Codon-optimized new generation regulatable oncolytic fusion-promoting herpes simplex virus 1 virus and methods of use

Abstract

Malignancies that are resistant to conventional therapies represent a major therapeutic challenge. Embodiments of the present invention provide a new generation of codon-optimized, regulatable, fusion-promoting oncolytic herpes simplex virus-1 that is more effective at selectively killing target cells, such as tumor cells. In various embodiments presented herein, the oncolytic viruses described herein are suitable for treating solid tumors as well as other cancers.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 936,776, filed November 18, 2019, pursuant to 35 U.S. SC §119(e), the contents of which are incorporated herein by reference in their entirety. Invention Field

[0003] This invention relates to compositions and methods for treating cancer using codon-optimized, tunable, fusion-promoting oncolytic herpes simplex virus-1 (HSV-1) virus.

[0004] background

[0005] Oncolytic virus therapy relies on the ability of viruses to multiply and lyse human cells, with this replication-dependent lysis preferentially targeting cancer cells. Advances in cancer biology and a detailed understanding of the roles of host factors and viral-encoded gene products in controlling viral production in infected cells have facilitated the use of several viruses as potential anticancer therapeutics (Aghi and Martuza, 2005; Parato et al., 2005). Herpes simplex virus (HSV) possesses several unique properties as an oncolytic agent (Aghi and Martuza, 2005). It can infect a wide range of cell types, leading to the replication of new viruses and cell death. HSV has a short replication cycle (9 to 18 hours) and encodes many non-essential genes, which, when deleted, greatly limit the virus's ability to replicate in non-dividing normal cells. Due to its large genome, multiple therapeutic genes can be packaged into the genome of oncolytic recombinants.

[0006] The use of replication-conditional strains of HSV-1 as oncolytic agents was first reported for the treatment of malignant gliomas (Martuza et al., 1991). Since then, various efforts have been made to try to expand their therapeutic efficacy and improve the virus' replication specificity in tumor cells. However, not surprisingly, genetic deletions that impair viral replication in normal cells also result in a significant reduction in the virus' oncolytic activity in targeting tumor cells (Advani et al., 1998; Chung et al., 1999). Currently, there are no oncolytic viruses that are able to kill tumor cells exclusively while leaving normal cells intact. Therefore, the therapeutic dose of existing oncolytic viruses is significantly limited (Aghi and Martuza, 2005). The availability of oncolytic viruses whose replication can be tightly controlled and pharmacologically adjusted would provide a greatly increased safety and therapeutic efficacy. Such regulatable oncolytic viruses would minimize unwanted replication in adjacent and distant tissues after the tumor has been eliminated and undesired overloading of progeny virus in the target area. This regulatory feature would also allow the oncolytic activity of the virus to be turned off rapidly upon detection of adverse effects (Aghi and Martuza, 2005; Shen and Nemunaitis, 2005). The work described herein presents a new generation of regulatable fusion-promoting variants of oncolytic HSV that are significantly more effective at killing cancer cells than other oncolytic HSV viruses. SUMMARY

[0008] In the present invention, we first describe the use of the HSV-2 immediate early promoter to drive efficient gene expression from a reporter gene in the context of an HSV-1 recombinant virus. Second, we construct a mammalian cell expression plasmid that encodes the developed codon-optimized dominant negative TGF-β mutant mmTGF-β2-7M under the control of a modified HSV-2 ICP4 promoter. Third, we establish a shuttle vector that allows efficient insertion of a gene of interest into the intergenic region of the HSV-1 UL26 gene and the UL27 gene by homologous recombination. Fourth, we construct a tetracycline-regulatable fusion-promoting HSV-1 oncolytic virus that encodes mmTGF-β2-7M under the control of an HSV-2 ICP4 promoter containing tetO. To facilitate secretion of mmTGF-β2-7M, the codon-optimized mmTGF-β2-7M is fused to the signal peptide of the HSV-1 gD gene.

[0009] Accordingly, one aspect described herein provides an oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises: (a) a gene of HSV-1 or HSV-2, namely a VP5 gene, operably linked to a VP5 promoter comprising a TATA element, and comprising a 5' untranslated region; (b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' of the TATA element, wherein the VP5 gene is 3' of the tetracycline operator sequence; (c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is at the ICP0 locus; (d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant, a glycoprotein B (gB) variant, a UL24 variant, and a UL20 gene variant; (e) a gene sequence encoding a functional ICP34.5 protein; and (f) a gene sequence operably linked to a modified HSV promoter, wherein the gene is in the intergenic region of the UL26 gene and the UL27 gene, wherein the oncolytic HSV does not encode a functional ICP0, and does not contain a ribozyme sequence in the 5' untranslated region of VP5.

[0010] Accordingly, one aspect described herein provides an oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises: (a) a gene of HSV-1 or HSV-2, namely a VP5 gene, operably linked to a VP5 promoter comprising a TATA element, and comprising a 5' untranslated region; (b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' of the TATA element, wherein the VP5 gene is 3' of the tetracycline operator sequence; (c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is at the ICP0 locus; (d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant; (e) a gene sequence encoding a functional ICP34.5 protein; and (f) a gene sequence operably linked to a modified HSV promoter, wherein the gene is in the intergenic region of the UL21 gene and the UL22 gene, wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence in the 5' untranslated region of VP5.

[0011] One aspect described herein provides an oncolytic herpes simplex virus (HSV) comprising a recombinant DNA, wherein the recombinant DNA comprises: (a) a 5' untranslated region and a gene of HSV-1 or HSV-2, a VP5 gene, operably linked to a VP5 promoter comprising a TATA element; (b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' of the TATA element, wherein the VP5 gene is located 3' of the tetracycline operator sequence; (c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is located at the ICP0 locus; (d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant; (e) a gene sequence encoding a functional ICP34.5 protein; and (f) a gene sequence operably linked to a modified HSV promoter, wherein the gene is located in an intergenic region of the UL21 gene, the UL22 gene, the UL26 gene, and the UL27 gene, wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence located in the 5' untranslated region of VP5.

[0012] In one embodiment of any aspect herein, the gene sequence of (f) is a LacZ gene sequence.

[0013] In one embodiment of any aspect herein, the gene sequence of (f) is a dominant negative TGF-beta mutant sequence.

[0014] In one embodiment of any aspect herein, the dominant negative TGF-beta mutant sequence is a mmTGF-beta2-7M fragment sequence.

[0015] In one embodiment of any aspect herein, the promoter of (f) is a modified HSV immediate early promoter, a HCMV immediate early promoter, or a human elongation alpha promoter.

[0016] In one embodiment of any aspect herein, the variant gene is a gK variant gene encoding an amino acid substitution selected from the group consisting of: an Ala to Thr amino acid substitution corresponding to amino acid 40 of SEQ ID NO: 2; an Ala to "x" amino acid substitution corresponding to amino acid 40 of SEQ ID NO: 2, wherein "x" is any amino acid; an Asp to Asn amino acid substitution corresponding to amino acid 99 of SEQ ID NO: 2; a Leu to Pro amino acid substitution corresponding to amino acid 304 of SEQ ID NO: 2; and an Arg to Leu amino acid substitution corresponding to amino acid 310 of SEQ ID NO: 2.

[0017] In one embodiment of any of the aspects herein, the tetracycline operator sequence comprises two Op2 repressor binding sites.

[0018] In one embodiment of any of the aspects herein, the VP5 promoter is a VP5 promoter of HSV-1 or HSV-2.

[0019] In one embodiment of any of the aspects herein, the immediate early promoter is an immediate early promoter of HSV-1 or HSV-2.

[0020] In one embodiment of any of the aspects herein, the HSV immediate early promoter is selected from the group consisting of: an ICP0 promoter, an ICP4 promoter, and an ICP27 promoter.

[0021] In one embodiment of any of the aspects herein, the recombinant DNA is part of a HSV-1 genome.

[0022] In one embodiment of any of the aspects herein, the recombinant DNA is part of a HSV-2 genome.

[0023] In one embodiment of any of the aspects herein, the oncolytic HSV further comprises a pharmaceutically acceptable carrier.

[0024] In one embodiment of any of the aspects herein, the oncolytic HSV further encodes at least one polypeptide capable of increasing the efficacy of the oncolytic HSV in inducing an anti-tumor specific immunity.

[0025] In one embodiment of any of the aspects herein, the at least one polypeptide encodes a product selected from the group consisting of: interleukin 2 (IL2), interleukin 12 (IL12), interleukin 15 (IL15), an anti-PD-1 antibody or antibody reagent, an anti-PD-L1 antibody or antibody reagent, an anti-OX40 antibody or antibody reagent, a CTLA-4 antibody or antibody reagent, a TIM-3 antibody or antibody reagent, a TIGIT antibody or antibody reagent, a soluble interleukin 10 receptor (IL10R), a fusion polypeptide between a soluble IL10R and an IgG-Fc domain, a soluble TGF beta type II receptor (TGFBRII), a fusion polypeptide between a soluble TGFBRII and an IgG-Fc domain, an anti-IL10R antibody or antibody reagent, an anti-IL10 antibody or antibody reagent, an anti-TGFBRII antibody or antibody reagent, and an anti-TGFBRII antibody or antibody reagent.

[0026] In one embodiment of any of the aspects herein, the oncolytic HSV further encodes a fusion promoting activity.

[0027] Another aspect described herein provides an oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises: (a) a gene of HSV-1 or HSV-2, VP5 gene, operably linked to a VP5 promoter comprising a TATA element, containing a 5' untranslated region; (b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' of the TATA element, wherein the VP5 gene is 3' of the tetracycline operator sequence; (c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is at the ICP0 locus; (d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant; (e) a gene sequence encoding a functional ICP34.5 protein; and (f) a dominant negative TGF-beta mutant sequence operably linked to a modified HSV-2 immediate early promoter, wherein the gene is in the intergenic region of the UL26 gene and the UL27 gene, wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence in the 5' untranslated region of VP5.

[0028] In one embodiment of any aspect described herein, the oncolytic HSV further encodes a fusion promoting activity.

[0029] Another aspect described herein provides an oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises: (a) a gene of HSV-1 or HSV-2, VP5 gene, operably linked to a VP5 promoter comprising a TATA element, containing a 5' untranslated region; (b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' of the TATA element, wherein the VP5 gene is 3' of the tetracycline operator sequence; (c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is at the ICP0 locus; (d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant; (e) a gene sequence encoding a functional ICP34.5 protein; and (f) a dominant negative TGF-beta mutant sequence operably linked to a modified HSV-2 immediate early promoter, wherein the gene is in the intergenic region of the UL26 gene and the UL27 gene, wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence in the 5' untranslated region of VP5.

[0030] In one embodiment of any of the aspects described herein, the oncolytic HSV further encodes a fusion-promoting activity.

[0031] Another aspect described herein provides an oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises: (a) a 5' untranslated region and a gene of HSV-1 or HSV-2, a VP5 gene, operably linked to a VP5 promoter comprising a TATA element; (b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' of the TATA element, wherein the VP5 gene is located 3' of the tetracycline operator sequence; (c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is located at the ICP0 locus; (d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant; (e) a gene sequence encoding a functional ICP34.5 protein; and (f) a dominant negative TGF-beta mutant sequence operably linked to a modified HSV-2 immediate early promoter, wherein the gene is located in the intergenic region of the UL21 gene, the UL22 gene, the UL26 gene, and the UL27 gene, wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence located in the 5' untranslated region of VP5.

[0032] Another aspect described herein provides an oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA does not encode a functional ICP0 gene or an ICP34.5 gene; and encodes a functional mmTGF-beta 2-7M fragment sequence.

[0033] An oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA does not encode a functional ICP0 gene and an ICP34.5 gene; and encodes a functional mmTGF-beta 2-7M fragment sequence.

[0034] Another aspect described herein provides an oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA does not encode a functional ICP0; and encodes a functional mmTGF-beta 2-7M fragment sequence.

[0035] Another aspect described herein provides an oncolytic virus encoding a functional mmTGF-beta 2-7M fragment sequence.

[0036] Another aspect described herein provides a recombinant virus encoding a functional mmTGF-beta 2-7M fragment sequence.

[0037] Another aspect described herein provides a composition comprising any of the viruses described herein.

[0038] In one embodiment of any aspect described herein, the composition further comprises a pharmaceutically acceptable carrier.

[0039] Another aspect described herein provides a cell expressing any of the viruses or compositions described herein.

[0040] In one embodiment of any aspect described herein, the cell is a mammalian animal.

[0041] In one embodiment of any aspect described herein, the cell is a cancer cell or an immune cell.

[0042] In one embodiment of any aspect described herein, the immune cell is a B cell or a T cell.

[0043] In one embodiment of any aspect described herein, the cell expresses a high level of mmTGF-β2-7M.

[0044] Another aspect described herein provides a method for treating a cancer, the method comprising administering to a subject having a cancer any of the viruses or compositions described herein.

[0045] In one embodiment of any aspect described herein, the cancer is a solid tumor.

[0046] In one embodiment of any aspect described herein, the tumor is benign or malignant.

[0047] In one embodiment of any aspect described herein, the subject is diagnosed with or has been diagnosed with a cancer selected from the group consisting of a carcinoma, a melanoma, a sarcoma, a germ cell tumor, and a blastoma.

[0048] In one embodiment of any aspect described herein, the subject is diagnosed with or has been diagnosed with a cancer selected from the group consisting of non-small cell lung cancer, bladder cancer, breast cancer, brain cancer, colon cancer, prostate cancer, liver cancer, lung cancer, ovarian cancer, skin cancer, head and neck cancer, kidney cancer, and pancreatic cancer.

[0049] In one embodiment of any aspect described herein, the cancer is metastatic.

[0050] In one embodiment of any aspect described herein, the method further comprises administering an agent that modulates a promoter of a tet operon.

[0051] In one embodiment of any of the aspects described herein, the agent is doxycycline or tetracycline. In one embodiment of any of the aspects described herein, the agent is administered locally or systemically. In one embodiment of any of the aspects described herein, the systemic administration is oral administration.

[0052] In one embodiment of any of the aspects described herein, the virus or composition is administered directly to the tumor.

[0053] Another aspect described herein provides a hybrid nucleic acid sequence comprising a therapeutic antibody and a sequence of a mmTGF-β2-7M, wherein the mmTGF-β2-7M is fused to the Fc domain of the therapeutic antibody.

[0054] In one embodiment of any of the aspects described herein, wherein the sequence of the therapeutic antibody is a sequence of an immunotherapeutic antibody.

[0055] In one embodiment of any of the aspects described herein, the sequence of the therapeutic antibody is a sequence selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-Tim3 antibody, an anti-CTLA4 antibody, and an anti-TDM-1 antibody, and an anti-TIGIT antibody.

[0056] Another aspect described herein provides a polypeptide encoded by any of the hybrid nucleic acids described herein.

[0057] Another aspect described herein provides a vector expressing any of the hybrid nucleic acids or polypeptides described herein.

[0058] Another aspect described herein provides a chimeric antigen receptor (CAR) polypeptide comprising at least one of: (a) an extracellular domain comprising a dominant negative TGF-β mutant sequence; (b) a transmembrane domain; (c) a costimulatory domain; and (d) an intracellular signaling domain.

[0059] Another aspect described herein provides a nucleic acid encoding any of the CAR polypeptides described herein.

[0060] Another aspect described herein provides a mammalian cell comprising: (a) any of the CAR polypeptides described herein; or any of the nucleic acids described herein.

[0061] In one embodiment of any of the aspects described herein, the cell is a T cell.

[0062] In one embodiment of any of the aspects described herein, the cell is a human cell.

[0063] In one embodiment of any of the aspects described herein, the cell further comprises at least a second CAR polypeptide.

[0064] In one embodiment of any of the aspects described herein, the at least second CAR polypeptide comprises an extracellular domain comprising a sequence of an immunotherapeutic antibody.

[0065] In one embodiment of any of the aspects described herein, the cell is obtained from an individual having or diagnosed with cancer.

[0066] Another aspect described herein provides a method of treating cancer in a subject in need thereof, the method comprising administering any of the cells described herein.

[0067] Another aspect described herein provides a method of treating cancer in a subject in need thereof, the method comprising: (a) engineering a T cell to comprise any of the CAR polypeptides described herein or any of the nucleic acids described herein on the surface of the T cell; and (b) administering the engineered T cell to the subject.

[0068] In one embodiment of any of the aspects described herein, the engineered T cell further comprises at least a second CAR polypeptide.

[0069] In one embodiment of any of the aspects described herein, the method further comprises administering at least one additional anti-cancer therapeutic agent.

[0070] Definitions

[0071] All references cited herein are incorporated by reference in their entirety as though fully set forth.

[0072] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this application belongs. It should be understood that this application is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The rd definitions of common terms in abnormal chemistry can be found in Singleton et al., Dictionary of Microbiology and Molecular Biology 3 thMichael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012); Jon Lorsch (ed.) Laboratory Methods in Enzymology: DNA, Elsevier, (2013); Frederick M. Ausubel (ed.), Current Protocols in Molecular Biology (CPMB), John Wiley and Sons, (2014); John E. Coligan (ed.), Current Protocols in Protein Science (CPPS), John Wiley and Sons, Inc., (2005); and Ethan M Shevach, Warren Strobe, (eds.) Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, John Wiley and Sons, Inc., (2003); each of which provides general guidance to those of skill in the art for many of the terms used in this application.

