HSV vectors having improved safety profiles

Abstract

Disclosed are improved Disabled Infectious Single Cycle (DISC) Herpes Simplex Virus (HSV) and their uses in the treatment of cancer or as a vaccine. Also disclosed are improved methods of making DISC HSV.

Description

HSV VECTORS HAVING IMPROVED SAFETY PROFILES FIELD OF THE INVENTION

[0001] This disclosure relates to HSV vectors having improved safety profiles for oncolytic, vaccine and gene therapy uses. BACKGROUND OF THE INVENTION

[0002] HSV is a promising vector system for vaccine, oncology and gene therapy applications. For vaccines, HSV-based vectors have been engineered in several ways to create live attenuated vaccine vectors for the prophylaxis of HSV-1 and HSV-2 infection in humans. These HSV-based vaccine vectors can also encode heterologous antigens to generate protective immune responses to other human pathogens, such as influenza, HIV, malaria, foot and mouth disease virus, and SARS-CoV-1. For oncology, oncolytic HSV (o-HSV) vectors have also been engineered in several ways to specifically infect and / or efficiently replicate in tumor cells compared to normal cells. For gene therapy, several multi-mutated vectors have been developed pre-clinically to deliver transgenes to cells.

[0003] The process by which HSV enters a target cell is intricate, involving multiple stages, with essential interactions and changes in structure across various viral glycoproteins such as gD, gH / gL, and gB. These proteins are part of the virus's outermost lipid bilayer, known as the envelope. The initial interaction with the cell is facilitated by the binding of gC and gB to heparan sulphate on the cell surface. Following this, gD binds to cell receptors nectin-1 or HVEM, leading to structural changes that set off a cascade of events, ultimately causing the viral envelope to merge with the cell membrane through the fusogenic action of gB.

[0004] HSV-based vaccine vectors have been developed using multiple engineering strategies. These generally involve mutating an essential viral function responsible for efficient lytic replication or the generation of infectious viral progeny. For example, mutations in the key viral transcription factor ICP4 ensure the virus is unable to complete its lytic replication program and produce the progeny virus necessary for spread from the site of inoculation. Another strategy is to ensure the virus efficiently completes itsreplication program only once and the progeny virus that are produced by cells infected at the site of inoculation are unable to spread. These are termed Disabled Infectious Single Cycle (DISC) vectors. One strategy to create a DISC HSV for vaccine purposes is to delete the essential viral glycoprotein gD from the vector genome. To generate an infectious drug product despite deletion of gD, these DISC viruses are pseudotyped with gD by manufacturing them using complementing cells encoding gD. These gD- complementing cells express gD when infected with a gD-null vector and gD localizes to the cell surface. Because the lipid bilayer of HSV is derived from the cellular membrane, gD is present on the surface of these manufactured viruses even though their genomes do not encode gD. However, HSV is notoriously recombinogenic and attenuated vectors deleted for essential viral genes are well known to capture these genes from complementing cells during production. This can result in contamination of the gD-deleted DISC drug product with pathogenic HSV.

[0005] o-HSVs have been genetically engineered in two general ways to achieve tumor- specific replication and spread. Firstly, the neuro-virulence gene g34.5 has been deleted or otherwise conditionally expressed using tumor-specific promoters. g34.5 is a multi- functional protein with roles in regulating host cell protein synthesis and autophagy. g34.5 promotes protein synthesis in virally infected cells by binding protein phosphatase 1a and directing it to dephosphorylate the critical host cell translation initiation factor eIF2a. By maintaining pools of de-phosphorylated eIF2a, g34.5 counteracts host cell TypeI Interferon responses that lead to protein synthesis shutdown in infected cells through the activation of the cellular kinase PKR, which phosphorylates eIF2a in order to block the synthesis of viral proteins and the subsequent assembly of progeny viruses necessary for spread through an infected tissue. o-HSV attenuated through g34.5 inactivation selectively replicates in tumor cells that have compromised TypeI interferon signaling and do not efficiently replicate in normal tissues, which maintain full responsiveness to TypeI Interferons. This ensures the virus is able to spread through groups of cancer cells, but not normal tissue, which would cause toxicity. o-HSV that encode g34.5 under the control of a tumor-specific promoter demonstrate similar selectivity for tumor cells over normal cells because g34.5 is not efficiently expressed by these vectors in normal cells. Inessence, the virus is phenotypically g34.5-null in normal tissues, so is unable to efficiently spread through normal tissues and cause toxicity.

[0006] The tropism of o-HSV can be engineered such that the vector is unable to infect cells through its cognate receptors such as Nectin-1 and HVEM. This is termed de- targeting and is achieved by deleting or otherwise inactivating the Receptor Binding Domain (RBD) of gD responsible for binding the cognate receptors Nectin-1 and HVEM. To facilitate infection of cancer cells, researchers have developed several ways of re- targeting de-targeted HSV to cancer cells. One way is to engineer ectopic binders such as scFv or VHH into the genome of de-targeted HSV-based vectors. These ectopic binders attach to cell surface Tumor Associated Antigens (TAA) such as Her2, EGFR, and PSMA, and mediate infection of cells expressing these proteins. However, many normal cells express low levels of TAAs and there remains the risk of uncontrolled viral spread through these normal tissues for de-targeted, TAA-re-targeted o-HSV.

[0007] To date, no one has been able to generate a therapeutic virus strategy for oncology where the virus replicates and spreads efficiently in both cancer cells and normal cells in the tumor, but not normal cells outside the tumor, which is necessary for these vectors to remain safe. We term this new class of therapeutic viruses for oncology “Tumolytic Viruses” or “Tumolytics” to differentiate from the class of “Oncolytic Viruses” or “Oncolytics,” which efficiently replicate only in cancer cells. Such “Tumolytics,” which are fully replication competent vectors directed to infect, efficiently kill, and spread through both cancer cells and normal cells in tumors, rather than just cancer cells, may result in better therapeutic viruses for the treatment of cancer. These viruses can also be used for vaccine and gene therapy purposes. BRIEF SUMMARY OF THE INVENTION

[0008] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, andadvantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.

[0009] The invention relates to novel therapeutic HSV viruses that have an improved safety profile due to the method in which the viruses are manufactured, and methods of preventing and / or treating various diseases and medical conditions involving the administration of such HSV viruses.

[0010] Specifically, as described herein is a method of treating a patient suffering from a cancer, wherein said method comprises: administering to the patient a Disabled Infectious Single Cycle (DISC) Herpes Simplex Virus (HSV) combined with a first targeting adapter, wherein said DISC HSV comprises a lipid bilayer comprising a tag and lacking a functional Receptor Binding Domain (“RBD”) of gD and a deleted Receptor Binding Domain (“ΔRBD”) HSV comprising a HSV genome lacking a functional RBD of gD and a nucleic acid sequence encoding a heterologous polynucleotide; and wherein said first targeting adapter comprises a first binding domain with binding specificity to the tag and a second binding domain with binding specificity to a first tumor associated antigen (“TAA”) localized on the surface of a cancer cell; and administering a second targeting adapter to the patient, wherein said second targeting adapter comprises a first binding domain with binding specificity to the tag and a second binding domain with binding specificity to a second TAA localized on the surface of the cancer cell.

[0011] For this method, the second targeting adapter can be administered at least 2 hours, at least 4 hours, at least 12 hours, at least 24 hours, daily, weekly, or monthly after administration of the DISC HSV and the first targeting adapter.

[0012] In this method, the first and the second TAA can be selected from prostate specific membrane antigen (PSMA), TMEFF2, ROR1, KLK2, HLA-G, CD70, PD-1, PD-L1, CTLA- 4, EGFR, FLT3, HER- 2, CD19, CD20, CD3, mesothelin (MSLN), prostate stem cell antigen (PCSA), B-cell maturation antigen (BCMA or BCM ), G-protein coupled receptor family C group 5 member D (GPRC5D), Interleukin-1 receptor accessory protein (IL1RAP), delta-like 3 (DLL3), carbonic anhydrase IX (CAIX), carcinoembryonic antigen(CEA), CD5, CD7, CD10, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD 138, epithelial glycoprotein-2 (EGP 2), epithelial glycoprotein- 40 (EGP-40), epithelial adhesion molecule (EpCAM), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor a and b (FRa and b), ganglioside G2 (GD2), ganglioside G3 (GD3), epidermal growth factor receptor (EGFR), epidermal growth factor receptor vIII (EGFRvIII), ERB3, ERB4, interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), k-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), LI cell adhesion molecule (LICAM), melanoma-associated antigen 1 (melanoma antigen family Al, MAGE-A1), Mucin-16 (Muc-16), Mucin 1 (Muc-1), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor R2 (VEGF-R2), type 1 tyrosine-protein kinase transmembrane receptor (ROR1), B7-H3 (CD276), B7-FI6 (Nkp30), chondroitin sulfate proteoglycan-4 (CSPG4), DNAX accessory molecule (DNAM-1), ephrin type A receptor 2 (EpHA2), fibroblast associated protein (FAP), Gpl 00 / HLA-A2, glypican 3 (GPC3), HA-1H, HERK- V, IL-llRa, latent membrane protein (LMPl), neural cell-adhesion molecule (N- CAM / CD56), and trail receptor (TRAIL R). Moreover, the first TAA and said second TAA can be the same or different proteins. If necessary a third or even a fourth TAA can be used.

[0013] In this method, the heterologous polynucleotide can encode an immunomodulatory polypeptide, such as a granulocyte macrophage colony-stimulating factor (GM-CSF), Flt3L, tumor necrosis factor (TNF)-alpha, CD40 ligand (CD40L), IL-1, IL-2, IL-3, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, G-CSF, IFN-α, IFN-β, IFN-γ, IL-20 (MDA-7), IL-21, IL-23, IL-24, or a co-stimulatory molecules, such as B7-1 (CD80) and B7-2 (CD86), a chemokine such as RANTES or a macrophage inflammatory protein (MIP) (for example, MIP-3), or an immune checkpoint blocker such as polypeptides that block signaling through PD-1, CTLA-4 or PDL-1.

[0014] Alternatively or additionally, the heterologous polynucleotide could also encode a prodrug converting enzyme, such as a cytosine deaminase enzyme, uracil phosphoribosyltransferase, or a HSV-1 thymidine kinase or even a matrix degradingenzyme, such as a matrix metalloproteinases, a collagenase, a gelatinase, a stromelysin, a relaxin, a bacterial collagenase or a chondroitinase ABC. The heterologous polynucleotide could also encode for a fusogenic protein, such as an envelope glycoprotein derived from gibbon ape leukemia virus (GALV), a human endogenous retrovirus W, a fusogenic F or H protein from measles virus, a vesicular stomatitis virus G protein, or a GALV fusogenic glycoprotein. Additionally the heterologous polynucleotide encodes for a messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), antisense RNA, guide RNA, microRNA (miRNA), small interfering RNA (siRNA), and cell-free RNA (cfRNA).

[0015] In other embodiments, the second targeting adapter is a masked targeting adapter, which preferably, is administered intravenously, intralymphatically, or subcutaneously.

[0016] To improve the half-life of the adapter, either the first targeting adapter and / or the second targeting adapter further comprises a half-life extender (“HLE”), such as a polypeptide that binds human serum albumin (HSA), an antibody Fc (Ig Fc), fusion of the targeting adaptor to HSA, albumin-binding domain fusion, lipidation, hyperglycosylation, PEGylation, synthetic PEG alternatives (e.g., polyglycerol, poly(thioglycidylglycerol), polysarcosine, poly(carboxybetaine), natural carbohydrates (e.g., PolyXen, HEPtune, HESylation, Chonylation, HAylation), recombinant PEG alternatives (e.g., PASylation, XTEN, PsTag, ELPylation, Ekylation), Gelatin-like polypeptides, GeneticPolymer, or HAPylation.

[0017] Cancer can be treated using the described methods. Examples of cancer include, but are not limited to ovarian cancer, a breast cancer, a cervical cancer, a lung cancer, a prostate cancer, a gastric cancer, a colon cancer, an esophageal cancer, a pancreatic cancer, a bile duct cancer, a cholangiocarcinoma, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, a head and neck cancer, a metastatic melanoma, a glioma, a glioblastoma, and other solid tumors, as well as a non-Hodgkin’s lymphoma (NHL), a chronic myelogenous leukemia (CML), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a multiple myeloma (MM), an acute myeloid leukemia (AML), and other liquid tumors.

[0018] Alternatively, as described herein is a method of vaccinating a patient in need thereof, wherein said method comprises administering to the patient a DISC HSV combined with a targeting adapter, wherein said DISC HSV comprises a lipid bilayer comprising a tag and lacking a functional RBD of gD; and a ΔRBD HSV comprising a HSV genome lacking a functional RBD of gD and a nucleic acid sequence encoding an antigen; and wherein said targeting adapter comprises a first binding domain with binding specificity to the tag and a second binding domain with binding specificity to a cellular protein localized on the surface of a cell. In preferred embodiment, the ΔRBD HSV further comprises a nucleic acid sequence encoding an antigen.

[0019] Examples of such antigens include those capable of causing an allergic response, such as a response caused by allergens from pollen (such as birch pollen (e.g., Bet v 1), ragweed pollen (e.g., Amb a 1), and grass pollen (e.g., Phl p 5)), dust mites, such as those from European house dust mites (e.g., Der p 1, Der p 2) and American house dust mites (e.g., Der f 1, Der f 2); animal dander, notably from cats (e.g., Fel d 1) and dogs (e.g., Can f 1, Can f 2); mold spores, including those from Alternaria alternata (e.g., Alt a 1) and Aspergillus fumigatus (e.g., Asp f 1); foods such as peanuts (e.g., Ara h 1, Ara h 2, Ara h 3), eggs (e.g., Gal d 1, Gal d 2), and milk (e.g., casein, beta-lactoglobulin); insect stings, specifically bee venom (e.g., Api m 1) and wasp venom (e.g., Ves v 5); medications like penicillin (e.g., benzylpenicilloyl polylysine) and aspirin, which, although not a protein, can trigger immune responses; and latex, such as natural rubber latex components (e.g., Hev b 1, Hev b 3, Hev b 5, Hev b 6.02). Alternatively, encoded antigens are capable of raising an immune response to an infection, preferably wherein the infection is caused by a HSV, a respiratory syncytial virus (RSV), adenovirus, anthrax, cholera, a measles, mumps, rubella, yellow fever, typhoid, rotavirus, BCG, varicella zoster, whole-cell pertussis, polio, influenza, Japanese encephalitis, hepatitis A, rabies, Diphtheria, tetanus, hepatitis B, meningococcal, pneumococcal, Human papillomavirus, Group B meningococcal, Haemophilus influenzae type B, ebola, SARS-CoV2, Mycobaterium tuberculosis, M. ulcerous, M. marinum, M. leprae, M. absenscens, Chlamydia trachomatis, Neisseria gonorrhoeae or Treponema pallidum.

