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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
  • C12N2710/16621—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
  • C12N2710/16632—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

• Oncolytic herpes simplex virus 1 (HSV-1) is an attractive agent for cancer immunotherapy (Peters & Rabkin (2015) Mol. Ther. Oncolytics 2:15010; Chiocca & Rabkin (2014) Cancer Immunol . Res. 2:295-300) .
• HSV-1 Upon infection, HSV-1 undergoes sequential gene expression, DNA replication, assembly and egress, resulting in tumor cell destruction. This is accompanied by release of danger signals and neo-antigens that activate adaptive antitumor immunity.
• a range of oncolytic HSV is under various stages of development (Peters & Rabkin (2015) Mol. Ther. Oncolytics 2:15010).
• the most clinically advanced agent is talimogene laherparepvec (T-VEC) approved by FDA for treating advanced melanoma (Andtbacka, et al . (2015) J.
• HSV gi34.5 contains a large amino-terminal domain (aa 1-146) and carboxyl-terminal domain.
• HSV-1 activates double-stranded RNA dependent kinase (PKR) that shuts off protein synthesis by phosphorylation of translation initiation factor 2a (eIF-2a) .
• PLR RNA dependent kinase
• the Yi34.5 protein redirects protein phosphatase 1 (PPl) to dephosphorylate elF-2a.
• site-specific disruption of the gi34.5-RR interaction abrogates viral virulence.
• HSV Yi34.5 is also reported to affect glycoprotein processing and viral spread. In addition, evidence suggests that the Yi34.5 protein bears additional functions.
• a major limitation in the use of attenuated, replication-competent viruses to directly destroy tumors continues to be the reduced growth in many cell types, including cancer cells.
• host defenses limit the viral vectors to replicate successfully for a long enough period of time to eradicate the entire population of neoplastic cells.
• oncolytic viral backbones with improved replication often dampen innate immune priming necessary for antitumor immunity. As such, the surviving cancer cells proliferate or re-establish their strangle-hold on the patient.
• viral anti-tumor agents that lyse cancer cells and activate systemic antitumor responses effectively.
• This invention provides a method for treating a subject with cancer by administering to the subject a therapeutically effective amount of a recombinant Herpes Simplex Virus-1 (HSV-1) that expresses only a C-terminal portion of gc34.5 protein, e.g a protein consisting of SEQ ID NO: 2, with no wild-type or intact gi34.5 protein expression thereby treating the subject's cancer.
• HSV-1 Herpes Simplex Virus-1
• the recombinant HSV-1 further includes a deletion of one or more non-essential genes or fragments thereof, e.g., UL2, UL3 , UL4, UL9.5, UL10, ULll, ULI2,
• the recombinant HSV-1 further includes replacement of one or more non- essential genes with one or more genes expressing a therapeutic protein (e.g., interferon alpha (IFN-a), interleukin-2 (IL-2), and granulocyte-colony stimulating factor (G-CSF) ) , enzyme, antibody (e.g., anti-programmed cell death protein 1 antibody (anti-PDl), anti-checkpoints T-lymphocyte-associated protein 4 antibody (anti-CTLA4 ) , anti-OX40 (anti-CD134) antibody, and anti-CD40 antibody) or nucleic acid for cancer therapy, wherein the non-essential genes are selected from UL2, UL3 , UL4, UL9.5, UL10, ULll,
• a therapeutic protein e.g., interferon alpha (IFN-a), interleukin-2 (IL-2), and granulocyte-colony stimulating factor (G-CSF)
• enzyme e.g., anti-programmed
• the cancer is a solid tumor and is optionally a cancer selected from breast, lung, liver, skin (melanoma) , brain, and colon cancer.
• the method may further include the administration of an effective amount of a second therapeutic agent useful for the treatment of cancer.
• mice were sacrificed on day 24 after the initiation of treatment and the lungs were collected and fixed in formalin. The number of lung metastases was quantified by counting under a light microscope. The results shown are from one of three independent experiments. Differences between the selected groups were statistically assessed by a two-tailed Student t test ( *, P 0.05; **, P 0.01).
• FIG. 3 provides data demonstrating viral growth in 4T1 tumors.