[0073] As used herein, "cancer" can refer to the excessive proliferation of cells, whose unique trait—loss of normal cell control—leads to unregulated growth, lack of differentiation, local tissue invasion, and metastasis, and can be a leukemia, lymphoma, multiple myeloma, or a solid tumor. Non-limiting examples of leukemias include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL). In one embodiment, the cancer is ALL or CLL. Non-limiting examples of lymphomas include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt's lymphoma, hairy cell leukemia (HCL). In one embodiment, the cancer is DLBCL or follicular lymphoma. Non-limiting examples of solid tumors include adrenocortical tumor, alveolar soft-part sarcoma, carcinoma, chondrosarcoma, colorectal cancer, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, endodermal sinus tumor, epithelioid hemangioendothelioma, Ewing's sarcoma, germ cell tumor (solid), giant cell tumor of bone and soft tissue, hepatoblastoma, hepatocellular carcinoma, melanoma, nephroma, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), osteosarcoma, paraspinal sarcoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, synovial sarcoma, and Wilms' tumor. A solid tumor can be found in a bone, muscle, or organ, and can be a sarcoma or a carcinoma. It is contemplated that any aspect of the technology described herein can be used to treat all types of cancers, including cancers not listed in the present application. As used herein, the term "tumor" refers to an abnormal growth of cells or tissue, for example, of a malignant type or a benign type.

[0074] As used herein, "subject" means a human or an animal. Typically, the animal is a vertebrate such as a primate, rodent, domestic animal, or game animal. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include, for example, cattle, horses, pigs, deer, bison, water buffalo, feline species (e.g., domestic cat), canine species (e.g., dog, fox, wolf), avian species (e.g., chicken, emu, ostrich), and fish (e.g., trout, catfish, and salmon). In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0075] Preferably, the subject is a mammal. The mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. Mammals other than humans can advantageously be used as subjects that represent animal models of a disease, such as cancer. The subject can be male or female.

[0076] The subject can be one that has been previously diagnosed with or identified as suffering from or having a condition (e.g., cancer) that requires treatment or one or more complications associated with such a condition, and optionally, has undergone treatment for the condition or the one or more complications associated with the condition. Alternatively, the subject can also be one that has not been previously diagnosed with such a condition or associated complications. For example, the subject can be one that exhibits one or more risk factors for the condition or one or more complications associated with the condition or one that does not exhibit risk factors.

[0077] As used herein, the terms“treat,”“treatment,” or“treating” or“amelioration” refer to therapeutic treatment wherein the object is to reverse, alleviate, improve, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder (e.g., cancer). The term“treatment” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally“effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is“effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of the progression or worsening of symptoms as compared to what would be expected without treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term“treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0078] In various embodiments described herein, it is also contemplated to encompass variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants and / or conservatively substituted variants of any particular polypeptide described. With respect to amino acid sequences, one of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alter, e.g., the single amino acid or a small percentage of the amino acids in the encoded sequence are "conservatively modified variants" where the alterations result in the substitution of an amino acid with a chemically similar amino acid and retain the desired polypeptide activity. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles which occur in nature.

[0079] A given amino acid can be replaced by a residue that has a similar biochemical property, e.g., an aliphatic residue is replaced with another aliphatic residue (e.g., Ile, Val, Leu, or Ala; or a polar residue is replaced with another polar residue (e.g., between Lys and Arg; between Glu and Asp; or between Gin and Asn). Other such conservative substitutions (e.g., substitutions of entire regions having similar hydrophobic properties) are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any of the assays described herein to confirm that the desired activity of the native or reference polypeptide is retained, e.g., ligan-mediated receptor activity and specificity.

[0080] Amino acids can be grouped according to the similarity of their side chain properties (A. L. Lehninger, in Biochemistry, 2nd ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into the following groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions would entail exchanging a member of one of these classes for a member from another. Specific conservative substitutions include, for example, Ala for Gly or for Ser; Arg for Lys; Asn for Gin or for His; Asp for Glu; Cys for Ser; Gin for Asn; Glu for Asp; Gly for Ala or for Pro; His for Asn or for Gin; He for Leu or for Val; Leu for He or for Val; Lys for Arg, for Gin, or for Glu; Met for Leu, for Tyr, or for He; Phe for Met, for Leu, or for Tyr; Ser for Thr; Thr for Ser; Trp for Tyr; Tyr for Trp; and / or Phe for Val, for He, or for Leu.

[0081] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a fragment or segment of a peptide that retains at least 50% of the activity of a wild-type reference polypeptide according to an assay known in the art or described herein below. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0082] In some embodiments, the polypeptides described herein can be variants of the polypeptides or molecules as described herein. In some embodiments, the variants are conservatively modified variants. For example, conservatively substituted variants can be obtained by mutations of the natural nucleotide sequence. A "variant" as referred to herein is a polypeptide substantially homologous to a natural or reference polypeptide, but having an amino acid sequence different from the natural or reference polypeptide due to one or more deletions, insertions, or substitutions. DNA sequences encoding variant polypeptides encompass sequences that, when compared to the natural or reference DNA sequence, contain one or more additions, deletions, or substitutions of nucleotides, but encode a variant protein or fragment thereof that retains the activity of the non-variant polypeptide. Various PCR-based site-specific mutagenesis methods are known in the art and can be applied by the ordinary skill in the art.

[0083] The variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the natural or reference sequence. For example, the degree of homology (percent identity) between a natural sequence and a mutated sequence can be determined by comparing the two sequences using a computer program freely available on the World Wide Web for this purpose (e.g., BLASTp or BLASTn with default settings).

[0084] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to those of skill in the art. For example, mutations can be introduced by in vitro mutagenesis of an isolated gene sequence, e.g., by synthesis of an oligonucleotide containing the desired change and use of the oligonucleotide in a recombinant DNA synthesis protocol. Alternatively, site-specific mutagenesis procedures can be used to provide an altered nucleotide sequence having a particular codon altered according to a desired substitution, deletion, or insertion. Techniques for making such alterations are well established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entireties. Any cysteine residues not involved in maintaining the proper conformation of a polypeptide can also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bonds can be added to polypeptides to improve their stability or facilitate oligomerization.

[0085] As used herein, the term "DNA" is defined as deoxyribonucleic acid. The term "polynucleotide" can be used interchangeably herein with "nucleic acid" to mean a polymer of nucleosides. Typically, polynucleotides consist of nucleosides naturally occurring in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) joined by phosphodiester bonds. However, the term encompasses molecules comprising nucleosides or nucleoside analogs containing chemical or biological modifications, whether or not they occur naturally in nucleic acids, and such molecules can be preferred for certain applications. Where the present application refers to polynucleotides, it is understood that DNA, RNA, and in each case single and double stranded forms (as well as the complement of each single stranded molecule) are provided. "Polynucleotide sequence" as used herein can refer to both the polynucleotide material itself and / or the sequence information (i.e., a succession of letters used as abbreviations for the bases), which biochemically characterizes a particular nucleic acid. Unless otherwise indicated, the polynucleotide sequences presented herein are presented in the 5' to 3' direction.

[0086] The term "operably linked" as used herein refers to the arrangement of various nucleic acid molecular elements relative to one another such that the elements are functionally connected and able to interact with one another. Such elements can include, but are not limited to, promoters, enhancers, polyadenylation sequences, one or more introns and / or exons, and coding sequences of a gene of interest to be expressed. When operably linked, the nucleic acid sequence elements can act together to modulate the activity of one another, and ultimately can affect the expression level of the gene of interest, including any of those encoded by the sequences described above.

[0087] The term "vector" as used herein refers to a vector nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. The nucleic acid sequence can be "foreign" in the sense that it is foreign to the cell into which the vector is introduced, or the sequence is homologous to sequences in the cell but is not normally found in the position in the host cell nucleic acid where it is placed. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those of skill in the art will be well equipped to construct vectors by standard recombinant techniques (see, e.g., Maniatis et al., 1988 and Ausubel et al., 1994, both incorporated herein by reference). Additionally, the techniques described herein and illustrated in the figures are instructive for effective vector construction as well.

[0088] The term "oncolytic HSV-1 vector" refers to a genetically engineered HSV-1 virus corresponding to at least a portion of the HSV-1 genome that is capable of infecting a target cell, replicating, and being packaged into an HSV-1 virion. The genetically engineered virus includes a deletion and / or mutation and / or insertion of nucleic acid that renders the virus oncolytic, such that the genetically engineered virus replicates in and kills tumor cells through oncolytic activity. The virus can be attenuated or non-attenuated. The virus can or can not deliver a transgene that is different from the HSV viral genome. In one embodiment, the oncolytic HSV-1 vector does not express a transgene to produce a protein that is foreign to the virus.

[0089] As used herein, "UL21" refers to envelope protein UL21 (e.g., from human alpha herpesvirus 1). Sequences for UL21 are known for multiple species, e.g., HSV-1 UL21 (NCBI Gene ID: 2703372) polypeptide (e.g., NCBI Ref Seq YP_009137095.1). UL21 can refer to HSV-1 UL21, including naturally occurring variants, molecules, and alleles thereof, and can refer to homologs (e.g., HSV-2).

[0090] As used herein, “UL22” refers to envelope glycoprotein H (e.g., from human alphaherpesvirus 1). Sequences for UL22 are known for a variety of species, e.g., HSV-1 UL22 (NCBI Gene ID: 24271466) polypeptide (e.g., NCBI RefSeq YP_009137096.1). UL22 can refer to HSV-1 UL22, including naturally occurring variants, molecules, and alleles thereof, and can refer to homologs (such as HSV-2).

[0091] As used herein, “UL26” refers to capsid maturation protease (e.g., from human alphaherpesvirus 1). Sequences for UL26 are known for a variety of species, e.g., HSV-1 UL26 (NCBI Gene ID: 2703453) polypeptide (e.g., NCBI RefSeq YP_009137100.1). UL26 can refer to HSV-1 UL26, including naturally occurring variants, molecules, and alleles thereof, and can refer to homologs (such as HSV-2).

[0092] As used herein, “UL27” refers to envelope glycoprotein B (e.g., from human alphaherpesvirus 1). Sequences for UL27 are known for a variety of species, e.g., HSV-1 UL27 (NCBI Gene ID: 24271469) polypeptide (e.g., NCBI RefSeq YP_009137102.1). UL27 can refer to HSV-1 UL27, including naturally occurring variants, molecules, and alleles thereof, and can refer to homologs (such as HSV-2).

[0093] The term "promoter" as used herein refers to a nucleic acid sequence that directly or indirectly regulates transcription of a corresponding nucleic acid coding sequence operably linked thereto. A promoter can function alone to regulate transcription or, in some cases, a promoter can act in conjunction with one or more other regulatory sequences such as enhancers or silencers to regulate transcription of a gene of interest. A promoter includes a DNA regulatory sequence where the regulatory sequences are derived from a gene that binds RNA polymerase and initiates the transcription of the downstream (3'-direction) coding sequences. A promoter generally includes a sequence that functions as the initiation site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters that lack a TATA box, such as for example the promoters of mammalian terminal deoxynucleotidyl transferase genes and the promoter of the SV40 late genes, a discrete element covering the initiation site itself helps fix the position of initiation. Additional promoter elements modulate the frequency of transcription initiation. Usually these are in the region 30-110 bp upstream of the initiation site, although many promoters have been shown to contain functional elements downstream of the initiation site as well. In order for a coding sequence to be "under the control" of a promoter, the 5' end of the transcription initiation site of the transcriptional reading frame of the selected promoter can be positioned "downstream" (i.e., 3' of) the coding sequence. An "upstream" promoter stimulates the transcription of DNA and facilitates the expression of the coding RNA.

[0094] The spacing between the promoter elements is generally flexible, such that the promoter function is maintained when the elements are inverted or moved relative to each other. Depending on the promoter used, the individual elements can function in concert or independently to activate transcription. The promoters described herein can or can not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the activation of transcription of a nucleic acid sequence, such as those listed herein for a gene or portion or functional equivalent thereof.

[0095] A promoter can be one naturally associated with a nucleic acid sequence, as can be obtained by isolation of 5' non-coding sequences from a coding segment and / or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer can be one associated with a nucleic acid sequence in its natural environment. Alternatively, certain advantages can be obtained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cells, and which are not "naturally occurring" with the nucleic acid sequence in its natural environment, i.e., different from the naturally occurring regulatory sequences encoding or associated with the expression of a gene, and / or mutations that alter expression, such as the promoter of the immediate early gene of HCMV, the beta-lactamase (penicillinase), the lactase and the tryptophan (trp) promoter system.

[0096] A "gene" or "sequence" encoding a particular protein is a nucleic acid molecule which, when placed under the control of appropriate transcriptional and translational regulatory sequences, is expressed in a host cell to produce a polypeptide in vitro or in vivo. A gene of interest can include, but is not limited to, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence is usually located 3' to the gene sequence. Typically, a polyadenylation signal is provided to terminate transcription of the gene inserted into the recombinant virus.

[0097] The term "polypeptide" as used herein refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically of about 2 to 60 amino acids in length. Polypeptides used herein generally contain the most common amino acids found in proteins, such as the 20 L-amino acids. However, other amino acids and / or amino acid analogs known in the art can be used. One or more amino acids in a polypeptide can be modified, e.g., by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc. A polypeptide with a non-polypeptide moiety covalently or non-covalently bound thereto is still considered to be a "polypeptide." Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology, or synthesized by chemical means such as conventional solid-phase peptide synthesis, etc. The term "polypeptide sequence" or "amino acid sequence" as used herein can refer to the polypeptide material itself and / or sequence information (i.e., a contiguous string of letters or three-letter codes used as abbreviations for amino acid names) that biochemically characterizes the polypeptide. Unless otherwise indicated, polypeptide sequences presented herein are presented in the N-terminal to C-terminal direction.

[0098] The term "transgene" refers to a particular nucleic acid sequence that encodes a polypeptide or a portion of a polypeptide to be expressed in a cell into which the nucleic acid sequence has been inserted. The term "transgene" is intended to include (1) a nucleic acid sequence that does not naturally occur in the cell (i.e., a heterologous nucleic acid sequence); (2) a nucleic acid sequence that is a mutated form of a nucleic acid sequence that naturally occurs in the cell into which it has been inserted; (3) a nucleic acid sequence that serves to add additional copies of the same (i.e., homologous) or similar nucleic acid sequence that naturally occurs in the cell into which it has been inserted; or (4) a naturally occurring or homologous nucleic acid sequence that is silenced whose expression is induced in the cell into which it has been inserted. A "mutated form" or "modified nucleic acid" or "modified nucleotide" sequence means a sequence that contains one or more nucleotides that are different from the wild-type or naturally occurring sequence, i.e., the mutated nucleic acid sequence comprises one or more nucleotide substitutions, deletions and / or insertions. In some cases, the gene of interest can also include a sequence encoding a leader peptide or signal sequence such that the transgene product can be secreted from the cell.

[0099] As used herein, the term "antibody reagent" refers to a polypeptide that comprises at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binds to a given antigen. An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments of any of the aspects, an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody. For example, an antibody can comprise a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody comprises two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, CDR, and domain antibody (dAb) fragments (see, e.g., de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39; incorporated herein by reference in its entirety)) as well as whole antibodies. Antibodies can have the structural features of IgA, IgG, IgE, IgD, or IgM (as well as subtypes and combinations thereof). Antibodies can be from any source, including mouse, rabbit, porcine, rat, and primate (human and non-human primates) as well as primatized antibodies. Antibodies also include midibodies, nanobodies, humanized antibodies, chimeric antibodies, and the like.

[0100] The term "oncolytic activity" as used herein refers to a cytotoxic effect exerted on tumor cells in vitro and / or in vivo, without any appreciable or significant deleterious effect on normal cells under the same conditions. Cytotoxic effect under in vitro conditions is detected by various means known in the art, for example by staining with selective staining agents for dead cells, by inhibition of DNA synthesis or by apoptosis. Detection of cytotoxic effect under in vivo conditions is performed by methods known in the art.

[0101] As used herein, a "biologically active portion" of a molecule refers to that portion of the larger molecule that is capable of performing a similar function of the larger molecule. By way of non-limiting example only, a biologically active portion of a promoter is any portion of the promoter that retains the ability to affect gene expression, even if only marginally. Similarly, a biologically active portion of a protein is any portion of the protein that retains the ability to perform one or more biological functions of the full-length protein (e.g., binding to another molecule, phosphorylation, etc.), even if only marginally.

[0102] As used herein, the term "administering" refers to the placement of a therapeutic composition or a pharmaceutical composition as disclosed herein in a subject by a method or route which results in at least partial delivery of an agent at the desired location. Pharmaceutical compositions comprising an agent as disclosed herein can be administered by any appropriate route which results in effective treatment in a subject.

[0103] The term "statistically significant" or "significantly" refers to statistical significance and generally means a difference of two standard deviations (2SD) or greater.

[0104] Except in the operating and spectral examples, or where otherwise explicitly indicated, all numerical quantities used herein are to be understood as modified by the term "about" in all instances. The term "about" when used in connection with a percentage can mean ±1% when used in connection with a percentage.

[0105] As used herein, the term "comprising" means that other elements can also be present in addition to the elements recited. "Comprising" is used to mean including, but not limited to, whatever follows the term. The term "consisting of' means including, and limited to, whatever follows the term. As used herein, the term "consisting essentially of' means including at least the elements recited, and limited to other elements of any kind, provided that the other elements do not materially alter the basic and novel or functional characteristic(s) of the technology described in the specification.

[0106] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0107] In some embodiments, numerical values expressed in the description and claims for the amounts of ingredients, properties such as molecular weight, reaction conditions, and the like that are indicative of certain embodiments of the application will be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, numerical parameters

[0108] With the foregoing preliminary description and definitions in mind, additional background is provided below to provide context for the development and evolution of the inventive vectors, compositions, and methods described herein. BRIEF DESCRIPTION OF DRAWINGS

[0110] Exemplary embodiments are illustrated in reference to the accompanying drawings. It is intended that the embodiments disclosed herein and the drawings be considered illustrative rather than restrictive.