[0020] In other embodiments, the cellular protein localized on the surface of a cell is Nectin-1, HVEM, FLT3, prostate specific membrane antigen (PSMA), TMEFF2, ROR1, KLK2, HLA-G, CD70, PD-1, PD-L1, CTLA-4, EGFR, HER- 2, FAP, Pit1, CD19, CD20, CD3, mesothelin (MSLN), prostate stem cell antigen (PCSA), B-cell maturation antigen (BCMA or BCM ), G-protein coupled receptor family C group 5 member D (GPRC5D), Interleukin-1 receptor accessory protein (IL1RAP), delta-like 3 (DLL3), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD47, CD49f, CD56, CD74, CD123, CD133, CD 138, epithelial glycoprotein-2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial adhesion molecule (EpCAM), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor a and b (FRa and b), ganglioside G2 (GD2), ganglioside G3 (GD3), epidermal growth factor receptor (EGFR), epidermal growth factor receptor vIII (EGFRvIII), ERB3, ERB4, interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), k-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), LI cell adhesion molecule (LICAM), melanoma-associated antigen 1 (melanoma antigen family Al, MAGE-A1), Mucin-16 (Muc-16), Mucin 1 (Muc-1), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), tumor-associated glycoprotein 72 (TAG- 72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor R2 (VEGF-R2), type 1 tyrosine-protein kinase transmembrane receptor (ROR1), B7-H3 (CD276), B7-FI6 (Nkp30), chondroitin sulfate proteoglycan-4 (CSPG4), DNAX accessory molecule (DNAM-1), ephrin type A receptor 2 (EpHA2), fibroblast associated protein (FAP), Gpl 00 / HLA-A2, glypican 3 (GPC3), HA-1H, HERK-V, IL-llRa, latent membrane protein (LMPl), neural cell-adhesion molecule (N-CAM / CD56), and trail receptor (TRAIL R).

[0021] The vaccination methods disclosed herein can be used to treat HSV, a respiratory syncytial virus (RSV), adenovirus, anthrax, cholera, a measles, mumps, rubella, yellow fever, typhoid, rotavirus, BCG, varicella zoster, whole-cell pertussis, polio, influenza, Japanese encephalitis, hepatitis A, rabies, Diphtheria, tetanus, hepatitis B, meningococcal, pneumococcal, Human papillomavirus, Group B meningococcal, Haemophilus influenzae type B, ebola, SARS-CoV2, Mycobaterium tuberculosis, M.ulcerous, M. marinum, M. leprae, M. absenscens, Chlamydia trachomatis, Neisseria gonorrhoeae or Treponema pallidum.

[0022] Alternatively, the vaccination methods disclosed herein can be used an allergic response, preferably caused by allergens from pollen (such as birch pollen (e.g., Bet v 1), ragweed pollen (e.g., Amb a 1), and grass pollen (e.g., Phl p 5)), dust mites, such as those from European house dust mites (e.g., Der p 1, Der p 2) and American house dust mites (e.g., Der f 1, Der f 2); animal dander, notably from cats (e.g., Fel d 1) and dogs (e.g., Can f 1, Can f 2); mold spores, including those from Alternaria alternata (e.g., Alt a 1) and Aspergillus fumigatus (e.g., Asp f 1); foods such as peanuts (e.g., Ara h 1, Ara h 2, Ara h 3), eggs (e.g., Gal d 1, Gal d 2), and milk (e.g., casein, beta-lactoglobulin); insect stings, specifically bee venom (e.g., Api m 1) and wasp venom (e.g., Ves v 5); medications like penicillin (e.g., benzylpenicilloyl polylysine) and aspirin, which, although not a protein, can trigger immune responses; and latex, such as natural rubber latex components (e.g., Hev b 1, Hev b 3, Hev b 5, Hev b 6.02).

[0023] Also disclosed is a method of making a tagged ΔRBD HSV, comprising; propagating a ΔRBD HSV in a null RBD (“ØRBD”) HSV manufacturing cell in the presence of a manufacturing adapter, wherein said ΔRBD HSV comprises a HSV genome lacking a functional RBD of gD; wherein said ØRBD HSV manufacturing cell lacks a functional RBD; wherein either the ΔRBD HSV or the ØRBD HSV manufacturing cell encodes a tag; and wherein said manufacturing adapter assists infection of the ΔRBD HSV with the ØRBD HSV manufacturing cell; and recovering the tagged ΔRBD HSV.

[0024] In these methods, the manufacturing adapter can be an artificial receptor or a bispecific antibody and the ΔRBD HSV can further comprise a nucleic acid sequence encoding a heterologous polynucleotide. The method of making can be extended to combining the recovered tagged ΔRBD HSV with a targeting adapter to produce the DISC HSV.

[0025] In preferred embodiments, the ØRBD HSV manufacturing cell is a Vero, a A549, a MRC, a 293 or a CHO cell.

[0026] In alternative embodiments, thea) protein expressed on the ØRBD HSV manufacturing cell’s surface comprises Nectin, HVEM, Pit1, CD47, EGFR; or a wildtype or engineered protein derived from a non-human species; b) the manufacturing adapter is an anti-GCN4 H6 scFv fused to the AA 146-517 of human Nectin-1 (Uniprot Q15223), anti-gD or anti-gH or anti-gB scFv / VHH fused to AA 146-517 of human Nectin; or c) the manufacturing adapter is capable of binding to (a) a GCN4 transcription factor or a fragment thereof; (b) a La protein or fragment thereof; (c) a leucine- zipper moiety; (d) HSA or (e) CD3.

[0027] As described herein, the manufacturing adapter can be exogenously provided, can be encoded by the ØRBD HSV manufacturing cell line, can be encoded by an extrachromosomal sequence in the ØRBD HSV manufacturing cell; or can be encoded by ΔRBD HSV.

[0028] In all embodiments, the ΔRBD HSV can lack a polynucleotide sequence encoding amino acids 6-38 of gD, a polynucleotide sequence encoding amino acids 6-24 of gD, a polynucleotide encoding amino acids 6-24 and 38 of gD; or a polynucleotide encoding a polypeptide comprising full-length gD or fragment thereof, wherein said fragment comprises amino acids 6-38 of gD.

[0029] As is described herein, the lipid bilayer can lack a polypeptide comprising amino acids 6-38 of gD, a polypeptide comprising amino acids 6-24 of gD, a polypeptide comprising amino acids 6-24 and 38 of gD; or a polypeptide comprising-full length gD or fragment thereof, wherein said fragment comprises amino acids 6-38 of gD.

[0030] In all embodiments, the ΔRBD HSV can further comprise a polynucleotide sequence that encodes for the tag. This tag can be inserted into the RBD of gD.

[0031] In preferred embodiments, the tag is selected from GCN4, Her2, CD3, CD28, PDLI, PD-1, CTLA4, TIGIT, OX40, CD40, ICOS, 4-1BB, CD47, TIGIT, VISTA, ROR1, HLA-G, HLA-E, TROP2, Tissue Factor, Nectin-4, Fibroblast Activation Protein (FAP), Folate receptor alpha, CD19, CD22, CD30, CD33, CD38, CD70, CD79b, BCMA, PSMA,KLK2, Mesothelin, NKG2A, MAGE-A1, MAGE-A4, HSA, or any other peptide that is capable of being bound by the targeting adapter. Other examples of targeting adapters can be found, for example, in US20240226207 which is hereby incorporated by reference in its entirety.

[0032] As described herein, the targeting adapter can be covalently linked to the tag, is a masked adapter, is combined with the tagged ΔRBD HSV immediately prior to the administration of the DISC HSV, is packaged in a single vial with the tagged ΔRBD HSV, is manufactured along with the tagged ΔRBD HSV; and / or is provided exogenously.

[0033] Also disclosed herein is a Disabled Infectious Single Cycle (DISC) Herpes Simplex Virus (HSV) comprising (a) a tagged ΔRBD HSV, wherein said tagged ΔRBD HSV comprises (i) a ΔRBD HSV, wherein said ΔRBD HSV comprises a HSV genome lacking a polynucleotide sequence encoding amino acids 6-38 of gD; and (ii) a lipid bilayer comprising a tag; and (b) a targeting adapter comprising a first binding domain with binding specificity to the tag and a second binding domain with binding specificity to a cellular protein localized on the surface of a cell. In embodiments, the lipid bilayer further lacks a polypeptide comprising amino acids 6-38 of gD or a polypeptide comprising wild- type gD or fragment thereof, wherein said fragment comprises amino acids 6-38 of gD.

[0034] In some embodiments, the ΔRBD HSV as described herein comprises: (a) the absence of a polynucleotide sequence encoding a fragment of gD, so long as the fragment comprises at least amino acids 6-38 of gD; (b) the absence of a polynucleotide sequence encoding the full-length wild type gD; (c) the absence of a polynucleotide sequence encoding the targeting adapter; (c) a polynucleotide sequence capable of encoding the tag; (d) the replacement of the polynucleotide encoding 6-38 of gD with a polynucleotide sequence capable of encoding the tag; (e) a polynucleotide sequence capable of encoding a manufacturing adapter; and / or (f) a polynucleotide sequence capable of encoding a heterologous polypeptide.

[0035] As described herein, the manufacturing adapter is a bispecific adapter that binds to an endogenous protein expressed on the cell surface of a ØRBD HSV manufacturing cell line and to the tag. Alternatively, the manufacturing adapter is a bispecific adapterthat binds to a heterologous protein expressed on the cell surface of a ØRBD HSV manufacturing cell line and to the tag. In either of these embodiments, the heterologous polypeptide can be derived from an antigen (such as an allergen or tumor associated antigen), an immunomodulatory polypeptide, a prodrug converting enzyme, a matrix degrading enzyme, and / or a fusogenic protein.

[0036] As is also described herein, the tag and / or the manufacturing adapter is expressed using an inducible promoter. Additionally, the endogenous protein expressed on the cell surface of a ØRBD HSV manufacturing cell line is selected from: Nectin, HVEM, Pit1, CD47, or EGFR. The heterologous protein expressed on the cell surface of a ØRBD HSV manufacturing cell line may also be an anti-GCN4 H6 scFv fused to the AA 146-517 of human Nectin-1 (Uniprot Q15223) separated by a G4S linker, anti-gD or anti-gH or anti- gB scFv / VHH fused to AA 146-517 of human Nectin. Alternatively, the heterologous protein expressed on the cell surface of a ØRBD HSV manufacturing cell line is any wildtype or engineered protein derived from a non-human species.

[0037] In certain embodiments, the DISC HSV comprises an inducible promoter in a rtTA, ARGENT, RheoSwitch, or an abscidic acid-inducible promoter. In other embodiments, the bispecific adapter is capable of binding to (a) a GCN4 transcription factor or a fragment thereof; (b) a La protein or fragment thereof; (c) a leucine-zipper moiety; (d) CD3; or (e) HSA.

[0038] In more specific embodiments, the GCN4 transcription factor (SEQ ID NO: 20) is targeted by an anti-GCN4 scFv, such as one comprising a VH comprised of HCDR1 (SEQ ID NO: 4), HCDR2 (SEQ ID NO: 5), and HCDR3 (SEQ ID NO: 6) and / or a VL comprised of LCDR1 (SEQ ID NO: 7), LCDR2 (SEQ ID NO: 8), and LCDR3 (SEQ ID NO: 9) or an anti-GCN4 scFv comprising a VH having a polypeptide sequence at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 10 and / or a VL having a polypeptide sequence at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 11.

[0039] In other embodiments, a La protein (SEQ ID NO: 3) or fragment thereof is targeted by an anti-La scFv, such as one comprising a VH comprised of HCDR1 (SEQ ID NO: 12),HCDR2 (SEQ ID NO: 13), and HCDR3 (SEQ ID NO: 14) and / or a VL comprised of LCDR1 (SEQ ID NO: 15), LCDR2 (SEQ ID NO: 16), and LCDR3 (SEQ ID NO: 17) or an anti-La scFv comprising a VH having a polypeptide sequence at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 18 and / or a VL having a polypeptide sequence at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 19.

[0040] In other embodiments, a leucine zipper moiety can be targeted by a synthetic leucine-zipper moiety RE (SEQ ID NO: 1) or synthetic leucine-zipper moiety ER (SEQ ID NO: 2); or a synthetic leucine-zipper moiety RE (SEQ ID NO: 1) and the second leucine- zipper moiety is synthetic leucine-zipper moiety ER (SEQ ID NO: 2), or the first leucine- zipper moiety is synthetic leucine-zipper moiety ER (SEQ ID NO: 2) and the second leucine-zipper moiety is synthetic leucine-zipper moiety RE (SEQ ID NO: 1).

[0041] In other embodiments, the targeting adapter comprises a first binding domain capable of binding to GCN4, Her2, CD3, CD28, PDLI, PD-1, CTLA4, TIGIT, OX40, CD40, ICOS, 4-1BB, CD47, TIGIT, VISTA, ROR1, HLA-G, HLA-E, TROP2, Tissue Factor, Nectin-4, Fibroblast Activation Protein (FAP), Folate receptor alpha, CD19, CD22, CD30, CD33, CD38, CD70, CD79b, BCMA, PSMA, KLK2, Mesothelin, NKG2A, MAGE-A1, MAGE-A4, HSA or any other peptide that is capable of being bound by the targeting adapter.

[0042] Additionally, the targeting adapter comprises a second binding domain. This second binding domain is capable of binding to a protein expressed on the surface of a cell, including a cancer cell, a normal cell, and / or both types of cells.

