Recombinant herpes simplex virus for cancer immunotherapy

Abstract

The present invention provides a method for treating a subject suffering from cancer by administering to the subject a therapeutically effective amount of a recombinant herpes simplex virus-1 (HSV-1), wherein the recombinant herpes simplex virus-1 expresses only the C-terminal portion of the γ134.5 protein (e.g., amino acid residues 147 to 263) without wild-type or complete γ134.5 protein expression. The recombinant HSV-1 of the present invention induces immune activation, selectively replicates in cancer cells, and resists clearance by interferon.

Description

[0001] introduction

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 62 / 682,202, filed on June 8, 2018, the contents of which are incorporated herein by reference in their entirety.

[0003] This invention was made with government support under Grant No. AI 112755 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. Background Art

[0004] Oncolytic herpes simplex virus 1 (HSV-1) is an attractive agent for cancer immunotherapy (Peters and Rabkin (2015) Mol. Ther. Oncolytics 2: 15010; Chiocca and Rabkin (2014) Cancer Immunol. Res. 2: 295-300). Once infected, HSV-1 undergoes sequential gene expression, DNA replication, assembly and release, leading to the destruction of tumor cells. This is accompanied by the release of danger signals and new antigens that activate acquired anti-tumor immunity. A series of oncolytic HSVs are in different stages of research and development (Peters and Rabkin (2015) Mol. Ther. Oncolytics 2: 15010). The most advanced agent in the clinic is talimogenelaherparepvec (T-VEC) approved by the FDA for the treatment of advanced melanoma (Andtbacka et al., (2015) J. Clin. Oncol. 33: 2780-88). Other examples of oncolytic HSV that have undergone clinical trials or are in clinical trials are G207, 1716 and ΔG47 (Markert et al., (2000) Gene Ther. 7:867-74; Rampling et al., (2000) Gene Ther. 357:525-6; Streby et al., (2017) Clin. Cancer Res. 23:3566-74; Fukuhara et al., (2016) Cancer Sci. 107:1373-79). Although there are differences in backbone design, these oncolytic HSV viruses have initially deleted the γ134.5 gene encoding virulence factors.

[0005] HSVγ134.5 contains a large amino-terminal domain (aa 1-146) and a carboxyl-terminal domain. In infected cells, HSV-1 activates double-stranded RNA-dependent kinase (PKR), which stops protein synthesis by phosphorylating translation initiation factor 2α (eIF-2α). Therefore, the γ134.5 protein redirects protein phosphatase 1 (PP1) to dephosphorylate eIF-2α. Notably, site-specific disruption of the γ134.5-PP interaction eliminates viral virulence. HSVγ134.5 has also been reported to affect glycoprotein processing and viral transmission. In addition, evidence suggests that the γ134.5 protein has other functions. These functions include inhibition of autophagy, induction of IFN through TANK-binding kinase 1, and accelerated dendritic cell maturation through Toll-like receptors and nuclear release. Although the γ134.5 protein shuttles between the nucleus and the cytoplasm, its precise interaction with host cells remains unclear.

[0006] Several lines of evidence demonstrate that HSV-1 mutants lacking the γ134.5 gene exhibit antitumor activity. This has been shown in immunodeficient as well as immunocompetent preclinical models for tumors including glioma, colon cancer, ovarian cancer, breast cancer, liver cancer, and melanoma (Mineta et al., (1995) Nat. Med. 1:938-43; Toda et al. (1998) Hum. Gene Ther. 9:2177-85; Chambers et al., (1995) Proc. Natl. Acad. Sci. USA 92:1411-15; Randazzo et al., (1995) Virology 211:94-101; Thomas and Fraser (2003) Mol. Ther. 8:543-51; Coukos et al., (2000) Clin. Cancer Res. 6:3342-53; Braidwood et al., (2014) J. Hepatocell. Carcinoma 1:149-61; Wang et al., (2016) Gene Ther. 23:135-43; WO 2017 / 013419 A1; US ​​2017 / 0319638 A1; US ​​7223593). However, the differences in treatment outcomes are very large. Although the underlying events are complex, the nature of the virus-host interaction seems to be decisive. It has been shown that activation of mitogen-activated protein kinase or RAS proto-oncogenes in tumor cells inhibits PKR and thereby allows viral replication. On the other hand, it has been reported that genetic or epigenetic inhibition of the stimulating interferon gene (STING) permits the γ134.5 null mutant to destroy tumors because it mediates type I IFN production. Therefore, active PKR, STING or IFN production within tumor cells is believed to reduce the efficacy of oncolytic HSV lacking the γ134.5 gene.

[0007] The main limitation of using attenuated replication-competent viruses to directly destroy tumors has always been the reduced growth in a variety of cell types including cancer cells. Despite the initial oncolytic wave, host defense limits the successful replication of viral vectors for a long enough period of time to eradicate the entire tumor cell population. In addition, the oncolytic virus backbone with improved replication often impairs the natural immune sensitization necessary for anti-tumor immunity. Therefore, surviving cancer cells proliferate or re-establish their control over patients. Therefore, it is necessary to lyse cancer cells and effectively activate viral anti-tumor agents that effectively activate systemic anti-tumor responses. Summary of the invention

[0008] The present invention provides a method for treating a subject suffering from cancer by administering to the subject a therapeutically effective amount of a recombinant herpes simplex virus-1 (HSV-1), wherein the recombinant herpes simplex virus-1 expresses only the C-terminal portion of the γ134.5 protein, such as a protein consisting of SEQ ID NO: 2, without expression of wild-type or complete γ134.5 protein, thereby treating the cancer in the subject. In some embodiments, the recombinant HSV-1 also comprises a deletion of one or more non-essential genes or fragments thereof, such as UL2, UL3, UL4, UL9.5, UL10, UL11, ULI2, UL13, ULI4, UL20, UL21, UL23, UL24, UL39, UL40, UL41, UL43, UL43.5, UL44, UL45, UL46, UL47, UL50, UL51, UL53, UL55, Us1, Us1.5, Us2, Us3, Us4, Us5, Us7, Us8, Us8.5, Us9, Us10, Us11, Us12 and ICP0. In other embodiments, the recombinant HSV-1 further comprises replacing one or more non-essential genes with one or more genes expressing therapeutic proteins (e.g., interferon α (IFN-α), interleukin-2 (IL-2), and granulocyte colony stimulating factor (G-CSF)), enzymes, antibodies (e.g., anti-programmed cell death protein 1 antibody (anti-PD1 antibody), anti-checkpoint T lymphocyte-associated protein 4 antibody (anti-CTLA4 antibody), anti-OX40 (anti-CD134 antibody) antibody, and anti-CD40 antibody) or nucleic acids for cancer therapy, wherein the non-essential genes are The gene is selected from UL2, UL3, UL4, UL9.5, UL10, UL11, ULI2, UL13, ULI4, UL20, UL21, UL23, UL24, UL39, UL40, UL41, UL43, UL43.5, UL44, UL45, UL46, UL47, UL50, UL51, UL53, UL55, Us1, Us1.5, Us2, Us3, Us4, Us5, Us7, Us8, Us8.5, Us9, Us10, Us11, Us12 and ICPO. In other embodiments, the cancer is a solid tumor and is optionally selected from breast cancer, lung cancer, liver cancer, skin cancer (melanoma), brain cancer and colon cancer. In addition to administering the recombinant HSV-1, the method can also include administering an effective amount of a second therapeutic agent useful for cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1Data demonstrating that ΔN146 reduces local tumor growth are provided. 4T1 cells were implanted subcutaneously into mice (day -7). On the 1st, 3rd and 6th days, the tumors formed were injected with PBS, Δγ134.5, ΔN146 or EUs11 suspended in PBS. Tumor size was measured periodically (x-axis) until the 24th day (n=6 per group). The average tumor volume over time is shown on the y-axis. The asterisk indicates the statistical significance obtained by nonparametric analysis. The results shown are from one of three independent experiments. The differences between the selected groups were statistically evaluated by a two-tailed Student t test (**, P<0.01).