[0111] Figure 1 A schematic representation of the QREOF-lacZ genome is shown. UL and US represent the unique long and unique short regions of the HSV-1 genome, which are flanked by their respective inverted repeats (open boxes). The DNA sequence encoding tetR (black box) and intron II, which replaces the ICP0 coding sequence, is shown on the map of the HSV-1 genome flanked by the rabbit beta-globin gene (diagonal striped box) encoding ICP0 sequence. ICP5TO represents the HSV-1 UL19 gene encoding ICP5 under the control of the HSV-1 Icp5 promoter carrying tetO. The amplified DNA fragment inserted in the intergenic region of the UL26 gene and the UL27 gene, which encodes the lacZ gene under the control of the modified HSV-2 ICP0 promoter (cross-hatched box).

[0112] Figure 2 H1299 cells seeded at 7.5 x 10e5 cells / 60 mm dish. Cells were infected with QREOF-lacZ at an MOI of 0.05 PFU / dish 48 h after cell seeding in the presence or absence of doxycycline. Infected cells were stained with X-Gal 48 h post-infection and photographed.

[0113] Figure 3U20S cells seeded at 1.5 x 10e6 cells / dish are shown. Twenty hours post-seeding, duplicate dishes of cells were transfected with 5 μg / dish of pICP6-eGFP or 5 μg / dish of pQUL2627-TGF-DN by lipofectamine 2000. Cell extracts were prepared 70 h post-transfection. Proteins from transfected cell extracts and media collected from transfected cells were separated on SDS-PAGE and immunoblotted with rabbit anti-TGF-β1 antibody (Abeam, ab179695).

[0114] Figure 4 The effect of blocking TGF-β1 signaling by mmTGF-β2-7M on U20S cell proliferation is shown. U20S cells were seeded at 1.5 x 10e6 cells / dish. Twenty hours post-seeding, triplicate dishes of cells were transfected with 5 μg / dish of pICP6-EGFP or 5 μg / dish of pQUL2627-TGF DN by lipofectamine 2000. Cells were collected 76 h post-transfection. Live cells were counted by trypan blue exclusion and plotted as the number of live cells per dish, expressed as mean ± SEM.

[0115] Figure 5 Expression of mmTGF-β2-7M in QREOF-DNT infected U20S cells is shown. U20S cells were mock infected or infected with QREOF-DNT or QREOF-lacZ at a MOI of 3 PFU / cell in the presence of doxycycline. Infected cell extracts were prepared 18 h post-infection. Proteins from mock infected and infected cell extracts were separated on SDS-PAGE and immunoblotted with monoclonal antibodies to anti-HSV-1 ICP27 (Santa Cruz) or rabbit anti-TGF-β1 antibody.

[0116] Figure 6 A schematic of the QREO5F genome is shown.

[0117] DETAILED DESCRIPTION OF THE INVENTION

[0118] Oncolytic viruses are genetically modified viruses that preferentially replicate in host cancer cells, leading to production of new viruses and ultimately cell death. Herpes simplex virus (HSV) has several unique properties that make it an attractive oncolytic agent. It can infect a broad range of cell types and has a short replication cycle (9 to 18 h). The use of a replication-conditional strain of HSV-1 as an oncolytic agent was first reported for the treatment of malignant glioma. Since then, various efforts have been made to try to expand its therapeutic efficacy and improve the replication specificity of the virus in tumor cells. However, not surprisingly, genetic deletions that impair viral replication in normal cells also result in a significant reduction in the oncolytic activity of the virus targeting tumor cells. Currently, there is no oncolytic virus that can kill only tumor cells while leaving normal cells intact. Therefore, the therapeutic dose of existing oncolytic viruses is significantly limited. The availability of an oncolytic virus whose replication can be tightly controlled and pharmacologically adjusted would provide a greatly increased safety and therapeutic efficacy. Such an adjustable oncolytic virus would minimize the risk of uncontrolled replication in adjacent and distant tissues and undesired overloading of progeny viruses in the target area after the tumor has been eliminated. This regulatory feature would also allow the oncolytic activity of the virus to be quickly turned off if adverse effects are detected.

[0119] Oncolytic HSV

[0120] HSV replicates in epithelial cells and fibroblasts and establishes a life-long latent infection in neuronal cell bodies within sensory ganglia of infected individuals. During productive infection, HSV genes are classified into three major categories based on the timing of their expression: immediate early (IE), early (E), and late (L) (Roizman, 2001). The HSV-1 viral proteins directly relevant to the present invention are the immediate early regulatory protein ICP0 and the viral major capsid protein ICP5 or VP5. While not essential for productive infection, ICP0 is required for efficient viral gene expression and replication at low multiplicity of infection in normal cells and for efficient reactivation from latency (Cai and Schaffer, 1989; Leib et al., 1989; Yao and Schaffer, 1995). ICP0 is required to stimulate viral mRNA translation in quiescent cells (Walsh and Mohr, 2004) and plays a fundamental role in countering the host’s innate antiviral response to HSV infection. In short, it prevents IFN-induced nuclear block to viral transcription, downregulates TLR2 / TLR9-induced inflammatory cytokine response to viral infection, suppresses TNF-a-mediated activation of the NF-κΒ signaling pathway and interferes with the DNA damage response to viral infection (Lanfranca et al., 2014). Given that tumor cells are impaired in multiple cellular pathways, such as DNA repair, interferon signaling and translational regulation (Barber, 2015; Critchley-Thorne et al., 2009; Kastan and Bartek, 2004; Li and Chen, 2018; Mohr, 2005; Zitvogel et al., 2015), it is not surprising that ICP0 deletion mutants replicate more efficiently in cancer cells than in normal cells, particularly in quiescent and terminally differentiated cells. Yao and Schaffer (Yao and Schaffer, 1995) were the first to demonstrate the oncolytic potential of ICP0 mutants, showing that plaque-forming efficiency of ICP0 null mutants was 100- to 200-fold higher in human osteosarcoma cells (U2OS) than in non-tumorigenic African green monkey kidney cells (Vero). It has recently been shown that a deficiency in the stimulator of interferon genes (STING) signaling pathway in U2OS cells results in their proven ability to efficiently support the growth of ICP0 null mutants (Deschamps and Kalamvoki, 2017).

[0121] Using the T-REx™ gene switch technology (Thermo Fisher / Invitrogen, Carlsbad, CA) invented by Dr. Yao Feng and a self-cleaving ribozyme, the first regulatable oncolytic virus KTR27 (U.S. Patent No. 8,236,941, incorporated by reference herein in its entirety) was generated in which the HSV-1 ICP0 gene was replaced by a DNA sequence encoding the tetracycline repressor (tetR) and the essential HSV-1 ICP27 gene was controlled by a self-cleaving ribozyme in the 5' untranslated region of the ICP27 promoter and ICP27 coding sequence carrying tetO. Recent DNA sequence analysis of a KTR27- derived fusogenic virus (designated KTR27-F) revealed that in addition to the deletion of two copies of the ICP0 gene, two copies of the HSV-1 ICP34.5 gene were also deleted from the KTR27-F virus. Furthermore, PCR analysis of KTR27 viral DNA with ICP34.5 gene-specific primers revealed that, similar to KTR27-F, KTR27 does not encode the ICP0 gene and the ICP34.5 gene. The ICP34.5 gene is located 5' to the ICP0 gene in the inverted repeat region of the HSV-1 genome that is flanked by unique long sequences of the HSV-1 genome. Various HSV-1 oncolytic viruses are based on the deletion of the ICP34.5 gene (Aghi and Martuza, 2005; Kaur et al., 2012; Lawler et al., 2017), including the recently FDA-approved talimogene laherparepvec (T-VEC) for the treatment of advanced melanoma (Rehman et al., 2016).

[0122] Based on the tet-dependent viral replication and tumor-selective features of KTR27 and the use of the self-cleaving ribozyme in the construction of KTR27 to achieve a higher degree of tet-dependent viral replication, and the notion that the small suboptimal expression of ICP27 significantly limits viral replication in cancer cells due to the IC P27, a new oncolytic virus, QREO5, based on tetR expression of an ICP0 null mutant, was recently developed, which encodes the late HSV-1 major capsid protein VP5 under the control of a tetO-containing VP5 promoter. Because VP5 is a late viral gene product whose expression is dependent on the expression of viral IE genes, it was hypothesized that the late kinetics of the tetO-carrying VP5 promoter would allow tighter control of VP5 expression by tetR expressed from the IE ICP0 promoter than ICP27 under the control of the tetO-carrying ICP27 promoter. Indeed, QREO5 exhibited significantly superior tet-dependent viral replication to KTR27 in infected H1299 cells and Vero cells. Moreover, because the QREO5 genome does not contain a self-cleaving ribozyme and encodes a wild-type ICP34.5 gene, it replicated 100-fold and 450-fold more efficiently than KTR27 in Vero cells and H1299 cells, respectively.

[0123] HSV-1 is a human neurotropic virus capable of infecting almost all vertebrate cells. Following natural infection, there is either a lytic, replicative cycle or establishment of latency (usually in peripheral ganglia), with DNA maintained indefinitely in a free state. HSV-1 contains a double-stranded linear DNA genome of approximately 152 kilobases in length, which has been completely sequenced by McGeoch (McGeoch et al., J. Gen. Virol. 69: 1531 (1988); McGeoch et al., Nucleic Acids Res 14: 1727 (1986); McGeoch et al., J. Mol. Biol. 181 : 1 (1985); Perry and McGeoch, J. Gen. Virol. 69: 2831 (1988); Szpara ML et al., J Virol. 2010, 84: 5303; Macdonald SJ et al., J Virol. 2012, 86: 6371). DNA replication and virion assembly occur in the nucleus of the infected cell. During the late stages of infection, concatemeric viral DNA is cleaved into genome-length molecules, which are packaged into virions. In the CNS, herpes simplex virus spreads across neurons, followed by intraneuronal transport (either retrograde or anterograde) to the nucleus where replication occurs.

[0124] Therefore, one aspect described herein provides an oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises: (a) a gene containing a 5' untranslated region and HSV-1 or HSV-2, namely the VP5 gene, which is operatively linked to a VP5 promoter containing a TATA element; (b) a tetracycline operon sequence located between 6 and 24 nucleotides at the 3' end of the TATA element, wherein the VP5 gene is located at the 3' end of the tetracycline operon sequence; and (c) a gene sequence encoding a tetracycline repressor operatively linked to an HSV immediate early promoter, wherein the gene sequence is located at... At the ICP0 locus; (d) a variant gene that increases syncytial formation compared to the wild type, wherein the HSV-1 or HSV-2 variant gene is selected from: glycoprotein K (gK) variant, glycoprotein B (gB) variant, UL24 variant, and UL20 gene variant; (e) a gene sequence encoding a functional ICP34.5 protein; and (f) a gene sequence operatively linked to a modified HSV promoter, wherein the gene is located in the intergenic region of the UL26 and UL27 genes, wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence located in the 5' untranslated region of VP5.

[0125] One aspect of this document provides an oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises: (a) a gene containing a 5' untranslated region and HSV-1 or HSV-2, namely the VP5 gene, which is operatively linked to a VP5 promoter containing a TATA element; (b) a tetracycline operon sequence located between 6 and 24 nucleotides at the 3' end of the TATA element, wherein the VP5 gene is located at the 3' end of the tetracycline operon sequence; and (c) a gene sequence encoding a tetracycline repressor operatively linked to an HSV immediate early promoter, wherein the gene sequence is located at the IC10 ... (d) A variant gene that increases syncytial formation compared to the wild type, wherein the HSV-1 or HSV-2 variant gene is selected from: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant; (e) a gene sequence encoding a functional ICP34.5 protein; and (f) a gene sequence operatively linked to a modified HSV promoter, wherein the gene is located in the intergenic region of the UL21 and UL22 genes, wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence located in the 5' untranslated region of VP5.

[0126] One aspect described herein provides an oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises: (a) a gene of HSV-1 or HSV-2, VP5 gene, operably linked to a VP5 promoter comprising a TATA element, containing a 5' untranslated region; (b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' of the TATA element, wherein the VP5 gene is 3' of the tetracycline operator sequence; (c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is at the ICP0 locus; (d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant; (e) a gene sequence encoding a functional ICP34.5 protein; and (f) a gene sequence operably linked to a modified HSV promoter, wherein the gene is in an intergenic region of the UL21 gene, the UL22 gene, the UL26 gene, and the UL27 gene, wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence in the 5' untranslated region of VP5.

[0127] Another aspect described herein provides an oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises: (a) a gene of HSV-1 or HSV-2, VP5 gene, operably linked to a VP5 promoter comprising a TATA element, containing a 5' untranslated region; (b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' of the TATA element, wherein the VP5 gene is 3' of the tetracycline operator sequence; (c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is at the ICP0 locus; (d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant; (e) a gene sequence encoding a functional ICP34.5 protein; and (f) a dominant negative TGF-beta mutant sequence operably linked to a modified HSV-2 immediate early promoter, wherein the gene is in an intergenic region of the UL26 gene and the UL27 gene, wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence in the 5' untranslated region of VP5.

[0128] Another aspect described herein provides an oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises: (a) a gene of HSV-1 or HSV-2, VP5 gene, operably linked to a VP5 promoter comprising a TATA element, containing a 5' untranslated region; (b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' of the TATA element, wherein the VP5 gene is 3' of the tetracycline operator sequence; (c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is at the ICP0 locus; (d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant; (e) a gene sequence encoding a functional ICP34.5 protein; and (f) a dominant negative TGF-beta mutant sequence operably linked to a modified HSV-2 immediate early promoter, wherein the gene is in the intergenic region of the UL21 gene and the UL22 gene, wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence in the 5' untranslated region of VP5.

[0129] Another aspect described herein provides an oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises: (a) a gene of HSV-1 or HSV-2, VP5 gene, operably linked to a VP5 promoter comprising a TATA element, containing a 5' untranslated region; (b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' of the TATA element, wherein the VP5 gene is 3' of the tetracycline operator sequence; (c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is at the ICP0 locus; (d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant; (e) a gene sequence encoding a functional ICP34.5 protein; and (f) a dominant negative TGF-beta mutant sequence operably linked to a modified HSV-2 immediate early promoter, wherein the gene is in the intergenic region of the UL21 gene, the UL22 gene, the UL26 gene, and the UL27 gene, wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence in the 5' untranslated region of VP5.

[0130] A distinguishing feature of the oncolytic viruses described herein is that the viral genome expresses a gene sequence that encodes a functional ICP34.5. The infected cell protein 34.5 (ICP34.5) is a protein (e.g., gene product) expressed from the gamma 34.5 gene in a virus, such as a herpes simplex virus. ICP34.5 is one of the HSV neurovirulence factors (C Chou J, Kern ER, Whitley RJ, and Roizman B, Science, 1990). One of the functions of ICP34.5 is to block the cellular stress response to viral infection, i.e., to block the double-stranded RNA-dependent protein kinase PKR-mediated antiviral response (Agarwalla, P.K., et al., Method in Mol. Bio., 2012).

[0131] The oncolytic viruses described herein are ICP0 null viruses. The infected cell polypeptide 0 (ICP0) is a protein encoded by the HSV-1 alpha 0 gene. ICP0 is produced during the immediate early phase of viral gene expression. ICP0 is synthesized and transported into the nucleus of the infected host cell, where it facilitates transcription from viral genes, disrupts cellular nuclear and cytoplasmic cellular structures (such as the microtubular network), and alters the expression of host genes. One of skill in the art can determine whether the ICP0 gene product has been deleted or whether the virus does not express a functional form of the gene product using a PCR-based assay to detect the presence of the gene in the viral genome or expression of the gene product, respectively, or using a functional assay to assess their function.

[0132] In one embodiment, the genes encoding these gene products contain mutations (e.g., inactivating mutations) that inhibit proper expression of the gene product. For example, the gene can encode a mutation in the gene product that inhibits proper folding, expression, function, etc. of the gene product. As used herein, the term "inactivating mutation" is intended to broadly mean a mutation or alteration to a gene in which expression of the gene is significantly reduced, or in which the gene product becomes non-functional, or in which its functional capacity is significantly reduced. The term "gene" encompasses the region encoding the gene product as well as regulatory regions of the gene (such as a promoter or enhancer), unless otherwise indicated.

[0133] Methods to achieve such alterations include: (a) any method that disrupts expression of the gene product, or (b) any method that renders the expressed gene non-functional. Numerous methods of disrupting gene expression are known, including altering the coding region of the gene or its promoter sequence by insertion, deletion, and / or base change. (See, Roizman, B and Jenkins, F.J., Science 229: 1208-1214 (1985)).

[0134] The essential feature of the DNA of the present application is the presence of a gene required for viral replication, which is operably linked to a promoter having a TATA element. The Tet operator sequence is located between 6 and 24 nucleotides 3' of the last nucleotide of the TATA element of the promoter and 5' of the gene. By using a version of the operator containing two op2 repressor binding sites (each such site having the nucleotide sequence: TCCCTATCAGTGATAGAGA (SEQ ID NO: 8) linked by a sequence of 2-20 (preferably 1-3 or 10-13) nucleotides, the strength of binding of the tet repressor to the operator sequence is enhanced. When the repressor binds to this operator, little or no transcription of the associated gene will occur. If the DNA having these features is present in a cell that is also expressing a tetracycline repressor, transcription of the gene will be blocked by the repressor bound to the operator, and viral replication will not occur. However, if, for example, tetracycline is introduced, it will bind to the repressor, causing it to dissociate from the operator, and viral replication will proceed.

[0135] During productive infection, HSV gene expression is divided into three major classes based on the temporal order of expression: immediate early (a), early (β), and late (γ), with the late genes further divided into two groups, γ1 and γ2. Expression of immediate early genes does not require de novo viral protein synthesis and is activated by the virion-associated protein VP16 and cellular transcription factors when the viral DNA enters the nucleus. The protein products of the immediate early genes are designated infection cell polypeptides ICPO, ICP4, ICP22, ICP27, and ICP47, and the promoters of these genes are preferred for directing expression of the tet repressor (tetR). Genes required for viral replication are controlled by promoters containing tetO, and these essential genes can be immediate early, early, or late genes, such as ICP4, ICP27, ICP8, UL9, gD, and VP5. In one embodiment, tetR has the sequence of SEQ ID NO: 9.