[0043] In further embodiments, the targeting adaptor comprises a second binding domain capable of binding to Nectin-1, HVEM, FLT3, prostate specific membrane antigen (PSMA), TMEFF2, ROR1, KLK2, HLA-G, CD70, PD-1, PD-L1, CTLA-4, EGFR, HER- 2, FAP, Pit1, CD19, CD20, CD3, mesothelin (MSLN), prostate stem cell antigen (PCSA), B- cell maturation antigen (BCMA or BCM ), G-protein coupled receptor family C group 5 member D (GPRC5D), Interleukin-1 receptor accessory protein (IL1RAP), delta-like 3 (DLL3), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7,CD10, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD47, CD49f, CD56, CD74, CD123, CD133, CD 138, epithelial glycoprotein-2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial adhesion molecule (EpCAM), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor a and b (FRa and b), ganglioside G2 (GD2), ganglioside G3 (GD3), epidermal growth factor receptor (EGFR), epidermal growth factor receptor vIII (EGFRvIII), ERB3, ERB4, interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), k-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), LI cell adhesion molecule (LICAM), melanoma-associated antigen 1 (melanoma antigen family Al, MAGE-A1), Mucin-16 (Muc-16), Mucin 1 (Muc-1), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor R2 (VEGF-R2), type 1 tyrosine-protein kinase transmembrane receptor (ROR1), B7-H3 (CD276), B7-FI6 (Nkp30), chondroitin sulfate proteoglycan-4 (CSPG4), DNAX accessory molecule (DNAM-1), ephrin type A receptor 2 (EpHA2), fibroblast associated protein (FAP), Gpl 00 / HLA-A2, glypican 3 (GPC3), HA-1H, HERK- V, IL-llRa, latent membrane protein (LMPl), neural cell-adhesion molecule (N- CAM / CD56), and trail receptor (TRAIL R).

[0044] In further embodiments, the targeting adapter is a masked targeting adapter.

[0045] Methods of making the tagged ΔRBD HSV are also included as described herein. For example, one method of making a tagged ΔRBD HSV that has an improved safety profile, including, propagating the ΔRBD HSV as described herein in a ØRBD HSV manufacturing cell; and recovering a tagged ΔRBD HSV. Examples of cells that can be used, include, but are not limited to a Vero, a A549, a MRC, a 293 or a CHO cell.

[0046] As described herein, the method of making the tagged ΔRBD HSV occurs where the polynucleotide encoding the tag is (a) expressed from an extrachromosomal sequence in the ØRBD HSV manufacturing cell; (b) inserted into the ØRBD HSV manufacturing cell genome; and / or (c) inserted in the ΔRBD HSV. Similarly, the polynucleotide encoding the bispecific adapter used in manufacturing can be (a) expressed from an extrachromosomal sequence in the ØRBD HSV manufacturing cell; and / or (b) inserted into the ØRBD HSV manufacturing cell genome; and / or (c) inserted inthe ΔRBD HSV. In other embodiments, the polynucleotide encoding the artificial receptor used in manufacturing is (a) expressed from an extrachromosomal sequence in the ØRBD HSV manufacturing cell; and / or (b) inserted into the ØRBD HSV manufacturing cell genome. Finally, as described herein, the targeting adapter is preferably produced separately from the tagged ΔRBD HSV.

[0047] As described herein, the tagged ΔRBD HSV produced by the described methods are also contemplated. These tagged ΔRBD HSV have improved safety profile by being generated using ØRBD HSV manufacturing cell lines.

[0048] In further embodiments, the tagged ΔRBD HSV is combined with a targeting adapter to produce the DISC HSV. In some embodiments, the targeting adapter is covalently linked to the tag, using covalent binders, such as SpyTagTMand SpyCatcherTM. In some embodiments, the targeting adapter is a masked adapter as described herein.

[0049] In some embodiments, (a) the targeting adapter and the tagged ΔRBD HSV are manufactured together; and / or (b) the targeting adapter and the tagged ΔRBD HSV are manufactured separately; and / or (c) the combining of the targeting adapter and the tagged ΔRBD HSV is performed immediately prior to administration of the DISC HSV to the subject; and / or (d) the tagged ΔRBD HSV and the bispecific adapter are packaged in a single vial.

[0050] The DISC HSV produced by the methods described herein are also contemplated.

[0051] Uses of the DISC HSV are also contemplated, such as for the use as a vaccine, an oncolytic or a tumolytic. For example, as a vaccine, the DISC HSV can be used to treat infection, preferably caused by a HSV, a respiratory syncytial virus (RSV), adenovirus, anthrax, cholera, a measles, mumps, rubella, yellow fever, typhoid, rotavirus, BCG, varicella zoster, whole-cell pertussis, polio, influenza, Japanese encephalitis, hepatitis A, rabies, Diphtheria, tetanus, hepatitis B, meningococcal, pneumococcal, Human papillomavirus, Group B meningococcal, Haemophilus influenzae type B, ebola, SARS-CoV2, Mycobaterium tuberculosis, M. ulcerous, M. marinum, M. leprae, M. absenscens, Chlamydia trachomatis, Neisseria gonorrhoeae or Treponema pallidum

[0052] Alternatively, the DISC HSV can also be used as a vaccine to treat an allergic response, preferably caused by allergens from pollen (such as birch pollen (e.g., Bet v 1), ragweed pollen (e.g., Amb a 1), and grass pollen (e.g., Phl p 5)), dust mites, such as those from European house dust mites (e.g., Der p 1, Der p 2) and American house dust mites (e.g., Der f 1, Der f 2); animal dander, notably from cats (e.g., Fel d 1) and dogs (e.g., Can f 1, Can f 2); mold spores, including those from Alternaria alternata (e.g., Alt a 1) and Aspergillus fumigatus (e.g., Asp f 1); foods such as peanuts (e.g., Ara h 1, Ara h 2, Ara h 3), eggs (e.g., Gal d 1, Gal d 2), and milk (e.g., casein, beta-lactoglobulin); insect stings, specifically bee venom (e.g., Api m 1) and wasp venom (e.g., Ves v 5); medications like penicillin (e.g., benzylpenicilloyl polylysine) and aspirin, which, although not a protein, can trigger immune responses; and latex, such as natural rubber latex components (e.g., Hev b 1, Hev b 3, Hev b 5, Hev b 6.02).

[0053] Additionally, as an oncolytic or tumolytic, the DISC HSV can be used to treat cancer. Cancers include, but are not limited to, ovarian cancer, a breast cancer, a cervical cancer, a lung cancer, a prostate cancer, a gastric cancer, a colon cancer, an esophageal cancer, a pancreatic cancer, a bile duct cancer, a cholangiocarcinoma, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, a head and neck cancer, a metastatic melanoma, a glioma, a glioblastoma, and other solid tumors, as well as a non-Hodgkin’s lymphoma (NHL), a chronic myelogenous leukemia (CML), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a multiple myeloma (MM), an acute myeloid leukemia (AML), and other liquid tumors.

[0054] The DISC HSV can be administered using an unmasked targeting adapter, which may or may not be covalently bound. Also, the DISC HSV can also be administered using a masked targeting adapter (covalently or non-covalently bound). Preferably, the masked targeting adapter is administered subsequent to the DISC HSV’s administration. For example, the masked targeting adapter can be administered at least 2 hours, at least 4 hours, or at least 12 hours after administration of the DISC HSV. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings.

[0056] FIG.1 illustrates the tagged ΔRBD HSV. The genome of HSV has been modified to lack a polynucleotide sequence encoding a functional RBD domain of gD (“ΔRBD HSV”). One way to accomplish this is to not have a polynucleotide sequence capable of encoding amino acids 6-38 of gD or amino acids 6-24 and 38 of gD. Additionally, the lipid bilayer comprises a tag (represented as a "star"). In embodiments, excluded from the lipid bilayer are proteins comprising amino acids 6-38 of gD or amino acids 6-24 and 38 of gD. Examples of such excluded proteins include fragments of gD that include amino acids 6- 38 of gD up to and including the full length gD.

[0057] FIG.2 illustrates the targeting adapter comprising a first binding domain specific to the tag on the lipid bilayer and a second binding domain specific to a cellular protein localized on a cell (represented as the grey shaded rectangle). Targeting adapters can further include half-life extender (“HLE”) modules, which extend the half-life of the targeting adapter after administration. Examples of such HLE include, but are not limited to a polypeptide that binds human serum albumin (HSA). The HLE can also be an antibody Fc (Ig Fc). Half-life extension can also be achieved by fusing the targeting adaptor to HSA. Other half life extension technologies include, but are not limited to: Albumin-binding domain fusion, lipidation, hyperglycosylation, PEGylation, synthetic PEG alternatives (e.g., polyglycerol, poly(thioglycidylglycerol), polysarcosine, poly(carboxybetaine), natural carbohydrates, PolyXen, HEPtune, HESylation, Chonylation, HAylation), recombinant PEG alternatives (e.g., PASylation, XTEN, PsTag, ELPylation, Ekylation), Gelatin-like polypeptides, GeneticPolymer, or HAPylation.

[0058] FIG. 3A-3B illustrates examples of manufacturing adapters. As used herein, a “manufacturing adapter” functions to assist infection of the ΔRBD HSV with the ØRBD HSV manufacturing cell during manufacturing. The manufacturing adapter can be expressed on the surface of the manufacturing cell as an artificial receptor (FIG. 3A). Alternatively, the manufacturing adapter is a bispecific adapter (FIG.3B) that is capableof binding to both the tag on the lipid bilayer of the tagged ΔRBD HSV and to a cell surface protein found on the ØRBD HSV manufacturing cell line. One example of the cell surface protein is a heterologous protein not encoded in human cells (e.g. a wildtype mouse cell surface protein such as mouse EGFR, or an engineered protein such as anti-GCN4 H6 scFv fused to the AA 146-517 of human Nectin-1 (Uniprot Q15223) separated by a G4S linker) encoded by a heterologous polynucleotide.

[0059] FIG. 4 illustrates the DISC HSV. A tagged ΔRBD HSV as described herein is combined with an exogenously provided targeting adapter to create the DISC HSV. Combination can occur as part of the manufacturing process or could be mixed just prior to administration to a patient. In certain embodiments, the targeting adapter can be covalently bound to the tagged ΔRBD HSV to create the DISC HSV.

[0060] FIG.5A-5B illustrates the ØRBD HSV manufacturing cell line lacking a functional RBD domain. A non-functional RBD domain can be obtained by many ways. First, the RBD domain is not expressed by the RBD because it lacks the ability to encode for a RBD. One example, would include any polynucleotide sequence capable of encoding amino acids 6-38 of gD. The null RBD ("ØRBD”) polynucleotide sequence that is absent includes just amino acids 6-38 of gD, or 6-24 and 38 of gD, as well as larger fragments of gD, up to and including the full length gD sequence, so long as the missing fragments comprise amino acids 6-38 of gD. Use of a ØRBD HSV manufacturing cell line is necessary to manufacture safe, tagged ΔRBD HSV and is a substantial and important new step in improving the safety of HSV based vectors for human therapy. Up until now, the current standard practice for manufacturing DISC HSV vectors for human use is to encode gD, including amino acids 6-38, in the vector genome along with secondary non- gD attenuating mutations (e.g. ICP4 deletion, ICP0 NLS deletion, g34.5 deletion) or grow a gD-null HSV vector without secondary non-gD attenuating mutations in an engineered cell line which expresses gD, or at least a gD fragment comprising the RBD. This cell line is often engineered to recombinantly express a heterologous gD while growing a HSV vector with some or all of gD deleted. See, for example, WO 2015 / 134368 and WO202345159. Deleting the gD sequence from the HSV vector genome, or deleting at least the sequence encoding amino acids 6-38, has been shown to prevent the HSVvector from infecting human cells and is a critical step in making DISC HSV vectors for human use.

[0061] However, using a manufacturing cell line engineered with gD still allows for the potential, even though at a very low rate, of homologous recombination between the HSV vector and the manufacturing cell line due to natural selection and evolutionary pressures. This potential for homologous recombination may not be observed at small scale production. However, even an extremely low rate of homologous recombination will be observed when scale up in manufacturing occurs, allowing for the HSV vector to recapture the gD sequence, making the recombined HSV vector no longer safe. This spontaneous recombination has the potential to contaminate (and make unusable) the entire scaled-up batch when detected by the release assays. In worse cases, if not detected during manufacturing and used to treat a patient, the spontaneously recombined HSV vectors have the potential to infect the patient with pathogenic HSV.

[0062] Thus, the invention disclosed herein elegantly solves this problem and goes against the standard practice. There is no potential for the tagged ΔRBD HSV to recapture the gD sequence from the ØRBD HSV manufacturing cell line. Manufacturing the DISC HSV having a substantially improved safety profile over the state of the art requires that the tagged ΔRBD HSV is produced using ØRBD HSV manufacturing cell as described herein.

[0063] FIG. 5A shows one embodiment where the ØRBD HSV manufacturing cell line expresses a manufacturing adapter on the cell surface, which is capable of binding to the tagged ΔRBD HSV. For example, the manufacturing adapter expressed on the surface of the ØRBD HSV manufacturing cell line is an artificial receptor, such as nectin where the region of nectin that binds WT HSV gD is replaced with a peptide that binds the tag on the lipid bilayer of tagged ΔRBD HSV (e.g. anti-GCN4 H6 scFv fused to the AA 146- 517 of human Nectin-1 (Uniprot Q15223) separated by a G4S linker).

[0064] FIG.5B shows an alternative embodiment where the manufacturing adapter is a bispecific adapter having two binding domains. The first binding domain is capable of binding to the tag on the lipid bilayer of the tagged ΔRBD HSV and a second bindingdomain capable of binding to a cell membrane protein expressed on the cell surface of the ØRBD HSV manufacturing cell line. In these embodiments, the manufacturing adapter is exogenously provided and the cell membrane protein can be an endogenous protein not encoded in human cells or an engineered protein encoded by a heterologous polynucleotide introduced into the cells used to derive the ØRBD HSV manufacturing cell

[0065] In other embodiments, the manufacturing adapter depicted in FIGS. 3B and 5B can be encoded by the ØRBD HSV manufacturing cell. In other embodiments, the manufacturing adapter depicted in FIGS. 3B and 5B is encoded by the tagged ΔRBD HSV. To prevent any potential homologous recombination, the targeting adapter is not encoded by the ΔRBD HSV or the tagged ΔRBD HSV.