[0010] Figure 2 Data demonstrating that ΔN146 reduces metastasis are provided. 4T1 cells were implanted subcutaneously into mice (day -7). On the 1st, 3rd and 6th days, the tumors formed were injected with PBS, Δγ134.5, ΔN146 or EUs11 suspended in PBS. Mice were killed on the 24th day after the start of treatment and the liver was collected and fixed in formalin. The number of liver metastases was quantitatively counted under an optical microscope. The results shown are from one of three independent experiments. The differences between the selected groups were statistically evaluated by a two-tailed Student t test (*, P<0.05; **, P<0.01).

[0011] Figure 3 Data demonstrating viral growth within 4T1 tumors are provided. Tumors treated with PBS, Δγ134.5, ΔN146, or EUs11 suspended in PBS were collected on day 9, and the infectious virus present in the tumor was quantified by plaque assay (n=6). The results shown are from three experiments performed with three replicates. The differences between the selected groups were statistically evaluated by a two-tailed Student t test (**, P<0.01).

[0012] Figure 4 In vitro comparative analysis of ΔN146 and EUs1 is shown. The effect of the virus on the expression of IFN-α1 and Cxc19 was analyzed. 4T1 cells were mock infected or infected with ΔN146 or EUs11 (5 pfu / cell). RNA samples were analyzed by quantitative polymerase chain reaction 6 hours after infection. Data represent three experiments and standard deviations of three replicate samples. DETAILED DESCRIPTION

[0013] The best intracellular environment for viral replication develops as the virus attaches to the cell membrane and begins to occur. Herpes simplex virus is followed by a series of events related to intracellular biochemistry, physiology and morphological changes after binding to the cell membrane receptor. After infection in sensitive cells, lytic replication is subject to the regulation and control of a series of coordinated gene transcriptions in time. Virus activates immediate early (IE or α) genes (ICP0, ICP4, ICP22, ICP27 and ICP47) to the binding of host cell membrane, which are transactivators, allowing the production of the next group of genes to be transcribed, i.e. early (β) genes. The expression of the immediate early gene product is followed by the expression of the protein encoded by the early gene and then the late (γ) gene. The cascade of whole gene activation and viral replication will spend about 18-24 hours in wild-type virus and always cause cell death. The recombinant HSV mutant of the present invention bypasses the protein synthesis arrest phenotype of the γ134.5 null virus and activates STING (interferon-stimulated gene) that mediates anti-tumor immunity, creating a more robust HSV mutant with targeted γ134.5 deletion.

[0014] It has now been found that recombinant HSV-1 expressing the C-terminal half of γ134.5 (ΔN146) replicates robustly and lyses malignant cells that are refractory to the γ134.5 null mutant (Δγ134.5). In infected cells, ΔN146, but not Δγ134.5, prevents phosphorylation of the translation initiation factor eIF-2α, ensuring the synthesis of viral proteins. Notably, ΔN146 also activates interferon regulatory factor 3 and IFN responses because it removes the γ134.5 inhibitory domain of STING, an immune factor known to elicit antitumor immunity. However, unlike Δγ134.5, ΔN146 is competent to replicate when exposed to IFN-α / β. This is attributed to the activity associated with the C-terminal half of γ134.5. Although EUs11 is competent to replicate, it inactivates the interferon regulatory factor IRF3. Therefore, its replication comes at the expense of immunosuppression. In the mouse 4T1 tumor model, ΔN146 reduced tumor growth and metastasis more effectively than Δγ134.5. Although similar in the reduction of tumor growth, ΔN146 reduced metastasis more effectively than EUs11. This is consistent with viral replication, IFN induction, and T cell infiltration in local tumors. ΔN146 is undetectable in normal tissues and is non-toxic in vivo. Therefore, selective editing of HSV-1 changes the virus-cell interaction, which leads to a unique anti-tumor platform, namely tumor selectivity, immune activation, and resistance to IFN clearance. Therefore, the present invention is a recombinant HSV-1 virus that expresses only half of the C-terminal of the γ134.5 protein without wild-type or complete γ134.5 protein expression and its use in cancer treatment.

[0015] As known in the art, γ134.5 is an HSV protein that promotes viral replication in peripheral tissues and invasion of the peripheral nervous system in experimental models (Whitley et al., (1993) J. Clin. Invest. 91: 2837-43; Perng et al., (1996) J. Virol. 70: 2883-93; Mao and Rosenthal (2003) J. Virol. 77: 3409-3417). In addition, it promotes HSV infection and replication in the central nervous system (Chou et al., (1990) Science 250: 1262-66; MacLean et al., (1991) J. Gen. Virol. 72: 631-39). HSVγ134.5 is known to contain a large amino-terminal domain (aa 1-146) and a carboxyl-terminal domain (aa 147-263) that binds to protein phosphatase 1α (He et al., (1998) J. Biol. Chem. 273: 20737-43). The nucleotide and amino acid sequences of wild-type γ134.5 can be obtained from: GENBANK accession number NC_001806.1, which provides the complete genome of HSV-1 strain 17; GENBANK accession number GU734771.1, which provides the complete genome of HSV-1 strain F; and GENBANK accession number GU734772.1, which provides the complete genome of HSV-1 strain H129. For illustrative purposes, wild-type or complete γ134.5 has the following amino acid sequence:

[0016] MARRRRHRGPRRPPGPTGAVPTAQSQVTSTPNSEPAVRSAPAAAPPPPPASGPPPSCSLLLRQWLHVPESASDDDDDDDWPDSPPPEPAPEARPTAAAPRPRSPPPGAGPGGGANPSHPPSRPF RLPPRLALRLRVTAEHLARLRLRRAGGEGAPEPPATPATPATPATPATPARVRFSPHVRVRHLVVWASAARLARRGSWARERADRARFRRRVAEAEAVIGPCLGPEARARALARGAGPANSV(SEQ ID NO:1).

[0017] As used herein, "recombinant HSV-1" refers to an engineered or modified human herpes simplex virus 1 that expresses only the C-terminal portion or half of the γ134.5 protein without the expression of the wild-type or complete γ134.5 protein. As used herein, the C-terminal portion or half of the γ134.5 protein refers to the following amino acid sequence of the γ134.5 protein or a variant thereof that retains or enhances anti-tumor activity:

[0018] RLRRAGGEGAPEPPATPATPATPATPATPARVRFSPHVRVRHLVVWASAARLARRGSWARERADRARFRRRVAEAEAVIGPCLGPEARARALARGAGPANSV (SEQ ID NO: 2).

[0019] In some aspects of the recombinant HSV-1 of the present invention, the endogenous γ134.5 gene has been modified so that both copies of the γ134.5 gene express only the C-terminal portion of the γ134.5 protein. In other aspects of the recombinant HSV-1 of the present invention, both copies of the endogenous γ134.5 gene are deleted and the nucleic acid encoding the C-terminal portion of the γ134.5 protein is inserted into one or more separate locations of the HSV-1 genome, such as in a non-essential gene. In this aspect, the HSV-1 genome has been modified so that the wild-type γ34.5 is non-functional, but the recombinant HSV-1 is still able to infect, replicate and lyse mammalian tumor cells.

[0020] Expression of the C-terminal portion of the γ134.5 protein can be driven by the γ134.5 protein promoter, another endogenous HSV-1 promoter, or a heterologous or exogenous promoter of viral or cellular origin. Exemplary promoters for use in the present invention include, but are not limited to, the immediate early promoters of herpes simplex virus α27, α4, α0, α22, and α47; from ICP8 (or U L 29), thymidine kinase (tk or U L 23) ICP6(U L 39) or the herpes simplex virus early promoter of any DNA replication gene; or a late promoter such as the Us11 promoter.

[0021] In certain embodiments, the recombinant HSV-1 further comprises a deletion of one or more non-essential genes of HSV-1. Non-essential genes are to be distinguished from essential genes in the absence of which the virus will not replicate. Non-essential genes may be beneficial genes, in which case replacement of such beneficial genes results in a virus that replicates at a much lower rate than the wild-type virus. Representative non-essential genes of HSV-1 include, but are not limited to, UL2, UL3, UL4, UL9.5, UL10, UL11, UL12, UL13, ULI4, UL20, UL21, UL23, UL24, UL39, UL40, UL41, UL43, UL43.5, UL44, UL45, UL46, UL47, UL50, UL51, UL53 and UL55 in the UL region; Us1, Us1.5, Us2, Us3, Us4, Us5, Us7, Us8, Us8.5, Us9, Us10, Us11 and Us12 in the Us region; and ICP0 in the inverted repeat region.