• the results shown are from three experiments with triplicate samples. Differences between the selected groups were statistically assessed by a two- tailed Student's t-test (**P ⁇ 0.01).
• FIG. 4 shows comparative analysis of DN146 and EUsll in vitro. Viral effects on the expression of IFN-al and Cxcl9 were analyzed. 4T1 cells were mock-infected or infected with DN146 or EUsll (5 pfu/cell) . At 6 hours post infection, RNA samples were analyzed by quantitative polymerase chain reaction. Data are representative of three experiments among triplicate samples with standard deviations .
• the optimal intracellular environment for virus replication develops through events that begin to take place with attachment of virus to the cell membrane.
• Binding of the herpes simplex virus to the cell membrane receptor (s) is followed by a cascade of events that are associated with biochemical, physiological, and morphological changes in the cells. Following infection in susceptible cells, lytic replication is regulated by a temporally coordinated sequence of gene transcription. Binding of the virus to a host cell membrane activates the immediate-early (IE or a) genes (ICPO, ICP4, ICP22, ICP27, and ICP47), which are transactivating factors allowing the production of the next group of genes to be transcribed, the early (b) genes. Expression of immediate-early gene products is followed by the expression of proteins encoded by the early and then, the late (g) genes.
• IE immediate-early
• the entire cascade of gene activation and viral replication in the wild-type virus takes about 18-24 hours and invariably results in cell death.
• the recombinant HSV mutant of the present invention circumvents the protein synthesis shutoff phenotype of gc34.5 null viruses and activates STING (interferon-stimulated genes) that mediate antitumor immunity, creating a more robust HSV variant with targeted Yi34.5 deletion.
• DN146 recombinant HSV-1, which expresses the C-terminal half of gi34.5 (DN146), robustly replicates in and lyses malignant cells that are refractory to the gi34.5 null mutant (7 ⁇ gi34.5).
• DN146 infected cells, DN146 but not Dgc34.5 precludes phosphorylation of translation initiation factor eIF2a, ensuing viral protein synthesis.
• DN146 also activates interferon regulatory factor 3 and the IFN response because it removes the gi34.5 inhibitory domain of STING, an immune factor known to prime immunity against tumor.
• DN146 replicates competently when exposed to IFN- a/b.
• DN146 reduces tumor growth and metastasis more effectively than Dgi34.5. While comparable in tumor growth reduction, DN146 reduces metastasis more effectively than EUsll. This coincides with viral replication, IFN induction and T cell infiltration in local tumors. DN146 is undetectable in normal tissues and avirulent in vivo.
• this invention is a recombinant HSV-1 virus that expresses only the C-terminal half of gi34.5 protein with no wild-type or intact gi34.5 protein expression and its use in the treatment of cancer.
• gc34.5 is an HSV protein that promotes viral replication in the peripheral tissues and penetration to the peripheral nervous systems in experimental models (Whitley, et al . (1993) J. Clin.
• HSV gc34.5 is known to include a large amino-terminal domain (aa 1-146) and carboxyl-terminal domain (aa 147- 263), which binds protein phosphatase la (He, et al . (1998)
• a wild-type or intact gc34.5 has the amino acid sequence:
• C-terminal portion of the gc34.5 protein can be driven by the gc34.5 protein promoter, another endogenous HSV-1 promoter, or heterologous or exogenous promoter of viral or cellular origin.
• exemplary promoters of use in the invention include, without limitation, the herpes simplex virus immediate-early promoters 27, a4 , aO, a22, and a47; the herpes simplex virus early promoters from ICP8 (or U L 29) , thymidine kinase (tk or U L 23), ICP6 (U L 39) or any of the DNA replication genes; or late promoter, e.g., the Usll promoter.
• one or more of non- essential genes has been replaced with one or more nucleic acids encoding and capable of expressing a therapeutic protein, enzyme, antibody, nucleic acid ⁇ e.g., a nucleic acid encoding said protein, enzyme, antibody, or a microRNA, ribozyme, and the like) , or the like for cancer therapy.
• a therapeutic protein refers to a functional protein (i.e., other than that of an enzyme or antibody), which has a therapeutic benefit in the treatment of cancer.
• suitable therapeutic proteins include, but are not limited to, rsCD40L (Eliopoulos et al . (2000) Mol.