[0066] For example, the manufacturing adapter can be inserted into and / or replace the polynucleotide sequence encoding amino acids 6-38 of gD in the tagged ΔRBD HSV. Alternatively, the manufacturing adapter can be encoded in the viral genome at a locus outside of the gD locus. When expressed from the ØRBD HSV manufacturing cell line the manufacturing adapter can be introduced into the cell genome or expressed from an extrachromosomal plasmid.

[0067] FIG.6A-B illustrates different approaches to manufacture the tagged ΔRBD HSV.

[0068] FIG.6A illustrates using an artificial receptor on the ØRBD HSV manufacturing cell line. In this example, the polynucleotide encoding the artificial receptor can be incorporated into the genome of the ØRBD HSV manufacturing cell line or expressed from an extrachromosomal plasmid. The artificial receptor can be constitutively expressed or inducibly expressed during manufacturing.

[0069] FIG.6B Illustrates the manufacturing of the tagged ΔRBD HSV using a bispecific adapter that recognizes a cell surface protein on the ØRBD HSV Manufacturing Cell Line. The bispecific adapter can be exogenously provided, or can be expressed by the ØRBD HSV manufacturing cell line. The polynucleotide encoding the bispecific adapter could be incorporated into the genome of the ØRBD HSV manufacturing cell line or expressed from an extrachromosomal plasmid. Alternatively, the bispecific adapter can be encodedby the tagged ΔRBD HSV using a viral promoter, heterologous promoter, or an inducible promoter which allows expression to be controlled during manufacturing.

[0070] FIG.7A-7B shows that in some embodiments, the targeting adapter is covalently attached to the tagged ΔRBD HSV using a covalent binder to create the DISC HSV. Examples of such covalent binders include SpyCatcherTM-SpyTagTMsystem or SnoopLigaseTM(Andersson, AM.C., Buldun, C.M., Pattinson, D.J. et al. SnoopLigase peptide-peptide conjugation enables modular vaccine assembly. Sci Rep 9, 4625 (2019), Setyo Utomo DI et al. “An Overview of Recent Developments in the Application of Antigen Displaying Vaccine Platforms: Hints for Future SARS-CoV-2 VLP Vaccines,” Vaccines (Basel) 2023 Sep 20:11(9):1506; Hatlem D et al. “Catching a SPY: Using the SpyCatcher- SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins,” Int J Mol Sci. 2019 Apr 30;20(9):2129)) (references hereby incorporated by reference in their entirety). FIG.7A shows the lipid bilayer of tagged ΔRBD HSV comprising the tag (grey circle, e.g. SpyTagTM) for the covalent binder (e.g. SpyCatcherTM). A non-covalent tag (star), for example a CD3 peptide tag, is also indicated. In FIG. 7A, the tagged ΔRBD HSV is a double tagged ΔRBD HSV, one tag for the attachment of manufacturing or targeting adapters through non-covalent interaction (star) and a second tag (grey circle) for the covalent attachment of targeting adapters. FIG.7B shows covalent binding of the covalent binder to the tag on the lipid bilayer. The covalent binder is attached to a second binding domain specific to a cellular protein localized on a cell. This covalently attaches to the lipid bilayer the second binding domain specific to a cellular protein localized on a cell.

[0071] For oncolytics and tumolytics, a double tagged ΔRBD HSV is used. In this situation, a covalently attached targeting binder using a covalent binder, such as SpyTagTM / SpyCatcherTMor other similar polypeptide-based covalent attachment systems, which is covalently bound to the covalent tag (grey circle). When this drug product is delivered to patients and infects target cells, the ability of progeny viruses produced from these cells can be controlled by administration of targeting adapters to a patient. In preferred embodiments, these targeting adapters attach non-covalently to the progeny viruses through the non-covalent tag (star). They can also attach covalently ornoncovalently through the covalent tag (grey circle). Also, in certain embodiments, a single tag, such as SpyTagTM, can be present on the ΔRBD HSV lipid bilayer and targeting adapters attached covalently to the SpyTagTMusing SpyCatcherTM(or other analogous systems) or non-covalently using scFv, VHH or other binders that non-covalently bind SpyTagTM.

[0072] FIG.8A-8B show examples of a "masked" targeting adapter used in combination with a double tagged ΔRBD HSV or DISC HSV. Here the first binding domain on the targeting adapter is capable of binding to a tagged ΔRBD HSV (labeled tag binder, e.g. anti-CD3) and the second binding domain on the targeting adapter is capable of binding to a protein on the cell surface (labeled anti-P1) (FIG.8A). This anti-P1 protein, and in some embodiments the tag binder, are "masked" and revealed when, in this example, a protease is brought in contact with the masked targeting adapter and cleaves at a specific protease cleavage site located on the targeting adapter. Examples of such masked targeting adapters include but are not limited to Probody® Platform from Cytomx, XPAT® from Amunix (stands for XTEN polypeptide fused Probody Activated T-cell Engagers), triTACs® from Harpoon Therapeutics (Trispecific T-cell Activating Construct), TRACTrTM (Tumor Activated T Cell Engager) from Janux, COBRATM (COnditional Bispecific Redirected Activation) from Maverick Therapeutics, all of which are hereby incorporated by reference in their entirety. This example also illustrates that the masked targeting adapter can also be covalently attached to the tag by a covalent binder (FIG.8B).

[0073] FIG.9A-9B shows examples of how a masked targeting adapter can be used in combination with a tagged ΔRBD HSV or DISC HSV, for example, to treat cancer. FIG. 9A shows a CD3 peptide tagged ΔRBD HSV combining with three different polypeptide binding domains included on the masked targeting adapters (in this example, EGFR, CD3 and FAP). Once the tagged ΔRBD HSV having the masked targeting adapters (in this example, the masked targeting adapters are non-covalently attached to the tagged ΔRBD HSV) are localized with the tumor microenvironment (“TME”), the masked targeting adapter is “unmasked” by proteases expressed by tumor cells in the TME. The unmasking of the targeting adapter allows the anti-CD3 binder of the targeting adapter to bind the CD3 peptide tag of tagged ΔRBD HSV. This then allows the tagged ΔRBD HSVto infect cancer cells using the targeting adapter that comprises an anti-EGFR binder and to infect normal cells, specifically cancer associated fibroblasts (CAFs), within the TME. The use of masked anti-CD3 containing targeting adapters not only facilitate infection of cells in the TME by tagged ΔRBD HSV, but also facilitate re-direction of infiltrating T-cells, especially the wave of anti-HSV T-cells that infiltrate tumors infected with therapeutic HSV vectors, to killing of both cancer cells and CAFs.

[0074] In FIG.9B, the tagged ΔRBD HSV is tagged with SpyTagTMand CD3. Two PDLI- tagged mono-specific adapters are encoded in the viral genome, one against EGFR and one against FAP. In this example, the tagged mono-specific adapters can’t bind the tagged ΔRBD HSV because their binding domains bind TAAs, so this virus can not infect cells and does not have a way to evolve the ability to infect. The drug product is directed to cancer cells through a targeting adapter where one binder covalently attaches to the tagged ΔRBD HSV (such as SpyTagTM-Catcher covalent linkage) and the other binder is an EGFR binding domain. To allow progeny virus produced in cells in the patient after administration of the EGFR targeted drug product to infect cells in the TME, a masked bi- specific where one binding domain binds CD3, so can bind the virus and T-cells, and the other binding domain binds PDLI, which will re-direct T-cells to PDLI expressing cancer cells in the TME and will also bind the tagged mono-specific adapters encoded by the virus and secreted into the TME to allow the virus to infect cancer cells through EGFR and CAFs through FAP.

[0075] In this example, masked targeting adapters can be exogenously administered after the tagged ΔRBD HSV has been administered and has been given sufficient time to infect target cells (e.g., after a couple of hours).

[0076] FIG. 10 illustrates different types of RBD Deletions. All ΔRBD HSV genomic structures comprise a deletion in the HSV genome of the polynucleotide encoding at least amino acids 6-38 of gD. Examples of the types of deletions that can be made, include, but are not limited to: • Deletion of the polynucleotide sequence encoding at least amino acids 6-38 of gD;• Deletion of polynucleotide sequences encoding larger fragments of gD, so long as all fragments comprise at least 6-38 of gD; • Deletion of the full-length wild type gD (e.g., the entire coding region). • Deletion of the polynucleotide sequence encoding at least amino acids 6-24 and 38 of gD.

[0077] FIG.11 illustrates different ways to express the tag from the ΔRBD HSV to create tagged ΔRBD HSV. In many embodiments, the ΔRBD HSV can further comprise a polynucleotide sequence capable of encoding the tag. The polynucleotide encoding this tag can be inserted into the ΔRBD HSV, at for example, positions 1, 2 or 3. These positions are exemplary for description purposes. In one embodiment, the polynucleotide encoding amino acids 6-38 of gD are replaced with a polynucleotide encoding the tag. In other embodiments, the polynucleotide encoding the tag is placed downstream or upstream of Us6(gD) using a 2A element to ensure it is expressed with kinetics similar to gD.

[0078] FIG. 12 illustrates different ways to express the tag and / or the manufacturing adapter from the ΔRBD HSV. In this example, the ΔRBD HSV comprises a polynucleotide sequence capable of encoding the tag and the manufacturing adapter. The polynucleotides encoding these sequences can be inserted into the ΔRBD HSV, at for example, positions 1, 2 or 3. These positions are exemplary for description purposes. In one embodiment, the polynucleotide encoding amino acids 6-38 of gD are replaced with a polynucleotide encoding the tag and the manufacturing adapter. Alternatively, the polynucleotide encoding the manufacturing adapter can be inserted, for example, at position 2 or 3. Preferably, the ΔRBD HSV does not encode for the targeting adapter.

[0079] FIG.13 illustrates an example of another ΔRBD HSV design. This example shows that amino acids 6-38 of gD are deleted and that a heterologous protein 1 can be expressed in either orientation from a promoter and can be terminated with a polyA adenylation signal. In this design, the polynucleotide encoding protein 1 is inserted between HSV US9 and US10. In certain embodiments, the amino acids 6-38 of RBD gDare replaced with a polynucleotide sequence encoding the tag to create tagged ΔRBD HSV. In other embodiments, the full length gD sequence is deleted.

[0080] FIG 14 illustrates another example of a ΔRBD HSV. This design shows as many as four different proteins (labeled protein 1, protein 2, protein 3 and protein 4 in the figure) are expressed using different promoters, in different orientations and using polyA adenylation signals. Each of these elements (promoter and polyA signal) can be substituted with functionally equivalent elements known in the art. The element labeled T2A represents ribosomal skip elements (“2A Elements”). The figure also shows an optional replacement of the polynucleotides encoding amino acids 6-38 of gD with the polynucleotide encoding the tag, although in other embodiments, the full length gD sequence is deleted.

[0081] FIG.15 illustrates an example of the ΔRBD HSV where the complete gD coding sequence has been deleted. This structure is preferred for vaccine constructs. Additional polypeptides directed to antigens, for example, can be encoded and expressed from this construct as described herein.

[0082] FIG.16 provides details of a tagged ΔRBD HSV constructed so that the RBD is replaced with a polynucleotide encoding a tag under the control of an inducible promoter that can be turned on during manufacturing, leading to the expression and display of the tag on the lipid bilayer. In this example, the inducible promoter from “RheoSwitch®” is used. See, for example, Chan et al., “Chapter 25 - Therapeutic Efficacy and Systemic Antitumor T Cell Immunity Induced by RheoSwitch-Regulated IL-12 Expression after Intratumoral Injection of Adenovirus Vector or Vector-Transduced Dendritic Cells,” Editor(s): Edmund C. Lattime, Stanton L. Gerson, Gene Therapy of Cancer (Third Edition), pages 363-376 (2014) (herein incorporated by reference in its entirety.

[0083] This construct also includes a miR Repression Array system that prevents leaky expression in human cells. Human cells constitutively express several miRNAs that are capable of binding the miR Repression Array in any Δ6-38 gD mRNA that happens to be expressed in human cells by the inducible promoter. With the inclusion of the miR Repression Array system, when the DISC HSV infects cells in the patient, very low to noexpression of Δ6-38 gD mRNA will occur due to the absence of small molecule inducer and host miRNA mediated degradation of leaky gD mRNA. As shown in this figure, the insertion of the polynucleotide encoding the RheoSwitch® and miR Repression Array are inserted between US5 and US7. However, these polypeptides can also be expressed anywhere in the HSV genome, such as from positions 2 and 3 as shown in Fig.11.

[0084] FIG.17 shows that the ΔRBD HSV described herein can also comprise additional elements. For example, the antigen presentation system (APS) is the major mechanism cells employ to tell the immune system that they are infected with a virus. Specifically, the APS mediates display of cellular and viral peptides in complex with MHC-I at the cell surface. By displaying viral peptides at the cell surface, the infected cell hoists a distinctive flag that tells CD8+ CTL the cell is infected with a virus. If the CD8+ CTL recognizes the viral peptide-MHC-I complex, it will kill the cell in order to stop the virus from continuing to use the cell to produce progeny viruses. The transporter associated with antigen processing (TAP) protein is responsible for carrying peptides from the cytoplasm into the endoplasmic reticulum (ER). Once inside the endoplasmic reticulum, these peptides are loaded on MHC-I for display at the cell surface. However, herpesviruses have evolved functions to inhibit TAP thereby blocking the display of viral peptides, which prevents CD8+ CTL from recognizing and killing virus infected cells. However, the endogenous HSV TAP inhibitor Us12 (aka ICP47) does not inhibit mouse TAP precluding preclinical evaluation of TAP inhibition in mouse models. Thus, the tagged ΔRBD HSV can further comprise a heterologous TAP inhibitor polypeptide that inhibits both mouse and human TAP as described in WO2013036795A2 (herein incorporated by reference in its entirety). This additional element functions to block the TAP in order to down regulate presentation of viral peptides on MHC / HLA, ideal for oncolytic use.