[0022] In an optional embodiment, one or more non-essential genes are replaced with one or more nucleic acids encoding and capable of expressing therapeutic proteins, enzymes, antibodies, nucleic acids (e.g., nucleic acids encoding the proteins, enzymes, antibodies or microRNAs, ribozymes, etc.) for cancer therapy. Therapeutic proteins refer to functional proteins (i.e., those other than enzymes or antibodies) that have therapeutic benefits in cancer treatment. Examples of suitable therapeutic proteins include, but are not limited to: rsCD40L (Eliopoulos et al., (2000) Mol. Cell. Biol. 20:5503-5515); Fas ligand (Sharma et al., (2000) Pharmacol. Ther. 88:333-347); TRAIL (Golstein (1997) Curr. Biol. 7:R750-753); TNF (Baker and Reddy (1996) Onc ogene 12:1-9; Theys et al., (1999) Appl. Environ. Microbiol. 65:4295-4300; Lammertyn et al., (1997) Appl. Environ. Microbiol. 63:1808-1813); GM-CSF for the treatment of melanoma, breast cancer, colorectal cancer, glioblastoma, neuroblastoma and prostate cancer (see, e.g., Eubank et al., (2009) Cancer Res. 69(5):2133-40); IFNα for the treatment of ovarian cancer and solid tumors (see, e.g., Goto et al., (1996) Br. J. Cancer 74:546–54); IL-2 for the treatment of neuroblastoma and ovarian cancer (see, e.g., Minor et al., (2017) Gynecol. Oncol. Rep. 22:43-44); and G-CSF for the treatment of breast cancer, bladder cancer, and ovarian cancer (see, e.g., Omura et al., (1996) Proc. Annu. Meet Am. Soc. Clin. Oncol. 15:A755).

[0023] Therapeutic enzymes refer to functional enzymes that have therapeutic benefits in cancer treatment. Special-purpose therapeutic enzymes include enzymes that can convert non-toxic prodrugs into toxic drugs that are cytotoxic to tumors. Examples of suitable therapeutic enzyme-prodrug pairs include, but are not limited to, herpes simplex virus thymidine kinase (HSV-TK) + ganciclovir (GCV) (Moolten (1986) Cancer Res. 46: 5276-5281); HSV-TK + A-5021 (1'S, 2'R) -9 {[1', 2'-bis (hydroxymethyl) cyclopropyl-1'-yl] methyl} guanine (Hasegawa et al., (2000) Cancer Gene Ther. 7: 557-562); horseradish peroxidase (HRP) + indole-3-acetic acid (IAA) (Greco et al., (2000) Cancer Gene Ther. 7: 1414-1420); bacterial enzyme carboxypeptidase G2 (CPG2) + 4-([2-chloroethyl][2-methylsulfonyloxyethyl]amino)benzoyl-L-glutamate (CMDA) or + 4-[N,N-bis(2-iodoethyl)amino]phenoxycarbonyl-L-glutamate (ZD2767P) (Spooner et al., (2000) Cancer Gene Ther. 7: 1348-1356; Webley et al., (2001) Br. J. Cancer 84: 1671-1676); human cytochrome P450 CYPIA2 + acetaminophen (Thatcher et al., (2000) Cancer Gene Ther. 7: 521-525); rabbit cytochrome P450 4Bl (CYP4Bl) + 4-sweet potato starch (4-IM) (Mohr et al., (2000) Cancer Gene Ther.7:1008-1014; Heuser et al., (2000) Cancer Gene Ther.7:806-12); rabbit cytochrome P450 4Bl (CYP2Bl) + oxaphosporines such as ifosfamide (IFO) (Kammertoens et al., (2000) Cancer Gene Ther.7:629-636); Escherichia coli nitroreductase (NTR) + CB1954 (Djeha et al., (2000) Cancer Gene Ther.7:721-731; Djeha et al., (2001) Mol. Ther.3:233-240); Escherichia coli cytosine deaminase (CD), Escherichia coli uracil phosphoribosyltransferase (UPRT) + 5-fluorocytosine (5-FC) (Kammertoens et al., (2000) Cancer Gene Ther.7:629-636; Block et al., (2000) Cancer Gene Ther.7:438-445; Bentires-Alj et al., (2000) Cancer Gene Ther. 7:20-6); cytochrome P450 enzymes + cyclophosphamide (CPA) (Huang et al., (2000) Cancer Gene Ther. 7:1034-42; Kan et al., (2001) Cancer Gene Ther. 8:473-82); rabbit carboxylesterase + 7-ethyl-10-[4-(1-piperidinyl)-1-piperidinyl]carbonyloxycamptothecin (CPT-II) (Meck et al., (2001) Cancer Res.61:5083-89); mushroom tyrosinase + bis-(2-chloroethyl)amino-4-hydroxyphenylaminoketone 28 (Jordan et al., (2001) Bioorg. Med. Chem.9:1549-58); Escherichia coli β-galactosidase + l-chloromethyl-5-hydroxy-1,2-dihydro-3H-benzo[E]indole (CC-1065) or + 1-(1'-chloroethyl)-5-hydroxy-l,2-dihydro-3H-benzo[E] Indole (Tietze et al., (2001) Chembiochem. 2: 758-765); mutants of carboxypeptidase G2 (CPG2, glutamate carboxypeptidase + 4-[bis(2-iodoethyl)amino]phenyloxycarbonyl-L-glutamate or + 3-fluoro-4-[bis(2-chloroethyl)amino]benzoyl-L-glutamate or + 3,5-difluoro-4-[bis(2-iodoethyl)amino]benzoyl-L-glutamate (Friedlos et al., (2002) Cancer Res. 62: 1724-1729).

[0024] Therapeutic antibodies refer to functional antibodies that have therapeutic benefit in cancer treatment.Examples of suitable therapeutic antibodies include, but are not limited to, atezolizumab (used to treat bladder cancer and breast cancer, NSCLC, and small cell lung cancer (SCLC)); avelumab (used to treat bladder cancer and Merkel cell carcinoma (MCC)); durvalumab (used to treat bladder cancer and NSCLC); nivolumab (used to treat bladder cancer, colon cancer, kidney cancer, liver cancer, NSCLC, metastatic SCLC, Hodgkin lymphoma, and melanoma); pembrolizumab (used to treat bladder cancer, cervical cancer, colorectal cancer, esophageal cancer, liver cancer, NSCLC, metastatic SCLC, Hodgkin lymphoma, and melanoma). LC, Hodgkin lymphoma, melanoma, and MCC); Bevacizumab (for glioblastoma, cervical cancer, colorectal cancer, renal cancer, non-small cell lung cancer (NSCLC), and ovarian cancer); Dinutuximab (for neuroblastoma); Pertuzumab (for breast cancer); Trastuzumab (for breast cancer and esophageal cancer); Cetuximab (for colorectal cancer); Panitumumab (for colorectal cancer), Ramucirumab (for colorectal cancer) cancer, and esophageal cancer); alemtuzumab (used to treat chronic lymphocytic leukemia (CLL)); blinatumomab (used to treat acute lymphoblastic leukemia (ALL)); obinutuzumab (used to treat CLL and non-Hodgkin's lymphoma); ofatumumab (used to treat CLL); rituximab (used to treat CLL and non-Hodgkin's lymphoma); necituzumab (used to treat NSCLC); ipilimumab (used to treat melanoma, pancreatic cancer, prostate cancer, and melanoma); daratumumab (used to treat multiple myeloma); elotuzumab (used to treat multiple myeloma); denosumab (used to treat bone cancer); olaratumumab (used to treat bone cancer); cemiplimab (used to treat Merkel cell carcinoma (MCC)); MEDI0562; GSK3174998; PF-04518600; CP-870893; dacetuzumab; ADC-1013; and ramucirumab (used to treat gastric or gastroesophageal cancer).