[0136] ICP0 plays a major role in enhancing reactivation of HSV from latency and confers a significant growth advantage to the virus at low multiplicities of infection. ICP4 is the major transcriptional regulatory protein of HSV-1, which activates expression of viral early and late genes. ICP27 is essential for productive viral infection and is required for efficient viral DNA replication and optimal expression of viral β genes and γ1 genes and a subset of viral γ2 genes. The function of ICP47 during HSV infection appears to be down-regulation of class I major histocompatibility complex (MHC) expression at the surface of infected cells.

[0137] The recombinant DNA can also include at least one (preferably at least two) sequences encoding tetracycline repressors, the expression of which is under the control of an immediate early promoter (preferably ICP0 or ICP4). The sequences of HSV ICP0, ICP4, and ICP27 promoters and their endogenous regulated genes are well known in the art (Perry et al., J. Gen. Virol. 67:2365-2380 (1986); McGeoch et al., J. Gen. Virol. 72:3057-3075 (1991); McGeoch et al., Nucl. Acid Res. 14: 1727-1745 (1986)) and procedures for making viral vectors containing these elements have been previously described (see U.S. published application 2005-0266564).

[0138] Not only are these promoters very active in promoting gene expression, but they are specifically induced by VP16, a transactivator released when HSV-1 infects a cell. Thus, transcription from the ICP0 promoter is particularly high when repressors are most needed to shut down viral replication. Once an appropriate DNA construct is made, it can be incorporated into HSV-1 virus using methods well known in the art. An appropriate procedure is described in US 2005-0266564, but other methods known in the art can also be employed.

[0139] Other promoters that can be used in the expression of genes in (f) of the present application include, but are not limited to, modified HSV immediate early promoters (e.g., HSV ICPO, ICP4, ICP27, ICP22, and ICP47 promoters / regulatory sequences), HCMV immediate early promoters (e.g., pWRG7128 (Roy et al., Vaccine 19, 764-778, 2001) and PbCl2 / CMV and pJW4303 mentioned in WO95 / 20660; which are incorporated herein by reference in their entirety), or the human elongation factor- 1 alpha (EF-1 alpha) promoter. These promoters are known in the art and one of skill in the art would be able to identify the sequences of these promoters to be used. In one embodiment, the promoter of (f) is a HSV-2 immediate early promoter with a tet operon.

[0140] In various embodiments, the variant gene comprises at least one amino acid change that deviates from the wild-type sequence of the gene. In one embodiment, the oncolytic HSV described herein can comprise two or more amino acid substitutions in at least one variant gene. The at least two amino acid substitutions can be found in the same gene, for example, a gK variant gene contains at least two amino acid substitutions. Alternatively, the at least two amino acid substitutions can be found in at least two different genes, for example, a gK variant gene and a UL24 variant gene each contain at least one amino acid substitution.

[0141] SEQ ID NO: 2 is the amino acid sequence encoding gK (strain KOS).

[0142] MLAVRSLQHLSTVVLITAYGLVLVWYTVFGASPLHRCIYAVRPT

[0143] GTNNDTALVWMKMNQTLLFLGAPTHPPNGGWRNHAHICYANLIAGRVVPFQVPPDATN

[0144] RRIMNVHEAVNCLETLWYTRVRLVVVGWFLYLAFVALHQRRCMFGVVSPAHKMVAPAT

[0145] YLLNYAGRIVSSVFLQYPYTKITRLLCELSVQRQNLVQLFETDPVTFLYHRPAIGVIV

[0146] GCELMLRFVAVGLIVGTAFISRGACAITYPLFLTITTWCFVSTIGLTELYCILRRGPA

[0147] PKNADKAAAPGRSKGLSGVCGRCCSIILSGIAMRLCYIAVVAGVVLVALHYEQEIQRR

[0148] LFDV (SEQ ID NO: 2)

[0149] Another distinguishing feature of the oncolytic viruses described herein is that the viral genome sequence does not comprise a ribozyme sequence, for example, in the 5' untranslated region of VP5. Ribozymes are RNA molecules that are capable of catalyzing biochemical reactions in a manner analogous to protein enzymes. Ribozymes are further described in, for example, Yen et al., Nature 431 :471-476, 2004, the contents of which are incorporated by reference in their entirety.

[0150] In one embodiment of various aspects, the oncolytic virus expresses the LacZ gene, which is well known in the art.

[0151] In an embodiment of various aspects, the oncolytic virus expresses a dominant negative TGF. As used herein, the term "dominant negative" refers to a mutated or modified protein that substantially prevents a corresponding protein with wild-type function from performing the wild-type function. For example, a dominant negative TGF will be able to inhibit the wild-type function of TGF in a cell expressing the dominant negative.

[0152] In an embodiment, the dominant negative TGF is able to inhibit the function (e.g., the ability to initiate TGF signaling) or expression level (e.g., mRNA or protein level) of wild-type TGF by at least 10%. In an embodiment, the dominant negative TGF is able to inhibit the wild-type function (e.g., the ability to initiate TGF signaling) or expression level (e.g., mRNA or protein level) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, as compared to an appropriate control. As used herein, an appropriate control refers to the function or expression level of TGF in a cell that has not been contacted with the dominant negative TGF. One of skill in the art can assess the function of TGF by, for example, assessing the level of TGF signaling in a cell, or the expression level of TGF by, for example, Western blot or PCR-based assays, respectively.

[0153] In an embodiment, the dominant negative TGF comprises, consists of, or consists essentially of a sequence that is at least 90% identical to wild-type TGF and is able to inhibit the wild-type function of TGF. In another embodiment, the dominant negative TGF comprises, consists of, or consists essentially of a sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to wild-type TGF and is able to inhibit the wild-type function of TGF.

[0154] In an embodiment, the dominant negative TGF is mmTGF-b2-7M fragment, having a nucleotide sequence of SEQ ID NO: 10.

[0155] ATGGCCCTGGACGCCGCCTACTGCTTCCGCAACGTGCAGGACAACTGCTGCCTGCGCCCCCTGTACATCGACTTCCGCAAGGACCTGGGCTGGAAGTGGATCCACGAGCCCAAGGGCTACAACGCCAACTTCTGCGCCGGCGCCTGCCCCTACCGCGCCAGCAAGAGCCCCAGCTGCGTGAGCCAGGACCTGGAGCCCCTGACCATCGTGTACTACGTGGGCCGCAAGCCCAAGGTGGAGCAGCTGAGCAACATGATCGTGAAGAGCTGCAAGTGCAGCTAA (SEQ ID NO: 10).

[0156] In one embodiment, the dominant negative TGFp is mmTGF-b2-7M fragment having the amino acid sequence of SEQ ID NO: 11.

[0157] ALDAAYCFRN VQDNCCLRPL YIDFRKDLGW KWIHEPKGYN ANFCAGACPY RASKSPSCVSQ DLEPLTIVY YVGRKPKVEQ LSNMIVKSCK CS (SEQ ID NO: 11).

[0158] In one embodiment, the dominant negative TGFp comprises, consists of, or consists essentially of a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identity to SEQ ID NO: 10 or SEQ ID NO: 11.

[0159] In one embodiment, the oncolytic HSV described herein further comprises at least one polypeptide encoding a product (e.g., a protein, a gene, a gene product, or an antibody or antibody reagent) that can increase the efficacy of the oncolytic HSV to induce anti-tumor specific immunity. Exemplary products include, but are not limited to, interleukin 2 (IL2), interleukin 12 (IL12), interleukin 15 (IL15), an anti-PD-1 antibody or antibody reagent, an anti-PD-L1 antibody or antibody reagent, an anti-OX40 antibody or antibody reagent, a CTLA-4 antibody or antibody reagent, a TIM-3 antibody or antibody reagent, a TIGIT antibody or antibody reagent, a soluble interleukin 10 receptor (IL10R), a fusion polypeptide between a soluble IL10R and an IgG-Fc domain, a soluble TGF-beta type II receptor (TGFBRII), a fusion polypeptide between a soluble TGFBRII and an IgG-Fc domain, an anti-IL10R antibody or antibody reagent, an anti-IL10 antibody or antibody reagent, an anti-TGF-beta 1 antibody or antibody reagent, and an anti-TGFBRII antibody or antibody reagent. In one embodiment, the product is a fragment of IL-2, IL-12, or IL-15 that comprises the same function as IL-2, IL-12, or IL-15, as described below. One of skill in the art can determine whether to use standard techniques in the art to induce anti-tumor specific immunity, which are further described in, e.g., Clay, TM, et al., Clinical Cancer Research (2001); Malyguine, A, et al., J Transl Med (2004); or Macchia I, et al., BioMed Research International (2013), each of which is incorporated by reference herein in its entirety.

[0160] Interleukin-2 (IL-2) is an interleukin, a type of cytokine signaling molecule in the immune system. IL-2 regulates the activity of white blood cells (e.g., leukocytes and lymphocytes) responsible for immunity. IL-2 is part of the body’s natural response to microbial infection, used to distinguish foreign “non-self” from “self.” It mediates its effects by binding to the IL-2 receptor expressed by lymphocytes. Sequences of IL-2 (also known as TCGF and Lymphokine) are known for many species, e.g., human IL-2 (NCBI Gene ID: 3558) polypeptide (e.g., NCBI Ref Seq NP_000577.2) and mRNA (e.g., NCBI Ref Seq NM_000586.3). IL-2 can refer to human IL-2, including naturally occurring variants, molecules, and alleles thereof. IL-2 refers to mammalian IL-2 of e.g., mouse, rat, rabbit, dog, cat, bovine, equine, porcine, etc. The nucleic acid sequence of SEQ ID NO: 5 includes nucleic acid sequences encoding IL-2.

[0161] SEQ ID NO: 5 is a nucleotide sequence encoding IL-2.

[0162]

[0163] Interleukin-12 (IL-12) is an interleukin naturally produced by dendritic cells, macrophages, neutrophils, and human B lymphoblastoid cells (NC-37) in response to antigenic stimulation. IL-12 is involved in the differentiation of naive T cells into Th1 cells. It is known as T-cell stimulating factor, which can stimulate the growth and function of T cells. It stimulates T cells and natural killer (NK) cells to produce interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), and reduces IL-4-mediated suppression of IFN-γ. Sequences of IL-12a (also known as P35, CLMF, NFSK, and KSF1) are known for many species, e.g., human IL-12a (NCBI Gene ID: 3592) polypeptide (e.g., NCBI Ref Seq NP_000873.2) and mRNA (e.g., NCBI Ref Seq NM_000882.3). IL-12 can refer to human IL-12, including naturally occurring variants, molecules, and alleles thereof. IL-12 refers to mammalian IL-12 of e.g., mouse, rat, rabbit, dog, cat, bovine, equine, porcine, etc. The nucleic acid sequence of SEQ ID NO: 6 includes nucleic acid sequences encoding IL-12a.

[0164] SEQ ID NO: 6 is a nucleotide sequence encoding IL-12a.

[0165]

[0166] Interleukin-15 (IL-15) is an interleukin secreted by mononuclear phagocytes (and some other cells) after being infected by a virus. This cytokine induces cell proliferation of natural killer cells; cells of the innate immune system whose primary role is to kill virus-infected cells. Sequences of IL-15 are known for many species, such as human IL-15 (NCBI Gene ID: 3600) polypeptide (e.g., NCBI Ref Seq NP_000585.4) and mRNA (e.g., NCBI Ref Seq NM_000576.1). IL-15 can refer to human IL-15, including naturally occurring variants, molecules, and alleles thereof. IL-15 refers to mammalian IL-15 of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, etc. The nucleic acid sequence of SEQ ID NO: 7 includes nucleic acid sequences encoding IL-15.

[0167] SEQ ID NO: 7 is a nucleotide sequence encoding IL-15.

[0168]

[0169]

[0170] Soluble or wild-type interleukin 10 receptor (IL10R) has been shown to mediate immunosuppressive signals of interleukin 10, leading to suppression of proinflammatory cytokine synthesis. The receptor has been reported to promote survival of myeloid progenitor cells through the insulin receptor substrate-2 / PI 3-kinase / AKT pathway. Activation of IL10R leads to tyrosine phosphorylation of JAK1 and TYK2 kinases. Two transcript variants of this gene have been found, one encoding a protein and the other not. Sequences of IL10R are known for many species, such as human IL10R (NCBI Gene ID: 3587) polypeptide (e.g., NCBI Ref Seq NP_001549.2) and mRNA (e.g., NCBI Ref Seq NM_001558.3). IL10R can refer to human IL10R, including naturally occurring variants, molecules, and alleles thereof. IL10R refers to mammalian IL10R of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, etc. The nucleic acid sequence of SEQ ID NO: 3 includes nucleic acid sequences encoding IL10R.

[0171] SEQ ID NO: 3 is a nucleotide sequence encoding IL10R.

[0172]

[0173]

[0174] Transforming growth factor beta receptor II (TGFBRII) in soluble or wild-type form is a protein encoded by this gene that, when bound to TGF-beta, forms a heteromeric complex with the type II TGF-beta receptor, transducing the TGF-beta signal from the cell surface to the cytoplasm. Sequences of TGFBRII are known for many species, such as human TGF-BRII (NCBI Gene ID: 7048) polypeptide (e.g., NCBI Ref Seq NP_001020018.1) and mRNA (e.g., NCBI Ref Seq NM_001024847.2). TGFBRII can refer to human TGFBRII, including naturally occurring variants, molecules, and alleles thereof. TGFBRII refers to mammalian TGFBRII of, e.g., mouse, rat, rabbit, dog, cat, bovine, equine, porcine, and the like. The nucleic acid sequence of SEQ ID NO: 4 includes nucleic acid sequences encoding TGFBRII.

[0175] SEQ ID NO: 4 is a nucleotide sequence encoding TGFBRII.

[0176]

[0177]

[0178] Antibodies or antibody reagents that bind to PD-1 or its ligand, PD-L1, are described, for example, in U.S. Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT Published Patent Application Nos. WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699; which are incorporated herein by reference in their entirety. In certain embodiments, the PD-1 antibodies include nivolumab (MDX 1106, BMS 936558, ONO 4538), a fully human IgG4 antibody that binds to PD-1 and blocks its activation by its ligands, PD-L1 and PD-L2; lambrolizumab (MK-3475 or SCH 900475), a humanized monoclonal IgG4 antibody against PD-1; CT-011, a humanized antibody that binds to PD-1; AMP-224, a fusion protein of B7-DC; an antibody Fc portion; BMS-936559 (MDX-1105-01), for PD-L1 (B7-H1) blockade. Agents that disrupt or block the interaction between PD-1 and PD-L1, such as high affinity PD-1 antagonists, are also specifically contemplated herein.

[0179] Non-limiting examples of PD-1 antibodies include pembrolizumab (Merck); nivolumab (Bristol Meyers Squibb); pidilizumab (Medivation); and AUNP12 (Aurigene). Non-limiting examples of PD-L1 antibodies can include atezolizumab (Genentech); MPDL3280A (Roche); MEDI4736 (AstraZeneca); MSB0010718C (EMD Serono); avelumab (Merck); and durvalumab (Medimmune).

[0180] Antibodies that bind OX40 (also known as CD134) are described, for example, in U.S. Patents No. US9006399, US9738723, US9975957, US9969810, US9828432; PCT published patent applications: WO2015153513, WO2014148895, WO2017021791, WO2018002339; and U.S. applications: US20180273632; US20180237534; US20180230227; US20120269825; which are incorporated by reference in their entirety.

[0181] Antibodies that bind CTLA-4 are described, for example, in U.S. Patents No. US9714290, US6984720, US7605238, US6682736, US7452535; PCT published patent applications: WO2009100140; and U.S. applications: US20090117132A, US20030086930, US20050226875, US20090238820; which are incorporated by reference in their entirety. Non-limiting examples of CTLA-4 antibodies include ipilimumab (YERVOY®, Bristol-Myers Squibb).

[0182] Antibodies that bind TIM3 are described, for example, in U.S. Patents No. US8552156, US9605070, US9163087, US8329660; PCT published patent applications: WO2018036561, WO2017031242, WO2017178493; and U.S. applications: US20170306016, US20150110792, US20180057591, US20160200815; which are incorporated by reference in their entirety.

[0183] Antibodies that bind TIGIT (also known as CD134) are described, for example, in U.S. Patents: US10017572, US9713641; PCT published patent applications: WO2017030823; and U.S. applications: US20160355589, US20160176963, US20150322119; which are incorporated by reference in their entirety.

[0184] Antibodies that bind interleukin 10 receptor (IL10R) (e.g., soluble or wild type) are described, for example, in U.S. Patent No. 7553932; and U.S. Applications: US20040009939, US20030138413, US20070166307, US20090087440, and US201000028450, which are incorporated by reference herein in their entireties.

[0185] Antibodies that bind TGFBRII (e.g., soluble or wild type) are described, for example, in U.S. Patent No. 6497729; and U.S. Applications: US2012114640, US20120021519, which are incorporated by reference herein in their entireties.

[0186] Another aspect provides an oncolytic herpes simplex virus (HSV) comprising a recombinant DNA that does not encode functional ICP0 or ICP34.5 and encodes a functional mmTGF-β2-7M fragment sequence.

[0187] An oncolytic herpes simplex virus (HSV) comprising a recombinant DNA, wherein the recombinant DNA does not encode functional ICP0 and ICP34.5 genes; and encodes a functional mmTGF-β2-7M fragment sequence.

[0188] Another aspect provides an oncolytic HSV comprising a recombinant DNA that does not encode functional ICP0 and encodes a functional mmTGF-β2-7M fragment sequence.

[0189] Another aspect provides an oncolytic HSV that encodes a functional mmTGF-β2-7M fragment sequence.