[0085] FIG 18 provides a specific example of a tagged ΔRBD HSV designed for oncolytic uses. The design shows four different encoded proteins: human CD40L, human Flt3L, an anti-CLTA4 Fc fragment and human IL12. The proteins are complementary and synergistic immunostimulatory cytokines effective in breaking tolerance in a tumor microenvironment. The figure also shows replacement of the polynucleotides encoding amino acids 6-38 of gD with the polynucleotide encoding the tag.DEFINITIONS

[0086] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0087] Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified materials or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting of the use of alternative terminology to describe the present invention.

[0088] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety for all purposes.

[0089] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a polypeptide" includes a mixture of two or more such polypeptide molecules or a plurality of such polypeptide molecules. Similarly, reference to a “polynucleotide” includes a mixture of two or more such polynucleotide molecules or a plurality of such polynucleotide molecules.

[0090] Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Likewise, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

[0091] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognizeor be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.

[0092] It should also be understood that the terms “about,” “approximately,” “generally,” “substantially,” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.

[0093] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0094] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrentapplicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0095] As used herein, the term “consists essentially of,” or variations such as “consist essentially of’ or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition. See M.P.E.P. § 2111.03.

[0096] As used herein, the term “isolated” means a biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. “Isolated” nucleic acids, peptides and proteins can be part of a composition and still be isolated if the composition is not part of the native environment of the nucleic acid, peptide, or protein. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. Elements of the DISC HSV

[0097] The term "Herpes Simplex Virus" or "HSV" as used herein encompasses both serotypes, HSV-1 and HSV-2. Both serotypes are applicable in the creation of ΔRBD HSV as discussed. Specifically, one embodiment of ΔRBD HSV is HSV-1. Additionally, the choice of HSV could include, but is not limited to, specific strains such as HSV1716 (also known as Seprehvir), G207, G47Delta, and Talimogene laherparepvec (also known as OncoVex GM-CSF), among others like NV1020, NV1023, NV1034, NV1042, rQNestin34.5, RP1, RP2, RP3, ONCR-148, ONCR-177, ONCR-152, ONCR-153, and VG161. Possible selections also include the wild-type HSV-1, strain 17, which is registered under GenBank Accession No. X14112. Furthermore, the HSV may be derivedfrom either laboratory or clinical ("non-laboratory") sources. Current laboratory strains include HSV-1 strain F, HSV-1 strain 17, HSV-1 strain KOS, and strain Patton, while clinical examples include strains such as HSV-1 strains Patton, JS1, and RH018A.

[0098] As used herein, a “Disabled, Infectious Single Cycle (DISC) HSV” is described in FIG.4 and its legend.

[0099] In this document, "gD" refers to Glycoprotein D, a 55 kDa glycoprotein located in the virion envelope, crucial for the entry of Herpes Simplex Virus (HSV) into host cells and vital for the virus's infectivity. Glycoprotein D initiates an essential series of interactions upon HSV's cellular entry, primarily by engaging with the gH / gL heterodimer. This interaction is a pivotal step in an activation cascade that also includes glycoproteins gH, gL, and gB, all integral to the virus's cell entry process. The cascade begins when gD binds to its receptors—nectin-1, HVEM, or modified heparan sulfates—which then signals to gH / gL, and ultimately to gB, which executes the fusion of HSV with the cell membrane. The gH / gL heterodimer interacts with the pre-fusion domain of gD, which becomes disengaged upon gD's binding to its receptors during the cell entry process. Specific regions within gD (termed the ”Receptor Binding Domain” or “RBD”) are targeted to its natural receptors like nectin-1 and HVEM, facilitating this entry mechanism. The amino acid sequence of gD is shown below with signal sequence underlined and the mature protein beginning with a Lys (“K”) at amino acid 25. signal sequence RBD mggtaarlgavilfvvivglhgvrgKYALADASLKMADPNRFRGKDLPVLDQLT DPPGVRRVYHIQAGLPDPFQPPSLPITVYYAVLERACRSVLLNAPSEAPQIVRG ASEDVRKQPYNLTIAWFRMGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNY YDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRAKGSCKY ALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPENQRTVAVYSLKIAGWHGPK APYTSTLLPPELSETPNATQPELAPEDPEDSALLEDPVGTVAPQIPPNWHIPSI QDAATPYHPPATPNNMGLIAGAVGGSLLAALVICGIVYWMHRRTRKAPKRIRLP HIREDDQPSSHQPLFY (SEQ ID NO: 21)

[0100] As used herein, a “RBD” refers to the “Receptor Binding Domain” of gD of HSV. The minimal defined region identified as RBD is shown bolded above and will be referred interchangeably herein as “amino acids 6-38 of gD” or “amino acids 6-38 of mature gD”.

[0101] As used herein, “ΔRBD HSV” refers to an HSV genome lacking a polynucleotide sequence encoding a functional gD. A non-functioning gD is preferably accomplished by the lack of a polynucleotide sequence in the HSV genome that is capable of encoding amino acids 6-38 of gD or amino acids 6-24 and 38 of gD.

[0102] As used herein, “tagged ΔRBD HSV” refers to a ΔRBD HSV surrounded by a lipid bilayer comprising a tag as is further defined in FIG.1 and its legend.

[0103] As used herein, a “tag” or “tag peptide” refers to peptide displayed on the lipid bilayer of the tagged ΔRBD HSV. The tag is capable of being bound by the first binding domain with binding in specificity to the tag present on the manufacturing adapter or targeting adapter. Examples of tags include, but are not limited to peptides derived from GCN4, Her2, CD3, CD28, PDLI, PD-1, CTLA4, TIGIT, OX40, CD40, ICOS, 4-1BB, CD47, TIGIT, VISTA, ROR1, HLA-G, HLA-E, TROP2, Tissue Factor, Nectin-4, Fibroblast Activation Protein (FAP), Folate receptor alpha, CD19, CD22, CD30, CD33, CD38, CD70, CD79b, BCMA, PSMA, KLK2, Mesothelin, NKG2A, MAGE-A1, MAGE-A4, HSA or any other peptide that is capable of being bound by the targeting adapter.

[0104] As used herein, a “lipid bilayer” makes up the envelope that surrounds the ΔRBD HSV and is derived from the ØRBD manufacturing cell line. The lipid bilayer comprises a tag, and in certain embodiments, the lipid bilayer does not comprise a polypeptide having the RBD domain of gD.

[0105] As used herein, a “targeting adapter” is described in FIG.2 and its legend.

[0106] As used herein, a “manufacturing adapter” is described in FIG.3 and its legend.

[0107] As used herein, “artificial receptor” is described in FIG.3 and its legend.

[0108] As used herein, a “bispecific adapter” is described in FIG.3 and its legend.

[0109] In the present invention, a “TAP inhibitor” is defined as a polynucleotide encoding a polypeptide that inhibits either TAP1 (Accession No. CAA47025) or TAP2 (Accession No. CAA47027), or both. Examples of preferred TAP inhibitors include UL49.5 polypeptide from bovine herpesvirus, CMV US6, HSV Us12 / ICP47, and / or EBV BNLF2a. Non-limiting examples of UL49.5 include, but are not limited to bovine herpesvirus (BHV),which is capable of inhibiting mouse and human TAP (van Hall et al., J. Immunology (2007) 178:657-662). UL49.5 polypeptides can also be derived from pseudorabies virus (PRV) and equine herpesvirus 1 and 4 (EHV-1 and EHV-4). These UL49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP and are active on rodent TAP, such as murine TAP. Other examples of TAP inhibitors include UL49.5 polypeptides from bubaline herpesvirus 1 (BuHV-1), cervid herpesvirus 1 (CvHV-1), felid herpesvirus 1 (FeHV-1), (see, Verweij et al., 2011 "Structural and functional analysis of the TAP-inhibiting UL49.5 proteins of varicelloviruses." Mol Immunol. Jul 15 Epub) and BNLF2a and ICP47. It is noted that UL49.5 functional homolog ICP47 from HSV-1 and HSV-2 does not inhibit rodent TAP [see, Koppers-Lalic, D. et al., (2008) PLoS; 4(5): el 000080]. ØRBD HSV Manufacturing Cell Line

[0110] As used herein, a “ØRBD HSV manufacturing cell line” refers to a cell used to manufacture the ΔRBD HSV. The ØRBD HSV manufacturing cell line lacks any polynucleotide sequence capable of encoding amino acids 6-38 of gD or amino acids 6- 24 and 38 of gD. This manufacturing cell line is referred to as a “null RBD” or "ØRBD” (rather than as ΔRBD) as the host cell may never had the ability to encode for the RBD. For example, the host cell may not endogenously produce gD. Thus, in this situation, the host cell does not require “deletion” of RBD. Alternatively, in situations where sequences encoding fragments of gD are still included in the host cell, the ØRBD HSV manufacturing cell line must lack any polynucleotide sequence encoding at least amino acids 6-38 of gD or amino acids 6-24 and 38 of gD, as well as larger fragments of gD, up to and including the full length gD sequence, so long as the fragments comprise amino acids 6-38 of gD. The cell that can be used to generate the ØRBD HSV manufacturing cell line can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, the ØRBD HSV manufacturing cell line is transfected, transduced, or infected with ΔRBD HSV. In another embodiment, the ØRBD HSV manufacturing cell line is a progeny or potential progeny of such cells. A progeny of a cell may or may not be identical to the parent cell, e.g., due to mutations or environmental influences that canoccur in succeeding generations or integration of the nucleic acid molecule into the host cell genome. Methods of Treating – Tumolytics and Vaccines

[0111] As used herein, “subject” or “patient” means any animal, preferably a mammal, most preferably a human. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc.

[0112] The terms "treat," "treating," and "treatment" as used herein encompass a broad range of therapeutic outcomes for diseases, disorders, or conditions, including cancer or as a vaccine. These terms refer to ameliorating or reversing at least one measurable physical parameter, which may not be initially noticeable in the subject but may become discernible. This encompasses causing regression of the disease, halting its progression, or at least decelerating its advancement. Specific interpretations include alleviating, preventing the onset, or shortening the duration of symptoms such as tumor growth, cancer-related issues, allergic responses, or infections. The terms also cover preventing recurrence, enhancing survival rates, or completely eradicating the disease in the subject. For instance, in cancer treatment, "treat" can imply relieving symptoms like tumor growth, metastasis, and enhancing the sensitivity of tumor cells to other treatments such as chemotherapy or radiation. Additionally, treatment may involve arresting or delaying the onset of cancer, reducing the risk of its development or progression, or killing tumor cells using methods like oncolytic viruses. The intended benefit of such treatments is to provide a statistically significant or perceptible improvement, recognized by the patient or physician. As a vaccine, the benefit of such treatment can prevent the occurrence, the spreading of infection and / or severity of symptoms as compared to the unvaccinated subject.

[0113] An "effective amount" includes doses that partially or completely achieve the desired therapeutic, prophylactic, and / or biological effect. The actual amount effective for a particular application depends on the condition being treated and the route ofadministration. The effective amount for use in humans can be determined from animal models.

[0114] As used herein the term "therapeutically effective" refers to that quantity that is sufficient to result in a desired activity upon administration to an animal in need thereof. Thus, within the context of the present invention, the term "therapeutically effective amount" refers to that quantity of a compound or composition that is sufficient to (a) treat at least one symptom of a cancer, such as but not limited to cancer cell proliferation, tumor growth, resistance to apoptosis, and angiogenesis, and / or to inhibit metastasis of a cancer cell or (b) treat at least one symptom of an allergic response and / or infection, such as but not limited to bacterial / viral proliferation or growth or inhibition of IgE mediated disease.

[0115] Examples of, a therapeutically effective amount refers to the amount of therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition to be treated or a symptom associated therewith; (ii) reduce the duration of the disease, disorder or condition to be treated, or a symptom associated therewith; (iii) prevent the progression of the disease, disorder or condition to be treated, or a symptom associated therewith; (iv) cause regression of the disease, disorder or condition to be treated, or a symptom associated therewith; (v) prevent the development or onset of the disease, disorder or condition to be treated, or a symptom associated therewith; (vi) prevent the recurrence of the disease, disorder or condition to be treated, or a symptom associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (viii) reduce hospitalization length of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (ix) increase the survival of a subject with the disease, disorder or condition to be treated, or a symptom associated therewith; (xi) inhibit or reduce the disease, disorder or condition to be treated, or a symptom associated therewith in a subject; and / or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy. The therapeutically effective amount or dosage can vary according to various factors, such as the disease, disorder or condition to be treated, the means of administration, the target site, thephysiological state of the subject (including, e.g., age, body weight, health), whether the subject is a human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.

[0116] When a combination of active ingredients is administered, an effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, for example, preventing or delaying the onset (or recurrence) of cancer, or reducing the likelihood of the onset (or recurrence) of cancer or cancer symptoms. Similarly, a prophylactically effective amount with respect to a vaccine is an amount sufficient to prevent or delay the onset (or recurrence) of allergic symptoms or infection, or reducing the likelihood of the onset (or recurrence) of allergic symptoms or infection. The full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0117] The phrase "enhancing an anti-tumor response" in the context of a DISC HSV means that the "anti-tumor" response induced following infection with a DISC HSV, as measured, for example, and without limitation, by decreased tumor growth, decreased frequency of or size of tumor metastases, increased tumor cell death, increased immune cell tumor infiltration, increased immune cell-mediated tumor cell killing, increased IFN-γ secretion by immune cells in the presence of living, apoptotic or dead tumor cells, increased levels of anti-tumor immune cells in the animal or human, and / or increased induction of anti-tumor immunity. By way of example, and without limitation, an anti-tumor response is enhanced by, for example, a DISC HSV (or the tagged ΔRBD HSV or the ΔRBD HSV) if the DISC HSV reduces tumor size by, e.g., at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30- fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, at least 1000-fold or more, compared to a control.

[0118] For example, the cancer can be selected from, but is not limited to, an ovarian cancer, a breast cancer, a cervical cancer, a lung cancer, a prostate cancer, a gastric cancer, a colon cancer, an esophageal cancer, a pancreatic cancer, a bile duct cancer, a cholangiocarcinoma, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, a head and neck cancer, a metastatic melanoma, a glioma, a glioblastoma, and other solid tumors, as well as a non-Hodgkin’s lymphoma (NHL), a chronic myelogenous leukemia (CML), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a multiple myeloma (MM), an acute myeloid leukemia (AML), and other liquid tumors.

