that EEV pre-treated tumors can increase NK cell infiltration in the tumor microenvironment. Such virotherapy pre-treatment “primes” the tumor, facilitating the entrance and persistence of NK cell therapies. CAR-NK cell therapies include, for example, CLDN6-CAR-NK Cells; NKG2D CAR-NK Cells; NKG2D-CAR-NK92 Cells; Anti-5T4CAR-NK Cells; Anti-Mesothelin CAR NK Cells; DLL3-CAR-NK Cells; NKG2D-Ligand Targeted CAR-NK Cells; ROBO1 CAR-NK Cells; Irradiated PD-L1 CAR-NK Cells; SZ003; ROBO1 Specific BiCAR-NK/T Cells; SZ011; Anti-PSMA CAR NK Cell (TABP EIC); NK-92/5.28.Z Cells; CAR.70-engineered EL15-transduced Cord Blood-derived NK Cells; Anti-5T4 CAR-raNK Cell; CCCR-NK92 Cells; MESO CAR NK cells; and those described in (Wang et al. Cell Death Discov. 2024; 10:40; Zhao et al Med Rev (ZWIV). 2023 Oct 24;3(4):305-320; and Blunt et a/. Immunother Adv. 2024; 4(1): ltad031).
Safety Switch To Inhibit Or Eliminate Virus
The viruses provided herein can be combined with an anti-viral agent, as a safety switch, to reduce systemic viral levels. Suitable anti-viral agents include, for example, acyclovir, brincidofovir gangcyclovir, rifampin, tecovirimat (ST-246; TPOXX®), Vaccinia immune globulin (VIG), anti-vaccinia monoclonal antibodies and anti-vaccinia virus agents described in (Wu et al. J Med Virol. 201991(11):2016-2024). Combinations and Kits
The EEV viruses, pharmaceutical compositions or combinations provided herein can be vided as combinations, such as packaged as kits. Kits optionally include one or more components such as instructions for use, devices and additional reagents, and components, such as tubes, containers, and syringes for practice of the methods. Exemplary kits can include an EEV virus provided herein and, optionally, include instructions for use, a device for detecting a vims in a subject, a device for administering the virus to a subject, or a device for administering an additional agent or compound to a subject.
Instructions typically include a tangible expression describing the EEV vims and, optionally, other components included in the kit, and methods for administration, including methods for determining the proper state of the subject, the proper dosage amount, and the proper administration method, for administering the vims. Instructions also can include guidance for monitoring the subject over the duration of the treatment time.
In another example, a kit can contain a device for detecting an EEV virus in a subject. Devices for detecting an EEV virus in a subject can include a low light imaging device for detecting light, for example, emitted from luciferase, or fluoresced from a fluorescent protein, such as a green or red fluorescent protein, a magnetic resonance measuring device such as an MRI or NMR device, a tomographic scanner, such as a PET, CT, CAT, SPECT or other related scanner, an ultrasound device, or other device that can be used to detect a protein expressed by the virus within the subject. Typically, the device of the kit will be able to detect one or more proteins expressed by the virus of the kit. Any of a variety of kits containing viruses and detection devices can be included in the kits provided herein, for example, a virus expressing luciferase and a low light imager or a virus expressing a fluorescent protein, such as a green or red fluorescent protein, and a low light imager.
Kits provided herein also can include a device for administering an EEV virus to a subject. Any of a variety of devices known in the art for administering medications, pharmaceutical compositions and vaccines can be included in the kits provided herein. Exemplary devices include, but are not limited to, a hypodermic needle, an intravenous needle, a catheter, a needle-less injection device, an inhaler and a liquid dispenser, such as an eyedropper. For example, an EEV virus to be delivered systemically, for example, by intravenous injection, can be included in a kit with a hypodermic needle and syringe. Typically, the device for administering a virus of the kit will be compatible with the virus of the kit; for example, a needle-less injection device such as a high-pressure injection device.
Kits provided herein also can include a device for administering an additional agent or compound to a subject. Any of a variety of devices known in the art for administering medications to a subject can be included in the kits provided herein. Exemplary devices include, but are not limited to, a hypodermic needle, an intravenous needle, a catheter, a needle-less injection device, an inhaler and a liquid dispenser, such as an eyedropper. Typically, the device for administering the compound of the kit will be compatible with the desired method of administration of the compound. For example, a compound to be delivered systemically or subcutaneously can be included in a kit with a hypodermic needle and syringe.
The kits provided herein also can include any device for applying energy to a subject, such as electromagnetic energy. Such devices include, but are not limited to, a laser, light-emitting diodes, fluorescent lamps, dichroic lamps, and a light box. Kits also can include devices to effect internal exposure of energy to a subject, such as an endoscope or fiber optic catheter.
J. METHODS OF TREATING CANCER AND OTHER PROLIFERATIVE
DISEASES, DISORDERS, AND CONDITIONS
Methods of Treatment
Provided herein are methods of treatment administering an EEV virus, such as for example an SR-EEV, S-R EEV or an IV-EEV. The viruses provided herein are designed and produced for systemic administration; they can be administered by any route known to those of the skill. The viruses provided herein advantageously can be administered systemically. The viruses can effect tumor selective lysis of multiple tumor sites and tumor metastasis, amplification and spread of the virus, promotion of an anti-tumor tumor microenvironment, and effect delivery of a vitally encoded payload, to treat a subject having a proliferative or inflammatory disease or condition. For example, the condition can be associated with immunoprivileged cells or tissues. A disease or condition associated with immunoprivileged cells or tissues can include, for example, proliferative disorders or conditions, including the treatment (such as inhibition) of cancerous cells, neoplasms, tumors, metastases, cancer stem cells, and other immunoprivileged cells or tissues, such as wounds and wounded or inflamed tissues. Exemplary of such methods, the EEV provided herein are administered by intravenous administration for systemic delivery. In other examples, the EEV provided herein are administered by intratumoral injection. In some embodiments, the subject has cancer. Any of the EEV provided herein can be used to provide virotherapy to subjects in need thereof.
The EEV provided herein can be administered by a single injection, by multiple injections, or continuously. For example, the EEV can be administered by slow infusion including using an intravenous pump, syringe pump, intravenous drip, or slow injection. For example, continuous administration of the EEV can occur over the course of minutes to hours, such as between or about between 1 minute to 1 hour, such as between 20 and 60 minutes.
Cancers amenable to the treatment and detection methods described herein also include cancers that metastasize. It is understood by those in the art that metastasis is the spread of cells from a primary tumor to a noncontiguous site, usually via the bloodstream or lymphatics, which results in the establishment of a secondary tumor growth. Examples of cancers contemplated for treatment include, but are not limited, to solid tumors and hematologic malignancies, such as, for example, melanoma, including choroidal and cutaneous melanoma, bladder cancer, non-small cell lung cancer, small cell lung cancer, lung cancer, head and neck cancer, breast cancer, pancreatic cancer, gum cancer, tongue cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, lymphoma, brain cancer, colon cancer, rectal cancer, choriocarcinoma, gliomas, carcinomas, basal cell carcinoma, biliary tract cancer, central nervous system (CNS) cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, gastric cancer, intraepithelial neoplasm, kidney cancer, larynx cancer, leukemia, liver cancer, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, myeloma, neuroblastoma, oral cavity cancer, retinoblastoma, rhabdomyosarcoma, cancer of the respiratory system, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, cancer of the urinary system, lymphosarcoma, osteosarcoma, mammary tumors, mastocytoma, adenosquamous carcinoma, carcinoid lung tumor, bronchial gland tumor, bronchiolar adenocarcinoma, fibroma, myxochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma, hepatocarcinoma, mesothelioma, astrocytoma, glioblastoma, Ewing's sarcoma, Wilms’ tumor, Burkitt's lymphoma, microglioma, osteoclastoma, oral neoplasia, fibrosarcoma, genital squamous cell carcinoma, transmissible venereal tumor, testicular tumor, seminoma, Sertoli cell tumor, hemangiopericytoma, histiocytoma, chloroma, granulocytic sarcoma, comeal papilloma, comeal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, stomach tumor, adrenal gland carcinoma, oral papillomatosis, hemangioendothelioma, cystadenoma, follicular lymphoma, intestinal lymphosarcoma, hepatocellular carcinoma, pulmonary adenomatosis, pulmonary sarcoma, Rous sarcoma, reticulo-endotheliosis, nephroblastoma, B-cell lymphoma, lymphoid leukosis, retinoblastoma, hepatic neoplasia, lymphosarcoma, plasmacytoid leukemia, swimbladder sarcoma (in fish), caseous lymphadenitis, lung carcinoma, insulinoma, sarcoma, neuroma, pancreatic islet cell tumor, gastric MALT lymphoma, gastric adenocarcinoma, pulmonary squamous cell carcinoma, leukemia, hemangiopericytoma, ocular neoplasia, preputial fibrosarcoma, ulcerative squamous cell carcinoma, preputial carcinoma, connective tissue neoplasia, and any other tumors or neoplasms that are metastasized or at risk of metastasis.
The subject of the methods provided herein can be any subject, such as an animal or plant subject, including mammal or avian species. For example, the animal subject can be a human or non-human animal including, but not limited to, domesticated and farm animals, such as a pig, cow, a goat, sheep, horse, cat, or dog. In some embodiments, the animal subject is a human subject. In some embodiments, the human subject is a pediatric patient.
The methods provided herein can further include one or more steps of monitoring the subject, monitoring the tumor, monitoring an EEV administered to the subject and/or monitoring a gene/payload expressed by the EEV. Any of a variety of monitoring steps can be included in the methods provided herein, including, but not limited to, monitoring tumor size, monitoring anti-(tumor antigen) antibody titer, monitoring the presence and/or size of metastases, monitoring the subject's lymph nodes, monitoring the subject's weight or other health indicators including blood or urine markers, monitoring anti-(viral antigen) antibody titer, monitoring viral expression of a detectable gene product, and directly monitoring viral titer in a tumor, tissue or organ of a subject.
The purpose of the monitoring can be for assessing the health state of the subject or the progress of therapeutic treatment of the subject, or can be for determining whether or not further administration of the same or a different EEV or virus is warranted, or for determining when or whether or not to administer a compound to the subject where the compound can act to increase the efficacy of the therapeutic method, such as for example an anti-cancer agent, or the compound can act to decrease the pathogenicity of the virus administered to the subject, such as a safety switch agent or an anti-viral agent.
Tumor and/or metastasis size can be monitored by any of a variety of methods known in the art, including external assessment methods or tomographic or magnetic imaging methods (e.g., whole body magnetic resonance imaging), such as the detection methods described herein. In addition, methods provided herein, for example, monitoring gene expression (e.g., viral gene expression and or virally encoded payloads), can be used for monitoring tumor and/or metastasis size.
Monitoring tumor size over several time points can provide information regarding the efficacy of the therapeutic methods provided herein. In addition, monitoring the increase or decrease in size of a tumor or metastasis, also can provide information regarding the presence (z.e., detection and/or diagnosis) of additional tumors and/or metastases in the subject. Monitoring tumor size over several time points can provide information regarding the development of a neoplastic disease in a subject, including the efficacy of treatments of a neoplastic disease in a subject, such as the treatments provided herein.
The methods provided herein also can include monitoring the antibody titer in a subject, including antibodies produced in response to the administration of an EEV. For example, the EEV administered in the methods provided herein can elicit an immune response to endogenous viral antigens, e.g., the EEV outer membrane proteins B5R, A33R, A56R and F13L can be targeted by anti-viral neutralizing antibodies.
The EEV administered in the methods provided herein also can elicit an immune response to exogenous genes expressed by an EEV. The EEV administered in the methods provided herein also can elicit an immune response to tumor antigens. Monitoring antibody titer against viral antigens, viral expressed exogenous gene products, or tumor antigens can be used in methods of monitoring the toxicity of a virus, monitoring the efficacy of treatment methods, or monitoring the level of gene product or antibodies for production and/or harvesting.
For example, monitoring antibody titer can be used to monitor the toxicity of a virus. Antibody titer against a virus can vary over the time period after administration of the EEV to the subject, where at some time points, a low anti-(viral antigen) antibody titer can indicate a higher toxicity, while at other time points a high anti-(viral antigen) antibody titer can indicate a higher toxicity. The viruses used in the methods provided herein are resistant to inactivation by humoral immunity, but can be immunogenic with respect to cellular immunity and can therefore elicit an immune response soon after administering the EEV to the subject.
Generally, a virus against which a subject's immune system can quickly mount a strong immune response can be a virus that has low toxicity when the subject's immune system can remove the virus from all normal organs or tissues. Thus, in some examples, a high antibody titer against viral antigens soon after administering the EEV to a subject can indicate low toxicity of a virus. In contrast, a virus that is not highly immunogenic can infect a host organism without eliciting a strong immune response, which can result in a higher toxicity of the virus to the host. Accordingly, in some examples, a high antibody titer against viral antigens soon after administering the EEV to a subject can indicate low toxicity of a virus.
In other examples, monitoring antibody titer can be used to monitor the efficacy of treatment methods. In the methods provided herein, antibody titer, such as anti-(tumor antigen) antibody titer, can indicate the efficacy of a therapeutic method such as a therapeutic method to treat neoplastic disease. Therapeutic methods provided herein can include causing or enhancing an immune response against a tumor and/or metastasis. Thus, by monitoring the anti-(tumor antigen) antibody titer, it is possible to monitor the efficacy of a therapeutic method in causing or enhancing an immune response against a tumor and/or metastasis.
The therapeutic methods provided herein also can include administering to a subject an EEV that can accumulate, amplify, and spread in a tumor and tumor metastasis, and can cause or enhance an anti-virus immune response. Accordingly, it is possible to monitor the ability of a host to mount an immune response against viruses accumulated in a tumor or metastasis, which can indicate that a subject has also mounted an anti-tumor immune response or can indicate that a subject is likely to mount an antitumor immune response, or can indicate that a subject is capable of mounting an antitumor immune response.
The methods provided herein also can include methods of monitoring the health of a subject. Some of the methods provided herein are therapeutic methods, including neoplastic disease therapeutic methods. Monitoring the health of a subject can be used to determine the efficacy of the therapeutic method, as is known in the art. The methods provided herein also can include a step of administering to a subject an EEV as provided herein. Monitoring the health of a subject can be used to determine the pathogenicity of an EEV, when administered to a subject. Any of a variety of health diagnostic methods for monitoring disease such as neoplastic disease, infectious disease, or immune-related disease can be monitored, as is known in the art. For example, the weight, blood pressure, pulse, breathing, color, temperature or other observable state of a subject can indicate the health of a subject. In addition, the presence or absence or level of one or more components in a sample from a subject can indicate the health of a subject. Typical samples can include blood and urine samples, where the presence or absence or level of one or more components can be determined by performing, for example, a blood panel or a urine panel diagnostic test. Exemplary components indicative of a subject's health include, but are not limited to, white blood cell count, hematocrit, or reactive protein concentration.