[0190] Yet another aspect provides a recombinant virus that encodes a functional mmTGF-β2-7M fragment sequence.

[0191] In one embodiment, any of the oncolytic HSVs described herein further encodes a fusion promoting activity.

[0192] One aspect of the application described herein provides a composition comprising any of the oncolytic HSVs described herein. In one embodiment, the composition is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to an active agent in combination with a pharmaceutically acceptable carrier (e.g., a carrier commonly used in the pharmaceutical industry).

[0193] In one embodiment, the composition further comprises at least one pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include aqueous solutions (such as physiological buffered saline) or other solvents or vehicles, such as glycols, glycerol, vegetable oils (e.g., olive oil), or injectable organic esters. Pharmaceutically acceptable carriers can be used to administer the compositions of the present application to either in vitro cells or in vivo subjects. Pharmaceutically acceptable carriers can contain physiologically acceptable compounds that act, for example, to stabilize the composition or to increase the absorption of the agent. Physiologically acceptable compounds can include, for example, carbohydrates such as glucose, sucrose, or dextrans; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins or other stabilizers or excipients. Other physiologically acceptable compounds include wetting agents, emulsifiers, dispersing agents or preservatives, which are particularly useful for preventing the growth or action of microorganisms. A variety of preservatives are well known and include, for example, phenol and ascorbic acid. Those of skill in the art will know that the selection of a pharmaceutically acceptable carrier, including physiologically acceptable compounds, depends, for example, on the route of administration of the oncolytic HSV.

[0194] Hybrid nucleic acid

[0195] Another aspect provided herein is a hybrid nucleic acid sequence comprising a sequence of a therapeutic antibody and a dominant negative TGFβ, wherein the dominant negative TGFβ is fused to the Fc domain of the therapeutic antibody.

[0196] Another aspect provided herein is a hybrid nucleic acid sequence comprising a sequence of a therapeutic antibody and a mmTGF-β2-7M fragment, wherein the mmTGF-β2-7M is fused to the Fc domain of the therapeutic antibody.

[0197] In one embodiment, the therapeutic antibody sequence is a sequence of an immunotherapeutic antibody. For example, the therapeutic antibody sequence is a sequence that can be selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-Tim3 antibody, an anti-CTLA4 antibody, and an anti-TDM-1 antibody and an anti-TIGIT antibody. Such therapeutic antibodies are described above.

[0198] Also provided herein is a polypeptide encoded by any of the hybrid nucleic acids described herein.

[0199] Also provided herein is a vector expressing any of the hybrid nucleic acids described herein.

[0200] Chimeric antigen receptor

[0201] The techniques described herein provide improved CARs for treating cancer. CARs and various improvements are discussed below.

[0202] The term "chimeric antigen receptor" or "CAR" as used herein refers to an engineered T cell receptor that grafts ligand or antigen specificity onto a T cell (e.g., a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immunoreceptors.

[0203] A CAR places a chimeric extracellular target binding domain that specifically binds a target (e.g., a polypeptide) expressed on the surface of a cell targeted by a T cell response onto a construct that includes a transmembrane domain and an intracellular domain of a T cell receptor molecule. In one embodiment, the chimeric extracellular target binding domain comprises an antigen binding domain of an antibody that specifically binds an antigen expressed on a cell targeted by a T cell response. The intracellular signaling domain of a CAR can vary as known in the art and as disclosed herein, but the chimeric target / antigen binding domain renders the receptor sensitive to signaling activation when the chimeric target / antigen binding domain binds a target / antigen on the surface of a targeted cell.

[0204] With respect to the intracellular signaling domain, so-called "first generation" CARs include those that provide only CD3 zeta signaling upon antigen binding. So-called "second generation" CARs include those that provide both a costimulatory (e.g., CD28 or CD137) and an activating (CD3 zeta) domain, and so-called "third generation" CARs include those that provide multiple costimulatory (e.g., CD28 and CD137) domains and an activating domain (e.g., CD3 zeta). In various embodiments, a CAR is selected to have high affinity or avidity for a target / antigen, e.g., an antibody-derived target or antigen binding domain will typically have a higher affinity and / or avidity for a target antigen than a naturally occurring T cell receptor. This property, combined with high specificity, can select antibodies to provide high specific T cell targeting of CAR T cells.

[0205] As used herein, "CAR T cell" or "CAR-T" refers to a T cell that expresses a CAR. When expressed in a T cell, a CAR has the ability to redirect T cell specificity and reactivity to a selected target in a non-MHC restricted manner, utilizing the antigen binding properties of a monoclonal antibody. Non-MHC restricted antigen recognition gives T cells expressing CARs the ability to recognize antigens that are independent of antigen processing, thereby bypassing a major mechanism of tumor escape.

[0206] As used herein, the term "extracellular target binding domain" refers to a polypeptide found outside of a cell that is sufficient to promote binding to a target. The extracellular target binding domain will specifically bind its binding partner (i.e., the target). As non-limiting examples, the extracellular target binding domain can include a sequence encoding a dominant negative peptide, an antigen binding domain of an antibody, or a ligand that recognizes and binds a cognate binding partner (e.g., a TGFp) protein.

[0207] In one embodiment, the CAR is a bispecific CAR. For example, the CAR comprises in its extracellular domain a sequence of a dominant negative TGFp sequence (e.g., mmTGF-β2-7M fragment); and a sequence of a therapeutic antibody (e.g., an anti-PD1 antibody, an anti-CTLA4 antibody, or an anti-TIM3 antibody).

[0208] Transmembrane domain

[0209] Each of the CARs described herein necessarily includes a transmembrane domain that links the extracellular target binding domain to the intracellular signaling domain.

[0210] As used herein, a "transmembrane domain" (TM domain) refers to the generally hydrophobic region of a CAR that crosses the plasma membrane of a cell. The TM domain can be a transmembrane region of a transmembrane protein (e.g., a Type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof, or a fragment thereof. While specific examples are provided herein and used in the examples, other transmembrane domains will be apparent to those of skill in the art and can be used in conjunction with alternative embodiments of the present technology. The selected transmembrane region or fragment thereof preferably does not interfere with the intended function of the CAR. As used with respect to a transmembrane domain of a protein or polypeptide, "fragment thereof" refers to a portion of the transmembrane domain sufficient to anchor or attach the protein to the surface of a cell.

[0211] In one embodiment, the transmembrane domain or fragment thereof of a CAR described herein comprises a transmembrane domain selected from the transmembrane domain of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0212] In an exemplary embodiment, the transmembrane domain or fragment thereof of the CAR is derived from or comprises the transmembrane domain of CD8. CD8 is an antigen found preferentially on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system and acts as a T cell co-receptor. CD8 is composed of alpha (CD8a) and beta (CD8b) chains. CD8a sequences are known for many species, e.g., human CD8a (NCBI Gene ID: 925) polypeptide (e.g., NCBI RefSeq NP_001139345.1) and mRNA (e.g., NCBI Ref Seq NM_000002.12). CD8 can refer to human CD8, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, CD8 can refer to CD8 of, e.g., dogs, cats, cows, horses, pigs, etc. Homologs and / or orthologs of human CD8 are readily identified by one of skill in the art for such species, e.g., using the NCBI Ortholog Search function, or searching for sequences similar to a reference CD8 sequence in available sequence data for a given species.

[0213] Co-stimulatory domain

[0214] A CAR described herein can comprise an intracellular domain or co-stimulatory domain of a co-stimulatory molecule. As used herein, the term “co-stimulatory domain” refers to the intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. Illustrative examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2CSLP76, TRIM, and ZAP70.

[0215] In an exemplary embodiment, the intracellular domain is the intracellular domain of 4-1BB. 4-1BBL is a type 2 transmembrane glycoprotein belonging to the TNF superfamily. 4-1BBL is expressed on activated T lymphocytes. 4-1BBL sequences are known for many species, e.g., human 4-1BBL (also known as TNFSF9) (NCBI Gene ID: 8744) polypeptide (e.g., NCBI Ref Seq NP_003802.1) and mRNA (e.g., NCBI Ref Seq NM_003811.3). 4-1BBL can refer to human 4-1BBL, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, 4-1BBL can refer to 4-1BBL of, e.g., dogs, cats, cows, horses, pigs, etc. Homologs and / or orthologs of human 4-1BBL are readily identified by one of skill in the art for such species, e.g., using the NCBI Ortholog Search function or searching sequence data available for the given species for sequences similar to the reference 4-1BBL sequence.

[0216] Intracellular signaling domain

[0217] A CAR as described herein can comprise an intracellular signaling domain. An “intracellular signaling domain” refers to a portion of a CAR polypeptide that is involved in transducing the information of effective CAR binding to a target antigen to the interior of an immune effector cell to cause effector cell function (e.g., activation, cytokine production, proliferation, and cytotoxic activity, including cytotoxic factor release to a CAR-bound target cell, or other cellular responses elicited after antigen binding to the extracellular CAR domain).

[0218] CD3 is a T cell co-receptor that facilitates T lymphocyte activation when performed simultaneously with the appropriate co-stimulation (e.g., binding of a co-stimulatory molecule). CD3 complex is composed of 4 different chains; CD3 of mammals is composed of one CD3 gamma chain, one CD3 delta chain, and two CD3 epsilon chains. These chains associate with a molecule known as T cell receptor (TCR) and CD3 zeta to produce an activation signal in T lymphocytes. The complete TCR complex includes TCR, CD3 zeta, and the complete CD3 complex.

[0219] In some embodiments of any of the aspects, a CAR polypeptide described herein comprises an intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif or ITAM from CD3 zeta. In some embodiments of any of the aspects, the ITAM comprises three motifs of the ITAM of CD3 zeta (ITAM3).

[0220] ITAMs are known as primary signaling domains that modulate primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary signaling domains that act in a stimulatory manner can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Non-limiting examples of ITAM-containing intracellular signaling domains of particular use in the present technology include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma,

[0221] In one embodiment, the CAR further comprises a linker domain. As used herein, "linker domain" refers to an oligo- or polypeptide region of about 2 to 100 amino acids in length that links any of the domains / regions of a CAR as described herein together. In some embodiments, the linker can comprise or consist of flexible residues such as glycines and serines, such that adjacent protein domains can move freely relative to one another. Longer linkers can be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. The linker can be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents thereof, and combinations thereof. In one embodiment, the linker region is T2A derived from Thosea asigna virus. Non-limiting examples of linkers useful in the present technology include P2A and F2A.

[0222] More detailed descriptions of CARs and CAR T cells can be found in Maus et al., Blood 2014 123:2624-35; Reardon et al., Neuro-Oncology 2014 16:1441-1458; Hoyos et al., Haematologica 2012 97:1622; Byrd et al., J Clin Oncol 2014 32:3039-47; Maher et al., Cancer Res 2009 69:4559-4562; and Tamada et al., Clin Cancer Res 2012 18:6436-6445; each of which is incorporated herein by reference in its entirety.

[0223] Another aspect provided herein is a nucleic acid encoding any of the CAR polypeptides described herein.

[0224] Cells

[0225] Provided herein are cells or populations thereof comprising any of the oncolytic or recombinant viruses described herein.​

[0226] Provided herein are cells or populations thereof comprising any of a hybrid nucleic acid, a polypeptide encoding a hybrid nucleic acid, or a vector expressing a hybrid nucleic acid or a polypeptide encoding a hybrid nucleic acid.

[0227] Also provided herein are cells or populations thereof comprising any of a CAR polypeptide described herein or any nucleic acid encoding a CAR polypeptide.

[0228] In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a non-human mammalian cell.

[0229] In one embodiment, the cell is a T cell. In one embodiment, the cell is a CAR T cell.

[0230] In one embodiment, the cell is an immune cell. As used herein, "immune cell" refers to a cell that plays a role in the immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the cell is a T cell; an NK cell; an NKT cell; a lymphocyte, such as a B cell and a T cell; and a myeloid cell, such as a monocyte, a macrophage, an eosinophil, a mast cell, a basophil, and a granulocyte.

[0231] In one embodiment, the cell is obtained from an individual having or diagnosed with a cancer.

[0232] In one embodiment, the cell is a CAR T cell.

[0233] In one embodiment, the cell is a bispecific CAR T cell, meaning comprising more than one CAR polypeptide. For example, the CAR T cell comprises a CAR polypeptide having an extracellular domain comprising a dominant negative TGFβ sequence (e.g., mmTGF-β2-7M fragment) and a second CAR polypeptide comprising an extracellular domain comprising a sequence of a therapeutic antibody (e.g., an anti-PD1 antibody, an anti-CTLA4 antibody, or an anti-TIM3 antibody).

[0234] In certain embodiments, the cell has a high level of dominant negative TGFβ, e.g., mmTGF-β2-7M. The level of expression of the dominant negative TGFβ can be determined by one of skill in the art, e.g., by evaluating the protein or mRNA level of the dominant negative TGFβ, respectively, by Western blot or PCR-based assays.

[0235] Methods of treatment

[0236] The oncolytic viruses, hybrid nucleic acids, and CAR T cells, or compositions thereof, described herein can be administered to a subject having a cancer. In certain embodiments, an agent that modulates the tet operator of an oncolytic virus is also administered with an oncolytic virus, or composition thereof, described herein, where appropriate. Exemplary agents include, but are not limited to, doxycycline or tetracycline.

[0237] One aspect provides a method of treating a cancer, the method comprising engineering a T cell to comprise any of the CAR polypeptides described herein, or a nucleic acid encoding a CAR polypeptide, on the surface of the T cell; and administering the engineered T cell to a subject.

[0238] In one embodiment, the cancer is a solid tumor. The solid tumor can be malignant or benign. In one embodiment, the subject is diagnosed with or has been diagnosed with a carcinoma, melanoma, sarcoma, germ cell tumor, and blastoma. Exemplary cancers include, but are in no way limited to: non-small cell lung cancer, bladder cancer, breast cancer, brain cancer, colon cancer, prostate cancer, liver cancer, lung cancer, ovarian cancer, skin cancer, head and neck cancer, kidney cancer, and pancreatic cancer. In one embodiment, the cancer is metastatic. These types of cancers are known in the art and can be diagnosed by a skilled clinician using standard techniques known in the art, such as blood analysis, blood cell count analysis, tissue biopsy, non-invasive imaging, and / or review of family history.

[0239] In cases where the tumor is easily accessible, such as a tumor of the skin, mouth, or a tumor that is accessible due to surgery, the virus can be applied locally. In other cases, it can be administered by injection or infusion. The agent used to modulate the tet operator and tetR interaction, such as doxycycline or tetracycline, can also be administered in this manner, prior to or at the time of infection, or it can be administered systemically, such as orally.

[0240] While certain routes of administration are provided in the foregoing description, any suitable route of administration of the vector can be modified in accordance with the present application, and thus the routes of administration described above are not intended to be limiting. Routes of administration can include, but are not limited to, intravenous, regional arterial infusion, oral, buccal, intranasal, inhalation, topical application to mucosa, or injection, including intratumoral, intradermal, intrathecal, intracisternal, intralesional, or any other type of injection. Administration can be continuous or intermittent, and will vary with the subject and condition to be treated. Those skilled in the art will readily understand that the various routes of administration described herein will allow the vectors or compositions of the present application to be delivered on, in, or near a tumor or target cancer cell. Those skilled in the art will also readily understand that the various routes of administration described herein will allow the vectors and compositions described herein to be delivered to the area near a tumor or individual cell to be treated. "Nearby" can include any tissue or bodily fluid in the subject that is in close enough proximity to a tumor or individual cancer cell such that at least a portion of the vector or composition administered to the subject reaches its intended target and exerts its therapeutic effect.

[0241] Prior to administration, the oncolytic virus can be suspended in any pharmaceutically acceptable solution, including sterile isotonic saline, water, phosphate buffered saline, 1,2- propylene glycol, polyethylene glycol mixed with water, Ringer's solution, and the like. The exact number of viruses to be administered is not critical to the present application, but should be an "effective amount," i.e., an amount sufficient to cause widespread enough cell lysis to mount an immune response to the released tumor antigens. Since the virus replicates in the cell after infection, the number initially administered will rapidly increase over time. Thus, widely varying amounts of virus initially administered can produce the same result by varying the time allowed for the virus to replicate, i.e., the time the cell is exposed to tetracycline. Generally, it is expected that the number of viruses (PFU) initially administered will be in the range of 1 x 105to 1 x 1011. 6 to lx 1011 10 .

[0242] Tetracycline or doxycycline will be administered locally or systemically at or 1-72 hours prior to infection to induce viral replication. The amount of tetracycline or doxycycline to be administered will depend on the route of delivery. In vitro, 1 pg / ml of tetracycline is sufficient to allow the virus to replicate in infected cells. Thus, when delivered locally, any solution containing 0.1 pg / ml to 100 pg / ml can be administered. However, much higher doses of tetracycline or doxycycline can be used if desired (e.g., 1-5 mg / ml). The total amount administered in a single local administration will depend on the size of the tumor or tumors being treated, but generally, 0.5 to 200 ml of tetracycline or doxycycline solution is expected to be used at a time. When administered systemically, higher doses of tetracycline or doxycycline will be administered, but the total amount required is expected to be significantly less than the amounts typically used to treat bacterial infections (e.g., in the case of doxycycline, 1-2 grams per day in adults, divided into 2-4 equal doses; 2.2-4.4 mg per kilogram of body weight per day in children, divided into at least 2 doses). In most cases, 5-100 mg per day is expected to be effective. The dosages of tetracycline and doxycycline are well known in the art and can best be determined by a skilled clinician for a given patient.

[0243] In some embodiments, the pharmaceutical compositions comprising CAR T cells as described herein can be parenteral dosage forms. Since administration of parenteral dosage forms generally bypasses the patient's natural defenses against contaminants, components other than the CAR T cells themselves are preferably sterile or capable of being sterilized prior to administration to the patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. Any of these can be added to a CAR T cell preparation prior to administration.