[0025] Methods for preparing recombinant viruses are known in the art. Briefly, to construct recombinant HSV, the target gene is cloned into a transfer plasmid. Subsequently, this plasmid is co-transfected into rabbit skin cells along with HSV-1 genomic DNA (the target gene is replaced by the HSV thymidine kinase gene). Progeny of the recombinant virus are selected and plaque purified on 143TK mutant cells in a culture medium containing a mixture 199V supplemented with 100 μg of bromodeoxyuridine / ml and 2% fetal bovine serum. Next, the thymidine kinase gene is restored by co-transfecting progeny viral DNA and a plasmid encoding the thymidine kinase gene in HAT medium. Preparation of the virus library and titration of infectivity are accomplished by Vero cells.

[0026] As demonstrated herein, recombinant HSV-1 expressing only the C-terminal half of the γ134.5 protein without wild-type or complete γ134.5 protein expression induces immune activation and robustly replicates and cleaves malignant cells that are refractory to the γ134.5 null mutant (Δγ134.5). Therefore, the present invention provides a method for treating a subject with cancer by administering to a subject, such as a human, a therapeutically effective amount of a recombinant HSV-1, wherein the recombinant HSV-1 expresses only the C-terminal portion of the γ134.5 protein (particularly SEQ ID NO: 2) without wild-type or complete γ134.5 protein expression, thereby treating the cancer of the subject. The recombinant HSV-1 can be administered as a sole anticancer therapy, or can be administered together with a therapeutically effective amount of a second anticancer agent, such as radiation and / or chemotherapy. In addition, the present method may also include the use of a target-specific portion (e.g., an antibody or cell marker) suitable for targeted administration of the recombinant HSV-1 of the present invention to the desired tissue.

[0027] As used herein, the term "treat" and the like refers to the elimination, reduction, alleviation, reversal and / or improvement of a disease or condition and / or symptoms associated therewith in the context of treating cancer. Solid and non-solid tumors that can be treated according to the methods herein include: bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, cervical cancer, including squamous cell carcinoma; adenocarcinomas, including thyroid cancer and skin cancer; leukemias, including acute lymphocytic leukemia, acute lymphoblastic leukemia, acute and chronic myeloid leukemia and promyelocytic leukemia; lymphomas, including B-cell lymphomas, T-cell lymphomas and Burkitt lymphomas; fibrosarcomas and rhabdomyosarcomas; melanomas; and neuroblastomas, astrocytomas and gliomas. In certain embodiments, the cancer treated according to the methods herein is a solid tumor. In other embodiments, the cancer is selected from breast cancer, liver cancer, lung cancer, skin cancer (melanoma), brain cancer and colon cancer.

[0028] Although not excluded, the treatment of a disease or condition does not require the complete elimination of the disease, condition or symptoms associated therewith, including the treatment of acute or chronic signs, symptoms and / or disorders. "Treatment" and the like may include "preventive treatment", which refers to reducing the probability of recurrence of a disease or condition or recurrence of a previously controlled disease or condition in a subject that does not have a disease or condition but has a risk of recurrence of the disease or condition or a recurrence of the disease or condition or is sensitive to recurrence of the disease or condition or a recurrence of the disease or condition. Therefore, "treatment" also includes relapse prevention or stage prevention. The term "treatment" and synonyms contemplate the administration of a therapeutically effective amount of the recombinant HSV-1 of the present invention to an individual in need of such treatment. Treatment can be symptomatically localized, for example, suppressing symptoms. Treatment can be performed in the short term, in the medium term, or can be a long-term treatment, for example, within the scope of maintenance therapy.

[0029] As used herein, the term "therapeutically effective amount" or "effective dose" refers to an amount of an active ingredient that, when administered to an individual in need thereof, is sufficient to effectively deliver the active ingredient to treat the condition or disease of interest. In the case of cancer or other proliferative disorders, the therapeutically effective amount of an agent may reduce (i.e., delay and preferably stop to some extent) unwanted cell proliferation; reduce the number of cancer cells; reduce tumor size; inhibit (i.e., delay and preferably stop to some extent) cancer cell infiltration into peripheral organs; inhibit (i.e., delay and preferably stop to some extent) cancer metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with cancer. To the extent that the administered active ingredient prevents the growth of existing cancer cells and / or kills existing cancer cells, it may be cytostatic and / or cytotoxic.

[0030] The recombinant HSV-1 of the present invention can be used as is, provided by a live carrier cell, or formulated in a pharmaceutical composition comprising a pharmaceutically acceptable excipient. The pharmaceutical composition provided by the present invention can be particularly formulated for intravenous administration in the form of a solid or liquid or for intravenous injection. Those skilled in the art can determine the best pharmaceutical composition based on, for example, the intended route of administration, delivery form, and desired dosage. See, for example, Remington's Pharmaceutical Sciences (19th edition, 1995).

[0031] The recombinant HSV-1 can be incorporated into conventional systemic dosage forms such as injection preparations. The dosage form can also include necessary physiologically acceptable carrier materials, excipients, lubricants, buffers, surfactants, antibacterial agents, bulking agents (such as mannitol), antioxidants (ascorbic acid or sodium bisulfite), etc.

[0032] The main carrier or excipient in the pharmaceutical composition can be natural aqueous or non-aqueous. For example, a suitable carrier or excipient can be water for injection, physiological saline solution or artificial cerebrospinal fluid, which may be supplemented with other materials commonly found in parenteral compositions. Neutral buffered saline or saline mixed with serum albumin is another exemplary vehicle. The pharmaceutical composition can include a Tris buffer of about pH 7.0-8.5 or an acetate buffer of about pH 4.0-5.5, and can also include sorbitol or a suitable substitute thereof. The pharmaceutical composition of the present invention can be prepared for storage by mixing the selected composition with the desired purity with an optional formulation agent (Remington's Pharmaceutical Sciences) in the form of a lyophilized cake or aqueous solution. In addition, the recombinant HSV-1 can be formulated into a lyophilized agent with a suitable excipient such as sucrose.

[0033] The administration routes of the recombinant HSV-1 or pharmaceutical composition of the present invention include intravenous injection, intraperitoneal injection, intracerebral injection (intraparenchymal injection), lateral ventricular injection, intramuscular injection, intraocular injection, intraarterial injection, intraportal injection or intralesional injection; through a sustained release system or an implant device. The composition can be administered by bolus injection or continuous infusion or by an implant device. The composition can also be administered locally by implanting a membrane, sponge or other suitable material on which the desired molecule has been absorbed or encapsulated. In the case of an implant device, the device can be implanted in any suitable tissue or organ, and the desired molecule can be delivered by diffusion, timed release bolus injection or continuous administration.

[0034] Compositions of the present invention can be delivered parenterally. When parenteral administration is envisioned, the therapeutic composition used by the present invention can be in the form of a pyrogen-free parenteral acceptable aqueous solution, and the aqueous solution comprises the required active ingredient in a pharmaceutically acceptable vehicle. The vehicle particularly suitable for parenteral injection is sterile distilled water, wherein the active ingredient is formulated into a sterile isotonic solution properly preserved. Preparation can include preparing the required active ingredient with a medicament such as injectable microspheres, bioerodible particles, polymer compounds (such as polylactic acid or polyglycolic acid), beads or liposomes that can provide controlled or sustained release of the active ingredient, which can be delivered by warehouse injection subsequently. Preparations containing hyaluronic acid have the effect of promoting the duration in circulation. Implantable drug delivery devices can be used to introduce the required active ingredient.

[0035] The present invention also includes a method of treating cancer by administering to an individual in need thereof a recombinant HSV-1 of the present invention and one or more second therapeutic agents useful for cancer treatment. The recombinant HSV-1 and the second therapeutic agent can be administered simultaneously or sequentially. In addition, the recombinant HSV-1 and the second therapeutic agent can be administered from a single composition or two separate compositions.

[0036] The second therapeutic agent is administered in an amount that provides its desired therapeutic effect. The effective dosage range for each second therapeutic agent is known in the art, and the second therapeutic agent is administered to an individual in need thereof within such established range.