[0119] Yet further disclosed herein is a pharmaceutical composition comprising a DISC HSV and a pharmaceutically acceptable carrier. The term “pharmaceutical composition” as used herein means a product comprising the DISC HSV, the tagged ΔRBD HSV, or the ΔRBD HSV together with one or more pharmaceutically acceptable carriers.

[0120] As used herein, the term “carrier” refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic material that does not interfere with the effectiveness of a composition according to the invention or the biological activity of a composition according to the invention. According to particular embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in a polynucleotide, polypeptide, host cell, virus, and / or engineered immune cell pharmaceutical composition can be used in the invention.

[0121] The pharmaceutical compositions can also be administered in any convenient manner known to those skilled in the art. For example, the pharmaceutical compositions of the invention can be administered to the subject by aerosol inhalation, injection, ingestion, transfusion, implantation, and / or transplantation. The pharmaceutical compositions comprising the DISC HSV can be administered transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary,intramuscularly, intrapleurally, by intravenous (i.v.) injection, or intraperitoneally. In certain embodiments, the pharmaceutical compositions of the invention can be administered with or without lymphodepletion of the subject. Ideally, the compositions described herein can be formulated to be suitable for inhalation, intravenous, subcutaneous, or intramuscular administration.

[0122] The pharmaceutical compositions comprising the DISC HSV, the tagged ΔRBD HSV, or the ΔRBD HSV, can be provided in sterile liquid preparations, typically isotonic aqueous solutions with cell suspensions, or optionally as emulsions, dispersions, or the like, which are typically buffered to a selected pH. The pharmaceutical compositions can comprise carriers, for example, water, saline, phosphate buffered saline, and the like, suitable for the integrity and viability of the DISC HSV, the tagged ΔRBD HSV, or the ΔRBD HSV, and for administration of the pharmaceutical compositions.

[0123] The DISC HSV, the tagged ΔRBD HSV, or the ΔRBD HSV can be administered in combination with other treatments, including, but not limited to, a chemotherapy, an anti- PD-1 mAb, an anti-PD-L1 mAb, an anti-CTLA-4 mAb, an anti-CD20 mAb, an anti-CD19 mAb, an anti-CD33 mAb, an anti-CD47 mAb, an anti-CD73 mAb, an anti-EGFR mAb, an anti-HER-2 mAb, an anti-LAG-3 mAb, an anti-TIM-3 mAb, an anti-DLL-3 mAb, an anti- apelin mAb, an anti-TIP-1 mAb, an anti-FOLR1 mAb, other immuno-oncology drugs, an antiangiogenic agent, a radiation therapy, an antibody-drug conjugate (ADC), a targeted therapy, or other anticancer drugs. It is understood that "administered in combination" does not necessitate simultaneous packaging or administration, but rather is part of an overall treatment protocol. Polynucleotides and Constructs

[0124] As used herein, the term “polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0125] A "polynucleotide" also refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single-stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term polynucleotide, and in particular a DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear (e.g., restriction fragments) or circular DNA molecules, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation.

[0126] The term "exogenous" or “heterologous” refers to a combination of elements not naturally occurring. Thus, for example, an "exogenous polypeptide" refers to a polypeptide not normally expressed by a cell. In one example, a polynucleotide encodingthe tag is introduced into the genome (or expressed from an extrachromosomal plasmid) of the ΔRBD HSV or ØRBD manufacturing cell line. In contrast, the term “endogenous” refers to a polynucleotide or polypeptide normally found / expressed in the ΔRBD HSV, ØRBD manufacturing cell line, or target cell. An example of an endogenous protein of HSV is gD.

[0127] The term “vector” means a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers that function to facilitate the duplication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The vector polynucleotides may be DNA or RNA molecules or a hybrid of these. Exemplary vectors include, without limitation, plasmids, cosmids, phage vectors, and viral vectors. The term “expression vector” means a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0128] The term “expression” as used herein, refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term can also encompas “translation” of RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications.

[0129] The terms "polynucleotide cassette" and "gene expression cassette" means a manipulatable fragment of DNA / RNA carrying, and capable of expressing, one or more genes of interest between one or more sets of restriction sites. It can be transferred from one DNA sequence (usually on a vector) to another by 'cutting' the fragment out using restriction enzymes and “pasting” it back into the new context. Typically, the DNA fragment (nucleic acid sequence) is operatively associated with expression control sequence elements which provide for the proper transcription and translation of the target nucleic acid sequence(s) (genes). Such sequence elements may include a promoter and a polyadenylation signal. The "polynucleotide cassette" may further comprise "vectorsequences". By "vector sequences" is meant any of several nucleic acid sequences established in the art which have utility in the recombinant DNA technologies of the invention to facilitate the cloning and propagation of the polynucleotide cassette including (but not limited to) plasmids, cosmids, bacterial artificial chromosomes, phage vectors, viral vectors, and yeast artificial chromosomes.

[0130] The term "gene", also called a "structural gene" means a polynucleotide sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or part of one or more polypeptides (e.g., proteins), and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription.

[0131] A coding sequence is "under the control of” or "operatively associated with" an expression control sequences when a signal for a RNA polymerase to transcribe the coding sequence into RNA, particularly mRNA, which is then spliced (if it contains introns) and translated into the polypeptide encoded by the coding sequence.

[0132] The term "expression control sequence" refers to a promoter and any enhancer or suppression elements that combine to regulate the transcription of a coding sequence. In a preferred embodiment, the element is a transcriptional promoter.

[0133] A sequence "encoding" an expression product, such as a polypeptide, is a minimum nucleotide sequence that, when expressed, results in the production of that polypeptide.

[0134] As used herein, the terms "mutant" and "mutation" refer to any detectable change in genetic material (e.g., DNA) or any process, mechanism, or result of such a change. This includes gene mutations, in which the structure (e.g., DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g., polypeptide) expressed by a modified gene or DNA sequence. As used herein, the term "mutating" refers to a process of creating a mutant or mutation.

[0135] The term "nucleic acid hybridization" refers to anti-parallel hydrogen bonding between two single-stranded nucleic acids, in which A pairs with T (or U if an RNA nucleic acid) and C pairs with G. Nucleic acid molecules are "hybridizable" to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with the complementary bases of another nucleic acid molecule under defined stringency conditions. Stringency of hybridization is determined, e.g., by (i) the temperature at which hybridization and / or washing is performed, and (ii) the ionic strength and (iii) concentration of denaturants such as formamide of the hybridization and washing solutions, as well as other parameters. Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated. Under "low stringency" conditions, a greater percentage of mismatches are tolerable (i.e., will not prevent formation of an anti-parallel hybrid). See Molecular Biology of the Cell, Alberts et al, 3rd ed., New York and London: Garland Publ, 1994, Ch.7.

[0136] Typically, hybridization of two strands at high stringency requires that the sequences exhibit a high degree of complementarity over an extended portion of their length. Examples of high stringency conditions include: hybridization to filter-bound DNA in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65 °C, followed by washing in O. lx SSC / 0.1% SDS at 68°C (where lx SSC is 0.15M NaCl, 0.15M Na citrate) or for oligonucleotide molecules washing in 6xSSC / 0.5% sodium pyrophosphate at about 37°C (for 14 nucleotide-long oligos), at about 48 °C (for about 17 nucleotide-long oligos), at about 55°C (for 20 nucleotide-long oligos), and at about 60 °C (for 23 nucleotide-long oligos)). Accordingly, the term "high stringency hybridization" refers to a combination of solvent and temperature where two strands will pair to form a "hybrid" helix only if their nucleotide sequences are almost perfectly complementary (see Molecular Biology of the Cell, Alberts et al, 3rd ed., New York and London: Garland Publ, 1994, Ch.7).

[0137] Conditions of intermediate or moderate stringency (such as, for example, an aqueous solution of 2XSSC at 65 °C; alternatively, for example, hybridization to filter- bound DNA in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.2 x SSC / 0.1% SDS at 42 °C) and low stringency (such as, for example, an aqueous solutionof 2XSSC at 55°C), require correspondingly less overall complementarity for hybridization to occur between two sequences. Specific temperature and salt conditions for any given stringency hybridization reaction depend on the concentration of the target DNA and length and base composition of the probe, and are normally determined empirically in preliminary experiments, which are routine (see Southern, J. Mol. Biol. 1975; 98: 503; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., vol.2, ch.9.50, CSH Laboratory Press, 1989; Ausubel et al., (eds.), 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p.2.10.3).

[0138] As used herein, the term "standard hybridization conditions" refers to hybridization conditions that allow hybridization of sequences having at least 75% sequence identity. According to a specific embodiment, hybridization conditions of higher stringency may be used to allow hybridization of only sequences having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity.

[0139] Polynucleotides that "hybridize" to any desired nucleic acids of the present invention may be of any length. In one embodiment, such nucleic acid molecules are at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, and at least 70 nucleotides in length. In another embodiment, nucleic acid molecules that hybridize are of about the same length as the particular desired nucleic acid.

[0140] As used herein, the term "homologs" refers to genes in different species that apparently evolved from a common ancestral gene by speciation and / or did not evolve through speciation but exhibit convergent evolution, which is encoding for a polypeptide that exhibits a similar activity or phenotype despite no significant similarity in their gene sequences. Normally, homologs that apparently evolved from a common ancestral gene by speciation exhibit the same function. Identification of homologs that apparently evolved from a common ancestral gene by speciation can provide reliable prediction of gene function in newly sequenced genomes. Sequence comparison algorithms that can be used to identify homologs that apparently evolved from a common ancestral gene by speciation include without limitation BLAST, FASTA, DNA Strider, and the GCG pileupprogram. Homologs often have high sequence similarity. In contrast, homologs that arose through convergent evolution display no significant sequence similarity, but exhibit similar activity or phenotype.

[0141] The terms "percent (%) sequence similarity", "percent (%) sequence identity", and the like, generally refer to the degree of identity or correspondence between different nucleotide sequences of nucleic acid molecules or amino acid sequences of polypeptides that may or may not share a common evolutionary origin (see Reeck et al., supra). Sequence identity can be determined using any of a number of publicly available sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin), etc.

[0142] To determine the percent identity between two amino acid sequences or two nucleic acid molecules, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are, or are about, of the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent sequence identity, typically exact matches are counted.

[0143] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1990, 87:2264, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1993, 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al, J. Mol. Biol.1990; 215: 403. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to sequences of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to protein sequences of theinvention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res.1997, 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationship between molecules. See Altschul et al., (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See ncbi.nlm.nih.gov / BLAST / on the World Wide Web.

[0144] Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS 1988; 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0145] In a preferred embodiment, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 1970, 48:444-453), which has been incorporated into the GAP program in the GCG software package (Accelrys, Burlington, MA; available at accelrys.com on the World Wide Web), using either a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package using a NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that can be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is a sequence identity or homology limitation of the invention) is using a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0146] Statistical analysis of the properties described herein may be carried out by standard tests, for example, t-tests, ANOVA, or Chi squared tests. Typically, statistical significance will be measured to a level of p=0.05 (5%), more preferably p=0.01, p=0.001, p=0.0001, p=0.000001.ΔRBD HSV Can Express Other Heterologous Polypeptides

[0147] The ΔRBD HSV can also comprise the ability to express multiple other polypeptides referred to herein as “heterologous polypeptides”. For example, the ΔRBD HSV can comprise polynucleotide sequences encoding for one, two, three, four, five and even more different polypeptides. These polypeptides can enhance DISC HSV’s use as anti-tumor agent or vaccine. Examples of such heterologous polypeptides include antigens, immunomodulatory polypeptides, prodrug converting enzyme, matrix degrading enzyme, and / or fusogenic protein.

[0148] As a vaccine, “antigens” encompass proteins / peptides capable of raising an immune response to an infection. Examples of microorganisms that can cause infections, include but are not limited to HSV, respiratory syncytial virus (RSV), adenovirus, anthrax, cholera, a measles, mumps, rubella, yellow fever, typhoid, rotavirus, BCG, varicella zoster, whole-cell pertussis, polio, influenza, Japanese encephalitis, hepatitis A, rabies, Diphtheria, tetanus, hepatitis B, meningococcal, pneumococcal, Human papillomavirus, Group B meningococcal, Haemophilus influenzae type B, ebola, SARS-CoV2, Mycobaterium tuberculosis, M. ulcerous, M. marinum, M. leprae, M. absenscens, Chlamydia trachomatis, Neisseria gonorrhoeae or Treponema pallidum.

[0149] Antigens can also include those capable of causing an allergic response (also known as “allergens”). Examples of antigens causing an allergic response include, but are not limited to pollen, such as birch pollen (e.g., Bet v 1), ragweed pollen (e.g., Amb a 1), and grass pollen (e.g., Phl p 5); dust mites, such as those from European house dust mites (e.g., Der p 1, Der p 2) and American house dust mites (e.g., Der f 1, Der f 2); animal dander, notably from cats (e.g., Fel d 1) and dogs (e.g., Can f 1, Can f 2); mold spores, including those from Alternaria alternata (e.g., Alt a 1) and Aspergillus fumigatus (e.g., Asp f 1); foods such as peanuts (e.g., Ara h 1, Ara h 2, Ara h 3), eggs (e.g., Gal d 1, Gal d 2), and milk (e.g., casein, beta-lactoglobulin); insect stings, specifically bee venom (e.g., Api m 1) and wasp venom (e.g., Ves v 5); medications like penicillin (e.g., benzylpenicilloyl polylysine) and aspirin, which, although not a protein, can triggerimmune responses; and latex, such as natural rubber latex components (e.g., Hev b 1, Hev b 3, Hev b 5, Hev b 6.02).