• GM-CSF for the treatment of melanoma, breast carcinoma, colorectal carcinoma, glioblastoma, neuroblastoma, and prostate carcinoma
• IFNa for the treatment of ovarian carcinoma and solid tumors
• IL-2 for the treatment of neuroblastoma and ovarian carcinoma
• G-CSF for the treatment of breast carcinoma, bladder carcinoma, ovarian carcinoma (see, e.g., Omura, et al. (1996) Proc. Annu . Meet Am. Soc. Clin. Oncol. 15. ⁇ 755) .
• a therapeutic enzyme refers to an enzyme, which has a therapeutic benefit in the treatment of cancer.
• Therapeutic enzymes of particular use include enzymes capable of converting a nontoxic prodrug into a toxic drug which is cytotoxic to a tumor.
• 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) cycloprop-1 ' -yl] methyl ⁇ guanine
• HRP Horseradish peroxidase
• IAA Indole-3-acetic acid
• CPG2 carboxypeptidase G2 + 4— ( [2— chloroethyl] [2-mesyloxyethyl] amino) benzoyl-L-glutamic acid (CMDA) or + 4- [N, N-bis ( 2-iodoethyl ) amino] phenoxycarbonyl
• CPG2 carboxypeptidase G2
• a therapeutic antibody refers to an antibody that which has a therapeutic benefit in the treatment of cancer.
• suitable therapeutic antibodies include, but are not limited to, Atezolizumab (for the treatment of bladder cancer and breast cancer, NSCLC, and small cell lung cancer (SCLC) ) ; Avelumab (for the treatment of bladder cancer and Merkel cell carcinoma (MCC) ) ; Durvalumab (for the treatment of bladder cancer and NSCLC) ; Nivolumab (for the treatment of bladder cancer, colorectal cancer, kidney cancer, liver cancer, NSCLC, metastatic SCLC, Hodgkin lymphoma, and melanoma) ; Pembrolizumab (for the treatment of bladder cancer, cervical cancer, colorectal cancer, esophageal cancer, liver cancer, NSCLC, Hodgkin lymphoma, melanoma, and MCC) ; Bevacizumab (for the treatment of glioblastoma, cervical cancer, colorectal cancer,
• Methods of preparing a recombinant virus are known in the art. Briefly, to construct recombinant HSV, a gene of interest is cloned into a transfer plasmid. This plasmid is then co-transfected with HSV-1 genomic DNA (with a target gene replaced with HSV thymidine kinase gene) into rabbit skin cells. The progeny of the recombinant virus are selected and plaque-purified on 143 TK mutant cells in medium including of mixture 199V supplement with 100 pg of bromodeoxyuridine/ml and 2% fetal calf serum.
• the thymidine kinase gene is restored by co-transfection of progeny viral DNA and a plasmid encoding the thymidine kinase gene in HAT medium. Preparation of viral stocks and titrations of infectivity are done with Vero cells.
• this invention provides a method for treating a subject with cancer by administering to the subject, e.g.
• the recombinant HSV- 1 can be administered as the sole anticancer therapy, or in conjunction with a therapeutically effective amount of a second anticancer agent, such as radiation and/or chemotherapy.
• the method can also include the use of a target-specific moiety (e.g. , antibody or cell marker) suitable for targeted administration of the recombinant HSV-1 of the present invention to the desired tissue.
• the terms “treat,” “treating,” “treatment,” and the like refer to eliminating, reducing, relieving, reversing, and/or ameliorating a disease or condition and/or symptoms associated therewith, in this case treating cancer.
• Solid and non-solid tumors that can be treated in accordance with the method herein, include cancers of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, including squamous cell carcinoma; carcinoma, including thyroid and carcinomas of the skin; leukemia, including acute lymphocytic leukemia, acute lymphoblastic leukemia, acute and chronic myelogenous leukemia and promyelocytic leukemia; lymphoma including B cell lymphoma, T cell lymphoma, and Burkitt lymphoma; fibrosarcoma and rhabdomyosarcoma; melanoma; and neuroblastoma, astrocytoma and glioma.
• the cancer being treated in accordance with the method herein is a solid tumor.