[0244] Suitable carriers that can be used to provide parenteral dosage forms of the CAR T cells disclosed in the art are well known to those skilled in the art. Examples include, but are not limited to: saline solutions; glucose solutions; aqueous vehicles, which include, but are not limited to, sodium chloride injec tion, Ringer's solution, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0245] In some embodiments, the CAR T cells described herein are administered as a monotherapy, i.e., another treatment for the condition is not simultaneously administered to the subject.

[0246] Pharmaceutical compositions comprising the T cells described herein can generally be administered in dosages ranging from 10 4Up to 10 9 Cells / kg body weight, in some cases 10 5 Up to 10 6 The T-cell composition may be administered at doses of 1 cell / kg body weight, including all integer values ​​within those ranges. If necessary, the T-cell composition may also be administered multiple times at these doses. Cells may be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[0247] In some cases, it may be necessary to administer CAR T cells to the subject before drawing blood (or performing apheresis) to activate the T cells as described herein, and then re-infuse the patient with these activated and expanded T cells. This process can be performed multiple times every few weeks. In some cases, T cells can be activated from blood draws ranging from 10cc to 400cc. In other cases, T cells can be activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc.

[0248] Administration methods may include, for example, intravenous (iv) injection or infusion. The compositions described herein may be administered to patients via artery, intratumoral, intranodal, or intramedullary routes. In some embodiments, the T-cell composition may be injected directly into the tumor, lymph node, or site of infection. In one embodiment, the compositions described herein are administered into a body cavity or fluid (e.g., ascites, pleural fluid, peritoneal fluid, or cerebrospinal fluid).

[0249] As used herein, the term "therapeutic effective dose" is intended to refer to the amount of carrier that exerts oncolytic activity, causes attenuation or inhibition of tumor cell proliferation, and leads to tumor regression. The effective dose will vary depending on the pathology or condition to be treated, the patient and their condition, and other factors well known to those skilled in the art. The effective dose can be readily determined by those skilled in the art. In some embodiments, the treatment range is a single introduction of 10... 3 Up to 10 12 Each plaque-forming unit. In some embodiments, therapeutic doses within the above-described therapeutic range are administered via intratumoral, intrathecal, convection-enhanced, intravenous, or intra-arterial routes at intervals ranging from daily to monthly.

[0250] Combination therapy

[0251] The oncolytic viruses and CAR T cells described herein can be used in combination with other known agents and therapies. In one embodiment, the subject is further administered an anti-cancer therapy. "Combination" administration as used herein means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or the other treatment has stopped for other reasons. In some embodiments, the delivery of one treatment still occurs at the initiation of the delivery of the second treatment, such that there is overlap in administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatments are more effective due to the combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is observed with less of the second treatment, or the second treatment reduces the symptoms to a greater extent, or the like, compared to administration of the second treatment in the absence of the first treatment. In some embodiments, the delivery is such that a reduction in symptoms or other parameter related to the disorder is greater than that observed for one treatment delivered in the absence of the other. The effects of the two treatments can be additive, wholly additive, or greater than additive. The delivery can be such that the effects of the first treatment delivered are still detectable when the second therapeutic agent is delivered. The oncolytic viruses or CAR T cells described herein and at least one additional therapeutic agent can be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the oncolytic viruses or CAR T cells described herein can be administered first, the additional agent can be administered second, or the order of administration can be reversed. The oncolytic viruses or CAR T cells and / or the other therapeutic agent, procedure or modality can be administered during active disorder or during remission or less active disease. The oncolytic viruses or CAR T cells can be administered prior to the other treatment, concurrently with the treatment, after the treatment, or during remission of the disorder.

[0252] When administered in combination, the oncolytic virus or CAR T cell and the additional agent (e.g., second or third agent) or all can be administered in amounts or dosages that are higher, lower, or the same as the amounts or dosages of each agent used alone (e.g., as monotherapy). In certain embodiments, the oncolytic virus or CAR T cell, additional agent (e.g., second or third agent) or all are administered in amounts or dosages that are lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amounts or dosages of each agent used alone. In other embodiments, the amounts or dosages of the oncolytic virus or CAR T cell, additional agent (e.g., second or third agent) or all that produce the desired effect (e.g., treatment of cancer) are lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amounts or dosages of each agent required to achieve the same therapeutic effect individually. In other embodiments, the oncolytic virus or CAR T cell described herein can be used in a treatment regimen in combination with surgery, chemotherapy, radiation, mTOR pathway inhibitors, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies or other immune depleting agents (e.g., CAMPATH, anti-CD3 antibodies, or other antibody therapies), cytotoxins, fludarabine, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, or peptide vaccines (such as described in Izumoto et al., 2008 J Neurosurg 108:963-971).

[0253] In one embodiment, the oncolytic virus or CAR T cell described herein can be used in combination with a checkpoint inhibitor. Exemplary checkpoint inhibitors include anti-PD-1 inhibitors (nivolumab, MK-3475, Pembrolizumas, pidilizumab, AMP-224, AMP-514), anti-CTLA4 inhibitors (ipilimumab and Tremelimumab), anti-PDL1 inhibitors (atezolizumab, Avelomab, MSB0010718C, MEDI4736, and MPDL3280A), and anti-TIM3 inhibitors.

[0254] In one embodiment, the oncolytic virus or CAR T cell described herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzumab, gemtuzumab, rituximab, tositumomab), antimetabolites (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine)), mTOR inhibitors, TNFR glucocorticoid-induced TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., acrinon A, a gliotoxin, or bortezomib), immunomodulators (such as thalidomide or thalidomide derivatives (e.g., lenalidomide)). General chemotherapeutic agents that are considered for combination therapy include anastrozole bicalutamide bleomycin sulfate busulfan injection capecitabine N4-pentoxylcarbonyl-5-deoxy-5-fluorocytidine, carboplatin carmustine chlorambucil cisplatin cladribine cyclophosphamide or ), cytarabine, cytosine arabinoside, ), cytarabine liposomal injection decarbazine (DTIC- ), dactinomycin (actinomycin D, ), daunorubicin hydrochloride daunorubicin citrate liposome injection dexamethasone, docetaxel doxorubicin hydrochloride etoposide fludarabine phosphate 5-fluorouracil flutamide tezacitibine, gemcitabine (difluorodeoxycitidine), hydroxyurea idarubicin ifosfamide irinotecan L-asparaginase Calcium folinate, melphalan 6-mercaptopurine Methotrexate Mitoxantrone Mylotarg, paclitaxel phoenix (Yttrium 90 / MX-DTPA), pentostatin, with carmustine implants Polyphenon E20, raloxifene Teniposide 6-thioguanine, thiotepa, tirapazamine Topotecan hydrochloride for injection Vinblastine Vincristine and vinorelbine Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes): uracil mustard (Aminouracil ), Uracil mustard Uracil mustard (Uracil nitrogen ), Chlormethine (mustine) Cyclophosphamide Revimmune TM ), Ifosfamide Melphalan Chlorambucil Pipobroman Triethylenemelamine Triethylenethiophosphoramine, temozolomide Thiotepa Busulfan Carmustine Lomustine Streptozocin and Dacarbazine (DTIC- ). Additional exemplary alkylating agents include, but are not limited to, oxaliplatin Temozolomide and ); dactinomycin (also known as actinomycin D, ); melphalan (also known as L-PAM, L- sarcolysin, and phenylalanine mustard, hexamethylmelamine (also known as hexamethylmelamine (HMM), carmustine bendamustine busulfan and cisplatin carmustine (also known as BCNU, cisplatin (also known as CDDP, and chlorambucil cyclophosphamide or dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC- hexamethylmelamine (also known as hexamethylmelamine (HMM), ifosfamide prednumustine; procarbazine mechlorethamine (also known as nitrogen mustard, mustine and mechlorethamine hydrochloride, streptozocin thiotepa (also known as thiofosfonamide, TESPA and TSPA, cyclophosphamide and bendamustine HCl Exemplary mTOR inhibitors include, for example, sirolimus ester; deforolimus (also known as torisel, (1R,2R,4S)-4-[(2R)-2][(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexanza-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinic acid, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03 / 064383); everolimus (also known as Certican® or RAD001); rapamycin (AY22989, or RAD001); rapamycin (AY22989, ); simapimod (CAS 164301-51-3); emsirolimus (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-(i]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[iraw5,4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-JJpyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholin-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartyl-L-serine-(SEQ ID NO: 29), the inner salt (SF1126, CAS 936487-67-1), and XL765. Exemplary immunomodulators include, for example, afutuzumab (available from ) ; pegfilgrastim lenalidomide (CC-5013, ) ; thalidomide actimid (CC4047); and IRX-2 (a mixture of human cytokines, including interleukin 1, interleukin 2, and interferon gamma, CAS 951209-71-5, available from IRX Therapeutics). Exemplary anthracyclines include, for example, doxorubicin (Adriamycin® and ) ; bleomycin daunorubicin (daunorubicin hydrochloride, daunomycin, and ruborycin hydrochloride, ) ; daunorubicin liposomal (daunorubicin citrate liposomal, ) ; mitoxantrone (DHAD, ) ; epirubicin (Ellence TM ) ; idarubicin Idamycin ) ; mitomycin C geldanamycin; herbimycin; ravidomycin; and deacetyl-ravidomycin. Exemplary vinca alkaloids include, for example, vinorelbine tartrate vincristine and vindesine vinblastine (also known as vinblastine sulfate / vincaleukoblastine and VLB, Alkaban- and ) ; and vinorelbine Exemplary proteasome inhibitors include bortezomib Carfilzomib (PX-171-007, (S)-4-methyl-N-((S)-1-((S)-4-methyl-1-((R)-2- methyloxiranyl-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2- (morpholinoacetamido)-4-phenylbutyramido)-pentanamide); Marizomib (NPT0052); Ixazomib (MLN-9708); Delanzomib (CEP-18770); and O-methyl-N-[(2-methyl-5- thiazolyl)carbonyl]L-serinyl-O-methyl-N-[(1 1S')-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1- (phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[0255] A person of skill in the art can readily identify chemotherapeutic agents for use (see, e.g., Physicians’ Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison’s Principles of Internal Medicine, 18thedition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff’s Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): The Cancer Chemotherapy Handbook, 4thedition, St. Louis, Mosby-Year Book, 2003).

[0256] In one embodiment, the oncolytic virus or CAR T cell described herein is administered to a subject in combination with a molecule that reduces targeting of GITR and / or modulates GITR function, a molecule that reduces Treg cell population, an mTOR inhibitor, a GITR agonist, a kinase inhibitor, a non-receptor tyrosine kinase inhibitor, a CDK4 inhibitor, and / or a BTK inhibitor.

[0257] Efficacy

[0258] Efficacy of the oncolytic virus or CAR T cell in, for example, treating a condition described herein or inducing a response as described herein (e.g., reduction in cancer cells, reduction in tumor size) can be determined by a skilled clinician. However, the term treatment as used herein is considered to be “effective treatment” if one or more signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced at least 10% for example, following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or morbidity of a condition treated according to the methods described herein or any other suitable measurable parameter. Treatment according to the methods described herein can reduce the level of a marker or symptom of a condition, for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0259] Efficacy can also be measured by the inability of an individual to worsen, as assessed by hospitalization or the need for medical intervention (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and / or described herein.

[0260] Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for treating a disease refers to an amount sufficient to effect beneficial treatment, as defined herein, of that disease in a subject in need thereof. Efficacy of an agent can be determined by assessing physical indicators of the disease or the desired response. It is well within the capabilities of one of skill in the art to monitor the efficacy of an administration and / or treatment by measuring any one of such parameters or any combination of parameters. Efficacy of a given method can be assessed in an animal model of a condition described herein (e.g., treatment of ALL). When an experimental animal model is used, efficacy of a treatment is demonstrated when a statistically significant change in a marker is observed.

[0261] All patents and other publications; including references, issued patents, published patent applications, and co-pending patent applications; to the extent that they are incorporated by reference for the description and disclosure of methods which can be used in connection with the technology described herein, are expressly incorporated herein by reference. The publications referred to are provided solely for their disclosure prior to the filing date of this application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such publications by virtue of prior disclosure or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0262] The description of embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise forms disclosed. While specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative implementations can perform functions in a different order, or functions can be performed substantially concurrently. The teachings of the present disclosure provided herein can be applied to other programs or methods. Various embodiments described herein can be combined to provide further embodiments. If desired, aspects of the present disclosure can be modified to adopt the constitutions, functions, and concepts of the above-mentioned references and applications to provide other embodiments of the present disclosure. Furthermore, some changes can be made in the protein structure without affecting the biological or chemical action in kind or amount, due to the consideration of biological functional equivalence. These and other changes can be made to the present disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0263] In other embodiments, particular elements of any of the foregoing embodiments can be combined or substituted elements. Moreover, while advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments can also exhibit such advantages, and not all embodiments necessarily exhibit the same advantages. Accordingly, the scope of the present disclosure is intended to embrace all such alternatives, modifications, and variations.

[0264] The technology described herein is further illustrated by the following examples, which are in no way to be construed as further limiting.

[0265] The application provided herein can be further described in the following numbered paragraphs.

[0266] 1. An oncolytic herpes simplex virus (HSV) comprising a recombinant DNA, wherein the recombinant DNA comprises:

[0267] a) a gene of HSV-1 or HSV-2, namely the VP5 gene, operably linked to a VP5 promoter comprising a TATA element, which contains a 5' untranslated region;

[0268] b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' to the TATA element, wherein the VP5 gene is located 3' to the tetracycline operator sequence;

[0269] c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is located at the ICP0 locus;

[0270] d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant;

[0271] e) a gene sequence encoding a functional ICP34.5 protein; and

[0272] f) a gene sequence operably linked to a modified HSV promoter, wherein the gene is located in the intergenic region between the UL26 gene and the UL27 gene,

[0273] wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence located in the 5' untranslated region of VP5.

[0274] 2. An oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises:

[0275] a) a gene of HSV-1 or HSV-2, namely the VP5 gene, operably linked to a VP5 promoter comprising a TATA element, which contains a 5' untranslated region;

[0276] b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' to the TATA element, wherein the VP5 gene is located 3' to the tetracycline operator sequence;

[0277] c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is located at the ICP0 locus;

[0278] d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant;

[0279] e) a gene sequence encoding a functional ICP34.5 protein; and

[0280] f) a gene sequence operably linked to a modified HSV promoter, wherein the gene is located in the intergenic region of the UL21 gene and the UL22 gene,

[0281] wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence located in the 5' untranslated region of VP5.

[0282] 3. An oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises:

[0283] a) a gene comprising a 5' untranslated region and a VP5 gene of HSV-1 or HSV-2 operably linked to a VP5 promoter comprising a TATA element;

[0284] b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' of the TATA element, wherein the VP5 gene is located 3' of the tetracycline operator sequence;

[0285] c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is located at the ICP0 locus;

[0286] d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant;

[0287] e) a gene sequence encoding a functional ICP34.5 protein; and

[0288] f) a gene sequence operably linked to a modified HSV promoter, wherein the gene is located in the intergenic region of the UL26 gene, the UL27 gene, the UL21 gene, and the UL21 gene,

[0289] wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence located in the 5' untranslated region of VP5.

[0290] 4. The oncolytic HSV of any preceding paragraph, wherein the gene sequence of (f) is a LacZ gene sequence.

[0291] 5. The oncolytic HSV of any preceding paragraph, wherein the gene sequence of (f) is a dominant negative TGF-β mutant sequence.

[0292] 6. The oncolytic HSV of any preceding paragraph, wherein the dominant negative TGF-β mutant sequence is a mmTGF-β2-7M fragment sequence.

[0293] 7. The oncolytic HSV of any preceding paragraph, wherein the promoter of (f) is a modified HSV immediate early promoter, a HCMV immediate early promoter, or a human elongated alpha promoter.

[0294] 8. The oncolytic HSV of any preceding paragraph, wherein the variant gene is a gK variant gene encoding an amino acid substitution selected from the group consisting of: an Ala to Thr amino acid substitution corresponding to amino acid 40 of SEQ ID NO: 2; an Ala to “x” amino acid substitution corresponding to amino acid 40 of SEQ ID NO: 2, wherein “x” is any amino acid; an Asp to Asn amino acid substitution corresponding to amino acid 99 of SEQ ID NO: 2; a Leu to Pro amino acid substitution corresponding to amino acid 304 of SEQ ID NO: 2; and an Arg to Leu amino acid substitution corresponding to amino acid 310 of SEQ ID NO: 2.

[0295] 9. The oncolytic HSV of any preceding paragraph, wherein the tetracycline operon sequence comprises two Op2 repressor binding sites.

[0296] 10. The oncolytic HSV of any preceding paragraph, wherein the VP5 promoter is a VP5 promoter of HSV-1 or HSV-2.

[0297] 11. The oncolytic HSV of any preceding paragraph, wherein the immediate early promoter is an immediate early promoter of HSV-1 or HSV-2.

[0298] 12. The oncolytic HSV of any preceding paragraph, wherein the HSV immediate early promoter is selected from the group consisting of: an ICP0 promoter, an ICP4 promoter, and an ICP27 promoter.

[0299] 13. The oncolytic HSV of any preceding paragraph, wherein the recombinant DNA is part of a HSV-1 genome.

[0300] 14. The oncolytic HSV of any preceding paragraph, wherein the recombinant DNA is part of a HSV-2 genome.

[0301] 15. The oncolytic HSV of any preceding paragraph, further comprising a pharmaceutically acceptable carrier.

[0302] 16. The oncolytic HSV of any preceding paragraph, further encoding at least one polypeptide capable of increasing the efficacy of the oncolytic HSV in inducing anti-tumor specific immunity.