[0150] Examples of antigens that are considered “tumor associated antigen (TAA)” include, but are not limited to, prostate specific membrane antigen (PSMA), TMEFF2, ROR1, KLK2, HLA-G, CD70, PD-1, PD-L1, CTLA-4, EGFR, HER- 2, CD19, CD20, CD3, mesothelin (MSLN), prostate stem cell antigen (PCSA), B-cell maturation antigen (BCMA or BCM), G-protein coupled receptor family C group 5 member D (GPRC5D), Interleukin- 1 receptor accessory protein (IL1RAP), delta-like 3 (DLL3), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, epithelial glycoprotein-2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial adhesion molecule (EpCAM), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor a and b (FRa and b), ganglioside G2 (GD2), ganglioside G3 (GD3), epidermal growth factor receptor (EGFR), epidermal growth factor receptor vIII (EGFRvIII), ERB3, ERB4, interleukin-13 receptor subunit alpha-2 (IL-13Ra2), k-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), LI cell adhesion molecule (LICAM), melanoma-associated antigen 1 (melanoma antigen family Al, MAGE-A1), Mucin-16 (Muc-16), Mucin 1 (Muc-1), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor R2 (VEGF-R2), type 1 tyrosine-protein kinase transmembrane receptor (ROR1), B7-H3 (CD276), B7-FI6 (Nkp30), chondroitin sulfate proteoglycan-4 (CSPG4), DNAX accessory molecule (DNAM-1), ephrin type A receptor 2 (EpHA2), fibroblast associated protein (FAP), Gpl 00 / HLA-A2, glypican 3 (GPC3), HA-1H, HERK-V, IL-llRa, latent membrane protein (LMPl), neural cell-adhesion molecule (N-CAM / CD56), and trail receptor (TRAIL R).

[0151] The ΔRBD HSV may also encode for an “immunomodulatory polypeptide.” As used herein, an "immunomodulatory polypeptide" includes immunostimulatory polypeptides, such as, but not limited to, IL-12, a granulocyte macrophage colony- stimulating factor (GM-CSF), Flt3L, tumor necrosis factor (TNF)-alpha (TNF-α), CD40ligand (CD40L), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, G-CSF, interferon alpha (IFN-α), IFN-β, IFN-γ, IL-20 (MDA-7), IL-21, IL-23, IL-24, or a co-stimulatory molecules, such as B7-1 (CD80) and B7- 2 (CD86), a chemokine such as RANTES or a macrophage inflammatory protein (MIP) (for example, MIP-3), or an immune checkpoint blocker such as polypeptides that block signaling through PD-1, CTLA-4 or PDL-1. An immunomodulary polypeptide can functions in cancer to recruit and / or activate immune cells to infiltrate tumors, process immunoactive molecules, recognize tumor cells and / or lyse tumor cells. The presence of an immunostimulatory polypeptide can also enhance immune recruitment to virally infected cells and tumors, has the potential to be deleterious to viral infection of tumor cells and viral spread throughout tumor tissue.

[0152] The ΔRBD HSV may additionally comprise a polynucleotide that encodes a “prodrug converting enzyme” which is defined as an enzyme that converts a molecule with less activity against a target into a molecule with more activity against a target, for example a cytosine deaminase. A prodrug activating polypeptide can be a cytosine deaminase enzyme, which is capable of converting the inactive prodrug 5-fluorocytosine to the active drug 5-flurouracil. Various cytosine deaminase genes are available including those of bacterial origin and of yeast origin. A second gene, typically a gene encoding a second enzyme, may be used to enhance the prodrug conversion activity of the cytosine deaminase gene. For example, the second gene may encode an uracil phosphoribosyltransferase. Another example of a prodrug converting enzyme is the HSV-1 thymidine kinase.

[0153] The ΔRBD HSV may additionally comprise a polynucleotide that encodes a “matrix degrading enzyme” which is an enzyme that degrades or modifies extra-cellular matrix components in order to facilitate viral spread through the tumor, for example, a matrix metalloproteinase. Non-limiting examples of matrix degrading enzymes are: matrix metalloproteinases such as collagenases, gelatinases and stromelysins, relaxin, bacterial collagenase and chondroitinase ABC I.

[0154] The ΔRBD HSV may additionally comprise a polynucleotide that encodes a “fusogenic protein” which is a polypeptide capable of causing cell to cell fusion may beused. Preferably the fusogenic protein is a modified retroviral envelope glycoprotein, such as an envelope glycoprotein derived from gibbon ape leukemia virus (GALV) or human endogenous retrovirus W, a fusogenic F or H protein from measles virus and the vesicular stomatitis virus G protein. A further example of a fusogenic protein is a GALV fusogenic glycoprotein (see, Simpson et al. (2006) "Combination of a Fusogenic Glycoprotein, Prodrug Activation, and Oncolytic Herpes Simplex Virus for Enhanced Local Tumor Control." Cancer Res; 66:9: 4835-4842).

[0155] It is specifically contemplated that functional variants of any of the recited antigens, immunomodulatory polypeptides, prodrug converting enzyme, matrix degrading enzyme, and / or fusogenic protein can be encoded by the ΔRBD HSV. For example, variants of at least 70%, of at least 80% of at least 90%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, and / or of at least 99% of any of the antigens, immunomodulatory polypeptides, prodrug converting enzyme, matrix degrading enzyme, and / or fusogenic protein can be included in the ΔRBD HSV. Binding Molecules

[0156] The ΔRBD HSV or the ØRBD HSV Manufacturing Cell Line can express a “binding fragment” or “binding domain.” As used herein, a “binding fragment” or “binding domain” refers to a portion of the manufacturing adapter or targeting adapter that binds a peptide or antigen, e.g., an antibody or an epitope binding peptide. Binding domains may be synthetic, enzymatically obtainable or genetically engineered polypeptides and include portions of a binding domain, such as an immunoglobulin that bind an antigen, such as the VH, the VL, the VH and the VL, Fab, Fab’, F(ab')2, Fd and Fv fragments, domain antibodies (dAb) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelized VH domains, VHH domains, minimal recognition units consisting of the amino acid residues that mimic the CDRs of an antibody, such as FR3-CDR3-FR4 portions, the HCDR1, the HCDR2 and / or the HCDR3 and the LCDR1, the LCDR2 and / or the LCDR3, alternative scaffolds that bind an antigen, and multispecific proteins comprising the binding domains. Antigen binding fragments (such as VH and VL) may belinked together via a synthetic linker to form various types of single antibody designs where the VH / VL domains may pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chains, to form a monovalent antigen binding domain, such as single chain Fv (scFv) or diabody. Antigen binding fragments may also be conjugated to other antibodies, proteins, antigen binding fragments or alternative scaffolds which may be monospecific or multispecific to engineer bispecific and multispecific proteins. Exemplary antigen binding fragments also include genetically engineered antibody mimetic proteins, such as DARPin.

[0157] In more specific embodiments, examples of a manufacturing adapter include, but are not limited to: (a) an anti-GCN4 scFv comprising a VH comprised of HCDR1 (SEQ ID NO: 4), HCDR2 (SEQ ID NO: 5), and HCDR3 (SEQ ID NO: 6) and / or a VL comprised of LCDR1 (SEQ ID NO: 7), LCDR2 (SEQ ID NO: 8), and LCDR3 (SEQ ID NO: 9); (b) an anti-GCN4 scFv comprising a VH having a polypeptide sequence at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 10 and / or a VL having a polypeptide sequence at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 11; (c) an anti-La scFv comprising a VH comprised of HCDR1 (SEQ ID NO: 12), HCDR2 (SEQ ID NO: 13), and HCDR3 (SEQ ID NO: 14) and / or a VL comprised of LCDR1 (SEQ ID NO: 15), LCDR2 (SEQ ID NO: 16), and LCDR3 (SEQ ID NO: 17); (d) an anti-La scFv comprising a VH having a polypeptide sequence at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 18 and / or a VL having a polypeptide sequence at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 19, (e) a synthetic leucine-zipper moiety RE (SEQ ID NO: 1) or synthetic leucine-zipper moiety ER (SEQ ID NO: 2); (f) a synthetic leucine-zipper moiety RE (SEQ ID NO: 1) and the second leucine-zipper moiety is synthetic leucine-zipper moiety ER (SEQ ID NO: 2), or the first leucine-zipper moiety is synthetic leucine-zipper moiety ER (SEQ ID NO: 2) and the second leucine-zipper moiety is synthetic leucine-zipper moiety RE (SEQ ID NO: 1).

[0158] Alternatively, instead of a mouse anti-GCN4 scFv a humanized anti-GCN4 scFv (such as those described in US Patent US11174306 herein incorporated by reference inits entirety) and provided herein as SEQ ID NO: 22. Other adapters that can function as manufacturing adapters are described in, for example, in US20240226207 which is hereby incorporated by reference in its entirety.

[0159] As used herein, a binding domain that “specifically binds” or with “binding specificity to” refers to a binding domain that binds a target, with a KD of 1 x 10-7 M or less, or 1 x 10-8M or less, or 5 x 10-9M or less, or 1 x 10-9M or less, or 5 x 10-10M or less, or 1 x 10-10M or less. The term "KD" refers to the dissociation constant, which is obtained from the KD values for a binding domain can be determined using methods in the art in view of the present disclosure. For example, the KD of a binding domain can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a Biacore® system, or by using bio-layer interferometry technology, such as an Octet RED96 system. The smaller the value of the KD is, the higher affinity the bonding specificity is.

[0160] As used herein, the term “antibody” is used in a broad sense and includes immunoglobulin or antibody molecules including human, humanized, composite and chimeric antibodies and antibody fragments that are monoclonal or polyclonal. In general, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. Antibody structures are well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG and IgM), depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl , IgG2, IgG3 and IgG4. Accordingly, the antibodies disclosed herein can be of any of the five major classes or corresponding sub-classes. In one embodiment, the antibodies disclosed herein are IgGl, IgG2, IgG3 or IgG4. Antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains. Accordingly, the antibodies of the invention can contain a kappa or lambda light chain constant domain. According to particular embodiments, the antibodies disclosed herein include heavy and / or light chain constant regions from rat or human antibodies. In addition to the heavy and light constant domains, antibodies contain an antigen-binding region that is made up of a light chain variable region and a heavy chain variable region, each of which containsthree domains (i.e., complementarity determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1 , HCDR2, and HCDR3.

[0161] As used herein, the term an “isolated antibody” refers to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds an epitope of the ligand peptide (e.g, GCN4 or La protein) or a TAA is substantially free of antibodies that do not bind the epitope of the ligand peptide or TAA). In addition, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0162] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies of the invention can be made by the hybridoma method, phage display technology, single lymphocyte gene cloning technology, or by recombinant DNA methods. For example, the monoclonal antibodies can be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, such as a transgenic mouse or rat, having a genome comprising a human heavy chain transgene and a light chain transgene.

[0163] As used herein, the term “single-chain antibody” refers to a conventional single chain antibody in the field. One exemplary single-chain antibody is single-chain variable fragment (scFv) comprising a heavy chain variable region and a light chain variable region connected by a short peptide (e.g., a peptide of about 5 to about 20 amino acids). Another exemplary single-chain antibody is single-chain antigen-binding fragment (scFab) comprising one constant and one variable domain of each of the heavy and the light chains. Yet another exemplary single-chain antibody is VHH (or so called nanobody) corresponding to the variable region of a heavy chain of a camelid antibody.

[0164] As used herein, the term “human antibody” refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibodyproduced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide.

[0165] As used herein, the term “humanized antibody” refers to a non-human antibody that is modified to increase the sequence homology to that of a human antibody, such that the antigen-binding properties of the antibody are retained, but its antigenicity in the human body is reduced.

[0166] As used herein, the term “chimeric antibody” refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable region of both the light and heavy chains often corresponds to the variable region of an antigen binding domain derived from one species of mammal (e g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capability, while the constant regions correspond to the sequences of an antigen binding domain derived from another species of mammal (e g., human) to avoid eliciting an immune response in that species. Kits

[0167] In another general aspect, provided herein are kits, unit dosages, and articles of manufacture comprising the DISC HSV and optionally a pharmaceutical carrier. In certain embodiments, the kit provides instructions for its use. In another particular aspect, provided herein are kits comprising (1) the ΔRBD HSV and (2) the targeting adapter as disclosed herein. The ΔRBD HSV and the targeting adapter may be included in the kits as separate component or as a pre-mix.

[0168] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

[0169] While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention.The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

WHAT IS CLAIMED 1. A method of treating a patient suffering from a cancer, wherein said method comprises: a) administering to the patient a Disabled Infectious Single Cycle (DISC) Herpes Simplex Virus (HSV) combined with a first targeting adapter, wherein said DISC HSV comprises a) a lipid bilayer comprising a tag and lacking a functional Receptor Binding Domain (“RBD”) of gD; and b) a deleted Receptor Binding Domain (“ΔRBD”) HSV comprising a HSV genome lacking a functional RBD of gD and a nucleic acid sequence encoding a heterologous polynucleotide; and wherein said first targeting adapter comprises a first binding domain with binding specificity to the tag and a second binding domain with binding specificity to a first tumor associated antigen (“TAA”) localized on the surface of a cancer cell; and b) administering a second targeting adapter to the patient, wherein said second targeting adapter comprises a first binding domain with binding specificity to the tag and a second binding domain with binding specificity to a second TAA localized on the surface of the cancer cell.

2. The method of the previous claim, wherein the second targeting adapter is administered at least 2 hours, at least 4 hours, at least 12 hours, at least 24 hours, daily, weekly, or monthly after administration of the DISC HSV and the first targeting adapter.