Types of Cancer Treated
The viruses and methods provided herein can be used to treat any type of cancer, including any stage, and including metastatic cancer. Included are solid tumors and hematologic malignancies. Tumors that can be treated by the methods disclosed herein include, but are not limited to a bladder tumor, breast tumor, prostate tumor, carcinoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer, CNS cancer, glioma tumor, cervical cancer, choriocarcinoma, colon cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, cancer of the head and neck, gastric cancer, intra-epithelial neoplasm, kidney cancer, larynx cancer, leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, melanoma, myeloma, neuroblastoma, oral cavity cancer, ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, renal cancer, cancer of the respiratory system, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and cancer of the urinary system, such as lymphosarcoma, osteosarcoma, mammary tumors, mastocytoma, brain tumor, melanoma, adenosquamous carcinoma, carcinoid lung tumor, bronchial gland tumor, bronchiolar adenocarcinoma, small cell lung cancer, non-small cell lung cancers, 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, granulocytic sarcoma, corneal papilloma, corneal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, stomach tumor, adrenal gland carcinoma, oral papillomatosis, hemangioendothelioma, cystadenoma, follicular lymphoma, intestinal lymphosarcoma, fibrosarcoma, and pulmonary squamous cell carcinoma, hemangiopericytoma, ocular neoplasia, preputial fibrosarcoma, ulcerative squamous cell carcinoma, preputial carcinoma, connective tissue neoplasia, mastocytoma, hepatocellular carcinoma, lymphoma, pulmonary adenomatosis, pulmonary sarcoma, Rous sarcoma, reticulo-endotheliosis, fibrosarcoma, nephroblastoma, B-cell lymphoma, lymphoid leukosis, retinoblastoma, hepatic neoplasia, lymphosarcoma, plasmacytoid leukemia, swimbladder sarcoma (in fish), caseous lymphadenitis, lung carcinoma, insulinoma, lymphoma, sarcoma, salivary gland tumors, neuroma, pancreatic islet cell tumor, gastric MALT lymphoma and gastric adenocarcinoma. The advantages of the viruses herein is that they contain high levels of EEV, including the EEV with the chimeric or fusion protein EEV transmembrane protein that displays a complement inhibitory or resistance protein, such as CD55. The EEV phenotype renders the viruses suitable for IV administration, and the display of the complement resistance or inhibitory protein, which is encoded in the virus, results in virus that when amplified in a tumor, retain the complement resistance, which, as shown herein, increases systemic dissemination of virus to tumors and metastases.
In some embodiments, the tumor is selected from metastatic melanoma; esophageal and gastric adenocarcinoma; cholangiocarcinoma (any stage); pancreatic adenocarcinoma (any stage); gallbladder cancer (any stage); high-grade mucinous appendix cancer (any stage); high-grade gastrointestinal neuroendocrine cancer (any stage); mesothelioma (any stage); soft tissue sarcoma; prostate cancer; renal cell carcinoma; lung small cell carcinoma; lung non-small cell carcinoma; head and neck squamous cell carcinoma; colorectal cancer; ovarian carcinoma; hepatocellular carcinoma; and glioblastoma. In some embodiments, the tumor is selected from: glioblastoma, breast carcinoma, lung carcinoma, prostate carcinoma, colon carcinoma, ovarian carcinoma, neuroblastoma, central nervous system tumor, and melanoma.
Companion Diagnostic
Provided herein are methods of identifying suitable tumors for treatment by EEV vaccinia viruses. It is shown here in that the complement resistance phenotype of EEV is dependent upon the expression of CD55, CD59, CD46 levels, and/or other complement inhibitor proteins in tumors or tumor metastasis. In practice, a tumor sample can be assessed for levels of one or more of these proteins; subjects whose tumors have levels above a pre-determined minimum, are candidates for treatment with oncolytic viruses, particularly with EEV viruses. As detailed herein, however, the EEV viruses can be modified so that their production does not depend on the type of tumor.
A predetermined level can be a level determined based on the levels in tumors that produce complement-resistant EEV or levels higher than those in a tumor produce that fewer than a pre-determined amount, such as 50%, 60%, 70%, or 80%, resistant EEV, such as EEV produced in cells lines that produce at least, for example, 30% of the amount of complement inhibitor or resistance protein compared to Hela cells. As another measure, the level can be selected as at least 40% or 50% of the virus particles produced are EEVs.
K. Examples
The following examples are included for illustrative purposes only and are not intended to limit the scope of the subject matter.
EXAMPLE 1
Selection of a new Vaccinia virus strain that produces high levels of EEV
FIGURE 1 outlines a protocol for selection of a vaccinia virus clone that that can survive blood circulation. In this example a clonal vaccinia virus with high capacity to produce Extracellular Enveloped Virions (EEVs) was isolated. The EEV virus provides a level of protection against humoral immunity.
To prepare a new strain, the starting material was a polyclonal vaccinia IHD strain (NR-52). The IHD NR-52 virus originally was obtained from BEI Resources (Biological and Emerging Infections Research Resources), which is a centralized research reagent repository of the National Institute of Allergy. The IHD (International Health Department) strain was used in the development of smallpox vaccines. It was developed through extensive propagation via intracerebral inoculation in mice; it has clones with higher production of Extracellular Enveloped Virions (EEVs) compared to other strains.
As described below, IHD NR-52 virus was propagated and clones were selected that retain high EEV production and were further selected to exhibit a high capacity to kill tumors. To select a clone that retains higher production of EEV, a series of passages in a PC3 tumor cell line with the virus cultured in a mixture of human serum containing neutralizing antibodies and active complement were performed to select the most resilient clone(s) capable of surviving under these conditions. To select the clone with these properties, supernatants from the PC3 cell line were incubated with human serum, and incubated again in PC3 cultures. The process was repeated a plurality of times to select virus progeny that can produce EEVs at the high level and that are resistant to humoral immunity.
After multiple rounds of selection, the clone with the highest capacity to kill tumor cells was selected as follows. PC3 cells were cultured at a density of 10,000 cells per well in a 96-well plate. The following day, serial dilutions of selected supernatants were added to the cells. After a 2-hour incubation, the medium was removed and replaced with fresh medium containing either 20% fetal bovine serum (FBS) or 20% human serum (HSA). Cytotoxicity was monitored using an XCELLIGENCE® plate (Agilent) for a period of 5 days. The wells with the highest virus dilution, which killed the most PC3 cells compared to the IMV CALI control virus, were selected and underwent another two rounds of serum selections. Among the clones selected was a clone designated RT-N2 (or N2 herein, also referred to as RT-00). RT-00 showed high tumor killing capacity as well as producing a high level of EEV. RT-01, derived from RT-00, has TK deletion and an insert in TK of nucleic acid encoding TurboFP. EXAMPLE !
As demonstrated and described herein, the vaccinia virus strain designated RT-01 produces abundant extracellular enveloped virions (EEVs) containing a second human cell-derived membrane. Methods for manufacturing EEVs that preserve the membrane during manufacture of the viruses are provided herein and described below. Preservation of the membrane enhances protection against systemic elimination.
The EEVs viruses provided herein, such as the RT-01 virus, derivatives thereof and similarly produced viruses selected/modified for high EEV production, exhibit high serum resistance to thereby reduce elimination by the immune system of the host. For example, the resistance of the manufactured enveloped RT- 01 (EnvRT-01) virus against human humoral immunity and its rapid spread was assessed by a plaque assay and the xCELLigence label-free system. In various xenograft and syngeneic models, the RT- 01 virus’ specificity in targeting tumors and therapeutic efficacy, alone and in combination with cell therapies, were evaluated. The amplification of virus-encoded red fluorescent protein (RFP) was monitored using the Ami HT Spectral Instruments Imaging system. Flow cytometry and immunohistochemistry (IHC) were used to analyze immune infiltration in localized subcutaneous-' tumors or metastases. The virus's toxicology was evaluated in multiple immune- compromised and immunocompetent mouse models. The RT-01 viruses demonstrate an impressive survival of 80% in the presence of active complement, resistance to neutralizing antibodies, and rapid viral expansion. In animal studies, a single systemic dose of EnvRT-01 selectively targeted three distinct human cancer indications (lung, melanoma, head and neck), resulting in tumor growth suppression across all three indications. In an immunocompetent syngeneic model, EnvRT-01 effectively reduced multiple lung tumors. EnvRT-01 not only targeted and expressed viral-encoded proteins in all tumor sites but also significantly modified the tumor immune microenvironment, facilitating-1 other innate-based cell therapies and chemotherapies. In metastasis models, EnvRT-01 specifically targets tumors, reducing metastatic tumor sites not only in the lungs but also in the metastasized liver, while drastically transforming the immune microenvironments from "cold" to "hot." Characterization of EEV production of the clone designated RT-01
To assess the enhanced extracellular enveloped virus (EEV) production capability of the newly selected clone (RT-01) (RT-01 has TK deletion and an insert in TK of nucleic acid encoding TurboFP), a comparative comet assay between the N2 and CALI (ACAM2000) viruses was conducted. FIGURE 2 shows spreading patterns of the CAL 1 (low producer of EEV) and the EEV high producer clone designated RT-01.
Methods
CV1 cells were plated at a density of 2 x 10^5 cells per well in 24-well Costar® plates and allowed to reach 75 to 90% confluence by the next day. Subsequently, the cells were infected with either the RT-01 or CALI vaccinia strain at a range of 200 to 2 plaque-forming units (PFU) per well for 60 minutes at 37°C in a 5% CO2 environment. Following infection, the cells were carefully washed, and fresh growth medium was added. During the 40-hour incubation period without disruption, plates were inclined at approximately a 15° angle to facilitate convection currents. After the designated incubation time, cells were stained with crystal violet.
Results
As depicted in Figure 2, distinct spreading patterns were observed for the two virus types. As shown on the right, RT-01 resulted in the formation of comet plaques, indicating the generation of high levels of EEV, whereas on the left, the CALI virus exhibited only rounded plaques, characteristic of very low EEV producer virus. This distinction highlights the unique properties of the two virus strains.
The genome of RT-00 is related to IHD-W but possesses distinguishing features. For example, 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 the host membrane of the EEV, facilitating the location of VCP on the surface of the enveloped viral particle.
Flow virometry is employed to analyze the expression of human surface proteins in the enveloped EEV vaccinia virus compared to the non-enveloped IMV vaccinia virus. The RT-05 virus can express (display) CD55 (more than about 95% (close to 100%) of the viruses), whereas the CAL2 virus enriched with the non-enveloped IMV does not express CD55. The production of EEVs by RT viruses provides a membrane, shown herein to be capable of displaying human proteins that confer protection against human complement or other such immune proteins, facilitate targeted tumor cell recognition, and enhance homing capabilities for precise therapeutic intervention. Examples below demonstrate the anti-tumor activity of these viruses.
EXAMPLE S
EEV particles, but not IMV particles, are resistant to human complement
EEV particles were collected from tumor cells infected with the RT-01 virus. To do so, the RT-00 virus and CALI virus each were modified to encode and express the fluorescent protein TurboFP635 at the TK locus, and designated RT-01 and CAL2 respectively.
Clones of the RT-01 virus, which encodes the TurboFP635 fluorescent protein, were isolated from the tumor cell line and were tested to assess resistance to human complement elimination and neutralizing antibodies. As a control CAL2 virus, (CALI viruses modified to express TurboFP635, encoding nucleic acid inserted into the TK locus), which propagates primarily as IMV particles, was used as a control.
Supernatants from MDA-MB-231 cells infected with each of the two viruses (MOI = 0.5) after a 24-hour incubation period were harvested. Equivalent titers of the two viruses were then exposed to 20% human serum for one hour at 37 degrees Celsius, with shaking every 15 minutes. Serial dilutions of each virus subsequently were used for titration via a standard plaque assay on CV1 cells, without staining with crystal violet. The results then were imaged using fluorescent microscopy 40 hours after infection and the plaque forming units were counted.
As depicted in FIGURE 3, a clear distinction is evident between the RT-01 and CAL2 viruses exposed to 20% human serum. The CAL2 virus exhibited only a 12.4% survival rate; whereas the RT-01 virus had a significantly higher survival rate of 79.3%. These findings demonstrate and underscore the distinct ability of the RT-01 virus to resist human serum compared to the CAL2 control virus.
Comparison of survival of IMV and EEV particles purified from RT-01
The RT-01 virus was modified in the TK locus to encode and express the TurboFP635 protein. EEV particles and IMV particles were collected from HELA S3 tumor cells infected with the RT-01 virus. The RT-01 IMV particles were isolated from the tumor cell line pellet, and the RT-01 EEV were isolated from tumor cell culture supernatants; each were tested to assess resistance to human complement elimination. Supernatants and pellets from HeLa S3 cells infected with RT-01 viruses (MOI = 0.5) after a 24-hour incubation period were harvested. Equivalent titers of the two virus particles then were exposed to 20% human serum for one hour at 37 degrees Celsius, with shaking every 15 minutes. Serial dilutions of each virus subsequently were used for titration via a standard plaque assay on CV1 cells, without staining with crystal violet. The results then were imaged using fluorescent microscopy 40 hours after infection and the plaque forming units were counted.
A clear distinction was evident between the RT-01 IMV and EEV exposed to 20% human serum. Only EEV but not IMV from the same virus showed a significantly higher survival rate. These findings demonstrate and underscore the distinct ability of the RT-01 (EEV particles) virus to resist human serum compared to the IMV particles.
EXAMPLE 4
Sequence of the RT-00 virus and genome modifications thereof NGS sequencing was performed on the RT-00 virus
Sequencing reveals that the RT-00 virus, with the sequence set forth as SEQ ID NO:01, has the A34R SNP that leads to the amino acid replacement K151E, which is a mutation that increases EEV production compared to the virus IHD-NR52. Comparison of the sequence of RT-00 with the complete sequence of IHD-W and the publicly available IHD-J sequence indicates RT-00 is distinct from each of IHD-J and IHD-W and thus is a different clone. RT-00 differs from IHD-W in a number of SNPs, and in contrast to IHD-W, has wildtype A56R in contrast to IHD-W. RT-00 differs from IHD-J in several SNPs in the available sequence RT-00 is a clone that also contains the A34R mutation.
Various individual knock-outs that enhance tumor selectivity have been introduced. These include knock-outs of TK, B8R, A46R, F1L and A52R. Knock-outs are prepared in VGF (vaccinia growth factor), B 18R (also knows an B19R in the Copenhagen strain and IHD-W 1 and referred to herein with respect to RT-00 (N2) B19R; this locus is B18R in the WR strain), and combinations of additional knock-outs from among TK, VGF, B19R or TK, VGF, F1L, and B19R and B8R are prepared. The combination of knockouts of these genes (by complete or partial deletions or insertions or transpositions so that functional product is not produced) results in viruses that have lower toxicity and/or spread in normal tissues. Sequence analysis
First, a selected vaccinia virus strain using CV-1 cells as the host cell line was amplified, resulting in the derivative RT-00. The virus was then amplified and purified before extracting its DNA. Next-generation sequencing (NGS) was used to determine the whole genome sequence of the new clone, which then was compared to 145 available vaccinia vims genomes and other orthopox viruses. The RT clone, selected from the IHD polyclonal (NR-52, deposited in 1963), was determined to be a unique clone that had not been previously described.
The IHD-RT (RT) genome was found to contain the A34R SNP (p.K151E), which is also present in IHD-W 1, IHD- J, and Rabbit poxviruses. RT was found to be related to IHD-W, but with key differences that make the vims unique. For instance, RT did not contain the truncated form of the A56R protein found in IHD-W (PMID: 25410873). The A56R protein has multiple functions, including regulating the presence of viral -encoded complement regulatory proteins (VCP) on the cell surface (PMID: 20719953, PMID: 21715594). The expression of A56R on tumor cells could protect the cell from complement attack, potentially leading to better spread of oncolytic virotherapy. A56R expression is also located in the host membrane of the EEV, facilitating the location of VCP on the surface of the enveloped viral particle (PMID: 21715594).
RT-00 contains the following Single Nucleotide Polymorphisms (SNPs) that lead to amino acid changes compared to IHDW1 :
1. p.A58T in VACWR016: The VACVWR016 protein in RT-00 is an ankyrin-like protein that is identical to many other vaccinia viruses (except IHDwl) and may affect host-range.
2. p.D16[2]in K7R: D is only repeated 2 times instead of 3. This repeat of 2 is not found in any other Orthopoxvirus and produces a protein that is one amino acid shorter. 3. p.L419F in VACVWR058: The VACVWR058 protein is an IEV morphogenesis protein identical to WR058 and COP-E2L.