[0303] 17. An oncolytic HSV according to any preceding paragraph, wherein the at least one polypeptide encodes a product selected from the group consisting of: interleukin 2 (IL2), interleukin 12 (IL12), interleukin 15 (IL15), an anti-PD-1 antibody or antibody reagent, an anti-PD-L1 antibody or antibody reagent, an anti-OX40 antibody or antibody reagent, a CTLA-4 antibody or antibody reagent, a TIM-3 antibody or antibody reagent, a TIGIT antibody or antibody reagent, a soluble interleukin 10 receptor (IL10R), a fusion polypeptide between a soluble IL10R and an IgG-Fc domain, a soluble TGF beta type II receptor (TGFBRII), a fusion polypeptide between a soluble TGFBRII and an IgG-Fc domain, an anti-IL10R antibody or antibody reagent, an anti-IL10 antibody or antibody reagent, an anti-TGFBRII antibody or antibody reagent, and an anti-TGFBRII antibody or antibody reagent.

[0304] 18. An oncolytic HSV according to any preceding paragraph, wherein the oncolytic HSV further encodes a fusogenic activity.

[0305] 19. An oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises:

[0306] a) a 5’ untranslated region and a gene of HSV-1 or HSV-2, namely a VP5 gene, operably linked to a VP5 promoter comprising a TATA element;

[0307] b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3’ of the TATA element, wherein the VP5 gene is located 3’ of the tetracycline operator sequence;

[0308] c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is located at the ICP0 locus;

[0309] d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant;

[0310] e) a gene sequence encoding a functional ICP34.5 protein; and

[0311] f) a dominant negative TGF-beta mutant sequence operably linked to a modified HSV-2 immediate early promoter, wherein the gene is located in the intergenic region between the UL26 gene and the UL27 gene,

[0312] wherein the oncolytic HSV does not encode functional ICP0 and does not contain a ribozyme sequence located in the 5' untranslated region of VP5.

[0313] 20. An oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises:

[0314] a) a gene of HSV-1 or HSV-2, namely a VP5 gene, operably linked to a VP5 promoter comprising a TATA element, and containing a 5' untranslated region;

[0315] b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' of the TATA element, wherein the VP5 gene is located 3' of the tetracycline operator sequence;

[0316] c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is located at the ICP0 locus;

[0317] d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant;

[0318] e) a gene sequence encoding a functional ICP34.5 protein; and

[0319] f) a dominant negative TGF-beta mutant sequence operably linked to a modified HSV-2 immediate early promoter, wherein the gene is located in the intergenic region between the UL21 gene and the UL22 gene,

[0320] wherein the oncolytic HSV does not encode functional ICP0 and does not contain a ribozyme sequence located in the 5' untranslated region of VP5.

[0321] 21. An oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises:

[0322] a) a gene of HSV-1 or HSV-2, namely a VP5 gene, operably linked to a VP5 promoter comprising a TATA element, and containing a 5' untranslated region;

[0323] b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' of the TATA element, wherein the VP5 gene is located 3' of the tetracycline operator sequence;

[0324] c) a gene sequence encoding a tetracycline repressor operably linked to a HSV immediate early promoter, wherein the gene sequence is located at the ICP0 locus;

[0325] d) a variant gene that increases syncytia formation compared to wild type, wherein the HSV-1 or HSV-2 variant gene is selected from the group consisting of: a glycoprotein K (gK) variant; a glycoprotein B (gB) variant; a UL24 variant; and a UL20 gene variant;

[0326] e) a gene sequence encoding a functional ICP34.5 protein; and

[0327] f) a dominant negative TGF-beta mutant sequence operably linked to a modified HSV-2 immediate early promoter, wherein the gene is located in the intergenic region of the UL21 gene, the UL22 gene, the UL26 gene, and the UL27 gene,

[0328] wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence located in the 5' untranslated region of VP5.

[0329] 22. The oncolytic HSV of any preceding paragraph, wherein the dominant negative TGF-beta mutant sequence is a mmTGF-beta2-7M fragment sequence.

[0330] 23. The oncolytic HSV of any preceding paragraph, wherein the HSV-2 immediate early promoter of (f) is selected from the group consisting of ICP0, ICP4, and ICP27.

[0331] 24. The oncolytic HSV of any preceding paragraph, wherein the HSV-2 immediate early promoter of (f) contains a tet operon.

[0332] 25. The oncolytic HSV of any preceding paragraph, wherein the HSV is a regulatable tetracycline or doxycycline.

[0333] 26. An oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA does not encode a functional ICP0 gene or an ICP34.5 gene; and encodes a functional mmTGF-beta2-7M fragment sequence.

[0334] 27. An oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA does not encode a functional ICP0; and encodes a functional mmTGF-beta2-7M fragment sequence.

[0335] 28. The oncolytic HSV of any preceding paragraph, wherein the HSV further encodes a fusion promoting activity.

[0336] 29. The oncolytic HSV of any preceding paragraph, wherein the HSV is a regulatable tetracycline or doxycycline.

[0337] 30. An oncolytic virus encoding a functional mmTGF-β2-7M fragment sequence.

[0338] 31. A recombinant virus encoding a functional mmTGF-β2-7M fragment sequence.

[0339] 32. A composition comprising the virus of any preceding paragraph.

[0340] 33. The composition of any preceding paragraph, further comprising a pharmaceutically acceptable carrier.

[0341] 34. A cell expressing any of the viruses of any preceding paragraph or the composition of claims 32-33.

[0342] 35. The cell of any preceding paragraph, wherein the cell is a mammalian cell.

[0343] 36. The cell of any preceding paragraph, wherein the cell is a cancer cell or an immune cell.

[0344] 37. The cell of any preceding paragraph, wherein the immune cell is a B cell or a T cell.

[0345] 38. The cell of any preceding paragraph, wherein the cell expresses high levels of mmTGF-β2-7M.

[0346] 39. A method for treating cancer, the method comprising administering to a subject having cancer the virus of any preceding paragraph or the composition of any preceding paragraph.

[0347] 40. The method of any preceding paragraph, wherein the cancer is a solid tumor.

[0348] 41. The method of any preceding paragraph, wherein the tumor is benign or malignant.

[0349] 42. The method of any preceding paragraph, wherein the subject is diagnosed with or has been diagnosed with a cancer selected from the group consisting of carcinoma, melanoma, sarcoma, germ cell tumor, and blastoma.

[0350] 43. The method of any preceding paragraph, wherein the subject is diagnosed with or has been diagnosed with a cancer selected from the group consisting of non-small cell lung cancer, bladder cancer, breast cancer, brain cancer, colon cancer, prostate cancer, liver cancer, lung cancer, ovarian cancer, skin cancer, head and neck cancer, kidney cancer, and pancreatic cancer.

[0351] 44. The method of any preceding paragraph, wherein the cancer is metastatic.

[0352] 45. The method of any preceding paragraph, further comprising administering an agent that modulates the promoter of the tet operon-containing.

[0353] 46. The method of any preceding paragraph, wherein the agent is doxycycline or tetracycline.

[0354] 47. The method of any preceding paragraph, wherein the agent is administered locally or systemically.

[0355] 48. The method of any preceding paragraph, wherein the systemic administration is oral administration.

[0356] 49. The method of any preceding paragraph, wherein the virus or composition is administered directly to the tumor.

[0357] 50. A hybrid nucleic acid sequence comprising a therapeutic antibody and a sequence of mmTGF-β2-7M, wherein the mmTGF-β2-7M is fused to the Fc domain of the therapeutic antibody.

[0358] 51. The hybrid nucleic acid sequence of any preceding paragraph, wherein the therapeutic antibody sequence is a sequence of an immunotherapeutic antibody.

[0359] 52. The hybrid nucleic acid sequence of any preceding paragraph, wherein the therapeutic antibody sequence is a sequence selected from the group consisting of an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-Tim3 antibody, an anti-CTLA4 antibody, and an anti-TDM-1 antibody, and an anti-TIGIT antibody.

[0360] 53. A polypeptide encoded by the hybrid nucleic acid of any preceding paragraph.

[0361] 54. A vector expressing any of the hybrid nucleic acids of any preceding paragraph or any of the polypeptides of any preceding paragraph.

[0362] 55. A chimeric antigen receptor (CAR) polypeptide comprising at least one of:

[0363] a. an extracellular domain comprising a dominant negative TGF-β mutant sequence;

[0364] b. a transmembrane domain;

[0365] c. a costimulatory domain; and

[0366] d. an intracellular signaling domain.

[0367] 56. The CAR polypeptide of any preceding paragraph, wherein the dominant negative TGF-β mutant sequence is a mmTGF-β2-7M fragment sequence.

[0368] 57. A nucleic acid encoding the CAR polypeptide of any preceding paragraph.

[0369] 58. A mammalian cell comprising:

[0370] a. the CAR polypeptide of any preceding paragraph; or

[0371] b. the encoding nucleic acid of any preceding paragraph.

[0372] 59. The cell of any preceding paragraph, wherein the cell is a T cell.

[0373] 60. The cell of any preceding paragraph, wherein the cell is a human cell.

[0374] 61. The cell of any preceding paragraph, further comprising at least a second CAR polypeptide.

[0375] 62. The cell of any preceding paragraph, wherein the at least a second CAR polypeptide comprises an extracellular domain comprising a sequence that binds a checkpoint inhibitor.

[0376] 63. The cell of any preceding paragraph, wherein the checkpoint inhibitor is selected from the group consisting of: PD-L1, PD-1, TIGIT, TIM3, and CTLA4.

[0377] 64. The cell of any preceding paragraph, wherein the cell is obtained from an individual having or diagnosed with a cancer.

[0378] 65. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject the cell of any one of any preceding paragraph.

[0379] 66. A method of treating a cancer in a subject in need thereof, the method comprising:

[0380] a. engineering a T cell to comprise the CAR polypeptide of any preceding paragraph or the encoding nucleic acid of any preceding paragraph on the surface of the T cell; and

[0381] b. administering the engineered T cell to the subject.

[0382] 67. The method of any preceding paragraph, wherein the engineered T cell further comprises at least a second CAR polypeptide.

[0383] 68. The method of any preceding paragraph, further comprising administering at least one additional anti-cancer therapeutic.

[0384] 69. An oncolytic herpes simplex virus (HSV) comprising a recombinant DNA, wherein the recombinant DNA does not encode functional ICP0 gene and ICP34.5 gene; and encodes functional mmTGF-β2-7M fragment sequence.

[0385] Example 1

[0386] INTRODUCTION

[0387] Immune checkpoint blockade (ICB) represents an exciting new paradigm for the treatment of various cancers. However, the response rate to ICB is typically 10-35% (Bellmunt et al., 2017; Powles et al., 2018; Zou et al., 2016), leaving a greater proportion of patients unresponsive to ICB therapy. Immunosuppressive tumor microenvironment is one of the main obstacles that significantly limits the effectiveness of ICB in cancer immunotherapy. TGF-β plays a key role in promoting and maintaining the immunosuppressive state of the tumor microenvironment (Bollard et al., 2002; Gorelik and Flavell, 2002; Loffek, 2018; Massague, 2008; Wrzesinski et al., 2007; Zhang et al., 2016). Overexpression of TGF-β has been detected in multiple human cancer types and is associated with poor prognosis (Calon et al., 2015; Dong and Blobe, 2006; Haque and Morris, 2017; Lin and Zhao, 2015; Mariathasan et al., 2018; Massague, 2008; Wikstrom et al., 1998; Wrzesinski et al. 2007).

[0388] Studies have revealed that TGF-β inhibits Th1 responses and CD8+ T cell activity, while promoting CD4 + CD25 +T-reg cell function (Chen et al., 2005; Fantini et al., 2004; Loffek, 2018; Mariathasan et al., 2018; Tauriello et al., 2018; Verrecchia and Redini, 2018). In addition, TGF-b inhibits dendritic cell maturation and antigen presentation, as well as anti-tumor activity of NK cells, Ml macrophages and Nl neutrophils (Fridlender et al., 2009; Gong et al., 2012; Kneta et al., 2017; Loffek, 2018; Luo et al., 2006; Verrecchia and Redini, 2018; Zhang et al., 2016; Zheng et al., 2017). Mariathasan et al. have recently shown that TGF-b attenuates tumor responses to PD-L1 immune blockade by preventing T cell infiltration, while blockade of TGF-b signaling in the tumor microenvironment leads to a strong enhancement of anti-tumor T cell responses and tumor regression (Mariathasan et al., 2018). Thus, inhibition of TGF-b signaling in the tumor microenvironment has gained significant interest in cancer immunotherapy (Bendle et al., 2013; Biswas et al., 2007; de Gramont et al., 2017; Haque and Morris, 2017; Hutzen et al., 2017; Knudson et al., 2018; Loffek, 2018; Muraoka et al., 2002; Strauss et al., 2018) (Ahn Myung-ju, Barlesi F et al., 2019 - J Clinical Oncology - Abstract; Strauss J et al. and Gulley J, 2019 - Cancer Res).

[0389] Over the years, various molecules have been developed that are capable of blocking TGF-β signaling, including small molecules, peptides, and soluble forms of the TGF-β type II receptor (TβRII) and anti-TGF-β1 antibodies (Biswas et al., 2007; Bottinger et al., 1997; de Gramont et al., 2017; Gil-Guerrero et al., 2008; Haque and Morris, 2017; Muraoka et al., 2002; Qin et al., 2016; Rowland-Goldsmith et al., 2001; Tian et al., 2015; Tojo et al., 2005). Recently, Kim et al. developed a new dominant-negative monomeric form of TGF-β polypeptide, mmTGF-β2-7M, which exhibits high affinity for the TGF-β type II receptor (TβRII) but is unable to bind to the TGF-β type I receptor (TβRI) (Kim et al., 2017). Furthermore, mmTGF-β2-7M produced and purified from E. Coli is highly effective in blocking TGF-β1, TGF-β2, and TGF-β3 signaling in TGF-β reporter cell lines (Kim et al., 2017). To date, no reports have described the expression of mmTGF-β2-7M in mammalian cells. Therefore, there remains a need to determine whether mmTGF-β2-7M expressed from mammalian cells can serve as an effective dominant-negative mutant capable of blocking TGF-β signaling.

[0390] QREO5-F is a second-generation fusion-promoting tetracycline-regulated oncolytic HSV-1 recombinant virus recently developed by the inventors of the present application. Infection of multiple human cancer cell types with QREO5-F results in 35,000- to 5 x 10 7 fold tetracycline-dependent progeny virus production, whereas little virus replication and virus-associated cytotoxicity is observed in infected growing and growth-arrested normal human fibroblasts. QREO5-F is highly effective in pre-established Hep1-6 liver cancer and CT26.WT colon cancer tumors in immunocompetent mice. Importantly, QREO5-F virus therapy can induce potent tumor-specific immunity that can prevent tumor growth upon re-challenge of tumor-free mice with the same type of tumor cells. Given the critical role of TGF-β signaling in tumor biology and its potential immunosuppressive activity, it is specifically contemplated that the therapeutic efficacy of QREO5-F in cancer immunotherapy can be further enhanced upon re-expression of mmTGF-β2-7M in the local tumor microenvironment.

[0391] Construction and characterization of QREOF-lacZ, a QREO5-F derived recombinant encoding the lacZ gene under the control of the HSV-2 ICP0 immediate early promoter in the intergenic region of the HSV-1 UL26 gene and the UL27 gene.

[0392] Description of plasmids pQUL2627-TO and pQUL2627-lacZ

[0393] pQUL2627-TO contains a synthetic DNA fragment consisting of: 1) HSV-1 DNA sequence consisting of 963 bp upstream of the HSV-1 UL26 poly A signal to 30 bp downstream of the UL26 poly A signal; 2) a DNA sequence containing a modified HSV-2 ICP0 promoter in which the HSV-2 TATA element is changed to HCMV TATATAA followed by two tandem tet operators as described by Yao et al. (Yao et al., 1998), a MCS and a SV40 poly A signal sequence; and 3) HSV-1 DNA sequence consisting of 59 bp downstream of the HSV-1 UL27 poly A signal to 935 bp upstream of the UL27 poly A signal. pQUL2627-v is a pQUL2627-TO derived plasmid without the tet operator sequence. pQUL2627-lacZ is a pQUL2627-v derived plasmid encoding the lacZ gene under the control of the modified HSV-2 ICP0 promoter.

[0394] Construction and characterization of QREOF-lacZ

[0395] QREOF-lacZ is a QREO5-F derived recombinant virus in which a lacZ gene under the control of a modified HSV-2 ICP0 promoter is inserted in the intergenic region of the UL26 gene and the UL27 gene Figure 1 ) of QREO5-F. U2OS cells were co-transfected with pQUL2627-lacZ linearized with Sap I / Xmn I and infectious QREO5-F viral DNA by Lipofectamine 2000-mediated transfection to produce QREOF-lacZ (Akhrameyeva et al., 2011). Viruses expressing lacZ were then selected in the presence of 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) and plaque purified on U2OS cells.

[0396] QREOF-lacZ is a QREO5-F derived recombinant virus of the third round of plaque purification that shows uniform blue fusion-promoting plaques in U2OS cells and in the ICP0 expressing Vero cell line Q0-19 cells. Figure 2The results shown indicate that both the expression of the lacZ gene and the replication of QREOF-lacZ can be strictly regulated in QREOF-lacZ infected cells.

[0397] Construction and characterization of QREO-DNT, a QREOF-lacZ derivative, encoding a dominant negative TGF-β mutant, mmTGF-β2-7M, under the control of a modified HSV-2 ICP4 immediate early promoter at the intergenic region of HSV-1 UL26 and UL27 genes.