• the cancer is selected from breast, liver, lung, skin (melanoma), brain, and colon cancer.
• a treatment can be orientated symptomatically, for example, to suppress symptoms. Treatment can be carried out over a short period, be oriented over a medium term, or can be a long-term treatment, for example within the context of a maintenance therapy .
• terapéuticaally effective amount refers to an amount of the active ingredient ( s ) that, when administered, is (are) sufficient, to efficaciously deliver the active ingredient ( s ) for the treatment of a condition or disease of interest to an individual in need thereof.
• the therapeutically effective amount of the agent may reduce (i.e., retard to some extent and preferably stop) unwanted cellular proliferation; reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., retard to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., retard to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve, to some extent, one or more of the symptoms associated with the cancer.
• the administered active ingredient ( s ) prevents growth and/or kills existing cancer cells, it may be cytostatic and/or cytotoxic.
• the recombinant HSV-1 of the invention can be used as is, provided via live carrier cells, or formulated in a pharmaceutical composition containing a pharmaceutically acceptable excipient.
• Pharmaceutical compositions provided herein can be specially formulated for intravenous administration in solid or liquid form or for intravenous injection.
• Optimal pharmaceutical compositions can be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences (19 th edition, 1995).
• the primary carrier or excipient in a pharmaceutical composition may be either aqueous or nonaqueous in nature.
• a suitable carrier or excipient may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
• Neutral-buffered saline or saline mixed with serum albumin are further exemplary vehicles.
• Pharmaceutical compositions can include Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor.
• Administration routes for the recombinant HSV-1, or pharmaceutical compositions of the invention include injection by intravenous, intraperitoneal , intracerebral (intra-parenchymal) , intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices.
• Compositions may be administered by bolus injection or continuously by infusion, or by implantation device.
• Compositions also can be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration .
• compositions of the invention can be delivered parenterally .
• the therapeutic compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution including the desired active ingredient ( s ) in a pharmaceutically acceptable vehicle.
• a particularly suitable vehicle for parenteral injection is sterile distilled water in which the active ingredient ( s ) is formulated as a sterile, isotonic solution, appropriately preserved.
• Preparation can involve the formulation of the desired active ingredient ( s ) with an agent, such as injectable microspheres, bio- erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid) , beads or liposomes, that may provide controlled or sustained release of the active ingredient ( s ) , which may then be delivered via a depot injection.
• an agent such as injectable microspheres, bio- erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid) , beads or liposomes, that may provide controlled or sustained release of the active ingredient ( s ) , which may then be delivered via a depot injection.
• Formulation with hyaluronic acid has the effect of promoting sustained duration in the circulation.
• Implantable drug delivery devices may be used to introduce the desired active ingredient ( s ) .
• This invention also includes methods for treating cancer by administering to an individual in need thereof the recombinant HSV-1 of the invention and one or more second therapeutic agents useful for the treatment of cancer.
• the recombinant HSV-1 and the second therapeutic agent can be administered simultaneously or sequentially.
• the recombinant HSV-1 and second therapeutic agent can be administered from a single composition or two separate compositions.
• the second therapeutic agent is administered in an amount to provide 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 ranges.
• the second therapeutic agent is an antibody.
• Suitable antibodies include, but are not limited to, Atezolizumab; Avelumab; Durvalumab; Nivolumab
• anti-PDl Pembrolizumab (anti-PDl) ; Bevacizumab; Dinutuximab; Pertuzumab; Trastuzumab; Cetuximab; Panitumumab; Ramucirumab; Alemtuzumab; Blinatumomab; Obinutuzumab; Ofatumumab; Rituximab; Necitumumab; Ipilimumab (anti-CTLA4) ; Daratumumab; Elotuzumab;
• the second therapeutic agent includes is a chemotherapeutic agent, radiotherapeutic agent, anti-angiogenic agent, apoptosis-inducing agent, anti-tubulin drug or a tumor-targeted chemotherapeutic agent, radiotherapeutic agent, anti-angiogenic agent, apoptosis-inducing agent or anti-tubulin drug.
• Exemplary second therapeutic agents include, but are not limited to, anti-angiogenic agents such as angiostatin, endostatin, vasculostatin, canstatin and maspin and anti-tubulin drugs such as colchicine, taxol, vinblastine, vincristine, vindescine, a combretastatin or a derivative or prodrug thereof.