4. p.L22[2] The RT virus contains a gene identical to VACV_TT9_147, with 2 repeats of Leucine in position 22 which is not found in IHD-W. VACV_TT9_147 is a hypothetical protein describe as AGJ91568.1. IHDW1 may have a similar protein that has not been described before with an p.L22[3] instead of p.L22[2], RT-00 encodes the following proteins that are not present in IHDW1:
1. RPXV102 (a cell surface-binding protein and carbonic anhydrase homolog), which does not occur 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 the target cell (PMID: 16227218).
2. Hypothetical protein VACV_IOC_B141_207 (ALF05177.1) with one SNP that leads to a change of amino acid p.H73Y.
3. RT virus contains two copies, one in each ITR, of ORF Wyethl 11 013 (a surface glycoprotein), which does not occur in IHD-W, but is present with an identical amino acid sequence in other clones from other strains including Wyeth (PMID: 17062162).
Figure 26 presents a graphic representation of major sequence differences between the virus designated RT-00 (N2 or IHD-RT) herein, and the virus designated IHD-W 1; functionalities that are not present in IHD-W 1 are in gray; SNPs that lead to changes in the amino acid sequence are in white.
Comparing A33R, A34R, A36R, A56R, B5R, F13L, A45R, A29L partial, A31R A30L, A32L partial, and A13L with the available IHD-J sequence, the RT-00 virus has 2 SNPs in A30L and 1 in A45R. All data demonstrate that the RT-00 virus is a new clone.
The RT-00 is modified to increase tumor-selectivity. RT-00 was modified to delete the J2R (TK) gene. Additional knockouts were generated, including knockouts of A46R, A52R, B8R, VGF, F1L, or B19R, and double knockouts as follows: a) TK, A46R; b) TK, A52R; c) TK, B8R; d) TK, VGF; e) TK, F1L; and f) TK, B19R. The effect of each knockout is described below.
Triple knockouts also are generated by combining the double knockouts with an additional knockout. For example, the following are generated: 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; and n) TK, F1L, B19R. The knockouts and combinations thereof increase one or more of tumor selectivity, anti-tumor activity, and modulation of the humoral immune response.
The following is a description of effects of each knockout.
A46R and A52R: Viruses have developed strategies to counteract signaling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes proteins that disrupt TLR signaling by interfering with TLR: adaptor interactions. The knockout of these genes, such as A46R and A52R, results in better elimination of the virus by the immune system in normal tissues.
The B18R/B19R protein has significant regions of homology with the a subunits of the mouse, human, and bovine type I IFN receptors. This binds human IFNa2 with high affinity and inhibits transmembrane signaling, as demonstrated by inhibition of Fc receptor factor yl/y2 and interferon-stimulated gene factor-3 formation, as well as inhibition of the IFNa antiviral response.
The vaccinia virus (W) Western Reserve (WR) gene B18R encodes a secreted protein with 3 immunoglobulin domains that functions as a soluble receptor for IFN-o/p. After secretion, B18R binds to uninfected and infected cells. The B18R protein present at the cell surface maintains the properties of the soluble receptor, binding IFN-o/p with high affinity and with broad species specificity and protects cells from the antiviral state induced by IFN-o/p. W strain Wyeth expressed a truncated Bl 8R protein lacking the C- terminal immunoglobulin domain. This protein binds IFN with lower affinity and retains its ability to bind to cells, indicating that the C-terminal region of B18R contributes to IFN binding. The replication of a W B18R deletion mutant in tissue culture was restricted in the presence of IFN-o, whereas the wild-type virus replicated normally. Binding of soluble recombinant B18R to cells protected the cultures from IFN and allowed VV replication. This represents a strategy of virus immune evasion in which secreted IFN-o/p receptors not only bind the soluble cytokine but also bind to uninfected cells and protect them from the antiviral effects of IFN-o/p, maintaining the cells' susceptibility to virus infections. The adaptation of this soluble receptor to block IFN-o/p activity locally will help W to replicate in the host and spread in tissues. The table below, also provided as Figure 24, which describes additional constructs and the resulting viruses, summarizes viruses derived from RT-00 (N2), their nomenclature, and modifications (knockouts and insertions):
Figure 29 also describes the viruses provided herein.
B19R exists in a soluble and a membrane-bound form. As a type I IFN receptor, B19R has a broad species specificity. It has high affinity for human IFN-alpha and also binds rabbit, bovine, rat, pig, and mouse IFN-alpha and IFN-beta. It has been shown that secreted B19R binds to uninfected and infected cells. It presents at the cell surface and protects cells from the antiviral state induced by IFN-alpha and IFN-beta. Binding of soluble recombinant Bl 9R (B18R in the WR strain) protects cultured cells from IFN and allows vaccinia virus replication. Knockout of this gene results in better elimination of the virus in normal tissues with an intact type I IFN system.
The B8R gene of VAC V encodes a secreted glycoprotein in the form of a homodimer in infected cells at the early stage of infection. This protein is homologous to the extracellular domain of the IFN-y receptor and inhibits IFN-y. The B8R protein binds to and neutralizes antiviral activity of several species of IFN-y, including human and rat IFN-y. Deleting the B8R gene results in viral attenuation compared to the wild-type strain. Knockout of this gene results in increased elimination of the virus in normal tissues by the immune system.
The thymidine kinase (TK) enzyme plays a crucial role in viral replication. It helps the virus use host cell nucleotides for its own DNA synthesis, enabling the virus to replicate efficiently within the host cell. This activity contributes to the overall infectivity and pathogenesis of the vaccinia virus. The knockout of this gene results in lower amplification of the virus in normal tissues that have low TK expression.
VGF is a secreted protein produced early in viral infection and acts as a mitogen to prime surrounding cells for vaccinia infection. Although the VGF gene is not essential for replication in vitro, it has an important role in vivo, because TK knock-outs have lower toxicity than the wild-type virus with intact VGF. The knockout of this gene results in lower spread of the virus in normal tissues.
The Vaccinia virus F1L protein is encoded by the F1L gene of the vaccinia virus. This protein plays a role in inhibiting apoptosis by interfering with the release of cytochrome c from the mitochondria. It thereby facilitates the survival of host cells, whereby the virus can amplify. In tumor cells, where apoptosis or programmed cell death already is inhibited, there is no need for the virus to further block apoptosis. Knockout of the F1L gene results in lower amplification of the virus in normal tissues that have an intact cell death program. The table above, also depicted in Figure 24 describes the viruses and nomenclature. Figure 6 presents a representative vaccinia virus genome map.
EXAMPLE S
The EEV virus done RT-01 spreads faster to neighboring cells than the CAL2 virus
IMV viruses exit a host cell through cell lysis or cell-to-cell contact. The EEV exits a host cell by budding. The virus acquires the additional outer membrane during budding from the cell membrane, which acquisition results in extracellular release of the virus without causing cell destruction. The outer membrane is derived from the transGolgi network and endosomes as the virus traverses the cell.
To assess and compare the distant spreading capability of the RT-01 virus, a high EEV producer, 10 plaque-forming units (PFU) of RT-01 (N2 encoding the TurboFP635) and CAL2 were employed to infect CV1 cells. Spreading patterns were observed at 24 hours and 48 hours post-infection using fluorescence microscopy. At 24 hours post-infection, the number of plaques between the two viruses was similar. RT-01 displayed large comet tails and viral spreading, a feature characteristic of EEV viruses, absent in the CAL2 virus plaques, a virus that mainly propagates cell-to- cell. By 48 hours, the infection pattern diverged significantly, with the almost entire CV1 plate infected by RT-01, in contrast to the less extensive infection observed with CAL2 virus.
EXAMPLE 6
Systemic administration of virus shows that purified EEV particles RT-01 survive human complement and reach tumors following systemic administration; the IMV virus particles do not
This Example shows that the EEV RT-01 virus targets human lung cancer xenograft model (A549-luc, a cell line that constitutively expresses luciferase, available from the ATCC) in the presence of human serum after systemic administration.
Methods
EEV and IMV particles from RT-01 were prepared by purifying EEV from supernatants, and IMV from pellets (no EEV purified) from infected MDA-MB-231 tumor cells.
Tumor implantation and treatment: Animals (athymic nude mice, 5-6-week- old) were anesthetized with isoflurane and inoculated subcutaneously with 100 pL of A549 cells (5 x 106 cells/animal) on both right and left flank regions. Tumor growth was confirmed by measuring with caliper and bioluminescent imaging. Seven days after tumor implantation, tumors were randomized (n=5) and r.o (retro orbital - systemic administration) treated with 105 PFU EEV RT-01 or IMV RT-01 viruses that had been pre-incubated with human serum CBD-002 (1 : 1 ratio) at 37°C for 1 hour. A PBS -treated group served as a control. Tumors were measured by caliper twice weekly and imaged once per week.
Results
As shown in Figure 5, five days post-treatment, mice that were treated with purified EEV exhibited noticeable TuiboFP635 expression signals on tumors located on both their left and right flanks. Conversely, the IMV viral particles obtained from the tumor pellet yielded a minimal signal in only one of the five mice, and the signal was not tumor specific. This observation underscores the value of injecting purified EEV particles in a clinical context, such as when oncolytic viruses are exposed to human complement, antibodies, and other factors before reaching the tumor site.
RT-01 also was administered systemically to a mouse model bearing three different human solid tumor types: head and neck squamous cell carcinoma, lung cancer, and melanoma, as xenograft tumors. Systemic administration of RT-01 resulted in the TurboFP635 expression signals at each tumor site. Average % tumor growth inhibition (n=5 tumors) 10 days following administration was 30% for head and neck squamous cell carcinoma, 92% for lung cancer, and 87% for melanoma. The collective results indicate that systemic administration of RT EEV viruses can survive human humoral immunity and can target and eliminate multiple tumors.
EXAMPLE ?
The EEV virus RT-02 has a high capacity to distribute inter-tumorally
It is a goal of oncolytic virus therapy to reach multiple tumors and metastases. For viruses that are injected intratumorally, it also is a goal that progeny viruses generated at the injection site can intravasate and reach distant tumors. After infection of the first tumor, virus replication, killing and releasing protected EEV virus (progeny) can circulate and target metastasis or newly forming tumors at different sides in the body. This occurs only where the vaccinia virus can intravasate, survive circulation, and target a new tumor; this requires high levels of EEV.
To show this, RT-02 (high EEV producer) and the CAL2 virus (low EEV producer) were injected into the right tumor to assess the capacity to travel to noninjected tumor on the other side in a dual A549 lung cancer model. Animals (athymic nude mice, 5-6 weeks-old) were anesthetized with isoflurane and inoculated subcutaneously with 100 pL of A549 cells (5 x 106 cells/animal) on both right and left flank regions. Tumor growth was confirmed by caliper measurements. 18 days after tumor implantation, mice were randomized and intratumorally treated with 5 x 105 PFU RT-02 (deletions in TK and A46R genes and insertion of the TurboFP635 gene of the N2 virus in the TK locus, and nucleic acid encoding eGFP in the A46R locus) or CAL2 virus in the right flank tumor. A PBS-treated group served as a control. Tumors were measured by caliper twice weekly.
17 days post-virus treatment, TurboFP635 expression was exhibited on all treated tumors (right flank) and four of five untreated tumors (left flank) of the RT-02 group, indicating robust virus expression within the treated tumors, and that the virus can reach distant tumors. In contrast, the CAL2 virus was primarily located at the injection site and not in the distant tumor.
On day 17 post-virus treatment, all five mice on the treated side (right) and four out of five mice on the untreated side (left) in the RT-02 group exhibited Turbo expression, indicating robust virus expression within the treated tumors, and that the virus circulated and to reach distant tumors. In contrast, the CAL2 virus, primarily was located at the injection site and not located in the distant tumor.
Real-time measurements of TurboFP635 fluorescence within the tumors substantiated the findings by confirming more efficient virus amplification of RT-02 compared to CAL2 in the A549 model over the long term. While the signal intensity of both viruses at the treated tumor was comparable at seven days post-injection, beyond this time point, RT-02 exhibited superior persistence and expression compared to the CAL2 virus in both the treated and untreated tumors.
By 24 days post-treatment, all animals were euthanized and tumors were collected for a plaque assay to assess virus amplification within the tumors. The findings show greater virus amplification with RT-02 in the treated and untreated groups relative to CAL2.
EXAMPLE S
The EW virus RT-01 amplifies in human and mouse tumor cells
An advantage of RT-01 is its ability to infect mouse cell lines, so that syngeneic mouse models and mouse models can be used to develop the therapy. FIGURE 10 shows that RT-02, which is RT-01 encoding a fluorescent protein amplifies and kills mouse cells.
The two image sets in FIGURES 11A-11B illustrate the amplification of RT-01 in B16-F10 melanoma cancer cells (FIG. 1 IB) and CT26 prostate cancer cells (FIG. 11 A) at various multiplicities of infection (MOIs).
For each cell line (B16-F10 or CT26), 10,000 cells were seeded per well in a 96- well xCELLigence® e-plate (Agilent). After 24 hours post seeding, cells were infected with RT-01 or CAL2 virus at different MOIs, and cytolysis was measured in real-time. As shown in the images, robust TurboFP635 expression was detected with RT-01, whereas CAL2 virus showed minimal expression. This observation indicates the favorable amplification of the RT-01 virus in syngeneic tumor cells compared to the CAL2 virus.
It is shown herein that viruses provided herein, such as RT-01, produce high levels of extracellular enveloped virions (EEVs), which contain a second human cell- derived membrane that include virally-encoded proteins, providing augmented protection against elimination by the immune system when administered systemically. As exemplified herein, with RT-01, the effect of the virus against human humoral immunity and its rapid spread were assessed ex vivo using a plaque assay. RT-01 was administered in various xenograft and syngeneic models to evaluate its specificity in targeting tumors and its therapeutic efficacy, either alone or in combination with cell therapies. The amplification of virus-encoded fluorescent protein was monitored using an imaging system, and flow cytometry and immunohistochemistry (IHC) were employed to analyze immune infiltration in both treated and untreated tumors.
RT-01 particles exhibited an approximately 80% survival rate in the presence of active human complement. In animal studies, a single systemic dose of RT-01 selectively targeted three distinct human cancer indications (lung, melanoma, head & neck), leading to the suppression of tumor growth for the three indications. Similarly, in an immunocompetent syngeneic lung tumor model, RT-01 effectively targeted and reduced multiple murine lung tumors with just a low single systemic dose of treatment. RT-01 targets and expresses virus-encoded proteins in all tumor sites and drastically modifies the tumor immune microenvironment to favor an anti-tumor immune phenotype, thereby facilitating other cell therapies, such as innate-based cell therapies.
EXAMPLE 9
Method of Manufacturing Extracellular Enveloped Virus (EEV) Particles
Methods that produce high amounts of EEVs are provided. The methods isolate EEVs from the culture medium. EEVs, unlike IMVs, are released from cells prior to cell lysis; the second membrane in EEVs is from the cell membrane in which the EEVs are produced. The methods herein exploit the proper timing to remove the cell culture medium before the cells lyse, but at a time when EEVs are maximally released into the medium. The particular timing is cell line dependent, but readily determined as exemplified herein for Hela cells. The methods additionally are gentle, and are shear force-free so that the second membrane is retained upon purification of the virus from the cell culture medium. To effect this a pump, originally designed for cardiac surgery, is employed and no steps or elements that employ or rely on shear forces are included. As a result, the amount of EEV produced is maximized.