[0398] Description of plasmid pQUL2627-TGFDN and in vitro expression of mmTGF-β2-7M determined by transient transfection

[0399] pQUL2627-TGF-DN was constructed by replacing the DNA fragment containing the HSV-2 ICP0 / lacZ gene in pQUL2627-lacZ with a synthetic DNA fragment consisting of the codon-optimized mmTGF-β2-7M with the HSV-1 gD signal peptide under the control of the HSV-2 ICP4 / TO promoter containing tetO. mmTGF-β2-7M consists of 92 amino acids. To assess the expression of mmTGF-β2-7M, U2OS cells were mock-transfected or transfected with pQUL26.27-TGF-DN or pICP6-eGFP, a plasmid encoding eGFP under the control of the HSV-1 ICP6 promoter. Figure 3 The Western blot analysis shown in Figure 6 indicates that although a protein with a MW close to 50 kDa, which represents the full-length TGF-β1 precursor (390 amino acids), is present in both pICP6-EGFP and pQUL26.27-TGF-DN transfected cell extracts, only pQUL26.27-TGF-DN transfected cells produce a protein with a MW between 11-12 kDa, which is strongly recognized by the TGF-β1 specific antibody. The mature form of TGF-β1 was not detected in eGFP transfected cell extracts. However, the mature TGF-β1 was detectable in the extracellular medium collected from both pICP6-eGFP and pQUL26.27-TGF-DN transfected cells.

[0400] When the above transfected cells were examined under phase contrast light and fluorescence microscopy, it was observed that about 35-40% of the cells in the pICP6-eGFP transfected dishes were eGFP positive at 40h post transfection. While the eGFP transfected cells were morphologically similar to mock transfected cells at 28h to 70h post transfection, the U2OS cells transfected with pQUL2627-TGF-DN exhibited flat, significantly enlarged and significantly stressed at 48h and 70h post transfection. In addition, it appeared that the dishes transfected with pQUL2627-TGF-DN contained significantly fewer cells than the pICP6-EGFP transfected dishes. These observations were further confirmed by Figure 4 independent experiments shown in FIG. 6, which demonstrated that the number of cells per dish in the pICP6-eGFP transfected dishes was about 1.6 times higher than the number of cells in the pQUL2627-TGF-DN transfected dishes, indicating that the mmTGF-β2-7M expressed from the transfected cells effectively blocked TGF-β signaling in U2OS cells, consistent with previous studies that TGF-β signaling is essential for the proliferation, migration and invasion of osteosarcoma cells, including U2OS cells (Li et al., 2014; Matsuyama et al., 2003; Verrecchia and Redini, 2018).

[0401] Construction and characterization of QREO-DNT

[0402] QREO-DNT is a recombinant virus derived from QREOF-lacZ in which the lacZ gene under the control of the modified HSV-2 ICP0 promoter is replaced by a DNA fragment encoding a codon-optimized mmTGF-β2-7M under the control of the HSV-2 ICP4 / TO promoter sequence.

[0403] QREO-DNT was generated by co-transfecting U2OS cells with Ndel / BBS I-linearized pQUL2627-TGF-DN and infectious QREOF-lacZ viral DNA using Lipofectamine 2000. Viruses expressing mmTGF-β2-7M were selected and plaque purified on U2OS cells in the presence of X-Gal. Briefly, transfected progeny viruses were screened for recombination replacement of the LacZ gene of QREOF-lacZ with the DNA sequence containing HSV2 ICP4TO / mmTGF-β2-7M by standard plaque assay. Plaques were stained with X-Gal 72 h post-infection. White plaques reflecting replacement of the LacZ gene with the mmTGF-β2-7M DNA coding sequence were isolated. Replacement of the lacZ gene with the mmTGF-β2-7M DNA sequence in the region between the UL26 gene and the UL27 gene was confirmed by PCR analysis using primers specific for the HSV2 ICP4TO promoter sequence and the UL27 flanking sequence. QREO-DNT is a recombinant virus encoding mmTGF-β2-7M that was plaque purified in a second round and showed uniform white plaque fusion promoting plaques in U2OS cells and the ICP0 expressing Vero cell line Q0-19 cells.

[0404] The ability of QREO-DNT to efficiently express a dominant negative form of TGF-β (TGF-DN) was evaluated in U2OS cells at a MOI of 3 PFU / cell in the presence of doxycycline. Figure 5 Western blot analysis shown in Figure 5 demonstrates that while similar levels of ICP27 were detected in QREOF-lacZ- and QREO-DNT infected cells, only QREO-DNT expressed a protein of approximately 11-12 kDa that strongly reacted with the anti-TGF-β1 specific antibody. As expected, the full-length precursor form of TGF-β1 was detectable in mock infected and infected cell extracts. Notably, a very faint protein band of slightly higher MW than mmTGF-β2-7M was detected in the mock infected cell extract that can represent the mature form of TGF-β1 (112 amino acids). Overall, the results shown in Figure 5 indicate that QREO-DNT is capable of expressing high levels of the dominant negative form of TGF-β1, mmTGF-β2-7M. Figure 5

[0405] References

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[0421] Kim, S. K., Barron, L., Hinck, C. S., Petrunak, E. M., Cano, K. E., Thangirala, A., Iskra, B., Brothers, M., Vonberg, M., Leal, B., et al. (2017). An engineered transforming growth factor beta (TGF-beta) monomer that functions as a dominant negative to block TGF-beta signaling. J Biol Chem 292, 7173-7188.

[0422] Knudson, K. M., Hicks, K. C., Luo, X., Chen, J. Q., Schlom, J., and Gameiro, S. R. (2018). M7824, a novel bifunctional anti-PD-L1 / TGFbeta Trap fusion protein, promotes anti-tumor efficacy as monotherapy and in combination with vaccine. Oncoimmunology 7, el426519.

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[0427] Matsuyama, S., Iwadate, M., Kondo, M., Saitoh, M., Hanyu, A., Shimizu, K., Aburatani, H., Mishima, H.K., Imamura, T., Miyazono, K., et al. (2003). SB-431542 and Gleevec inhibit transforming growth factor-beta-induced proliferation of human osteosarcoma cells. Cancer Res 63, 7791-7798.

[0428] Muraoka, R.S., Dumont, N., Ritter, C.A., Dugger, T.C., Brantley, D.M., Chen, J., Easterly, E., Roebuck, L.R., Ryan, S., Gotwals, P.J., et al. (2002). Blockade of TGF-beta inhibits mammary tumor cell viability, migration, and metastases. J Clin Invest 109, 1551-1559. Powles, T., Duran, I., van der Heijden, M.S., Loriot, Y., Vogelzang, N.J., De Giorgi, U., Oudard, S., Retz, M.M., Castellano, D., Bamias, A., et al. (2018). Atezolizumab versus chemotherapy in patients with platinum-treated locally advanced or metastatic urothelial carcinoma (IMvigor211): a multicentre, open-label, phase 3 randomised controlled trial. Lancet 391, 748-757.

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[0440] Example 2

[0441] DNA sequences and amino acid sequences

[0442] Transcription cassette targeting a gene of interest to the HSV-1 UL26 / UL27 locus under the control of a modified HSV-2 ICP0 promoter carrying tetO:

[0443]

[0444]

[0445] DNA sequence consisting of 963 bp upstream of the UL26 polyA signal to 30 bp downstream of the UL26 polyA signal:

[0446]

[0447]

[0448] DNA sequence consisting of 59 bp downstream of the UL27 polyA signal to 935 bp upstream of the UL27 polyA signal:

[0449]

[0450] Modified HSV-2 ICP0 promoter carrying TetO plus a designed mcs followed by a sv40 polyA signal sequence:

[0451]

[0452]

[0453] SV40 polyA signal sequence:

[0454] CAGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTG (SEQ ID NO: 16)

[0455] HSV-1 gD signal peptide codon-optimized mmTGF-β2-7M under the control of the HSV-2 ICP4 / TO promoter sequence:

[0456] ggcgcgccgg gccggcgggg gc aacgggagc gcggggccgg catctcatt accacgaacc cggaagggca ggggagcgag cccgcccgcg agggctcatt agcatcgcgg gcggaagcg gaagccgccc gcgccgggcg ct aatgagatg ccgcgcgggc ggagcggcg gcggcgcga ccaacgggcc gccgccacg gacgcggacg cgcgggcgtc ggggcggggc cgcgcataat gcggttccac ctgggggcgg aaccccggcg agccggggcg cggcggcgtc gatcgctcct cctccgcgtc ctcctccttt cccccccgc c cgcgcgc c c c gaggacTATATGAGCCGAGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATCGAGCTCGCGTGTGCATCGCGTATCACCCAAGCTTgccaccATGGGCGGCGCCGCCGCCCGCCTGGGCGCCGTGATCCTGTTCGTGGTGATCGTGGGCCTGCACGGCGTGCGCGGCGCCCTGGACGCCGCCTACTGCTTCCGCAACGTGCAGGACAACTGCTGCCTGCGCCCCCTGTACATCGACTTCCGCAAGGACCTGGGCTGGAAGTGGATCCACGAGCCCAAGGGCTACAACGCCAACTTCTGCGCCGGCGCCTGCCCCTACCGCGCCAGCAAGAGCCCCAGCTGCGTGAGCCAGGACCTGGAGCCCCTGACCATCGTGTACTACGTGGGCCGCAAGCCCAAGGTGGAGCAGCTGAGCAACATGATCGTGAAGAGCTGCAAGTGCAGCTAAgaattc (SEQ I D NO: 17)

[0457] HSV-2 ICP4 / TO promoter sequence:

[0458] ggcgcgccgg gccggcgggg gcacaacggg agcgcggggc cgcatctcat tacgaacccg gaagggcagg ggagcgagcc cgcccgcgac gagggtctca ttagcatcgc gggcggaagc ggaagccgcc cgccgggcgc taatgagatg ccgcgcgggc ggagcggcgg cggcgaccaa cgggccgccc ccacggacgc ggacgcgcgg gcgtcggggc gggccgcgca taatgcggtt ccacctgggg gcggaacccc ggcgagccgg ggcgcggcgg cgtcgatcgc tcctcctccg cgtcctcctc cttccccccc cccgcgcgcg ccccgaggac TATATGAGC CGAGCTCTC CTATCAGTGA TAGAGATCT CCCTATCAGT GATAGAGAT CGAGCTCGC GTGTGCATC GCGTATCAC CCAAGCTT (SEQ ID NO: 18)

[0459] Because the HSV ICP4 promoter is subject to autorepression by ICP4, the ICP4 binding site in the HSV-2 ICP4 promoter was deleted from the TETO-containing HSV-2 ICP4 / TO promoter described above.

[0460] Codon-optimized mmTGF-β2-7M with an HSV-1 gD signal peptide at the N-terminus:

[0461] gccaccATGGGCGGCGCCGCCGCCCGCCTGGGCGCCGTGATCCTGTTCGTGGTGATCGTGGGCCTGCACGGCGTGCGCGGCGCCCTGGACGCCGCCTACTGCTTCCGCAACGTGCAGGACAACTGCTGCCTGCGCCCCCTGTACATCGACTTCCGCAAGGACCTGGGCTGGAAGTGGATCCACGAGCCCAAGGGCTACAACGCC

[0462] AACTTCTGCGCCGGCGCCTGCCCC

[0463] TACCGCGCCAGCAAGAGCCCCAGCTGCGTGAGCCAGGACCTGGAGCCCCT

[0464] GACCATCGTGTACTACGTGGGCCGCAAGCCCAAGGTGGAGCAGCTGAGCA

[0465] ACATGATCGTGAAGAGCTGCAAGTGCAGC TAA gaattc (SEQ ID NO: 19)

[0466] Codon-optimized gD signal peptide sequence:

[0467] ATGGGCGGCGCCGCCGCCCGCCTGGGCGCCGTGATCCTGTTCGTGGTGATCGTGGGCCTGCACGGCGTGCGCGGC (SEQ ID NO: 20)

[0468] atezolizumab (USAN / INN); atezolizumab (genetic recombinant) (JAN); Tecentriq (TN)

[0469] Heavy chain (448 amino acids):

[0470]

[0471]

[0472] Light chain (214 amino acids):

[0473]

[0474] At ezolizumab heavy chain fused to the C-terminus of the heavy chain consisting of mmTGF-β2-7M with GGGGGGS linker:

[0475]

[0476] The fusion protein can be either without linker or with 2-4 copies of the linker. The linker can also be GGGGS (SEQ ID NO: 27) or GGGGGS (SEQ ID NO: 28) or other linkers commonly used to fuse 2 different functional proteins.

[0477] KEGG DRUG: (e.g., pembrolizumab; pembrolizumab (USAN); pembrolizumab (genetic recombinant) (JAN); Keytruda (TN)

[0478] Heavy chain (447 amino acids)

[0479]

[0480] Light chain (218 amino acids)

[0481]

[0482] Pembrolizumab heavy chain consisting of mmTGF-β2-7M with GGGGGGS linker fused to the C-terminus of the heavy chain:

[0483]

[0484] The fusion protein can be either linkerless or have 2-4 copies of the linker. The linker can also be GGGGS (SEQ ID NO: 27) or GGGGGS (SEQ ID NO: 28).

Claims

1. An oncolytic herpes simplex virus (HSV) comprising recombinant DNA, wherein the recombinant DNA comprises: a) a 5' untranslated region and a gene of HSV-1 or HSV-2, a VP5 gene, operably linked to a VP5 promoter comprising a TATA element; b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' of the TATA element, wherein the VP5 gene is located 3' of the tetracycline operator sequence; c) a gene sequence encoding a tetracycline repressor operably linked to an HSV immediate early promoter, wherein the gene sequence is located at the ICP0 locus; d) a variant gene that increases syncytia formation compared to wild type, wherein the variant gene is a glycoprotein K (gK) variant; e) a gene sequence encoding a functional ICP34.5 protein; and f) a dominant negative TGF-β mutant gene sequence operably linked to an HSV promoter, wherein the gene is located in the intergenic region between the UL26 gene and the UL27 gene, wherein the dominant negative TGF-β mutant gene sequence is a mmTGF-β2-7M fragment sequence, wherein the oncolytic HSV does not encode a functional ICP0 and does not contain a ribozyme sequence located in the 5' untranslated region of VP5.

2. The oncolytic HSV of claim 1, wherein the HSV promoter of f) is an HSV immediate early promoter.

3. The oncolytic HSV of claim 1, wherein the gK variant gene encodes an amino acid substitution of Ala to Thr corresponding to amino acid 40 of SEQ ID NO:

2.

4. The oncolytic HSV of claim 1, wherein the tetracycline operator sequence comprises two Op2 repressor binding sites.

5. The oncolytic HSV of claim 1, wherein the VP5 promoter is a VP5 promoter of HSV-1 or HSV-2.

6. The oncolytic HSV of claim 2, wherein the HSV immediate early promoter is an immediate early promoter of HSV-1 or HSV-2.

7. The oncolytic HSV of claim 2, wherein the HSV immediate early promoter is selected from the group consisting of: an ICP0 promoter, an ICP4 promoter, and an ICP27 promoter.

8. The oncolytic HSV of claim 1, wherein the recombinant DNA is a portion of an HSV-1 genome.

9. The oncolytic HSV of claim 1, wherein the recombinant DNA is a portion of an HSV-2 genome.

10. The oncolytic HSV of claim 1, in combination with a pharmaceutically acceptable carrier.

11. The oncolytic HSV of claim 1, further encoding at least one polypeptide capable of increasing the efficacy of the oncolytic HSV in inducing an anti-tumor specific immunity. ​ ​ ​ ​ ​ ​ ​ ​ 12. The oncolytic HSV of claim 11, wherein the at least one polypeptide encodes a product selected from the group consisting of interleukin 2 (IL2), interleukin 12 (IL12), interleukin 15 (IL15), an anti-PD-1 antibody or antibody reagent, an anti-PD-L1 antibody or antibody reagent, an anti-OX40 antibody or antibody reagent, a CTLA-4 antibody or antibody reagent, a TIM-3 antibody or antibody reagent, a TIGIT antibody or antibody reagent, a soluble interleukin 10 receptor (IL10R), a fusion polypeptide between a soluble IL10R and an IgG-Fc domain, a soluble TGFp type II receptor (TGFBRII), a fusion polypeptide between a soluble TGFBRII and an IgG-Fc domain, an anti-IL10R antibody or antibody reagent, an anti-IL10 antibody or antibody reagent, and an anti-TGFBRII antibody or antibody reagent.

13. A composition comprising the virus of any one of claims 1-12.

14. The composition of claim 13, further comprising a pharmaceutically acceptable carrier.

15. A cell expressing any one of the viruses of any one of claims 1-12 or the composition of claim 13 or 14, wherein the cell is a cancer cell or an immune cell.

16. The cell of claim 15, wherein the cell is a mammalian cell.

17. The cell of claim 15, wherein the immune cell is a B cell or a T cell.

18. The cell of any one of claims 15-17, wherein the cell expresses a high level of mmTGF-β2-7M.

19. Use of the virus of any one of claims 1-12 or the composition of any one of claims 13-14 in the manufacture of a medicament for treating cancer in a subject having cancer.

20. The use of claim 19, wherein the cancer is a solid tumor.

21. The use of any one of claims 19-20, wherein the subject is diagnosed with a cancer selected from the group consisting of melanoma, sarcoma, germ cell tumor, and blastoma.

22. The use of any one of claims 19-20, wherein the subject is diagnosed with a cancer selected from the group consisting of bladder cancer, breast cancer, brain cancer, colon cancer, prostate cancer, liver cancer, lung cancer, ovarian cancer, skin cancer, head and neck cancer, kidney cancer, and pancreatic cancer.

23. The use of any one of claims 19-20, wherein the cancer is metastatic.

24. The use of any one of claims 19-20, wherein the medicament further comprises an agent that modulates a promoter containing a tet operon.

25. The use of claim 24, wherein the agent is doxycycline or tetracycline.

26. The use of claim 24, wherein the agent is administered locally or systemically.

27. The use of claim 26, wherein the systemic administration is oral administration.

28. The use of any one of claims 19-20, wherein the virus or composition is administered directly to a tumor.

29. The use of claim 22, wherein the lung cancer is non-small cell lung cancer.

30. The use of claim 19, wherein the subject is diagnosed with a cancer.

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