[0037] In certain embodiments, the second therapeutic agent is an antibody. Suitable antibodies include, but are not limited to, atezolizumab, avelumab, durvalumab, nivolumab, pembrolizumab, bevacizumab, dinutuximab, pertuzumab, trastuzumab, cetuximab, panitumumab, ramucirumab, alemtuzumab, blinatumomab, obinut ... Ofatumumab; Rituximab; Necitumumab; Ipilimumab (anti-CTLA4 antibody); Daratumumab; Elotuzumab; Denosumab; Olaratumumab; Cemiplimab; MEDI0562 (anti-OX40 antibody); GSK3174998 (anti-OX40 antibody); PF-04518600 (anti-OX40 antibody); CP-870,893 (anti-CD40 antibody); dacetuzumab (anti-CD40 antibody); ADC-1013 (anti-CD40 antibody); and Ramucirumab.

[0038] In other embodiments, the second therapeutic agent comprises a chemotherapeutic agent, a radiotherapeutic agent, an anti-angiogenic agent, an apoptosis-inducing agent, an anti-tubulin drug, or a tumor-targeted chemotherapeutic agent, a radiotherapeutic agent, an anti-angiogenic agent, an apoptosis-inducing agent, or an anti-tubulin drug. Exemplary second therapeutic agents include, but are not limited to, anti-angiogenic agents such as angiostatin, endostatin, angiostatin, angiostatin, and maspin; and anti-tubulin drugs such as colchicine, paclitaxel, vinblastine, vincristine, vindescine, combretastatin, or a derivative or prodrug thereof. Other examples of second therapeutic agents include, but are not limited to, alkylating agents, nitrogen mustards, cyclophosphamide, trofosfamide, chlorambucil, nitrosoureas, carmustine (BCNU), lomustine (CCNU), alkyl sulfonates, busulfan, treosulfan, triazenes, plant alkaloids, vinca alkaloids (vincristine, vinblastine, vindescine, vinorelbine), taxanes, DNA topoisomerase inhibitors, epipodophyllotoxin epipodophyllins, 9-aminocamptothecin, camptothecin, crisnatol, mitomycins, mitomycin C, antimetabolites, antifolates, DHFR inhibitors, trimetrexate, IMP dehydrogenase inhibitors, mycophenolic acid, tiazofurin, ribavirin, EICAR, ribonucleotide reductase inhibitors, hydroxyurea, deferoxamine, pyrimidine analogs, uracil analogs, floxuridine, doxifluridine, ratitrexed, cytosine analogs, arabinoside C), cytosine arabinoside, fludarabine, purine analogs, mercaptopurine, thioguanine, DNA antimetabolites, 3-HP, 2'-deoxy-5-fluorouracil, 5-HP, α-TGDR, aphidicolin glycine, ara-C, 5-aza-2'deoxycytidine, β-TGDR, cyclocytidine, guanazole (inosine sugar dialdehyde), macebecin II (macebecinII), pyrazoloimidazoles, hormone therapy, receptor antagonists, antiestrogens, tamoxifen, raloxifene, megestrol acetate, LHRH agonists, goserelin, leuprolide acetate, antiandrogens, flutamide, bicalutamide, retinoids / deltoides, cis-retinoic acid, vitamin A derivatives, all-trans retinoic acid (ATRA-IV), vitamin D3 analogs, CB1093, ICH1060, photodynamic therapy, vertoporfin, BPD-MA, phthalocyanine, photosensitizer Pc4, demethoxyhypocrellin A (2BA-2-DMHA), cytokines, interferon alpha, interferon beta, interferon gamma, tumor necrosis factor, angiogenesis inhibitors, angiostatin (plasminogen fragment), antiangiogenic antithrombin UI, angioenzyme, ABT-627, Bay 12-9566, benefin, BMS-275291, cartilage-derived inhibitor (CDI), CD59 complement fragment, CEP-7055, Col 3, combretastatin A-4, endostatin (collagen XVIII fragment), fibronectin fragment, Groβ, halofuginone, heparinase, heparin hexose fragment, HMV833, human chorionic gonadotropin (hCG), IM-862, interferon-induced protein, interleukin-12, kringle5 (plasminogen fragment), marimastat, metalloproteinase inhibitor (UMP), 2-methoxyestradiol, MMI270 (CGS27023A), neovastat, NM-3, panzem, PI-88, placental ribonuclease inhibitor, plasminogen activator inhibitor, platelet factor-4 (PF4), prinomastat, prolactin 161, proliferation-related protein (PRP), retinoids, solimastat, squalamine, SS3304, SU5416, SU6668, SU11248, tetrahydrocortin-S, tetrathiomolybdate, thalidomide, thrombospondin-1 (TSP-1), TNP-47 0, transforming growth factor-β, angiostatin, angiostatin (calreticulin fragment), ZD6126, ZD6474, farnesyl transferase inhibitors (FTI), bisphosphonates, antimitotic agents, allocolchicine, halichondrin B, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, thiocolchicine, tritylcysteine, isoprenoid inhibitors, dopaminergic neurotoxins, cell cycle inhibitors, staurosporine, actinomycins, actinomycin D, dactinomycin, bleomycins, bleomycin A2, bleomycin B2, pyromycin, anthracycline, doxorubicin, epirubicin, pyridoxine, doxycycline, mitoxantrone, MDR inhibitors, verapamil, Ca21 ATPase inhibitors, and thapsigargin.

[0039] The following non-limiting examples are provided to further illustrate the present invention.

[0040] Example 1: Materials and Methods

[0041] Cells and viruses: Vero, HT-29, SW480, C32, A375, MDA-MB-231, 4T1, HepG2, and A549 cells were obtained from the American Type Culture Collection. Vero, SW480, C32, A375, MDA-MB-231, and A549 cells were passaged in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. HT-29, 4T1, and HepG2 cells were passaged in RPMI1640 medium supplemented with 10% fetal bovine serum. HSV-1 (F) is the prototype HSV-1 strain used in this study (Ejercito et al., (1968) J. Gen. Virol. 2:357-364). In the recombinant virus Δγ134.5, a 1 kb fragment from the coding region of the γ134.5 gene was deleted (Chou et al., (1990) Science 250:1262-1266). In ΔN146, the sequence encoding amino acids 1 to 146 in the γ134.5 gene was deleted (Ma et al., (2012) J. Virol. 86:2188-2196). In EUS11, the γ134.5 gene was deleted but there was a Us11 gene driven by the α-47 promoter (Liu et al., (2018) J. Virol. 92). The preparation of the virus library and the titration of infectivity were performed as previously described (Ma et al., (2012) J. Virol. 86:2188-2196).

[0042] Viral infection: Viral infection was performed at the specified multiplicity of infection (Verpooten et al., (2009) J. Biol. Chem. 284: 1097-1105). Cells were then collected and processed for immunoblotting, real-time PCR analysis, or viral growth analysis (Ma et al., (2012) J. Virol. 86: 2188-96; Wu et al., (2016) J. Virol. 90: 10414-22). Cells were analyzed by CELLTITER- Luminescent cell viability assay (Promega) measures cell viability. For interferon analysis, Vero and MDA-MB-231 cells were treated or not treated with human interferon-α (Sigma), and 4T1 cells were treated with mouse interferon-α (Sigma) for 20 hours. Virus yield was then measured in 48 hours after infection with virus infected cells.

[0043] Immunoblot analysis and ELISA: Cells were harvested, rinsed with phosphate-buffered saline (PBS), and washed on ice with ice-cold buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1.0% Triton TM X-100 and protease inhibitor cocktail). After centrifugation, the supernatant was mixed with disruption buffer (50 mM Tris-HCl pH 6.8, 2% (weight / volume) SDS, 0.1% bromophenol blue, 10% glycerol and 100 nM β-mercaptoethanol) and boiled. The samples were then subjected to electrophoresis on a denaturing polyacrylamide gel, transferred to a nitrocellulose membrane, and reacted with the following antibodies: anti-gC antibody (Jing et al., (2004) J. Virol. 78:7653-66), anti-γ134.5 antibody (Cheng et al., (2002) J. Virol. 76:9434-45), anti-ICP27 antibody (Virusys Inc.), anti-ICP0 antibody (Santa Cruz), anti-eIF-2α antibody (Cell Signaling Technology, Inc.), anti-phospho-eIF-2α antibody (Cell Signaling Technology, Inc.), anti-IRF3 antibody (Cell Signaling Technology, Inc.), anti-phospho-IRF3 antibody (Cell Signaling Technology, Inc.), and anti-β-actin antibody (Sigma). The membranes were rinsed in PBS and reacted with donkey anti-rabbit or anti-mouse immunoglobulins conjugated to horseradish catalase and developed using the enhanced chemiluminescent Western Blot Probe System kit (Amersham Pharmacia Biotechnology, Inc.) For enzyme-linked immunosorbent assay (ELISA), cell culture supernatants were collected and analyzed for IFNα and Cxcl9 according to the manufacturer's protocol (R&D Systems).