To produce viruses that comprise a high level of EEVs, the process is designed to 1) isolate the viruses that are EEV, and 2) minimize disruption of the fragile second membrane. For any virus and cell line in which the virus is produced, the amount of EEVs that are produced is fixed; the methods herein are designed to maximize the amount of EEV isolated. In brief, the virus is cultured for a time sufficient for the EEVs to be released into the culture medium, but before the virus lyses the cell, which releases IM Vs. Culture medium, containing the EEVs is harvested and processed to purify the EEV virus. The methods herein propagate virus and isolate the virus in conditions that protect the second membrane. This is achieved by culturing the virus under shear force- free or minimal conditions, and for a time sufficient to produce EEV virus that is released into the cell culture medium, but prior to lysis of the cells by the virus, which would release IMV into the medium. The medium, which contains the EEVs, is harvested and the EEV virus purified therefrom, by gentle steps that minimize disruption of the second membrane; each step of the purification is a close to shear force-free as possible. As exemplified, concentration of the virus is effect by filtration, such as TFF, with gentle pumps, such as heart pumps used in humans. The methods provided herein result in a high percentage of EEV relative to total virus, and can retain close to 100% intact second membranes in the EEV. The percent of EEV isolated is a function of the ability to produce EEV, which is a function of the vims and cells and method of production; and, also, complement or serum resistance of the resulting vims, which has detailed herein, is a function of the cells in which the vims is propagated and/or the modifications of the virus, such as expression vitally encoded fusion proteins (chimeric proteins) between the vitally encoded membrane protein and a CRP or other humoral immunity modulating protein. As shown herein, some cell lines cannot produce EEV or produce very low levels, (see, e.g., Example 10 below). Some cell lines do not produce EEV with complement resistance conferred by cell line, for example, because of low expression of CDS 5 by the cell line (see, e.g., Example 11 below).
As detailed herein, the second membrane is derived from the cell in which vims is produced; some cells do not express complement resistance proteins in the cell membrane. As detailed and exemplified herein, viruses produced and provided herein can be modified to encode and express and display humoral immunity resistance proteins, such as CRPs, so that viruses as propagated have increased serum stability independent of the cell in which the viruses propagate, including in vivo so that viruses propagated in tumors, regardless of the tumor type, express the encoded humoral immunity resistance protein, on the second membrane, resulting in viruses that disseminate systemically and display serum resistance. The methods in this Example are for propagating virus in vitro to maximize the amount of EEV isolated. Combining the methods for production (manufacturing) with EEV that express humoral resistance proteins by virtue of the cell line in which they are produced and/or the modifications of the virus to increase serum stability, results in viral particles that exhibit a stealth of at least 60%, 70%, 80%, 90% and greater resistance to serum or serum stability, referred to as steal.
In practicing the methods of production/manufacture of virus, low shear force, measured in shear force/second, is employ throughout all steps, which includes tubing, pumps for gentle pumping (such as the Levitronix pump, and bearingless pumps), which do not destroy the second membrane, the cell culture medium, such as high glucose and other such components, no wheel, biomagnetic viability. Cell membranes are damaged or destroyed under shear forces of 100 shear/ seconds or more. Hence the components and steps are selected and performed to exert a shear force of less than 100 shear/seconds. For example, the pumps are selected that exert 10-100 shear/seconds, generally the pumps exert a force closer to 10 shear force/second. Pumps are selected for this low force, ingredients that lower shear force are added to the medium. There is some balancing of lowering shear force vs. purification time; longer purification times decreases yield of virus. Typically, the methods employ components so that purification takes about 6 hours. Tubing is generally hollow fibers. The formulation buffer is designed to improve freezing/thawing. Exemplary formulation buffer is a biocompatible pH 7-7.5, such as lOmM Tris/HCl, containing sucrose, trehalose, mannitol, glycine, and human albumin to preserve the integrity of the viruses. An exemplary buffer contains 10 mM Tris/HCl, 1% sucrose, 2% trehalose, 5% mannitol, and 300 mM glycine, 0.1% recombinant human albumin. The skilled person can prepare similar formulation buffers.
Thus, method provided herein is a shear force-free process (low shear force) for purification of the EEVs. The final product can be provided as a ready-to-use injection solution. It is a reproducible and fast procedure. The end product, as produced, is ready for IV injection or for storage for later use.
Virus is propagated in a cell line. For EEVs, the cell line or cells for propagation, generally is one that is designed to or selected to or known to produce a high level of EEVs, and, where the viruses are not modified as detailed herein to encode complement resistance proteins in the second membrane, cell lines that express CD55 (or other such protein) in relatively high amounts are used so that the resulting EEVs are serumresistant by virtue of the CD55(or other such protein). Exemplary cell lines that express high levels of CDS 5 include Hela S3, MDA-MB-231, and iPSC, particularly as detailed elsewhere herein . In general, a cell line that produces at least 5%, 10%, 15%, 20%, 25%, 30%, or at least 40%, or at least 50%, or more, EEV particles is selected for production of EEV viruses. As exemplified herein, EEV production using HeLa cells and the RT viruses, production is at least 5% to about 10%. A goal in selection of a cell line production is the resistance to serum provided by the cell line (“stealth activity” complement resistance or resistance to humoral immunity) so that the resulting particles have a stealth of at least 60%, 70%, 80%, 90% or more, meaning that upon exposure to serum, at least the recited percentage will not be inactivated by serum.
As described herein and exemplified below, modified EEV viruses that encode in the viral genome as a chimera with a transmembrane second membrane protein can be propagated independent of cell line or amplified in any tumor following administration to produce the S-R EEVs (also referred to herein as IV-EEVs). For viruses that are modified as described herein to be IV-EEVs (or S-R EEVs) so that they express a complement inhibitory or resistance protein, such as CD55, CD46 or CD59 in the second membrane of EEV, any host cell line can be used, such as, for example A549, CV-1, Vero, Hek293, stem cells, include iPSCs, particularly those modified to produce EEVs that exhibit increased serum resistance. Upscale of EEV production is achieved using a suspension cell line in a spinner or a bioreactor or other such device, adapted or produced for use with the methods herein, in which cell counts of up to 2 million cells per milliliter can be obtained. Alternatively, culturing can be effected using microcarriers with adherent cells to adapt the culture for growth in a bioreactor in suspension, or other bioreactors or growing adherent cells. The time point of harvesting is for Hela cells infected 1-2 xl0e6 cells/mL with IMV at MOI 0.5 result in a high level of EEV particles in the supernatant and low IMV, low impurities of host cell DNA, and protein derived from the suspension cells.
EEVs are pre-cleared from supernatants using a 1.2 pm filtration step to separate EEV particles from cells and cell debris. Different filter materials like, PP3, cellulose acetate or glass fiber can be used. To increase EEV particle recovery and to avoid damaging during the filtration step, 3% - 10% sucrose can be added.
In the downstream processing system, a shear-force less pump (such as, for example, a Levitronics pump) is used in combination with a Tangential Flow Filtration (TFF) device. The use of a shear-forceless pump ensures that the very shear force sensitive EEV particles are not destroyed. During this step hollow fibers made of different materials, such as Polysulfone or mixed cellulose are used to concentrate and purify the precleared virus solution by a factor of 10-500. Hollow fibers, material PS (such as Polysulfon material) and ME (Mixed Cellulose Ester) with a pore size from 0.05 pm up to 0.1 pm provide rapid concentration of the virus particles and removal most of impurities derived from the upstream process. Following buffer exchange against a formulation buffer that does not inhibit benzonase activity and is supplemented with 5mM MgCh, which protects of EEV particles, and de-aggregates the particles, host cell DNA is removed by benzonase enzyme (or other such DNAase) digest after virus concentration by tangential flow filtration (TFF). After the Benzonase enzyme digest another buffer exchange via TFF is performed to further increase purity and EEV stability for long term storage and/or direct IV administration.
In summary the steps were performed as follows:
Step 1) HeLa cell culture in suspension spinner flasks achieved HeLa-S cell densities of 2xl0e6 cells per mL. Culture conditions were 37°C 5% CO2.
Step 2) Direct infection in the reactor with IMV crude lysates with an MOI of 0.5 virus particles. The optimal harvest time point for 1-2 x 10e6 suspension HeLa cells after infection with MOI 0.5 is 44 +/- 4 hours, which results in hardly any dead cells and resulting in pure secreted EEV particles in the supernatant. As described above, an important aspect of the method is to produce EEV particles, which are released from the cells before the cells lyse to avoid contamination of the supernatant by IMV. EEV particles, not IMV particles, are released into the cell culture medium. The particular timing is a function of the cell line, starting virus, and MOI, which the skilled person can be empirically determined, and which is exemplified herein using the RT-00 virus and HeLa cells.
Step 3) Pre-filtration with a 1.2pm filter to separate cells/cell material from viruses, which reduces shear forces; 5-10% sucrose is added before filtration is performed. The combination of the 5-10% reduces shear forces to which the virus particles are exposed.
Step 4) Benzonase digestion to reduce host cell DNA present in the medium.
Step 5) Nearly shear force free concentration of viruses by a TFF (pore size 0.05pm, to 0.1 pm).
Step 6) Nearly shear force free re-buffering of the viruses in a storage and IV injectable formulation buffer, containing lOmM Tris/HCl, 1% sucrose, 2% trehalose, 5% mannitol, and 300 mM glycine, 0.1% recombinant human albumin.
Step 7) Filling and storage at -80°C (or, depending on storage time and intended use -20°C).
After thawing, EEVs were assessed by analyzing the resistance and/or survival to active human serum. The assays tests survival following 1 hour pre-incubation at 37°C in 12% human active complement and another incubation for about 2 days, at 37°C in 4% human active complement. The in vitro assay is tool investigating the resistance/survival of the manufactured EEV to human humoral immunity. Increased potency of EEV directly correlates with a higher likelihood of EEV reaching tumor sites following intravenous (IV) administration.
The results and effects on yield of EEVs that are serum resistant are shown in Figure 25. Bar one (#1) shows IMVs from RT-01 purified via sucrose gradient ultra centrifugation after release by lysis from infected HeLa host cells. This viruses produced by this method exhibit a low serum resistance, falling below the 20% survival of active particles.
Serum resistance of purified EEV viruses using the manufacturing processes as provided here are shown in bars #2 - #4, in which EEVs are produced by methods with increasing improvements to low-shear force in addition to isolation of viruses from the cells that are released in die culture medium prior to cell lysis. The results show increasing amounts of serum resistant EEV as indicated by increased potency (serum resistance) of the resulting EEV.
#2 run was performed without any benzonase digestion, which leads to higher viral particle aggregation and downstream purification needs around 2 days (HeLa cells)
#3 Reduced harvest time point to obtain purer EEV particles, sucrose was added before pre-clearing filtration was performed. Downstream purification was reduced to around 26 hours. (HeLa)
#4 genetically engineered vaccinia virus overexpressing CD55 in HeLa, 5% sucrose was added directly to the reactor, spinner speed was removed to 30 rpm. Harvest timepoint was reduced to 45 hours.
Figure 25 shows the potency, percent serum resistance of EEV viruses following various the above manufacturing procedures. Bar one (#1) represents EEVs from a standard manufacturing process (pellet purification) for the primary virus fraction released from infected host cells. This initial process exhibits lower than 20% survival of active particles. EEVs produced with improvements in the manufacturing process are represented by bars #2 - #4, each improvement described above. Increasing amounts of EEV are produced by methods with increasing improvements, particularly in reducing shear forces.
In another example, the EXPi293F™ cell line (Thermofisher) was employed as host cell line. Cells were cultured in a 3-liter spinner flask with a serum-free medium, achieving cell densities ranging from 3.0 to 9.0 x 10^6 cells per milliliter and maintaining a viability of over 90%. The population doubling time per day was approximately 0.9. The cells were infected with RT virus (unmodified for increased complement resistance) at a multiplicity of infection (MOI) of 0.5, and the optimal harvest time for maximal extracellular enveloped virus (EEV) content was determined to be between 21 to 24 hours post-infection, yielding around 1-4 particles per cell, which is approximately .0 x 10A7 plaque-forming units per milliliter. The resistance of these EEV particles against active complement was noted to about 12%, indicating a relatively lower resistance compared to other cell lines. Higher complement resistance is achieved using viruses modified to display a humoral immunity resistance proteins, such as a CRP. The methods/processes of manufacture can be conducted in various culture devices, including spinner flasks, suspension cultures, and in bioreactors. All share the common aspects of culturing and processing under low shear-force conditions and all share culturing of cells and harvesting culture medium prior to lysis, where the culturing conditions and timing and other parameters are selected so that EEVs are produced and released into the medium and isolated prior to lysis of the cells. Figures 36A-D summarize some exemplary processes or methods. Figure 36A depicts the process in a spinner flask; Figure 36B depicts the process 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.
EXAMPLE 10
Production of complement resistant EEV vaccinia virus particles is tumor dependent
As shown in previous Examples, exemplary vaccinia virus N2 extracellular enveloped virus (EEV) particles produced using Hela cells and released in cell culture supernatants exhibit significant resistance to rapid elimination by the human humoral immune system. This virus was prepared to produce an EEV virus that possesses the properties of EEV viruses, and that also has high anti-tumor activity. The methods exemplified in the previous examples to generate the N2 virus and derivatives thereof can be applied to other starting EEV viruses to generate viral clones that have high antitumor activity. The methods also can be applied to any known vaccinia virus, particularly those that were produced for anti-tumor therapy. The viruses can be modified, such as by modification of the A34R gene/locus, or B5R to be EEV viruses, or can be propagated and clones selected for high EEV-produdng clones. The viruses also can be modified as described in the Examples below to express complement inhibitor proteins in the EEV thereby produce EEVs independent of the cell line or tumor in which the virus replicates.
EEVs are crucial for vaccinia virus dissemination from cell to cell and for long- range spread within a host. Survival of the EEV particle depends on their capacity to resist quick inactivation by humoral immunity. This resistance varies depending on the cell line used to generate the EEV particles. Protection of enveloped viruses against human complement can be influenced by the expression of human complement inhibitors, such as CD46, CD55, and CD59, in the cell in which the virus is produced and in which the EEV forms. These inhibitors are encoded by the host cell line and incorporated into the virus membrane during virus assembly in the second membrane. In this example, the capacity of EEV generated by different tumor and non-tumor cell lines to survive complement inactivation is assessed.
Methods
Human Tumor cell lines, MDA-MB-231, BT549, and HeLa cells, Human nontumor cell lines, VP001 (Adipose-derived Mesenchymal stem cells, used as vaccinia virus carriers) or Non-human primate cells CV-1 (Used as host cell line for Vaccinia virus) were seeded in 5xl05 cells in 6-well plates. Cells were then infected with RT-01 vaccinia virus at MOI 0.5. After 2 hours of incubation, the culture medium was exchanged to eliminate virus from the cell culture. 24h post infection, supernatant was collected and kept on ice for titration and tested for resistance to human serum.
Resistance to human serum of viral particles was measured by incubating cell culture supernatant with 20% healthy donor serum (CBD-02) or fetal bovine serum for 1 hour at 37 degrees Celsius. The titer of infectious viral particles was analyzed by plaque assay.
Results
As shown in the table, the percentage of released resistant EEV viral particles was different in different cell lines. Tumor cell lines MDA-MB-231 and HeLa produce the most serum-resistant viral particles, >80%. The cell line BT-549 produced only 31% resistant viral particles in the supernatant. Non-tumor cell lines produced fewer than 10 % particles resistant to human serum. For administration, a high percentage of protected (EEV) virus is advantageous. These data also indicate that after administration of EEV virus, resistance of the EEV generated at the tumor site, depends on the tumor type for general EEV viruses. Production of EEV viruses, prepared as described herein that to encode complement resistance proteins or effective portions thereof or other such proteins, does not depend on the type of tumor. Hence, viruses provided herein that encode transmembrane proteins so that they express complement resistance proteins in the second membrane (EEV) to exhibit humoral immunity, following delivery to tumors and/or the tumor microenvironment and upon replication result in high levels of EEV in the tumors and tumor microenvironment. EEV viruses can spread systemically to other tumors.