• anti-angiogenic agents such as angiostatin, endostatin, vasculostatin, canstatin and maspin
• anti-tubulin drugs such as colchicine, taxol, vinblastine, vincristine, vindescine, a combretastatin or a derivative or prodrug thereof.
• second therapeutic agents include, but are not limited to, alkylating agents, nitrogen mustards, cyclophosphamide, trofosfamide , chlorambucil, nitrosoureas, carmustine (BCNU) , lomustine (CCNU) , alkylsulphonates , busulfan, treosulfan, triazenes, plant alkaloids, vinca alkaloids (vineristine, vinblastine, vindesine, vinorelbine) , taxoids, DNA topoisomerase inhibitors, epipodophyllins , 9-aminocamptothecin, camptothecin, crisnatol, mitomycins, mitomycin C, anti metabolites, anti-folates, DHFR inhibitors, trimetrexate, IMP dehydrogenase inhibitors, mycophenolic acid, tiazofurin, ribavirin, ElCAR, ribonuclotide reductas,
• Vero, HT-29, SW480, C32, A375, MDA-MB-231, 4T1, HepG2 and A549 cells were obtained from the American Type Culture Collection. Vero, S 480, C32, A375, MDA-MB-231 and A549 cells were propagated in Dulbecco ' s modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. HT-29, 4T1 and HepG2 cells were propagated in RPMI1640 supplemented with 10% fetal bovine serum. HSV-l(F) is a prototype HSV-1 strain used in this study (Ejercito, et al. (1968) J. Gen. Virol.
• Viral Infections were carried out at indicated multiplicities of infection (Verpooten, et al. (2009) J. Biol. Chem. 284:1097-1105). Cells were then harvested and processed for immunoblot, 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) . The cell viability was determined by CELLTITER-GLO® Luminescent Cell Viability Assay (Pro ega) according to the manufacture protocols.
• Vero and MDA-MB-231 cells were untreated or treated with human interferon-a (Sigma) , and 4T1 cells were treated with mouse interferon- (Sigma) for 20 hours. Cells were then infected with viruses and viral yields were determined at 48 hours post-infection .
• the membranes were rinsed in PBS and reacted with either donkey anti-rabbit or anti-mouse immunoglobulin conjugated to horseradish peroxidase and developed with an enhanced chemiluminescence western blot detection system kit (Amersham Pharmacia Biotechnology, Inc.).
• ELISA enzyme-linked immunosorbent assays
• supernatants of cell culture were collected to analyze IFN-a and Cxcl9 according to the manufacturer's instructions (R&D Systems).
• RMA Robust Multi-array Average
• Quantitative Real-Time PCR Assay Cells were mock- infected or infected with viruses. At 6 hours after infection, total RNA was harvested from cells using an RNase plus mini kit (Qiagen) and subjected to DNase I digestion (New England BioLabs) . cDNA was synthesized using a high capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative real-time PCR was performed using an Applied Biosystems ABI Prism 7900HT instrument with ABI SYBR® green master mix (Applied Biosystems) . Gene expression levels were normalized to endogenous control 18S rRNA. Relative gene expression was determined by the 2 DDet method (Schmittgen & Livak (2008) Nat. Protoc. 3:1101-8). Primers for each gene were chosen according to the recommendation of the qPrimerDepot database. Primer sequences are provided in Table 1.
• mice Five-week-old mice BALB/c mice were purchased from Harlan Sprague Dawley Inc. and housed under specific-pathogen-free conditions in a biosafety level 2 containment. All experimental procedures involving animals were approved by the institutional animal care and use committee of University of Illinois at Chicago. At 6 weeks of age, 1x10 s viable 4T1 cells suspended in 0.1 ml of PBS were inoculated subcutaneously into the right flank of mice (day -7) . When the tumor reached a volume of approximately 100 mm 3 eight days after, mice were randomly assigned into three groups for intra-tumor injections of Dgi34.5, DN146 or PBS on days 1, 3 and 6.
• Each tumor was injected slowly with a total of lxlO 7 PFU of virus or PBS in a volume of 0.1 ml.