[0044] Transcriptome analysis: Monolayers of 4T1 cells were mock infected or infected with virus (5 pfu / cell). Six hours after infection, RNA was extracted from cells using the RNase plus mini kit (Qiagen) and treated with DNase I (New England BioLabs). Duplicate RNA samples were analyzed using the Clariom TM SAffymetrix array processing. TMThe raw data generated by the S mouse array were processed in R using the Oligo package. The feature intensity values ​​from each CEL file were converted to normalized expression values ​​using the Robust Multi-array Average (RMA) with default settings. All positive and negative control probes and Affymetrix reporter genes (RPTR) were removed before downstream analysis. PCA (principal component analysis) plots were generated to check for any batch effects. Differential gene expression analysis was performed using the limma package. Significantly expressed genes were filtered with an adjusted p-value <0.05. Heat maps were generated from the raw data (normalized expression values) using the R package "pheatmap" v1.0.8.

[0045] Quantitative real-time PCR analysis: Cells were mock infected or infected with virus. At 6 hours post-infection, total RNA was collected from cells using RNase plus mini kit (Qiagen) and subjected to DNase I (New England BioLabs) digestion. cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Applied Biosystems ABI Prism 7900HT instrument and ABI Quantitative real-time PCR was performed using green master mix (Applied Biosystems). Gene expression levels were normalized to endogenous control 18S rRNA. -ΔΔCT Relative gene expression was determined by the qPrimerDepot method (Schmittgen and Livak, (2008) Nat. Protoc. 3: 1101-8). Primers for each gene were selected according to the recommendations of the qPrimerDepot database. Primer sequences are provided in Table 1.

[0046] Table 1

[0047]

[0048] Mouse studies: 5-week-old BALB / c mice were purchased from Harlan Sprague Dawley Inc. and housed in biosafety level 2 isolators under specific pathogen-free conditions. All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of the University of Illinois at Chicago. At 6 weeks of age, 1x10 5 Live 4T1 cells were subcutaneously inoculated on the right flank of mice (day -7). After 8 days, when the tumor volume reached about 100 mm 3At the time of the experiment, the mice were randomly divided into three groups and injected with Δγ134.5, ΔN146 or PBS intratumorally on days 1, 3 and 6, respectively. Each tumor was slowly injected with 1×10 7 PFU of virus or PBS. Tumor growth was monitored every other day by measuring two perpendicular tumor diameters with a digital caliper. Tumor volume was calculated using the following formula: volume = (length x width x height) / 2. On day 24 after tumor inoculation, mice were euthanized by CO2 inhalation.

[0049] Tissue analysis: Six mice from each treatment group were sacrificed on selected days after the last intratumoral injection to collect tumors, lungs, livers, spleens, and blood. To measure viral load, samples were crushed, homogenized, and beads were beaten, frozen and thawed three times, and sonicated in DMEM. After centrifugation, tumor supernatants were used for plaque assays. Supernatants from lungs, livers, spleens, and blood were used for real-time quantitative PCR analysis. In brief, the supernatants were suspended in a buffer containing 1% SDS, 50mM Tris (pH 7.5), and 10mM EDTA. After incubation with proteinase K (50 μg / ml) at 37°C, viral DNA was extracted and quantified by real-time PCR using HSV-1gD specific primers: TACAACCTGACCATCGCTTG (SEQ ID NO: 21) and GCCCCCAGAGACTTGTTGTA (SEQ ID NO: 22).

[0050] For metastatic formation analysis, lungs from mice were minced and fixed in formalin. The number of lung metastases was quantified by counting under a light microscope.

[0051] Immunohistochemical analysis: Tissue sections were processed and HSV-1 antigens were detected with anti-HSV-1 antibodies (Dako). CD4 (Cell Signaling Technology, Inc.) and CD8 (Cell Signaling Technology, Inc.) antibodies were used according to the manufacturer's protocol. Samples were incubated with the primary antibody, followed by the addition of biotinylated anti-rabbit immunoglobulin secondary antibodies, avidin-horseradish peroxidase, and 3,3′-diaminobenzidine tetrahydrochloride (0.04%) as a chromogen in 0.05 M Tris HCl (pH 7.4) and 0.025% H2O2 (Ventana Medical Systems, Tucson, AZ).

[0052] Example 2: Replication of the ΔN146 mutant in tumor cells

[0053] It has been shown that the HSVγ134.5 mutant containing only amino acids 147-263 (ΔN146) substantially impairs viral growth in normal cells or tissues (Ma et al., (2012) J. Virol. 86:2188-2196; Ma et al., (2017) Sci. Rep. 7:41461; Pan et al., (2018) J. Virol. 92:e01015-18). To determine the activity of this mutant in malignant cells, viral replication was assessed. This analysis showed that in 4T1 (mouse breast cancer) cells, wild-type HSV-1 replicated to 1x10 7 pfu / ml, while the γ134.5 null mutant (Δγ134.5) only reached 1x10 3 However, ΔN146 grew to 1x10 6 pfu / ml, indicating robust replication. A similar trend was observed in MDA-MB-231 (human breast cancer) cells, where ΔN146 replicated 100-fold more robustly than Δγ134.5. In addition, these phenotypes were recapitulated in a range of other tumor cells, including human HT29 (colon), SW480 (colon), HepG2 (liver), C32 (melanoma), A375 (melanoma), and A549 (lung).

[0054] Subsequently, the kinetics of viral growth were examined. As infection progressed, HSV-1 grew steadily in wild-type 4T1 cells, with the titer increasing to 1x10 at 72 h post-infection. 7 pfu / ml. Although the level was slightly lower, ΔN146 replicated to 1x10 6 pfu, while Δγ134.5 barely replicated, with a titer of 1x10 throughout the infection. 3 pfu / ml. A similar trend was observed in MDA-MB-231 cells, where ΔN146 replicated 100-fold more potently than Δγ134.5. To assess the cytolytic activity of the virus, cell viability was determined. This analysis showed that, similar to the wild-type virus, ΔN146 lysed almost 95% of 4T1 cells by 72 h with slightly delayed kinetics, while Δγ134.5 destroyed approximately 40% of the cells. This effect was also replicated in MDA-MB-231 cells. Together, these results indicate that ΔN146 replicates and lyses tumor cells more efficiently than the γ134.5 null mutant.

[0055] Example 3: Expression of the C-terminal portion of γ134.5 inhibits phosphorylation of eIF2α

[0056] With the sequential expression of α, β and γ genes, HSV infection progresses in a time manner. In the absence of γ134.5, the initiation of viral DNA replication leads to the cessation of protein synthesis (Chou and Roizman (1992) Proc.Natl.Acad.Sci.USA 89:3266-70). In order to evaluate the impact of ΔN146, the expression of representative proteins ICP27 (α protein) and gC (γ protein) was measured because the expression of these proteins depends on viral DNA replication. Simulate infection or infect cells with HSV-1, Δγ134.5 or ΔN146 viruses, and let the samples undergo Western blot analysis 12 hours after infection. The analysis shows that the wild-type virus expresses both ICP27 and gC in infected 4T1 and MDA-MB-231 cells. Although Δγ134.5 expresses ICP27 in 4T1 or MDA-MB-231 cells, a very small amount of gC is detected. Under the same conditions, ΔN146 expressed similar levels of ICP27 and gC as wild-type HSV-1, indicating its ability to block translational arrest caused by viral DNA replication.