Vaccinia particles released into the supernatant produced by different cell lines showed different levels of resistance to humoral immunity. These data indicate that in general for EEV viruses (other than the viruses exemplified below and described herein that are IV-EEV and independent of the host cells), the manufacturing host cell line should be selected to be one that produces high level of EEVs that exhibit serum resistance to ensure resistance to quick clearance at time of administration of EEV viral particles. Examples below demonstrate production and spread of EEVs resistant to humoral immunity that can be manufactured in any cell line.
EXAMPLE 11
Every tumor type and patient tumor has different levels of CD55.
As shown every tumor type and patient tumor have different levels of CD55; the level is not specific to a tumor type. As shown and described herein, vaccinia EEV particles resistance to complement can depend on the presence of CD55 and other anticomplement 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. As shown in this Example, the production of CD55, and hence complement resistance/resistance to humoral immunity, is not dependent on tumor type, but is specific to particular cell lines and tumors.
An analysis of CD55 expression, at the RNA level, on multiple tumor types using an RNA TCGA dataset (source: proteinatlas.com database) showed that every tumor type exhibited different levels of CD55 when compared among them. Glioma, head and neck, liver, renal, prostate, testis, breast, among other tumor types showed very low levels of CD55 (see, Figure 12). Every cancer patient sample of the same tumor type showed variations at the RNA level expression of RNA encoded CD55.
For example, comparing the expression level of CD55 in a plurality of established tumor cell lines, (source: proteinatlas.org/ENSG00000196352-CD55/cell+line) reveals a drastic difference of expression, at the RNA level, among tumors. As an example, Hela cells (originally from a cervical cancer) express very high levels of CD55 mRNA, while C33-A cells (also from a cervical cancer) have almost no detectable levels of CD55 mRNA. MDA-MB-231 cells (breast cancer) have considerable levels of CD55 mRNA, whereas BT549 do not. FIGURES 13A and 13B show the levels of RNA encoding CDS 5 in cervical cancers lines and in breast cancers (TPM= transcripts per million of protein encoding genes). The data indicate that CD55 expression is not a property of the type of the tumor, and, hence, is not a property of the tumor type in a subject.
In general, vaccinia EEV particles are manufactured in a cell line that has high levels of CD55, such as HeLa cells. Upon administration of such EEVs, there is a secondary problem that arises when the viral particles reach a target tumor site and start generating viral progeny. If the tumor is one that does not that express high CD55 or other complement inhibitor, the resulting progeny will not be resistant to complement inhibition; the complement resistance phenotype does not propagate. EEV particles generated at a tumor site depend on expression of anti-complement proteins at the tumor site, and such expression is not consistent among tumors of the same type. Tumors with low levels of CDS 5 generate EEV particles with low protection against complement; low protection results in elimination of the virus, thereby limiting the spread of the virus and elimination of the EEV. Other complement inhibitor proteins, such as CD59 and CD46 confer similar properties. CDS 5 in a tumor result in viruses that amplify in such tumors so that they are protected against complement; such viruses will disseminate because they are EEV viruses and will propagate because they are resistant to complement.
The results also indicate that CD55, CD59, and CD46 levels, and/or other complement inhibitor proteins can be biomaikers for companion diagnostics to assess the probability or likelihood of clinical success of general vaccinia virus-based therapies or other enveloped virus-based therapies for treatment of particular subjects. In practice, a tumor sample can be assessed for levels of one or more of these proteins; subjects whose tumors have levels above a pre-determined minimum, are candidates for treatment with oncolytic viruses, particularly with EEV viruses. A predetermined level can be a level determined based on the levels in tumors that produce complement-resistant EEV or levels higher than those in a tumor produce that fewer than a pre-determined amount, such as 50%, 60%, 70%, or 80%, resistant EEV (see, Figures 12 and 13A-B), such as EEV produced in cells lines that produce at least, for example, 30% of the amount of complement inhibitor or resistance protein compared to Hela cells. As another measure, the level can be selected as at least 40% or 50% of the virus particles produced are EEVs.
EXAMPLE 12
Generation of a recombinant vaccinia virus encoding and expressing a fusion protein B5R-CD55
As shown, the presence of the human-encoded anti-complement protein CDS 5 and/or other complement inhibitor in the second membrane of viral particles confers resistance to human complement in the EEV. This requires: 1) starting with an EEV virus; and 2) manufacturing the virus in a cell line that has high levels of CD55 expression.
A solution to this problem is provided herein. The solution also ensures that EEV with humoral immunity are produced in vivo and do not depend upon the tumor in which the viruses replicate. Provided are viruses and methods and viruses to ensure the expression of complement inhibitory or resistance protein, such as CD55, CD46, or CD59, or a complement-resistance conferring potion thereof, on the EEV membrane. The resulting virus is an IV-EEV that has higher resistance to humoral immunity than IMV and in contrast to EEV propagates to produce EEV with humoral resistance independently of the cells in which it propagates.
This is exemplified herein with CD55 expressed in the transmembrane protein B5R. It is understood that other such resistance-conferring proteins can be expressed, and that they can be expressed in other transmembrane proteins that are on the EEV second membrane.
It is shown herein, that encoding and expression of a protein, such as CD55, in enveloped vaccinia viruses, regardless of the host or tumor cell line or tumor type, or subject in which the virus is propagated, exhibit resistance to complement. The resulting viruses thereby can systemically disseminate upon administration and also following amplification in a tumor. Hence, provided are EEV viruses that encode a complement inhibiting protein, such as CD55 or other complement resistance proteins, as a fusion protein with a transmembrane protein so that the complement inhibiting protein, such as CDS 5, or a sufficient portion thereof to exhibit complement inhibiting activity) is displayed on the surface of the EEV. It is shown herein that this results in an EEV virus that can be propagated and retain complement resistance in any cell line, and can propagate in tumors in vivo and retain the EEV and complement resistant phenotype. This has heretofore not been achieved. Prior to this the upon propagation in cells with low levels of complement inhibiting proteins, the viruses were eliminated by complement, and thereby unable to disseminated in a subject.
The fusion protein, encoded by the virus, comprises a complement inhibiting protein or portion thereof sufficient to inhibit complement inserted into the extracellular domain (ECD) of a virally encoded (generally a native viral protein, but can be a recombinant or heterologous transmembrane protein) in the transmembrane domain of any transmembrane viral protein that is displayed on the second membrane. Exemplary of such fusion protein is the viral protein B5R fused to CD55 lacking the signaling peptide. The fusion protein is expressed in an EEV virus. The B5R transmembrane (TM) (SEQ ID NO: 234) portion of the protein directs the expression of the CD55 to the second membrane of the EEV virus, thereby by producing an EEV displaying a CD55 protein in the second membrane. The fusion protein ensures high levels of expression of the anti-complement protein or an active domain thereof in the second membrane of the virus and of the infected cell, thereby eliminating dependence on the cell for production of EEV viruses, and resulting in a virus that will propagate and produce EEV virus progeny in vitro and in vivo.
Also provided, as described herein, and demonstrated in Examples below, 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 also permit use of other host cell lines, including nontumor cell lines, such as, for example, stem cells, and CD55 high-expressing cells, in addition to cells such Hela cells and other cells (see, e.g., Figures 12 and 13A-B and accompanying text) that produce higher levels of CD55, thereby generating serumresistant EEVs in vitro. Other cell lines include 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.
Also provided are EEVs that encode and express the full sequence of CD55, or other full receptors like CD46 or CD59 without the B5R signal. Use of a transmembrane protein transmembrane domain, such as the B5R transmembrane domain, as a fusion protein with the complement inhibitor, ensures the location/orientation of any domain into the desired location in the EEV transmembrane protein. This results in expression in vivo of the complement inhibitor and production of EEVs at a tumor site. Figure 14 depicts the display of CD55 on the EEV second membrane.
Methods
Nucleic acid encoding the B5R protein without four short consensus repeat (SCR) domains (AIX99103.1) was used for preparation of nucleic acid encoding the fusion protein. B5R (STC) was linked to hCD55 without the signal peptide, by a linker to express hCD55 on the surface membrane on EEV vaccinia enveloped virus. The nucleic acid encoding the B5R was codon optimized for vaccinia virus using IDT codon optimization software with the final sequence (SEQ ID NO:252) as follows and the construct encoding the fusion protein depicted below (see Figure 15).
The ORF of the fusion protein CD55-B5R was built under control of the pSEL, pSE or pSL promotor. The ORF was flanked by homologous A46R regions left and right and cloned into pUC-GW-Kan (Genewiz) to generate donor vector pUC_A46R_hCD55- B5R (See, SEQ ID NOs:6-8) Recombinant viruses encoding the fusion protein were generated by homologous recombination of parental virus (RT-02) and donor plasmid pUC_A46R_hCD55-B5R. Negative selection for eGFP and positive for TurboFP635 (TuiboFP635 was introduced into TK locus as a marker gene) and then 3-5 rounds of virus purification on CV1 cells resulted in purified virus encoding and expressing hCD55. The resulting viruses was designated RT-05, RT-06, and RT-07. See Figure 24 for a list of viruses and phenotypes and particulars thereof.
The purified virus was confirmed with PCR. Figure 15 show the structure of A46R-hCD55-B5R as constructed and inserted into the parental virus to produce RT-05.
Discussion
As described in the detailed description, others have genetically engineered the vaccinia virus to express CDS 5 domains or similar proteins in an the IMV viral particle; none of have done so in an EEV. IMV particles, without a second membrane are easier to manufacture than the EEV. The double membrane is usually eliminated during the manufacturing process and/or cryopreservation. The other manufacturing methods purify viruses from host cell pellets in which the IMV particles are located.
As shown herein, the use of EEVs and not IMVs advantageously results in better spread of the virus. EEVs, produced in cell lines, contain other host cell line proteins in the acquired second membrane that confer further functionality to the EEV. For example, EEV particles express CD44, integrins, and other human receptors or immunomodulators in their second membrane that can help EEVs to inhibit immune system activation and to specifically target or accumulate in tumors.
EEVs, not IMVs, are the only particles that are actively secreted during the virus life cycle, thereby releasing virus particles into systemic circulation early during the viral cycle. The approach herein of expression of the CD55 in the second membrane provides retention of the full functionality of the EEV in vitro and in vivo.
A difficulty with respect to manufacture of EEVs, which have a fragile second membrane, is solved herein. Provided are manufacturing protocols that maintains the integrity of the second membrane. Also provided are EEVs that can be manufactured in any cell line and can be also manufactured (propagated) in situ in a treated subject. In such instances, virus is delivered, for example, using a cell-based platform to deliver viruses or by local administration into the tumor. The tumor becomes, in effect, a bioreactor that produces EEV, propagating and spreading EEV in vivo to systemically treat tumors.
EXAMPLE 13
Low CD55 expressing tumor cells infected with RT-05 Vaccinia viruses armed with B5R-CD55 express high levels of CD55 in the external membrane.
This Example demonstrates and describes production of EEV viruses that can be manufactured in any cell line. The levels of protein expression of CD55 on MDA-MB- 231 and BT549 cells lines was analyzed before and after infection with vaccinia virus armed with the B5R-CD55 fused protein. MDA-MB-231 and BT549 cells infected with vaccinia virus armed with CD55-B5R. RT-05 differs from RT-03 because it has a knock- out at B8R and is not armed with CD55-B5R; RT-05 encodes CD55-B5R in the A46R locus. The cells were examined using surface staining and intracellular staining. The results are depicted in FIGURE 16, which shows a histogram overlay. The results demonstrate that RT-05 virus infection leads to an increase in CDS 5 expression, especially in the CD55-negative (low expression) 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 vims (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 notable expression of CDS 5. In another experiment, EEV particles isolated from supernatants also exhibited expression of CD55 in the second membrane.
EXAMPLE 14
RT-05 EEV particles generated on tumor cells lines expressing B5R-CD55 viral- encoded protein are more resistant to human complement induced inactivation.
Each of human Tumor cell lines BT549 (negative for CDS 5) and non-human primate cells CV-1 (negative for CD55) were seeded in 5x10s cells in 6-well plates. Cells then were infected with RT-02 (backbone vaccinia virus control) or RT-05 (expressing B5R-CD55) at MOI 0.5. After 2 hours of incubation the medium was exchanged to eliminate virus from cell culture. At 24h post infection, supernatant was collected and kept on ice for titration and tested for resistance to human serum. Resistance to human serum of viral particles was measured by incubating cell culture supernatant with 20% healthy donor serum (CBD-02) or Fetal bovine serum for 1 hour at 37 degrees Celsius. Titer of infectious viral particles was analyzed by plaque assay.
Results
As shown in the table, the percentage of release resistant RT-05 viral particles “EEV” progeny from were highly resistant to human serum-induced inactivation even where generated in cells expressing lower levels of CD55. Released RT-05 viral particles, B5R-CD55-expressing vaccinia virus, were 300% more resistant to human complement than the backbone EEV vaccinia virus RT-02. Almost all viral particles released from B5R-CD55-expressing vaccinia virus (RT-05) from CD55 negative tumor cell lines were resistant to human complement.
EXAMPLE ISA Systemic administration of RT-05 EEV particles targeted or accumulated in and transformed all tumor microenvironments in immunocompetent mouse.
Manufactured RT-05 EEV particles as described in Example 9 were injected intravenously into immunocompetent EMT-6 tumor-bearing mouse models. Every animal had 2 tumors, one on the left flank and another on the right flank. After administration of one single dose intravenously of 3.5e6 PFU, imaging of TurboFP635 was analyzed in the whole animal using AMI HT equipment and was compared to untreated control. Analysis of TurboFP635 expression, day 3 and 6 post inoculation, indicated that the virus survived circulation and can specifically target every single tumor in the mouse. The viruses replicated and amplified in every tumor by day 6. The results are depicted in FIGURE 17, which shows the amounts of virus in the injected right flank tumors and in the un-injected left flank tumors on days 3 and 6. Controls show no virus in the tumors.
On day 7, animals were sacrificed, and all tumors (2 tumors per mouse) were analyzed for changes in immune infiltrates or TILs by flow cytometry. Seven (7) days after administration every tumor had increased levels of CDS T cells, CD4 effector T cells and lower CD4 T regulatory cells defined as (CD4+, CD25+, Foxp3+). The ratio of CD8/T regulatory cells showed a drastic increase in every single tumor of the mouse, indicating that the EEV particles targeted every tumor and quickly and specifically changed the tumor microenvironment. FIGURE 18 depicts the types of immune cells in the tumors, indicating a conversion to an anti-tumor phenotype.
In another experiment, a multiplex immunohistochemistry image shows high CD4+ and CD8+ T-cell infiltration and a decreased CD1 lb+ myeloid population in the tumor microenvironment following treatment with RT-01 (see, Example 15C) indicating remodeling of the tumor microenvironment.
EXAMPLE 15B
Resistance of enveloped CD55 viruses to human serum.
As shown in Figure 29, inclusion of all or a sufficient portion of a complement resistance protein (or inhibitory protein) on the surface of the EEV membrane confers increased resistance to human serum of high EEV viruses compared to the same EEV virus that does not include the complement resistance protein To demonstrate this, BT549 cells were seeded in 6-well plates and infected the following day at a multiplicity of infection (MOI) of 0.5 for 3 hours. Following infection, cells were washed and incubated for 24 hours to allow for the generation of extracellular enveloped virus (EEV) in the supernatant. EEV samples then were harvested and combined with either fetal bovine serum (FBS) as a control or 20% human serum. After a 1-hour incubation period, the samples underwent serial dilution for a viral plaque assay (VP A) using monolayer CV1 cells seeded in 24-well plates. The resistance percentage of each virus in the presence of human serum was calculated relative to the control in FBS. The results demonstrate significantly increased protection conferred by the engineered enveloped CD55 virus compared to the parental control.