• the tumor growth was monitored every other day by measuring two perpendicular tumor diameters with a digital caliper.
• mice were euthanized by C0 2 inhalation.
• Tissue Analysis On selected days after the last intratumor injection, six mice from each treatment group were sacrificed to collect the tumor, lung, liver, spleen and blood. To measure viral load, the samples were minced, homogenized and bead-beaten, freeze-thawed three times, and sonicated in DMEM. After centrifugation, the tumor supernatants were used for plaque assays. The supernatants from the lung, liver, spleen and blood were used for quantitative real-time PCR assay. Briefly, the supernatants were suspended in buffer containing 1% SDS, 50 mM Tris (pH 7.5), and 10 mM EDTA.
• viral DNA was extracted and quantified by real-time PCR using HSV-1 gD-specific primers: TACAACCTGACCATCGCTTG (SEQ ID NO: 21) and GCCCCCAGAGACTTGTTGTA (SEQ ID NO: 22) .
• lungs from mice were excised, and fixed in formalin.
• the number of lung metastases was quantified by counting under a light microscope .
• Example 3 Expression of the C-terminal Portion of gi34.5 Inhibits eIF2a Phosphorylation
• HSV infection proceeds in a temporal manner, with sequential expression of , b, and g genes.
• Onset of viral DNA replication invokes the cessation of protein synthesis in the absence of gi34.5 (Chou & Roizman (1992) Proc. Natl. Acad. Sci. USA 89:3266-70) .
• To assess the impact of DN146 expression of representative proteins ICP27 (a protein) and gC (g protein) was measured as the expression of these proteins relies on viral DNA replication.
• Cells were mock- infected or infected with HSV-1, Dgi34.5 or DN146 virus and at 12 hours post-infection, samples were subjected to western blot analysis.
• transcriptome analysis in 4T1 cells was carried out. It was observed that numerous genes in diverse cellular pathways were expressed differentially in 4T1 cells mock infected and infected with viruses. Of note, many genes in the innate immune pathways were evidently up-regulated in response to DN146. Among the 46 genes tested, most remained unchanged or marginally expressed in cells mock infected or infected wild-type virus. However, they were upregulated in cells infected with Dgi34.5, albeit to a different extent. Notably, gene induction was more pronounced in cells infected with DN146, indicating that DN146 has a propensity to stimulate the inflammatory response.
• Type I IFN is necessary to prime immunity against a tumor. On the other hand, it mediates antiviral responses.
• DN146 consistently replicated 500- to 1000-fold higher than Dgi34.5 in the presence of exogenous IFN-a.
• amino-acids 147-263 from Yi34.5 are sufficient to confer viral resistance to IFN.
• Example 6 DN146 Reduces Primary Tumor Growth and
• Tumors established subcutaneously in the flank of mice were thrice injected with PBS, Dgi34.5, DN146 or EUsll (lxlO 7 pfu) on days 1, 3, and 6. Tumor size was then monitored. As illustrated in FIG. 1, control tumors treated with PBS grew at a faster rate over time. Treatment with gi34.5 null virus marginally reduced local tumor growth. However, intra-tumor inoculation with DN146 or EUsll markedly slowed tumor growth and a reduction in tumor size became more apparent as treatment progressed. On day 24, DN146 as well as EUsll reduced the tumor size by nearly 45% as compared to the mock control or Dgi34.5. Hence, while comparable to EUsll, DN146 displayed superior activity against primary tumors when compared with Dgi34.5.
• FIG. 2 shows that pulmonary metastasis was readily detectable in control mice, with an average of 25 nodules per animal as measured by microscopic analysis.
• Treatment with Dgi34.5 or EUsll reduced incidence, with an average of 15 nodules per animal.
• DN146 further reduced metastatic burden, with an average of 10 nodules.

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• 2019
  • 2019-06-07 US US17/054,338 patent/US12329794B2/en active Active
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  • 2019-06-07 CN CN201980038180.3A patent/CN112243378B/zh active Active
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  • 2019-06-07 EP EP19733912.0A patent/EP3801583B1/en active Active
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  • 2019-06-07 WO PCT/US2019/035922 patent/WO2019236931A1/en not_active Ceased
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