[0057] Because phosphorylation of eIF2α is coupled to protein synthesis, eIF2α phosphorylation caused by stress kinases PKR, PERK or GCN2 was monitored in 4T1 and MDA-MB-231 tumor cells. This analysis showed that the expression of eIF2α was similar in mock- or virus-infected tumor cells. Interestingly, phosphorylated eIF2α was present in mock-infected cells, probably due to oncogenic stress. Although wild-type HSV-1 abolished eIF2α phosphorylation in 4T1 and MDA-MB-231 tumor cells, Δγ134.5 aggravated it, while ΔN146 completely abolished eIF2α phosphorylation. Therefore, the region spanning the C-terminal part of γ134.5 is sufficient to inhibit eIF2α phosphorylation in tumor cells.

[0058] Example 4: ΔN146 stimulates interferon response in tumor cells

[0059] To evaluate the response of tumor cells to viral infection, transcriptome analysis was performed in 4T1 cells. A large number of genes in multiple cellular pathways were observed to be differentially expressed in mock-infected and virus-infected 4T1 cells. Notably, many genes in innate immune pathways were significantly upregulated in response to ΔN146. Of the 46 genes tested, most remained unchanged or were expressed in small amounts in cells infected with mock-infected or wild-type virus. However, they were upregulated in cells infected with Δγ134.5, albeit to varying degrees. Notably, gene induction was more pronounced in cells infected with ΔN146, indicating that ΔN146 has a propensity to stimulate inflammatory responses.

[0060] To confirm these results, the expression of selected cytokines and interferon-stimulated genes was determined by real-time PCR. As expected, the wild-type virus stimulated very little expression of IFN-α1, IFIT1, Ccl5, and Cxcl9, while Δγ134.5 or ΔN146 induced these genes significantly. This was supported by the levels of cytokine production in ELISA assays. To dissect its molecular basis, the interferon regulatory factor (IRF3), which activates the immune response, was analyzed. IRF3 was unphosphorylated in 4T1 cells infected with mock infection or wild-type HSV-1. In contrast, it became phosphorylated in cells infected with Δγ134.5 or ΔN146. This was not due to differences in viral infectivity, as shown by the normal expression of ICP0 and ICP27. These results were confirmed in multiple experiments, and the phenotype was also observed in human MDA-MB-231 cells. In conclusion, similar to Δγ134.5, ΔN146 is immunostimulatory when infecting malignant cells.

[0061] Example 5: ΔN146 is resistant to IFN

[0062] Type I IFN is required for eliciting anti-tumor immunity. On the other hand, it mediates antiviral responses. To determine whether ΔN146 is resistant to IFN clearance, viral growth was examined. As proof of concept, the viral response to IFN was first performed in Vero cells lacking the IFN-α / β genes. IFN-α treatment had little effect on the replication of HSV-1(F), but significantly reduced the replication of Δγ134.5 by about 1000-fold. However, IFN-α only modestly reduced the replication of ΔN146. Moreover, similar trends were observed when tested in 4T1 and MDA-MB-231 cells. Although IFN-α reduced viral replication overall, it had little effect on wild-type HSV-1 or ΔN146. In fact, in the presence of exogenous IFN-α, the replication of ΔN146 was consistently 500 to 1000 times higher than that of Δγ134.5. Therefore, amino acids 147 to 263 from γ134.5 are sufficient to confer viral resistance to IFN.

[0063] Example 6: ΔN146 reduces the growth and metastasis of primary tumors in vivo

[0064] Based on the results presented in Examples 2 to 5, it is assumed that the ability of ΔN146 to replicate and activate inflammation will enhance tumor destruction in vivo. To demonstrate this, invasive 4T1 breast cancer that spontaneously metastasizes was selected, and the spontaneous metastasis process is similar to human breast tumors. As a comparison, Δγ134.5 was also used because it is similar to HSV1716 (Rampling et al., (2000) Gene Ther. 7:859-866; Streby et al., (2017) Clin. Cancer Res. 23:3566-3574). In addition, recombinant HSV EUs11 (Liu et al., (2018) J. Virol. 92) was also included because the virus is structurally equivalent to the oncolytic backbone of talimogenelaherparepvec (Liu et al., (2003) Gene Ther. 10:292-303). On days 1, 3, and 6, mice were injected with PBS, Δγ134.5, ΔN146, or EUs11 (1×10 7 pfu) 3 times. Tumor size was then monitored. Figure 1 As shown in , control tumors treated with PBS grew at a faster rate over time. Treatment with γ134.5 null virus slightly reduced local tumor growth. However, intratumoral inoculation with ΔN146 or EUs11 significantly slowed tumor growth, and the reduction in tumor size became more and more obvious as treatment progressed. At day 24, ΔN146 and EUs11 reduced tumor size by nearly 45% compared to mock control or Δγ134.5. Therefore, although similar to EUs11, ΔN146 exhibited superior anti-priming tumor activity when compared to Δγ134.5.

[0065] To evaluate the effect of the virus on metastasis, lung tumor formation was analyzed on day 24. Figure 2 It was shown that lung metastases were readily detected in control mice, with an average of 25 nodules per animal measured by microscopic analysis. Treatment with Δγ134.5 or EUs11 reduced the incidence, with an average of 15 nodules per animal. Notably, ΔN146 further reduced the metastatic burden, with an average of 15 nodules. These results suggest that viral Δγ134.5 reduced lung metastasis; however, ΔN146 presented a more significant effect.

[0066] Example 7: ΔN146 replicates in primary tumors but not in normal tissues

[0067] To assess viral replication, virus yield was determined in primary tumors collected on day 9. This analysis showed that Δγ134.5 was expressed at 1x10 2pfu / g tumor tissue average titer replication ( Figure 3 ). On the other hand, EUs11 has 7x10 3 pfu / g tumor tissue. Similarly, ΔN146 was grown at an average titer of 5x10 3 pfu / g tumor tissue. Apparently, similar to EUs11, ΔN146 replicated 50-fold more strongly than Δγ134.5. Consistent with this, viral antigens were detected in thin sections of this tumor bed, with ΔN146 and EUs11 being more widely dispersed than Δγ134.5. This correlated with the degree of necrosis of the tumor tissue.

[0068] To assess whether the virus spreads to normal tissues, the presence of Δγ134.5, ΔN146 and EUs11 in the lungs, blood, liver and spleen was determined by qPCR assays. This analysis showed that the viruses were undetectable in these tissues at day 9, although they were readily found in tumors. These results suggest that, similar to Δγ134.5 or EUs11, the replication of ΔN146 is restricted to tumor tissues in vivo.

[0069] To confirm that viral replication was indeed actively occurring in the tumor, three treatments of 4T1 primary tumors were performed and viral yields were measured on days 7, 9, and 15. This analysis showed that approximately 2x10 2 pfu / g tumor tissue. As treatment progressed, the amount of Δγ134.5 remained constant at first and then decreased to 1x10 pfu / g tumor tissue on day 15. However, under the same conditions, the level of ΔN146 increased to 1x10 pfu / g tumor tissue on day 9. 4 pfu / g tumor tissue, and then decreased to 1x10 3 pfu / g tumor tissue. EUs11 showed a similar growth pattern. Therefore, unlike Δγ134.5, ΔN146 and EUs11 are able to replicate in tumors in vivo.

[0070] Example 8: ΔN146 induces CD4+ and CD8+ T cell infiltration into primary tumors

[0071] Previous evidence suggests that oncolytic HSV with a γ134.5 deletion activates systemic antitumor immunity (Thomas and Fraser (2003) Mol. Ther. 8:543-51; Toda et al., (1999) Hum. Gene Ther. 10:385-93). Because intratumoral viral injection reduces both local tumor growth and metastasis formation, it was determined whether there was induction of adaptive immunity. Therefore, CD4+ and CD8+ T cells were evaluated by immunohistochemical analysis. Primary tumors collected on day 24 were sliced ​​and stained for the presence of CD4+ and CD8+ T cells. In mock-infected tumors, some CD4+ and CD8+ T cells were detected. + or CD8 + T cells (<4%). However, in tumors treated with Δγ134.5, CD4 + T cells increased to 12% and CD8 + T cells rose to 7%. Similarly, ΔN146 accounted for 15% of CD4 + T cells and 8% CD8 + Although EUs11 triggered immune cell infiltration, the observed effect was not significant for CD4 + T cells (10%) and CD8 + T cells (5%) were both decreased. These results suggest that, similar to Δγ134.5, ΔN146 induces T cell infiltration, whereas EUs11 appears to impair this process.