EXAMPLE 15C
Systemic administration of Enveloped RT-01 Targets Tumors and Metastasized Tumors.
Data shown herein and additional data from other such experiments, summarized as follows, demonstrate dramatic remodeling of the tumor microenvironment (TME) to anti-tumor phenotypes.
Lung One million (le6) LL2 cells metastatic lung cancer cells were implanted subcutaneously into the right flanks of C57/BL6 immunocompetent mice. 5 days post implantation, a single dose of 3.5e6 plaque-forming unites (PFU) of RT-01 was intravenously (i.v.) injected, alongside a PBS control. Tumors were collected 7 days post virus injection and tumor tissues were harvested for analysis. Multiplexed immunohistochemistry (IHC) was conducted to identify changes in the cellular composition of the tumor microenvironment using antibodies against the markers CDS and CD4 to identify these t cell population, an antibody against vaccina virus to identify RT-01, and DAPI to stain cells. IHC antibodies for CD8+ and CD4+ T cells and for vaccinia virus were used to access the infiltration and tissue localization of RT-01 and of these T cell populations in the tumor microenvironment. Representative multiplex IHC images of RT-01 treated LL2 lung tumors implanted subcutaneously show an infiltration of CD8+ and CD4+ T cells into the tumor microenvironment. CD8+ T cells localized at or near RT-01 infected tumors and tissues while CD4+ T cells localized throughout the RT-01 infected tumor microenvironment.
Tumor tissues (n=5 per group) also were collected for flow cytometry and assessed for levels of leukocytes, tumor infiltrating lymphocytes (TILs), T cells, T regs, myeloid cells, and macrophage subsets (Ml and M2). Systemic administration of RT-01 significantly increased the populations of leukocytes (as a percentage of live cells), TILs (as a percentage of leukocytes), and T cells (as a percentage of leukocytes) in the LL2 mouse lung tumors. RT-01 significantly decreased the populations of Tregs (as a percentage of leukocytes) and myeloid cells (as a percentage of leukocytes) in the tumors. RT-01 treatment significantly increased the Ml macrophages as a percentage of macrophages, indicating macrophage polarization towards the pro-inflammatory/anti- tumor Ml subset from the anti-inflammatory/pro-tumor M2. Systemic administration of RT-01 induces an anti-tumor or immunogenic tumor microenvironment in primary tumor microenvironments.
A second experiment was conducted to test the anti-tumor activity of RT-01 in a model of metastatic lung cancer.
One million (le6) LL2 -luciferase (luc) expressing metastatic lung cancer cells were intravenously administered via the tail vein of C57ZBL6 immune-competent mice. Two weeks after tumor injection, a single dose of 3.5e6 plaque-forming units (PFU) of RT-01 was intravenously injected, alongside a PBS control. Six days post-virus injection, mice were imaged for luciferase-expressing tumors and then dissected and imaged for luciferase expression and TurboFP635-expressing virus. Bioluminescent emission (luciferase signal) measurements localized to the lungs of control mice indicating LL2 lung cancer cells colonized the lungs following intravenous administration. Treatment with RT-01 demonstrated a reduction in luciferase signal indicating an overall reduction in tumor burden within the lungs of mice treated with RT-01, 6 days post-virus treatment. Lung tissues from control and RT-01 treated mice were then harvested and imaged by fluorescent and bioluminescence imaging. Lung tissue taken from control treated mice demonstrated luciferase signal throughout the lung tissues whereas lung tissue taken from RT-01 treated mice demonstrated markedly less luciferase signal. Fluorescent imaging for TurboFP635, a marker for RT-01, demonstrated co-localization to lung tumor sites demonstrating that the RT-01 specifically targets the lung tumor cells. As depicted in Figure 30A, the virus (highlighted in red) specifically targeted lung tumors (shown in rainbow colors).
Lung tissue also was analyzed by multiplexed IHC using antibodies for CD8+ and CD4+ T cells and vaccinia virus access the infiltration and tissue localization of RT- 01 and of these T cell populations in the tumor microenvironment. Multiplex IHC images of RT-01 treated LL2 lung tumors demonstrated that RT-01 specifically targeted tumors and induced the recruitment of immune CD8+ and CD4+ T cells into the tumor microenvironment.
Liver tissue taken from control treated mice demonstrated luciferase signal indicating the presence of metastatic tumors. Liver tissue taken from RT-01 treated mice demonstrated little to no luciferase signal demonstrating that the virus significantly reduced the metastatic burden in the liver compared to the control. As shown in the Figure 3 OB, the virus significantly reduced the metastatic burden in the liver compared to the control (left panel). Thus, the systemically administered viruses such as RT-01 effectively colonize tumors and reduce metastases. Systemic administration of RT-01 can induce an anti-tumor or immunogenic tumor microenvironment in both primary and metastatic tumor microenvironments.
Dramatic changes in TME after RT-01 administration in CT26 colon cancer model (Immunocompetent). 2.5e5 CT26 cells were implanted subcutaneously into the right flanks of Balb/c mice, followed by intravenous (i.v.) treatment of 3.5e6 PFU of RT-01 5 days post implantation or a PBS control. Tumors were harvested and analyzed by multiplex IHC using the markers for CD3, CDS, CD4, CD49, CD1 lb, vaccinia, and DAPI. A representative tumor section demonstrates the presence of myeloid cells and CD4+ 1 cells localized in the tumor microenvironment of a control treated tumor. Systemic RT-01 treatment showed significant increase in the number of CD4+ and CD8+ T cells alongside a decrease in CD11+ myeloid cells at the tumor site following RT01 treatment. (No significant difference was observed with CD49+ NK cell numbers).
Dramatic changes in TME after RT-Oladministration in EMT6 breast cancer model (Immunocompetent).
Ie6 EMT 6 cells were implanted subcutaneously into the right flanks of Balb/c mice, followed by intratumoral i.t. treatment of 3.5e6 PFU RT-01 or a PBS control 5 days post implantation. Tumors were harvested and analyzed by multiplex IHC using the markers for CD3, CDS, CD4, CD49, CD1 lb, vaccinia, and DAPI. A representative tumor section demonstrates the presence of myeloid cells and CD4+ 1 cells localized in the tumor microenvironment of a control treated tumor. Intratumoral treatment with RT- 01 led to the infiltration of CD8+ 1 cells co-localizing at or around RT-01 infection tissue sites.
As shown herein, administration of the virus results in T cell infiltration of the tumors leading to remodeling of the tumor microenvironment. Data herein shows propagation and trafficking of virus to other tumors and metastases. High levels of cytokines from CD4+ and CD8+ T cells can lead to changes in the tumor microenvironment, such as upregulation of MHC. Remodeling of the tumor microenvironment improves the anti-tumor response of the immune system and renders tumors susceptible to immunotherapy. CD8+ T cells are lymphocyte cells, responsible for inhibiting tumor proliferation and disrupting metastasis by directly recognizing and killing tumor cells via intracellular antigens. For example, cytotoxic CD4+ T cells attack the tumors recognize MHC-H-positive tumor cells via their T-cell receptor and use contact-mediated delivery to release perforin (Prf) and granzyme B (GrB) to cause tumor cell death. EXAMPLE 16
Combination therapy with immunomodulatory drugs and vaccinia virus resists cellular and humoral immunity, resulting in sustained virus persistence at the tumor lesions
Upon intravenous systemic administration of vaccinia virus, the virus traffics to multiple tumor locations from the distant injection site. Subsequently vaccinia virus lyses the cells and by a cascade of cellular immune responses converts the tumor microenvironment in an anti-tumor phenotype. The triggered cellular immunity, however, drastically decreases viral replication and persistence even though this can serve as a safety mechanism to eliminate virus-induced toxicity. To address this problem, a method is provided herein to timely increase virus persistence against immune cell clearance by administering an immunomodulatory agent to modulate the immune response, but not eliminate it. For example, administration of cyclophosphamide at high doses depletes lymphocytes, thereby eliminating anti-virus (and hence anti-tumor) immunity. When used at moderate doses in combination with the virus, it activates the immune system in a conducive manner.
Alkylating agents, such as cyclophosphamide, at high doses are potent cytotoxic and lympho-ablative drugs, used as immunosuppressive regimens in the oncological and internal medicine. Low doses have immunostimulatory and/or antiangiogenic effects (see, e.g., Sistigu etal. ((2011) Semin Immunopathol 33:369-383, DOI 10.1007/s00281- 011-0245-0). Combination therapy of the virus and low dose chemotherapy or other such immunomodulating agent can solve the problem of virus clearance by the immune system, by reducing such clearance but not eliminating the immune response that is an important aspect of the anti-tumor response. The problem and a solution is illustrated in FIGURE 19. As discussed, administration of an oncolytic virus, such as a vaccinia virus, alters the tumor microenvironment by rendering it more susceptible to therapy with the virus and with immunotherapy agents. Combining the therapy with an agent the reduces the anti-viral response of the immune system to the virus, but that does not deplete the immune system allows replication of the viruses and transformation of the tumor microenvironment, but does not eliminate the immune system, which elimination renders the virus toxic. FIGURE 19 shows and describes the resistance of the RT-01 (N2) virus to humoral immunity but not to immune cell-mediated clearance. The virus lyses the cells and by a cascade of cellular immune responses and converts the tumor microenvironment into an anti-tumor 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 etal. (2011) Semin Immunopathol 33369-3%3, 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.
Methods le6 EMT 6 cells were implanted in right flank of BALB/c mice (5-6-week-olds). 5 days after the tumor implantation, 3.5e6 PFU of RT-01 virus (expressing TurboFP635 at TK locus) was intravenously injected once into the tail vain. Multiple time points imaging of TurboFP635 were performed to follow the virus amplification into the tumors using the Ami-HT Imaging System (Spectral) system.
Results
As depicted in Figure 19, there was a noticeable trend in virus amplification over the observed period. Virus amplification increased from day 2 to day 6, subsequently showing a decline on day 12, and stabilizing on day 15 and 19. This pattern underscores the virus's capacity to persist in the bloodstream, eventually reaching the dimensions of the tumor. The virus propagated and expanded/amplified expansion within the tumor, a phenomenon consistently observed around one week after intravenous injection.
The presence of the virus in the body, can be curtailed by the adaptive immune response 1-2 weeks following treatment. The timeline depends on various factors including the specific subject under consideration, the growth rate of distinct tumor cell lines within a living organism, and the permittivity of virus to the cancer cells. Timely clearance of the virus is indicative of its safety profile. As described herein, combining viral treatment with immune-depleting drug, such as low dose chemotherapy, can enhance the anti-tumor effects by muting or eliminating the adaptive immune response. (see, e.g., Sistigu etal. ((2011) Semin Immunopathol 33:369-383, DOI 10.1007/s00281- 011 -0245-0)) describes that the “immunostimulatory and/or antiangiogenic effects of low dosing cyclophosphamide (CTX [or CPA herein]) influences dendritic cell homeostasis and promotes IFN type I secretion, contributing to the induction of antitumor cytotoxic T lymphocytes and/or the proliferation of adoptively transferred T cells, to the polarization of CD4+ T cells into TH1 and/or TH17 lymphocytes affecting the Treg/Teffector ratio in favor of tumor regression. CTX has intrinsic “pro-immunogenic" activities on tumor cells, inducing the hallmarks of immunogenic cell death on a variety of tumor types.” EXAMPLE 17
Combination therapy with the virus and an immunomodulatory agent effectively modulates the immune cell system, enhancing treatment efficacy, promoting the persistence of the virus, and increasing the expression of viral payload genes.
As discussed above, administration of an immunomodulatory agent prolongs virus persistence in a timely manner, and dramatically alters tumor microenvironment in combination with the vaccinia virus. High doses of chemotherapy agents, such as cyclophosphamide (CPA) can effect lymphodepletion. For example, for cyclophosphamide, a dose greater than 100 mg/kg was reported to deplete lymphocytes such as T and B cells in mice, whereas lower dose (10-40mg/kg) showed downregulation of Treg population (Sistigu et cd. Semin Immunopathol. (2011) 33(4):369-83. doi: 10.1007/s00281-011 -0245-0). As another example, 10-40mg/kg in mice and about 250mg/m2 iv in humans are considered low dose or "metronomic dosing" where the immunostimulatory attribute of CPA was highlighted. (Sistigu et al. Semin Immunopathol. (2011) 33(4):369-83; Zitvoge et al. Semin Immunopathol 2011).
In this example, chemotherapy, in combination with vaccinia virus, totally transformed tumor immune microenvironment, creating a favorable setting for virus amplification and persistence, increasing anti-tumor responses by inducing high level of activated T cell infiltration and reducing Tregs. Combination with immunomodulatory agents such as low dose chemotherapy, e.g., with CPA, can enhance virus amplification, persistence, leading to improved expression of the viral payload genes.
In this Example, antitumor therapeutic efficacy of the combinatorial treatment strategy consisting of oncolytic virus RT-01 and CPA in syngeneic EMT6 breast cancer model is studied. The results are shown in FIGURE 20, which shows that administration of RT-01, which is the N2 EEV virus with the turboFP635 inserted into TK, and subsequent administration of chemotherapy with low dose cyclophosamide (CPA). This combination increased the anti-tumor response showing increased tumor regression, higher persistence of virus linked to immunodepletion, and an anti-tumor phenotype in the tumor microenvironment. Figure 20 shows the results of treatment with RT-01 virus and CPA treatment. The results show improved tumor regression, persistence of virus, and favorable changes in the tumor microenvironment.
Methods
Animals (BALB/c, 6-8 weeks old) were anesthetized with isoflurane and inoculated subcutaneously with 100 pL of EMT6 cells (1 x 106 cells/animal) on the right flank regions. Tumor growth was confirmed by measuring with caliper. Three (3) days after the tumor implantation, tumors were randomized (n=5) and i.v. treated with 3.5 x 106PFU RT-N2 viruses followed by i.p. treatment of 150 mg/kg CPA at day 7. RT-01 or CPA monotherapy treated groups were served as a single treatment control whereas PBS treated group was included as no treatment control. Tumors were measured by caliper twice per week and imaged once per week to confirm virus location/amplification. At day 7 after RT-N2 treatment (10 days after tumor implantation), tumors were collected, dissociated, stained, and analyzed with flow cytometer for the TIL analysis.
Results of treatment with viruses resistant to humoral immunity in combination with chemotherapy (low dose to achieve immunomodulation)
Treatment with RT-01 and chemotherapy combination therapy achieved significantly enhanced therapeutic efficacy showing higher tumor regression rates and better survival of the mice compared to the no treatment or monotherapy groups. Increased virus persistence also was observed with the combination therapy; changes in the tumor infiltrating lymphocyte (TIL) composition in the tumors were observed. The combination therapy significantly increased IFNy-producing CD8+ cytotoxic T cells and decreased regulatory T cell and myeloid cell populations in the tumors. Thus, the treatment with the IV-EEV viruses (viruses resistant to humoral immunity as described herein), in combination with a treatment that is immunomodulatory such that the virus persists and effects conversion of the tumor microenvironment so that it has an antitumor phenotype, but the response of the immune system is dampened. Exemplary of such immunomodulatory treatments are alkylating agents, platinum-based agents, and other such chemotherapeutic agents that, at high doses, can ablate the immune system, but at lower doses are immunomodulatory by reprogramming the tumor microenvironment such that anti-tumor immune cells/immunogenic immune cells are increased (e.g., IFNy-producing T cells and CD8+ cytotoxic T cells) while pro-tumor immune cells/immunosuppressive immune cells regulatory T cells are decreased. Low dose chemotherapy administered after systemic oncolytic viral administration can support viral amplification, viral persistence, spread and virus-mediated anti-tumor reprogramming the tumor microenvironment.