3. The method of either one of the previous claims, wherein: a) the first and the second TAA is selected from prostate specific membrane antigen (PSMA), TMEFF2, ROR1, KLK2, HLA-G, CD70, PD-1, PD-L1, CTLA- 4, EGFR, FLT3, HER- 2, CD19, CD20, CD3, mesothelin (MSLN), prostate stemcell antigen (PCSA), B-cell maturation antigen (BCMA or BCM ), G-protein coupled receptor family C group 5 member D (GPRC5D), Interleukin-1 receptor accessory protein (IL1RAP), delta-like 3 (DLL3), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD 138, epithelial glycoprotein-2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial adhesion molecule (EpCAM), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor a and b (FRa and b), ganglioside G2 (GD2), ganglioside G3 (GD3), epidermal growth factor receptor (EGFR), epidermal growth factor receptor vIII (EGFRvIII), ERB3, ERB4, interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), k-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), LI cell adhesion molecule (LICAM), melanoma-associated antigen 1 (melanoma antigen family Al, MAGE-A1), Mucin-16 (Muc-16), Mucin 1 (Muc-1), NKG2D ligands, cancer- testis antigen NY-ESO-1, oncofetal antigen (h5T4), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor R2 (VEGF-R2), type 1 tyrosine- protein kinase transmembrane receptor (ROR1), B7-H3 (CD276), B7-FI6 (Nkp30), chondroitin sulfate proteoglycan-4 (CSPG4), DNAX accessory molecule (DNAM-1), ephrin type A receptor 2 (EpHA2), fibroblast associated protein (FAP), Gpl 00 / HLA-A2, glypican 3 (GPC3), HA-1H, HERK-V, IL-llRa, latent membrane protein (LMPl), neural cell-adhesion molecule (N- CAM / CD56), and trail receptor (TRAIL R); b) said first TAA and said second TAA are the same; c) said heterologous polynucleotide encodes an immunomodulatory polypeptide, such as a granulocyte macrophage colony-stimulating factor (GM-CSF), Flt3L, tumor necrosis factor (TNF)-alpha, CD40 ligand (CD40L), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, G-CSF, IFN-α, IFN-β, IFN-γ, IL-20 (MDA-7), IL-21, IL-23, IL-24, or a co-stimulatory molecules, such as B7-1 (CD80) and B7-2 (CD86), a chemokine such as RANTES or a macrophage inflammatory protein (MIP) (for example, MIP-3), or an immune checkpoint blocker such as polypeptides that block signaling through PD-1, CTLA-4 or PDL-1; d) said heterologous polynucleotide encodes a prodrug converting enzyme, such as a cytosine deaminase enzyme, uracil phosphoribosyltransferase, or a HSV- 1 thymidine kinase; e) said heterologous polynucleotide encodes a matrix degrading enzyme, such as a matrix metalloproteinases, a collagenase, a gelatinase, a stromelysin, a relaxin, a bacterial collagenase or a chondroitinase ABC; f) said heterologous polynucleotide encodes a fusogenic protein, such as an envelope glycoprotein derived from gibbon ape leukemia virus (GALV), a human endogenous retrovirus W, a fusogenic F or H protein from measles virus, a vesicular stomatitis virus G protein, or a GALV fusogenic glycoprotein; and / or g) said heterologous polynucleotide encodes a messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), antisense RNA, guide RNA, microRNA (miRNA), small interfering RNA (siRNA), and cell- free RNA (cfRNA).

4. The method of any one of the previous claims, wherein the second targeting adapter is a masked targeting adapter.

5. The method of any one of the previous claims, wherein the second targeting adapter is administered intravenously, intralymphatically, or subcutaneously.

6. The method of any one of the previous claims, wherein the first targeting adapter or said second targeting adapter further comprises a half-life extender (“HLE”), such as a polypeptide that binds human serum albumin (HSA), an antibody Fc (Ig Fc), fusion of the targeting adaptor to HSA, albumin-binding domain fusion,lipidation, hyperglycosylation, PEGylation, synthetic PEG alternatives (e.g., polyglycerol, poly(thioglycidylglycerol), polysarcosine, poly(carboxybetaine), natural carbohydrates (e.g., PolyXen, HEPtune, HESylation, Chonylation, HAylation), recombinant PEG alternatives (e.g., PASylation, XTEN, PsTag, ELPylation, Ekylation), Gelatin-like polypeptides, GeneticPolymer, or HAPylation.

7. The method of any one of the previous claims, wherein the cancer is an ovarian cancer, a breast cancer, a cervical cancer, a lung cancer, a prostate cancer, a gastric cancer, a colon cancer, an esophageal cancer, a pancreatic cancer, a bile duct cancer, a cholangiocarcinoma, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, a head and neck cancer, a metastatic melanoma, a glioma, a glioblastoma, and other solid tumors, as well as a non- Hodgkin’s lymphoma (NHL), a chronic myelogenous leukemia (CML), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a multiple myeloma (MM), an acute myeloid leukemia (AML), and other liquid tumors.

8. A method of vaccinating a patient in need thereof, wherein said method comprises administering to the patient a DISC HSV combined with a targeting adapter, wherein said DISC HSV comprises: a) a lipid bilayer comprising a tag and lacking a functional RBD of gD; and b) a ΔRBD HSV comprising a HSV genome lacking a functional RBD of gD and a nucleic acid sequence encoding an antigen; and wherein said targeting adapter comprises a first binding domain with binding specificity to the tag and a second binding domain with binding specificity to a cellular protein localized on the surface of a cell.

9. The method of the previous claim, wherein the ΔRBD HSV further comprises a nucleic acid sequence encoding an antigen.

10. The method of the previous claim, whereina) the antigen is capable of causing an allergic response, such as a response caused by allergens from pollen (such as birch pollen (e.g., Bet v 1), ragweed pollen (e.g., Amb a 1), and grass pollen (e.g., Phl p 5)), dust mites, such as those from European house dust mites (e.g., Der p 1, Der p 2) and American house dust mites (e.g., Der f 1, Der f 2); animal dander, notably from cats (e.g., Fel d 1) and dogs (e.g., Can f 1, Can f 2); mold spores, including those from Alternaria alternata (e.g., Alt a 1) and Aspergillus fumigatus (e.g., Asp f 1); foods such as peanuts (e.g., Ara h 1, Ara h 2, Ara h 3), eggs (e.g., Gal d 1, Gal d 2), and milk (e.g., casein, beta-lactoglobulin); insect stings, specifically bee venom (e.g., Api m 1) and wasp venom (e.g., Ves v 5); medications like penicillin (e.g., benzylpenicilloyl polylysine) and aspirin, which, although not a protein, can trigger immune responses; and latex, such as natural rubber latex components (e.g., Hev b 1, Hev b 3, Hev b 5, Hev b 6.02); b) the antigen is capable of raising an immune response to an infection, preferably wherein the infection is caused by a HSV, a respiratory syncytial virus (RSV), adenovirus, anthrax, cholera, a measles, mumps, rubella, yellow fever, typhoid, rotavirus, BCG, varicella zoster, whole-cell pertussis, polio, influenza, Japanese encephalitis, hepatitis A, rabies, Diphtheria, tetanus, hepatitis B, meningococcal, pneumococcal, Human papillomavirus, Group B meningococcal, Haemophilus influenzae type B, ebola, SARS-CoV2, Mycobaterium tuberculosis, M. ulcerous, M. marinum, M. leprae, M. absenscens, Chlamydia trachomatis, Neisseria gonorrhoeae or Treponema pallidum; and / or c) the cellular protein localized on the surface of a cell is Nectin-1, HVEM, FLT3, prostate specific membrane antigen (PSMA), TMEFF2, ROR1, KLK2, HLA- G, CD70, PD-1, PD-L1, CTLA-4, EGFR, HER- 2, FAP, Pit1, CD19, CD20, CD3, mesothelin (MSLN), prostate stem cell antigen (PCSA), B-cell maturation antigen (BCMA or BCM ), G-protein coupled receptor family Cgroup 5 member D (GPRC5D), Interleukin-1 receptor accessory protein (IL1RAP), delta-like 3 (DLL3), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD47, CD49f, CD56, CD74, CD123, CD133, CD 138, epithelial glycoprotein-2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial adhesion molecule (EpCAM), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor a and b (FRa and b), ganglioside G2 (GD2), ganglioside G3 (GD3), epidermal growth factor receptor (EGFR), epidermal growth factor receptor vIII (EGFRvIII), ERB3, ERB4, interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), k-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), LI cell adhesion molecule (LICAM), melanoma-associated antigen 1 (melanoma antigen family Al, MAGE-A1), Mucin-16 (Muc-16), Mucin 1 (Muc-1), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), tumor- associated glycoprotein 72 (TAG-72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor R2 (VEGF-R2), type 1 tyrosine-protein kinase transmembrane receptor (ROR1), B7-H3 (CD276), B7-FI6 (Nkp30), chondroitin sulfate proteoglycan-4 (CSPG4), DNAX accessory molecule (DNAM-1), ephrin type A receptor 2 (EpHA2), fibroblast associated protein (FAP), Gpl 00 / HLA-A2, glypican 3 (GPC3), HA-1H, HERK- V, IL-llRa, latent membrane protein (LMPl), neural cell-adhesion molecule (N- CAM / CD56), and trail receptor (TRAIL R).

11. The method of any one of claims 8-10, wherein said vaccination is to treat: a) HSV, a respiratory syncytial virus (RSV), adenovirus, anthrax, cholera, a measles, mumps, rubella, yellow fever, typhoid, rotavirus, BCG, varicella zoster, whole-cell pertussis, polio, influenza, Japanese encephalitis, hepatitis A, rabies, Diphtheria, tetanus, hepatitis B, meningococcal, pneumococcal, Human papillomavirus, Group B meningococcal, Haemophilus influenzae type B, ebola, SARS-CoV2, Mycobaterium tuberculosis, M. ulcerous, M.marinum, M. leprae, M. absenscens, Chlamydia trachomatis, Neisseria gonorrhoeae or Treponema pallidum; or b) an allergic response, preferably caused by allergens from pollen (such as birch pollen (e.g., Bet v 1), ragweed pollen (e.g., Amb a 1), and grass pollen (e.g., Phl p 5)), dust mites, such as those from European house dust mites (e.g., Der p 1, Der p 2) and American house dust mites (e.g., Der f 1, Der f 2); animal dander, notably from cats (e.g., Fel d 1) and dogs (e.g., Can f 1, Can f 2); mold spores, including those from Alternaria alternata (e.g., Alt a 1) and Aspergillus fumigatus (e.g., Asp f 1); foods such as peanuts (e.g., Ara h 1, Ara h 2, Ara h 3), eggs (e.g., Gal d 1, Gal d 2), and milk (e.g., casein, beta- lactoglobulin); insect stings, specifically bee venom (e.g., Api m 1) and wasp venom (e.g., Ves v 5); medications like penicillin (e.g., benzylpenicilloyl polylysine) and aspirin, which, although not a protein, can trigger immune responses; and latex, such as natural rubber latex components (e.g., Hev b 1, Hev b 3, Hev b 5, Hev b 6.02).

12. A method of making a tagged ΔRBD HSV, comprising; a) propagating a ΔRBD HSV in a null RBD (“ØRBD”) HSV manufacturing cell in the presence of a manufacturing adapter, wherein said ΔRBD HSV comprises a HSV genome lacking a functional RBD of gD; wherein said ØRBD HSV manufacturing cell lacks a functional RBD; wherein either the ΔRBD HSV or the ØRBD HSV manufacturing cell encodes a tag; and wherein said manufacturing adapter assits infection of the ΔRBD HSV with the ØRBD HSV manufacturing cell; and b) recovering the tagged ΔRBD HSV.

13. The method of claim 12, wherein the manufacturing adapter is an artificial receptor.

14. The method of claim 12, wherein the manufacturing adapter is a bispecific antibody.

15. The method of any one of claims 12-14, wherein the ΔRBD HSV further comprises a nucleic acid sequence encoding a heterologous polynucleotide.

16. The method of any one of claims 12-14, further comprising combining the recovered tagged ΔRBD HSV with a targeting adapter to produce the DISC HSV.

17. The method of any one of claims 12-16, wherein the ØRBD HSV manufacturing cell is a Vero, a A549, a MRC, a 293 or a CHO cell.

18. The method of any one of claims 12-17, wherein a) the protein expressed on the ØRBD HSV manufacturing cell’s surface comprises Nectin, HVEM, Pit1, CD47, EGFR; or a wildtype or engineered protein derived from a non-human species; b) the manufacturing adapter is an anti-GCN4 H6 scFv fused to the AA 146-517 of human Nectin-1 (Uniprot Q15223), anti-gD or anti-gH or anti-gB scFv / VHH fused to AA 146-517 of human Nectin; or c) the manufacturing adapter is capable of binding to (a) a GCN4 transcription factor or a fragment thereof; (b) a La protein or fragment thereof; (c) a leucine- zipper moiety; (d) HSA or (e) CD3.

19. The method of any one of claims 11-18, wherein the manufacturing adapter is: a) exogenously provided; b) encoded by the ØRBD HSV manufacturing cell line; c) encoded by an extrachromosomal sequence in the ØRBD HSV manufacturing cell; or d) encoded by ΔRBD HSV.

20. The method of any one of the previous claims, wherein the ΔRBD HSV lacks:a) a polynucleotide sequence encoding amino acids 6-38 of gD; b) a polynucleotide sequence encoding amino acids 6-24 of gD; c) a polynucleotide encoding amino acids 6-24 and 38 of gD; and d) a polynucleotide encoding a polypeptide comprising full-length gD or fragment thereof, wherein said fragment comprises amino acids 6-38 of gD.

21. The method of any of the previous claims, wherein the lipid bilayer lacks: a) a polypeptide comprising amino acids 6-38 of gD; b) a polypeptide comprising amino acids 6-24 of gD; c) a polypeptide comprising amino acids 6-24 and 38 of gD; or d) a polypeptide comprising-full length gD or fragment thereof, wherein said fragment comprises amino acids 6-38 of gD.

22. The method of any of the previous claims, wherein the ΔRBD HSV comprises a polynucleotide sequence that encodes for the tag.

23. The method of the previous claim, wherein the polynucleotide sequence is inserted into the RBD of gD.

24. The method of any of the previous claims, wherein the tag is selected from GCN4, Her2, CD3, CD28, PDLI, PD-1, CTLA4, TIGIT, OX40, CD40, ICOS, 4-1BB, CD47, TIGIT, VISTA, ROR1, HLA-G, HLA-E, TROP2, Tissue Factor, Nectin-4, Fibroblast Activation Protein (FAP), Folate receptor alpha, CD19, CD22, CD30, CD33, CD38, CD70, CD79b, BCMA, PSMA, KLK2, Mesothelin, NKG2A, MAGE-A1, MAGE-A4, HSA, or any other peptide that is capable of being bound by the targeting adapter.

25. The method of any one of the previous claims, wherein the targeting adapter: a) is covalently linked to the tag; b) is a masked adapter;c) is combined with the tagged ΔRBD HSV immediately prior to the administration of the DISC HSV; d) is packaged in a single vial with the tagged ΔRBD HSV; e) is manufactured along with the tagged ΔRBD HSV; and / or f) is provided exogenously.

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