[0072] Example 9: ΔN146 and EUs11 interact differently with tumor cells

[0073] To determine whether ΔN146 and EUs11 interact differently with tumor cells, in vitro assays were performed. Figure 4 As shown in , ΔN146 infection stimulated the transcription of IFN-α1 and cxcl9 genes. In contrast, EUs11 suppressed gene expression. This was paralleled by the production levels of cytokines measured by ELISA. Consistently, ΔN146 stimulated the phosphorylation of IRF3, while EUs11 could not, indicating that EUs11 mediated immune suppression after viral infection.

[0074] To assess the ability of the viruses to destroy tumor cells, cell viability was measured. This analysis showed that, similar to EUS11, ΔN146 lysed approximately 95% of 4T1 cells by 74 hours. Therefore, both ΔN146 and EUs11 efficiently lysed tumor cells. In addition, viral replication within 4T1 cells with or without IFN treatment was determined. This analysis showed that in the absence of IFN-α, both ΔN146 and EUs11 replicated efficiently, reaching titers of approximately 1x106 pfu / ml. The addition of exogenous IFN-α moderately reduced viral replication of ΔN146 and EUs11, with a titer of 5x10 4 pfu / ml, indicating that they were equally resistant to type I IFN. Sequence Listing <110> The Board of Trustees of the University of Illinois <120> Recombinant herpes simplex virus for cancer immunotherapy <130> UIC0078WO <150> US 62 / 682,202 <151> 2018-06-08 <160> twenty two <170> PatentIn version 3.5 <210> 1 <211> 248 <212> PRT <213> Herpes simplex virus 1 <400> 1 Met Ala Arg Arg Arg Arg His Arg Gly Pro Arg Arg Pro Arg Pro Pro 1 5 10 15 Gly Pro Thr Gly Ala Val Pro Thr Ala Gln Ser Gln Val Thr Ser Thr 20 25 30 Pro Asn Ser Glu Pro Ala Val Arg Ser Ala Pro Ala Ala Ala Pro Pro 35 40 45 Pro Pro Pro Ala Ser Gly Pro Pro Pro Ser Cys Ser Leu Leu Leu Arg 50 55 60 Gln Trp Leu His Val Pro Glu Ser Ala Ser Asp Asp Asp Asp Asp Asp 65 70 75 80 Asp Trp Pro Asp Ser Pro Pro Pro Glu Pro Ala Pro Glu Ala Arg Pro 85 90 95 Thr Ala Ala Ala Pro Arg Pro Arg Ser Pro Pro Pro Gly Ala Gly Pro 100 105 110 Gly Gly Gly Ala Asn Pro Ser His Pro Pro Ser Arg Pro Phe Arg Leu 115 120 125 Pro Pro Arg Leu Ala Leu Arg Leu Arg Val Thr Ala Glu His Leu Ala 130 135 140 Arg Leu Arg Leu Arg Arg Ala Gly Gly Glu Gly Ala Pro Glu Pro Pro 145 150 155 160 Ala Thr Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro Ala 165 170 175 Arg Val Arg Phe Ser Pro His Val Arg Val Arg His Leu Val Val Trp 180 185 190 Ala Ser Ala Ala Arg Leu Ala Arg Arg Gly Ser Trp Ala Arg Glu Arg 195 200 205 Ala Asp Arg Ala Arg Phe Arg Arg Arg Val Ala Glu Ala Glu Ala Val 210 215 220 Ile Gly Pro Cys Leu Gly Pro Glu Ala Arg Ala Arg Ala Leu Ala Arg 225 230 235 240 Gly Ala Gly Pro Ala Asn Ser Val 245 <210> 2 <211> 102 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 2 Arg Leu Arg Arg Ala Gly Gly Glu Gly Ala Pro Glu Pro Pro Ala Thr 1 5 10 15 Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro Ala Arg Val 20 25 30 Arg Phe Ser Pro His Val Arg Val Arg His Leu Val Val Trp Ala Ser 35 40 45 Ala Ala Arg Leu Ala Arg Arg Gly Ser Trp Ala Arg Glu Arg Ala Asp 50 55 60 Arg Ala Arg Phe Arg Arg Arg Val Ala Glu Ala Glu Ala Val Ile Gly 65 70 75 80 Pro Cys Leu Gly Pro Glu Ala Arg Ala Arg Ala Leu Ala Arg Gly Ala 85 90 95 Gly Pro Ala Asn Ser Val 100 <210> 3 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 3 gccttgacac tcctggtaca aatgag 26 <210> 4 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 4 cagcacattg gcagaggaag acag 24 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 5 caaggcaggt ttctgaggag 20 <210> 6 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 6 aagcagattc tccatgacct g 21 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 7 ctgctgcttt gcctacctct 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 8 cacttcttct ctgggttggc 20 <210> 9 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 9 tccttccttc cttccttcct tcc 23 <210> 10 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 10 aggctctttt tcaccctgtc tgg 23 <210> 11 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 11 ggccttgacc tttgctttac tg 22 <210> 12 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 12 cacagagcag cttgacttgc a 21 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 13 cctccttggg ttcgtctaca 20 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 14 agtggctgat atctgggtgc 20 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 15 cctgctgctt tgcctacatt 20 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 16 acacacttgg cggttctttc 20 <210> 17 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 17 ccctgtttct tccacagtgc cta 23 <210> 18 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 18 gagacaatgg tctggttgcc atc 23 <210> 19 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 19 cctgcggctt aatttgactc 20 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 20 aaccagacaa atcgctccac 20 <210> twenty one <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> twenty one tacaacctga ccatcgcttg 20 <210> twenty two <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> twenty two gcccccagag acttgttgta 20

Claims

1. Use of a recombinant herpes simplex virus-1 (HSV-1) expressing only the C-terminal portion of the γ134.5 protein without the expression of wild-type or complete γ134.5 protein in the preparation of a medicament for treating a subject suffering from cancer, wherein the C-terminal portion of the γ134.5 protein consists of SEQ ID NO: 2, and wherein the cancer is breast cancer.

2. The use according to claim 1, wherein the recombinant HSV-1 further comprises a deletion of one or more non-essential genes or fragments thereof.

3. The use according to claim 2, wherein the non-essential gene is selected from the group consisting of UL2, UL3, UL4, UL9.5, UL10, UL11, ULI2, UL13, ULI4, UL20, UL21, UL23, UL24, UL39, UL40, UL41, UL43, UL43.5, UL44, UL45, UL46, UL47, UL50, UL51, UL53, UL55, Us1, Us1.5, Us2, Us3, Us4, Us5, Us7, Us8, Us8.5, Us9, Us10, Us11, Us12 and ICP0.

4. The use according to claim 1, wherein the recombinant HSV-1 further comprises replacing one or more non-essential genes with one or more genes expressing therapeutic proteins, enzymes, antibodies or nucleic acids for cancer therapy.

5. The use according to claim 4, wherein the non-essential gene is selected from the group consisting of UL2, UL3, UL4, UL9.5, UL10, UL11, ULI2, UL13, ULI4, UL20, UL21, UL23, UL24, UL39, UL40, UL41, UL43, UL43.5, UL44, UL45, UL46, UL47, UL50, UL51, UL53, UL55, Us1, Us1.5, Us2, Us3, Us4, Us5, Us7, Us8, Us8.5, Us9, Us10, Us11, Us12 and ICP0.

6. The use according to claim 4, wherein the therapeutic protein is selected from interferon α, interleukin-2 and granulocyte colony stimulating factor.

7. The use according to claim 4, wherein the antibody is selected from anti-programmed cell death protein 1 antibody, anti-checkpoint T lymphocyte-associated protein 4 antibody, anti-OX40 antibody and anti-CD40 antibody.

8. The use according to claim 1, wherein a second therapeutic agent useful for cancer treatment is administered in combination with the recombinant HSV-1.

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