Various regimens employing the virus and immunomodulatory treatments can be employed. The regimens, and/or virus treatment, can be combined or, as necessary, with an anti-viral, as a safety switch, to reduce systemic viral levels. Immunomodulatory agents include low dose chemotherapeutic agents, such as alkylating agents, and platinum-based agents.
Anti-virals include acyclovir, brincidofovir, gangcyclovir, rifampin and tecovirimat (ST-246; TPOXX®), and other such anti-virals (see, e.g., Wu et al. (2019) “Screening and evaluation of potential inhibitors against vaccinia virus from 767 approved drugs,” J Med Virol 91.2016-2024, doi: 10.1002/jmv.25544). Other agents include, for example, vaccinia virus immune globulin (VIG), anti-vaccinia virus monoclonal antibodies. Exemplary treatment regimen for use with systemic viral administration includes: 1) treatment with an immunomodulatory agent; then 2) treatment with virus; and then 3) clear virus with ST-246 or other anti-viral. Alternatively, a step of immunomodulation can be included after treatment with the virus, not before.
EXAMPLE 18 Priming tumors with vaccinia virus drastically enhances immune cell therapy
The systemic administration of the virus facilitates priming of the tumor microenvironment, creating an environment conducive for the application of cell therapy. NK cells are a subset of innate immune cells and play a crucial role in combating virus- infected cells by recognizing those with decreased type I MHC. This Example assesses the infiltration of adoptively transferred allogeneic NK cells into the tumors. The results show that vaccinia virus transforms the tumor microenvironment resulting in increased immune cell infiltration. This Example, using NSG mice with lesional administration of the vaccinia vims, demonstrates significant benefits for immune cell therapy resulting from vims priming. As described and shown in FIGURES 21A, 21B and 21C pre-treatment by administration of vaccinia vims enhances NK infiltration in a solid tumors. Figure 21A depicts the protocol; Figure 21B shows a graph of vims presence in the tumors; Figure 21C shows the amount of infiltrated NK cells in the tumors measured by fluorescence signals, the images and a graphical depiction of the results.
Methods
5E6 A549-Luc cells were implanted in right flanks of NSG mice (5-6-week-old). When tumors reached 60-80 mm3, 5e5 PFU of RT-01 virus (expressing TurboFP635 at IK locus) was intratumorally injected. Four days after vims treatment, 5E6 DiR-labeled NK cells were intravenously administered. Multiple time points imaging of TurboFP635 and DiR were performed using Ami-HT Imaging System (Spectral) to trace NK cells in the animals and monitor the vims amplification at the tumor sites. Three days after NK cell administration, tumors were collected and imaged ex-vivo to assess the presence of NK cells and vims.
Results
Intra-tumoral RT-01 (N2) vims treatment prior to the NK cell administration significantly increased NK cell infiltration to the tumor sites as shown by in vivo and ex vivo. Following pre-treatment of the tumors with vims, NK cells exhibited faster infiltration into the tumor lesions. Pre-treatment of the tumors with vims, thus, can improve the response to immune cell therapy, including NK cell therapy.
Many cell therapies require pretreatment with dmgs, such as fhidarabine or cyclophosphamide, to suppress the immune system of the subject and create a more favorable environment for the infused cells to engraft and expand. In an exemplary regimen, provided herein, for systemic viral administration in combination with cell therapies, the vims can be systemically administered with such an agent, before, after, or with the cell therapy. Regimens include, for example, 1) treatment with an immunomodulatory agent; 2) treatment with vims; and then cell therapy. In other examples, immunomodulatory steps are not administered. In other example, the regimen can include an anti-viral, generally after treatment, to modulate vims levels. In some instances, the cell therapy reduces or eliminate the vims so that an anti-viral is not needed. In another example after virus treatment, antibodies, such as anti-vaccinia antibodies, are administered. In some instances, the subject already has anti-vaccinia virus antibodies, which can enhance the efficacy of cell therapies.
EXAMPLE 19
Generation of a systemic platform by administering EEVs provides expression of any selected antigen or antigens in the tumor microenvironment to combine with targeted anti-tumor therapies, including ADCs (Antibody-Drug Conjugates), CAR- T cells, CAR-NK cells, and others
The EEV viruses provided herein can be modified to encode target antigens for expression in the tumors to provide targets for combination therapy with immune cell and other antigen-targeted therapies. The virus is modified to encode and express selected antigens in the tumor cells. The virotherapy, which results in expression of target antigens on the cell surface, can be treated with antibody drug conjugates or CAR- T or other such therapies.
The viruses modified as provided herein exhibit improved dissemination and propagation when administered. The expression of the target antigens provides targeting of the infected cells by other cell therapy modalities (ADC or CAR-T). This is depicted in FIGURE 22, which depicts a tumor cell infected with a vaccinia virus that encodes a target antigen so that the antigen is expressed on the surface of the tumor cell.
Viruses armed with the B5R-CD55 fusion protein (TV-EEVs) and encoding BFP (blue fluorescent protein) and target antigens CD20 or HER2 were manufactured. MDA- MB-231 target cells were infected. After 24 hours of infection, expression of the virally- encoded products were confirmed by flow cytometry analysis. DAUDI or SKBR3 cell lines were included as positive controls for CD20 and HER2 expression, respectively. Hence by administering virally-encoded antigens, any targeted therapeutic product, such as any available antibody-drug conjugated ADC, antibody or CAR-T drug, can be repurposed to treat other tumors that initially did not express the antigen.
FIGURES 23A and 23B show target antigens expressed on the surface of the infected tumor cell. Figure 23A shows that virally-encoded CD20 is expressed, and Figure 23B shows that virally encoded HER2 is expressed. Such tumor cells express the targeted antigens for treatment with therapeutic products that target the antigens. Figure 23A table
Figure 23B table
For example, the non-human viral protein, A56, was expressed on the tumor cell membrane following infection with a vaccinia virus. A chimeric antigen receptor (CAR) construct that targets the A56 protein expressed on the tumor cell membrane was prepared and administered in a rodent model. The results demonstrated an antitumor response, achieved through the combined treatment strategy with the vaccinia virus, A56-targetingCAR T cells, and hydroxyurea (see, Cho et al. (2024) iScience doi.org/10.1016/j.isci.2024.109256). Such treatment regimens can be effected by employing a vaccinia virus that can be systemically administered. Hence, a combination treatment regimen employing the IV-EEV modified so that the A56 protein or other such unique viral protein is expressed on tumor cells following systemic administration. CAR-T cells that target the protein can be prepared, and administered with or following systemic administration of the virus. An immunomodulator, such as a chemotherapeutic agent can be administered with, or serially with the CAR-T cells to reduce or mitigate the effects of the immune system of the host on the virus.
EXAMPLE 20
Combination of systemic viruses with ADC against the virus
The viruses provided here can be used in therapeutic regimens in which viruses that, upon systemic administration, target all disseminated tumors. The therapeutic effect can be further enhanced by combining it with a drug that is highly specific to the tumor type or location, thereby delivering potent toxins or other targeted therapeutic products. For example, Antibody-Drug Conjugates (ADCs) are prepared against any antigen encoded by the virus, including native viral proteins of the virus. Once the ADC targets the tumor infected with the virus the potent toxin eliminates a large portion of the tumor. As the ADC is designed to target cells infected with the virus, it does not affect other tissues where the virus is not present, thus minimizing off-target effects. This strategy can improve the therapeutic use of ADCs in the clinic, many of which have serious adverse events due to their targeting of human antigens. This combination renders them more specific for tumors by targeting the virus, which infects the tumors.
EXAMPLE 21
Expression of encoded payloads in tumors
RT vaccinia viruses can express vaccinia virus-encoded proteins on the surface of infected tumor cells. These proteins can serve as target antigens for ADC and other antigen targeted therapies. In an example, it was demonstrated that tumor cells infected with RT vaccinia viruses express viral proteins within the infected cell membrane. An analysis was conducted on the levels of protein expression of E3L, A33R, and L1R in infected MDA-MB-231, BT549, and A549 cell lines, both before and after infection with vaccinia virus. The results showed that RT-01 and other variants of vaccinia virus infection lead to an increase in E3L and A33R on the surface of the cells. This indicates that vaccinia virus proteins can be selected to generate antibody-drug conjugates that specifically target only infected cells, avoiding any side effects due to off-target toxicity. The target is specific to tumor cells infected with the virus, and not present on any other human tissues. Systemic virotherapy, thus, allows for the combination with other targeted drugs to specifically kill only tumor cells.
In another example, it was demonstrated that the RT vaccinia viruses can deliver and secrete encoded payload at the tumor sites. RT virus was engineered to encode and secrete a selected payload in the tumor microenvironment FLT3L, IL- 15 superagonist (IL-15/IL-15R alpha chain complex) or anti-VEGF single chain antibody. Table A, below, shows the secretion of FLT3L when Hela cells were infected with the RT vaccinia virus encoding FLT3L at a MOI of 1 for 24 or 48 hours. The Hela cells infected with the RT vaccinia virus show high level of payload secretion in the supernatant collected from the culture plates. The payload secretion also was confirmed with supernatant collected from Hela cells infected with RT virus encoding IL-15 superagonist or RT- encoding(anti-VEGF scAb with Flag tag) by ELISA detecting the Flag tag (Table B). In vivo payload delivery also was confirmed by ELISA performed on the tumor explants. LL2 lung tumor cells were subcutaneously implanted into the right flanks of C57/BL6 immunocompetent mice. When tumors reached a substantial volume, a single dose of 5e6 PFU of RT virus carrying FLT3L payload was intravenously (i.v.) injected. Tumors were collected 6 days post virus injection and lysed for the FLT3L ELISA analysis. Table C shows FLT3L production detected in the tumors systemically treated with RT-66 virus (eGFP(TK")/FLT3L-BFP(B19R-)), whereas control virus did not produce any payload in the tumors.
EXAMPLE 22
Sequences of various transfer vectors and/or descriptions of components of the resulting virus (see, Figure 24 for the relationships and derivation of each vector and inserts in each, and description herein). As is apparent, some virus constructs were prepared using the same gene/transfer vectors but have a different viral backbone or other variations and inserts/deletions. The transfer vectors, which contain vaccinia virus flanking sequences introduce the noted sequences, including promoters to produce the viruses (designated by “RT’ numbers). Some of the RT viruses were produced by Cre Lox integration rather than homologous recombination with a transfer vector. The transfer vectors are pUC-derived vectors (see, e.g., Figure 28B and the accompanying sequence). See SEQ ID NOs: See SEQ ID NOs: 2-21, 518-523, and 628-634. pUC-Turbo (TK) ( 1-4748 ) SEQ ID NO : 2 Promoter (underlined) : pSEL ( 841-880 ) HR Si te ^italicized) : J2R flanking sequence ( 255-812 , 1652- 2345 ) Transgene (bold) : TurboFP635 ( 899-1606)
EXAMPLE 23
EEVs produced by hiPSCs are resistant to complement-mediated vaccinia virus clearance.
Systemic injection of an oncolytic virus leads to viral clearance by the host immune system, including complement, thereby reducing therapeutic efficacy. The EEV virus offers protection by expressing complement-regulatory proteins on its second membrane or extracellular envelop. Targeting, homing receptors, and other immune regulatory proteins can be expressed in the extracellular membrane. These immune- protective proteins can be incorporated into the EEV membrane during the manufacturing stage by selecting appropriate host cell lines. Hela cells have been documented to exhibit high expression of complement-regulatory proteins. The immunogenic nature of this cell line, and being of tumor origin cell lines, poses challenges for development as a human cell therapy. In this example, virus is manufactured in hiPSCs, a non-tumor cell, which express these proteins on the EEV membrane and to provide protection against complement-mediated virus clearance when used as a host cell line. hiPSCs express other immunomodulatory and receptors that will be expressed on or in the extracellular envelop of the virus.
Methods:
Human iPSCs (hiPSCs; DYS0530 derived from skin) were seeded to reach 70% confluency at the time of infection, while Hela cells were seeded at 50,000 cells per 6- well plate a day before infection. The cells were infected with RT-01 in infection medium containing 2% FBS, which was removed after 3 hours. 24 hours post-infection, media containing EEV (hiPSC EEV and Hela EEV) were collected by removing pellets. Freshly collected hiPSC EEV and Hela EEV were incubated with human serum from three different donors (CBD13, CBD27, AB2391) to induce complement-mediated vims neutralization for one hour. Neutralized EEVs were serially diluted for VP A analysis as previously described. FBS was included as a serum complement control, and IMV was included as a neutralization-prone vims control.
Results: The results shown in Figures 33 A-C show that hiPSCs produce a comparable number of viruses in the supernatant when compared to Hela cells. hiPSC EEVs survived at 37% compared to 12.7% in the IMV control when neutralized with CBD13 serum for one hour, while Hela EEVs survived at 57%. hiPSC EEVs survived at 59% compared to 21.6% in the IMV control when neutralized with CBD27, whereas Hela EEVs survived at 68%. Finally, hiPSC EEVs survived at 69% compared to 4.9% in the IMV control when neutralized with CBD27, while Hela EEVs survived at 84.6%. These results demonstrate that hiPSCs can be used as a manufacturing host cell line to produce EEVs that are protective against complement neutralization. This data indicates that hiPSCs can be used to manufacture enveloped viruses, incorporating hiPSC functionality into the virus envelope. This example shows the protections against serum-induced inhibition, and other functionalities, such as stealth, immunomodulation, targeting, and tumor homing from different hiPSCs can be added to the extracellular membrane of enveloped viruses. 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
EXAMPLE 24
Regression of lung metastatic tumors was observed following systemic administration of vaccinia virus.
As detailed below, a highly lung-metastatic tumor cell line was generated by isolating metastasized LL2 tumors from the lungs after tail vein administration of LL2- Luc cells in C57/B6 mice. LL2-Luc cells are a luciferase-expressing mouse cell for use for in vivo in vivo imaging in xenograft models; they are available from the ATTC asCRL-1642-LUC2 ™. The isolated LL2-Luc cell line exhibits high rates of lung engraftment and was designated a lung-metastatic LL2 tumor model. Use of this cell line allows generation of a reproducible metastasis model in syngeneic animals. This example shows by use this model that RT-52 has remarkable therapeutic efficacy after systemic administration.
Methods le6 lung-metastatic LL2-Luc cells were administered via tail vein of C57/B6 mice (5-6 week-olds). Eight days after the tumor colonization of the lungs, mice were randomized based on BLI signals of the LL2-luc tumors (n=4) 1 day prior (-1) day of treatment administration (day 0). 5e6 PFU of RT-52 was intravenously injected into the tail vein 1 or three times. Some animals additionally received an intraperitoneal administration of 75 mg/kg CPA (cyclophosphamide) two days after each virus treatment, and immunomodulatory agents often use to prime patients prior to immunotherapy treatments. The treatment was repeated three times every 3-4 days. The RT-52 virus alone treatment group was included for comparison as were virus and CPA only control groups. Multiple time-point imaging of BLI and TurboFP635 were performed to follow metastatic tumor growth and virus amplification in the tumors using Ami-HT Imaging System (Spectral).
Results
As depicted in the Figure 34, there was a noticeable trend of tumor regression with the virus treatment (RT-52 alone) when total bioluminescent emission was measured. The tumor burdens of untreated control group have significantly increased from treatment day 0 to day 9, at which time point the animals had to be sacrificed due to clinical symptoms. The virus-treated group showed slower tumor growth during this observation period. Data indicate virus reach metastatic sites and efficiently kill tumor cells, express payload (in this example TurboFP635). The RT-52 virus therapy achieved significantly improved durable therapeutic efficacy when animals were immunomodulated with CPA, as observed before in the subcutaneous syngeneic tumor models. Even a single dose of therapy resulted in improved therapeutic efficacy compared to virus alone or CPA controls for longer period of times. Prolonged virus presence at the tumor sites, as well as changes in the tumor immune microenvironments can account for this. RT-52 virotherapy did not contain any additional therapeutic payload and the backbone alone was able to significantly impact tumor growth alone with more durable therapeutic response when animals were treated with CPA.
EXAMPLE 25
Generation and Selection of RT-00-derived tumor selective knock-outs.
The clone, designated RT-00, exhibits high EEV expression. To enhance tumor specificity and minimize off-target effects after systemic administration, genetic modifications were introduced. Knock-outs were generated to improve tumor selectivity. As detailed above, the following variants were generated: single knockout RT-01 (TK-), and double knockouts RT-17 (TK-, B19R-), RT-21 (TK-, VGF-), and RT-23 (TK-, A46R-). All backbone variants expressed TurboFP635.
To evaluate tumor selectivity, intravenous injections were administered to immunocompromised, tumor-bearing nude mice with all aforementioned variants. Upon sacrifice, selected organs were subjected to ex vivo imaging to assess the expression of TurboFP635, which indicated virus tumor-specific targeting.
Methods
5x10^ A549 lung cancer cells were subcutaneously injected into the right flank of 5-6-week-old immunocompromised nude mice. Twelve days post-injection, when tumors reached a volume of 60-100 mm3, mice were randomized and intravenously administered with the indicated viruses at low (5x10A5 PFU) or high (5x10A6 PFU) single dose. Ex vivo fluorescence imaging of virus-encoded TurboFP635 was performed on selected organs (n=5) using the Ami-HT Imaging System (Spectral). Signal intensity was analyzed with Aura software and visualized using application GraphPad (Prism).
Results
As shown in Figures 32A and 32B, which provide heat maps representing the intensity of fluorescence signal by organ (n=5), single and double knockout viruses, result in significant viral amplification within tumor tissues. The TK-only knockout show poorer tumor preferential amplification to increased off-target effects, with higher viral replication observed in organs such as the lungs, kidneys, and ovaries compared to the double knockout strains. The RT variant amplifies in mouse tissue, showing that the this is a valid model to analyze virus targeting and selectivity. The IK- + B 19R- and IK- + VGF- double knockouts exhibit lower levels of off-target effects compared to the TK- + A46R- double knockout. These trends were consistent across low and high doses of viral administration.
To further minimize off-target effects and improve preferential amplification of the virus in tumor tissues, triple-knockout viruses designated RT-51 (TK-/B19R-/A46R-) and RT-52 TK-/VGF-/A46R-) were generated and tested in an A549 lung tumor-bearing immunocompromised nude mouse model.
Following the same methodology described above, ex vivo fluorescent imaging of organs shows in Figure 32C that both triple-knockout viruses exhibited significantly reduced off-target amplification in non-tumor tissues while maintaining potent tumor targeting and amplification of the payload. Thus, triple-knockout viruses, such as those designated RT-51 and RT-52, are among the variants that not only have high EEV production, increased serum resistance, but also increased tumor targeting and amplification.
EXAMPLE 26
Lung tumors by RT virus and CPA combo treatment
As described in Example 17, a high dose of CPA can lead to the lymphodepletion and related toxicity when combined with virus treatment. This Examples tests whether an engineered RT virus with improved tumor specificity can overcome off-site toxicity issues with high dose CPA and enhance therapeutical efficacy.
Methods
One million (le6) LL2 lung cancer cells were implanted subcutaneously into both flanks of C57ZBL6 immunocompetent mice (n=10). 5 days post implantation, a single dose of 5e6 PFU of RT-64 (TK-, A46R-, B19R-) or RT-65 (TK-, A46R-, VGF-) was intravenously injected followed by intraperitoneal injection with 150mg/kg of cyclophosphamide (CPA) 2 days after the virus treatment. Tumor volumes were measured twice weekly with calipers and multiple time point imaging of TurboFP635 was performed to follow the virus amplification using the Ami-HT Imaging System. Data was visualized with the GraphPad (Prism) application.
Results
The RT-64 and RT-65 viruses trafficked to and amplified at the tumor sites, which started to clear after day 5 post treatment. CPA treatment prolonged virus presence at the tumor sites, as shown in Figure 37A. The virus treatment led to reduced tumor volumes. RT-65 showed significantly higher tumor regression even when used as monotherapy (Figure 37B) When combined with 150 mg/kg CPA, RT-64 and RT-65 showed significantly improved efficacy. In previous studies, this dose of CPA was not tolerable with virus treatment, but it was well tolerated with RT-64 and RT-65 due to improved tumor specificity. The enhanced efficacy was confirmed by a mouse survival curve where two mice achieved complete remission with the RT-65 and CPA combination treatment. (Figure 37C)
EXAMPLE 27 Biodistribution of RT-52 virus in mice conditioned with CPA.
Example 25 shows that modified RT viruses exhibit tumor specificity with reduced off-target toxicity. To ensure toxicity profile of the modified RT viruses, we evaluated which organs they biodistributed and how rapidly they clear from the body using C57/B6 syngeneic mice.
Methods
One million (le6) LL2 lung cancer cells were implanted subcutaneously into the right flanks of C57/BL6 immunocompetent mice. 5 days post implantation, a single dose of 5e6 PFU of RT-52 (TK-, A46R-, VGF-) was intravenously injected followed by i.p. injection with 75mg/kg CPA 2 days after the virus treatment, alongside a PBS control. The following organs were collected from tumor-bearing and non-tumor bearing mice at 1, 7, 14, 42 days post virus treatment along with blood and tumor: Brain, heart, kidney, lung, liver, ovary, spleen, lymph node. Harvested organs were homogenized and DNA were extracted for quantification of vaccinia virus by qPCR for A56R gene.
Results
The Table below (A) shows biodistribution in the tumor-bearing mice. High level of RT-52 (copies/ug of DNA, n=3) was detected in the tumors right after the virus treatment (day 1) with some levels of RT-52 also detected in the organs such as ovary, lung, lymph node. The virus preferentially replicate in the tumors compared to the other organs when observed on day 7 after treatment. A reduced quantity of viruses was detected at 14 days post treatment in all the organs including tumors although a high level of the virus was still present in the tumors. CPA treatment resulted in increased virus. In the disease-free mouse model, RT-52 was detected in the ovary with relatively higher level of virus detected with CPA + RT-52 combination treatment as observed in the tumor-bearing mouse model. However, regardless of CPA treatment viruses were completely cleared from all the organs by day 42 post treatment, when analyzed in nontumor bearing mice (Figure 34).
TABLE A
TABLE B
EXAMPLE 28
Tumor cell lines infected with RT-77 carrying CD55-A33R payload exhibit high expression of CD55 on the surface of the cells.
The level of CD55 expression after RT-77 infection was confirmed in cell lines with varying degrees of CDS 5 expression. Three cell lines were included as target cells: CV1 cells (CD55 negative cell line), BT-549 (a low CD55-expresser cell line), and HeLa cells (a high CD55-expresser cell line). Briefly, cells were seeded at 5x10s cells per well in 6-well plates and infected with RT-65 (tKO backbone vaccinia virus control) or RT-77 (tKO vaccinia virus expressing A33R-CD55) at an MOI of 0.05. 24 hours after infection, cells were collected, surface-stained with a CD55 antibody, and analyzed using a Cytoflex flow cytometer to measure CDS 5 expression on the surface of target cells after respective vaccinia vims infection.
The results in the Table below demonstrated good infection with both RT-65 (TurboFP+) and RT-77 (TurboFP+ and eGFP+) vaccinia virases as confirmed by the reporter gene expression. RT-77 vims infection led to an increase in CDS 5 expression, especially in the CD55-negative (CV1) and CD55 low-expresser (BT-549) cell lines. CV1 cells did not express CD55 with control RT-65 vims infection. However, they showed moderate expression of CD55 after infection with RT-77 vims, demonstrating that the CD55 payload can be expressed on the surface of the target cells. This was also confirmed with the CD55 low-expresser BT-549 cell line. The BT-549 cell line expressed a moderate amount of CDS 5 as a baseline with the control RT-65 vims, which increased to the higher level after. It exhibited high after infection with RT-77 vims. We did not observe much difference in CD55 expression in HeLa cells, as this cell line already had high CDS 5 expression.
EXAMPLE 29
RT-77 EEV particles expressing A33R-CD55 are more resistant to human complement-mediated inactivation of virus.
Hela cells (a high CD55-expressing tumor cell line) were seeded at 5x10s cells per well in 6-well plates, while hiPSCs were seeded 4 days earlier to reach 70-80% confluency on the day of infection. 24 hours after seeding, cells were infected with RT- 65 (tKO backbone vaccinia virus control) or RT-77 (expressing A33R-CD55) at an MOI of 0.5. IMV virus was also included as a control since its lack of a second membrane makes it more prone to complement-mediated vims elimination. After 3 hours of incubation with the RT viruses, the media were changed to eliminate the vims from the infected cell culture. At 24 hours post-infection, EEV supernatant was collected and tested for resistance to human serum complement. Briefly, the resistance of each EEV supernatant to the human serum was measured by incubating them with 20% healthy donor serum (CBD27 or AB2391) or control fetal bovine serum for 1 hour at 37°C. The titer of infectious viral particles was estimated by a virus plaque assay.
Results
As shown in FIGURES 38A and 38B, the EEVs released from RT-77-infected Hela or hiPSCs were highly resistant to human serum complement-mediated inactivation compared to EEVs from RT-65. For example, EEVs produced by RT-77-infected Hela cells showed almost 100% protection against complement from serum from donor CBD27, whereas EEVs produced by its control virus (RT-66) showed around 30% protection from complement-mediated virus clearance. The resistance of RT-77 EEVs was confirmed by testing against two different types of human sera (CBD27 and AB2391).
EXAMPLE 30
Comparison of dKO and tKO RT viruses in tumor cell killing.
For each cell line (MDA-MB-231, BT-549, Hela), 20000 cells were seeded per well in a 96-well XCELLIGENCE® e-plate (Agilent). 24 hours after seeding, cells were infected with dKO RT viruses (RT-17 (TK-, B19R-), RT-21 (TK-, VGF-), RT-23 (TK-, A46R-) or tKO RT viruses (RT-64 (TK-, A46R-, B19R-), RT-65 (TK-, A46R-, VGF-) at different MOIs (0.01, 0.1, 1) and cytolysis was monitored in real-time. As shown in the tables, all the dKO and tKO RT viruses showed high cytolysis capabilities in all three target cell lines. For some of the cell lines, dKO and tKO clones showed even higher cytolysis compared to the single KO RT virus (RT-01).
EXAMPLE 31
Screens of NCI-60 cancer cell line panel for cytolysis with RT-65 virus
NCI-60 cancer cell lines were tested for their permissivity to RT-65 virus infection/replication in vitro. NCI-60 panel constitutes with cancer cell lines of diverse lineage derived from nine distinct tissues (breast, colon, central nervous system, renal, lung, melanoma, ovarian, prostate, and blood) previously characterized extensively by the National Cancer Institute. In addition to the NCI-60 panel, we also included 6 pancreatic cell lines.
Result
PFU 50 and 500 as low and high doses of RT-65 viruses, respectively, were tested. RT-65 exhibited high permissivity in most of the NCI-60 cancer cell lines, including colon, non-small cell lung cancer, melanoma, ovarian, renal, prostate, and breast cancer cell lines. Leukemia is the only cancer cell type that lower cytotoxicity to RT-65 (Table xx). Variability of cytotoxicity was observed between cell lines even if they were of the same tumor type. No correlation between cell doubling time and RT-65 cytotoxicity was observed.
EXAMPLE 32
Canine cell lines are permissive to RT viruses
D-17 (osteosarcoma canine cell line) and CMT-U27 (breast cancer canine cell line) cells were seeded at 20000 cells per well in a 96-well XCELLIGENCE® e-plate
(Agilent). 24 hours after seeding, cells were infected with RT-01 (TurboFP (TK-)) or
RT-05 (TurboFP (TK-), CD55-B5R (A46R-) viruses at different MOIs (0.01, 0.1, 1, 10), and cytolysis was monitored in real-time. As shown in the Figures 39A-B, 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 post-infection. The CMT-U27 cell line showed resistance at lower MOI, with cytolysis only observed at the highest MOI (MOI 10).
These data demonstrate that canine cell lines are permissive to the RT viruses, see
FIGURES 39A and B.
EXAMPLE 33
Therapeutic efficacy of the RT virus carrying superagonist payload The vaccinia viruses can deliver therapeutic immunomodulatory payloads directly to tumors. The ability of IL-15 superagonist payload (encoded into the virus genome) to alter immune tumor microenvironment and enhance therapeutic efficacy of RT-viruses was assessed in LL2 lung syngeneic tumor model. IL-15 and IL-15/IL-15R alpha chain complex (referred to IL-15 superagonist herein) are well known proteins, modified forms thereof are known in the art, and exemplary sequences are set forth herein; muteins and modified forms thereof that have increased activity, such as increased binding to targeted cells/receptors (see, e.g., Zhu et al. (2009) J. Immunol. 183:3598-3612; U.S. Patent No. 11,318,201; Mae/ al. (2021) Cancer Res 81:3635-3648. doi: 10.1158/0008-5472.CAN-21-0035; Kowalsky etal., (2018) Molecular Therapy 26:2476-2485).
Methods
One million (le6) LL2 lung cancer cells were implanted subcutaneously into both flanks of C57ZBL6 immunocompetent mice (6-8 weeks old, n=16). 4 days post implantation, mice were treated with a single dose of 5e6 PFU of RT-65 (TK-, A46R-, VGF- ) or RT-96 (RT-65 with IL- 15 superagonist payload). Tumor volumes were measured twice weekly with calipers. At day 6 after RT treatment, 6 tumors were collected, dissociated, stained, and analyzed with Cytoflex flow cytometer for the TIL analysis. The payload expression also was confirmed from the tumor explants by IL- 15 ELISA as described in previous Examples.
Results
The results of the production of the payload (IL15 superagonist) in the tumors from the RT-96-treated mice at 6 days post treatment (Figure 40 A) were confirmed. TIL analysis shows dramatic changes in TME with RT-65 and RT-96 treatment at 6 days post treatment. Both treatments significantly increased lymphocyte and T cell infiltration, with RT-96 exhibiting higher number of leukocyte and T cells in the tumors compared to ones from its parent virus, RT-65. The RT-65 and RT-96 treatments also changed myeloid compartment, increasing inflammatory Ml macrophages and neutrophiles while decreasing anti-inflammatory M2 macrophages. RT-65 treatment decreased dendritic cell numbers in the tumors, but this decreased pattern was reversed with RT-96 treatment. Overall, TIL analysis shows that RT virus treatments induce favorable immune cell changes in TME, which is further enhanced by IL-15 superagonist expression. The favorable change in the tumor microenvironment (TME) resulted in significant therapeutic efficacy. RT-65 and RT-96 demonstrated significantly enhanced therapeutic efficacy compared to the untreated animals on days 11 and 13 post-treatment, as evidenced by higher tumor growth inhibition. Specifically, the average tumor size was smaller in the RT-96-treated mice compared to the RT-65-treated ones, making RT-96 group still significantly different from the untreated group on day 17 post-treatment (note that 3 animals from untreated group were euthanized due to ulceration at this time point). This indicates that IL- 15 further enhances the therapeutic efficacy of RT-65 leading to a durable response. (Figure 40C). RT-96 still exhibited therapeutic efficacy on day 19 post-treatment; at which time tumor growth inhibition was no longer observed following RT-65 treatment Results are shown in Figures 40A-C.
Since modifications will be apparent to those of skill in the art, it is intended that this invention be limited only by the scope of the appended claims.